Tdt magazine 2 by femsim

Tumors of the Digestive Tract The underexposed facts and unresolved questions Edition 2, volume 1, February 2019 In this issue Diet and gastrointestinal cancer

The gut microbiome and cancer

Treatment of pancreatic cancer

Contents Volume 2 Issue 1 | TDT Feb 2019

Editor-in-chief Femke Doubrava-Simmer Assistant Editor Tanya Bisseling Associate Editors Erik Aarntzen Annemarie Boleij Manon van den Berg Alina Vrieling Editorial Office Radboudumc Department of Pathology P.O. Box 9101 6500 HB Nijmegen Internal post 824 Geert Grooteplein Zuid 10 The Netherlands T +31 (0) 24 3614361

Diet and gastrointestinal cancer The effect of prehabilitation in patients with colorectal cancer - a literature review Lieke Schaeken, Marceline Sanders, Brechtje Vanderfeesten Dipeptide distress: a brief review of oncogenic properties of aspartame Manus van Dongen, Viktor Yurevych The epigenetic and biochemical effects of vitamins b and d, and calcium on prevention of colorectal cancer development Emre Dilmen, Iris Teunissen van Manen, Daphne Roelofs The gut microbiome Microbes with a sense of tumor: a study to evaluate the effect of gut microbiota on immune checkpoint inhibition Viktor Yurevych, Manus van Dongen The impact of bacterial toxins on the hallmarks of cancer - a review Lieke Schaeken, Marceline Sanders, Brechtje Vanderfeesten The ultimate future organoid model: incorporating the nervous and immune system in an intestinal organoid model to study microbiome-gut interactions in the context of colorectal cancer Emre Dilmen, Iris Teunissen van Manen, Daphne Roelofs Treatment of pancreatic cancer Nab-paclitaxel to improve the overall survival in pancreatic cancer Emre Dilmen, Iris Teunissen van Manen, Daphne Roelofs Cracking the stromal shell: a brief insight into metabolic reprogramming of cancerassociated fibroblasts in pancreatic cancer. Viktor Yurevych, Manus van Dongen Making the step into clinical trials: nanocarrier-modulated RNAi therapeutics for pancreatic ductal adenocarcinoma Lieke Schaeken, Marceline Sanders, Brechtje Vanderfeesten

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The Effect of Prehabilitation in Patients With Colorectal Cancer A literature review Lieke Schaeken, s125623; Marceline Sanders, s4018087; Brechtje Vanderfeesten, s1025772. BMS69 Tumors of the Digestive Tract. MSc Biomedical Sciences, Radboud University.

Abstract

Introduction

Introduction: Prehabilitation programs aim to decrease postoperative complications after colorectal cancer treatment. This study aims to review the effectiveness of prehabilitation on clinical outcome in patients undergoing colon cancer treatment.

Colorectal cancer (CRC) is the worldâ&#x20AC;&#x2122;s fourth most deadly cancer (1). CRC is most frequently diagnosed in older patients between 65 and 74 years old, with the mortality rate being the highest in patients between 75 and 84 years old (2). Different treatment options are currently being applied, however, surgery remains the golden standard in CRC patients. Surgery, especially in older patients, carries the risk of postoperative complications, which in turn lead to higher mortality rates, a longer hospital stay and a higher risk of readmission (3).

Methods: A systematic literature review of all comparative studies on prehabilitation patients undergoing colorectal cancer surgery was performed in PubMed screening publications within the last ten years. Results: Eight studies were included in this study. The 6-min walk test (6MWT) was used in 5 studies to measure functional capacity as primary outcome. Three studies used length of stay as primary outcome. The studies included four unimodal-, one bimodal- and three trimodal prehabilitation programs. Conclusion: Trimodal prehabilitation appears to be the most promising approach in decreasing postoperative complications and improving the fitness of patients after treatment. However, more homogeneity among studies is needed to provide more evidence for multimodal prehabilitation to implement it as standard care in the future. 7-2-2019 Radboud University Nijmegen, The Netherlands

Development of approaches to improve the postoperative risks is a high priority. Surgery brings a 20 to 40% reduction in physiological and functional capacity, even in the absence of complications (4). Since CRC patients are often elderly patients, these patients already have a compromised physical function (sarcopenia) and nutritional status. Cancer and its treatment worsen this even further and this can lead to fatigue, loss of appetite, nausea, and pain. There is evidence that a compromised nutritional status and sarcopenia are positively correlated with postoperative complications and mortality after CRC surgery (5). The role of preoperative interventions, better known as prehabilitation, with regard to clinical outcomes in patients with CRC undergoing treatment are not clarified.

Prehabilitation programs are designed to enhance functional capacity and psychological health and aim to enable patients to withstand an upcoming stressor, e.g. surgery (6). The programs used can be of unimodal, bimodal, or multimodal origin. Multimodal prehabilitation often is multidisciplinary involving physical exercise, nutritional intervention and/or anxiety reduction. Overall, prehabilitation should diminish the postoperative complications (7). The aim of this study is to summarize evidence on effectiveness of prehabilitation, including physical exercise, possibly combined with nutritional counselling and anxiety reduction strategies, on clinical outcome in patients undergoing colon cancer treatment.

Methods An electronic literature search was conducted through the PubMed database in February 2019. The search strategy was based on the following terms: colorectal cancer; anticancer treatment; prehabilitation/physical activity/ nutritional intervention/anxiety reduction; and clinical outcome. The full search strategy is described in appendix A. A filter was added to merely obtain articles published in the last 10 years. Eligible studies were limited to those

that assessed the effect of prehabilitation, existing of physical and/or nutritional interventions and anxiety reduction, on clinical outcome in patients undergoing colon cancer treatment. Editorials, review articles, nonEnglish articles and non-relevant articles were excluded.

Results Study selection The initial digital search in PubMed identified 155 articles. After reading the title and abstract of these articles, 133 articles were excluded, and 22 full articles were assessed for inclusion. Finally, eight prehabilitation studies met the inclusion criteria and were included in this review. References and citations of the included studies yielded no additional studies. Figure 1 shows the flow diagram for the selection of articles. Of the eight studies that were included in this review (8â&#x20AC;&#x201C;15), four were randomized controlled trials (10,13,14), of which one was a pilot study (9). One study was a nonrandomized control trial (15). Moreover, two prospective observational cohort studies (11,12) and one observational pilot study (8) were included in this review.

â&#x17E;˘ Figure 1. Flow chart for the selection of articles for this literature review

Study characteristics Details of study characteristics are reported in Table 1. Five studies reported the mean age, which varied from 54.3 to 71.1 years. Three studies reported median age, which varied from 67 to 81 years. In two studies (13,14), the prehabilitation group was compared with a rehabilitation group as control. Three studies looked at differences between prehabilitation and standard care (8,12,15) and three other studies compared different exercise intensities (9–11).

Composition of prehabilitation program Prehabilitation programs used in the studies were either tri-, bi- or unimodal. A trimodal prehabilitation approach was defined as physical exercise, nutrition counseling and anxiety reduction strategies, and was assessed in three studies (8,13,14). One study evaluated a bimodal prehabilitation program with nutrition in addition to physical exercise (12), while four studies used physical prehabilitation exclusively (9–11,15).

Outcome measures One of the primary outcome measures assessed was walking capacity as measured by the 6-minute walking test (6MWT) (8,10,13– 15). The 6MWT evaluates the ability of an individual to maintain a moderate level of walking for a period of time, reflecting activities of daily living (16). Another primary outcome measure was length of hospital stay (LOS) (9,11,12). Secondary outcome measures that were assessed in the included studies are quality of life (QoL) (8,14,15), parameters of feasibility (9), physical and mental recovery (11,15) and re-operations or re-admissions (11).

Unimodal exercise

prehabilitation:

physical

Four of the studies included in this review evaluated prehabilitation with preoperative

physical activity as intervention before colorectal surgery (9–11,15). Dronkers et al (9) investigated the effect of a short-term intensive preoperative exercise program for elderly patients in a randomized controlled pilot study. The program consisted of resistance, inspiratory, and aerobic exercises, executed under supervision in a training center for a mean of 5 training sessions (intervention group; n=22), and was compared to homebased exercise advice (control group; n=20). The intensive therapeutic exercise program improved the respiratory function (P<0.01) but did not significantly change preoperative aerobic capacity and functional capacity of patients (P>0.05). Also, the postoperative course (postoperative complications; LOS) of both groups did not differ significantly (P>0.05). Charli and colleagues (10) evaluated the extent to which a structured prehabilitation regimen of stationary cycling and strengthening (bike/strengthening group; n=58) optimized recovery of functional walking capacity after surgery, compared with a simpler regimen of walking and breathing exercises (walk/breathing group; n=54) in patients scheduled for colorectal surgery. They found no significant differences between the groups in mean functional walking capacity, as measured by the 6MWT, over the prehabilitation period or at postoperative follow-up (P>0.05). Onerup et al. (11) performed a prospective observational cohort study with 115 patients, where they assessed self-reported levels of preoperative physical activity (PA) and compared these to measures of recovery. They found that the preoperative level of PA was associated with a faster selfassessed physical recovery 3 weeks after colorectal cancer surgery (P=0.038), compared to physical inactivity. They could not detect any significant associations between the primary outcome measure length of hospital stay or any of the other outcome measures (self-assessed mental recovery, re-admittances and re-

RCT (pilot)

RCT

Prospective observational cohort study

Non RCT

Prospective observational cohort

Observational pilot

RCT

Dronkers (9)

Carli (10)

Onerup (11)

Lin (15)

Chia (12)

Li (8)

BousquetDion (13)

Gillis (14)

Physical exercise + nutrition + anxiety

Physical exercise and nutrition

Physical exercise

Com ponents of prehab program

Mean (SD): Prehab: 65.7 (13.6); rehab: 66.0 (9.1)

Median (IQR): prehab: 74 (67.578); rehab: 71 (54.5-74.5)

Mean (SD): control: 66.4 (12); prehabilitation: 67.4 (11)

Median (range): control: 81 (75– 97); Intervention 79 (65–93)

Mean (SD): supervisedexercise group: 59.0 (9.5); usual care group : 54.3 (10.6)

Median: group 1: 75; group 2: 71; group 3-4: 67

Mean (SD): bike/strengthening group: 61 (16); w alk/breathing group: 60 (15)

Mean (SD): intervention: 71.1 (6.3); control: 68.8 (6.4)

Age, y

Prehabilitation vs. rehabilitation

supervised prehabilitation vs. standard rehabilitation

prehabilitation vs. control

2 w k preoperative + 2–6 w k postoperative vs. standard of care

Combined aerobic and resistance exercise program (supervisedexercise group) vs. usual care

Physical activity (level 1-4)

bike/strengthening group vs. w alk/breathing group

short-term intensive therapeutic exercise program vs. home-based exercise advice

Study design

38 (prehab); 39 (rehab)

41 (prehabilitation); 39 (rehabilitation)

42 (intervention); 45 (control)

57 (intervention); 60 (control)

21 (supervisedexercise group); 24 (usual care)

18 (group 1); 74 (group 2); 17 (group 3-4)

58 (bike/strengthening group); 54 (w alk/breathing group)

22 (intervention); 20 (control)

Primary: functional w alking capacity (6MWT); secondary: self reported physical activity; LOS; complications rate

Primary: functional w alking capacity (6MWT). secondary: self reported physical activity, LOS, health-related QoL

LOS, complications, 30-day mortality, recovery of functional status

Primary: QoL; secondary: muscle strength, cardiorespiratory fitness (6MWT), emotional distress, physical activity, fatigue, and sleep quality

Primary: LOS; secondary: return to w ork, physical and mental recovery, re-operations and readmissions

Functional w alking capacity (6MWT)

Preoperative functional capacity (respiratory muscle endurance) and postoperative course, LOS

Outcom e parameter

Prehabilitation did not further enhance postoperative w alking capacity w hen compared to standard rehabilitation. LOS and complications rate w as similar betw een both groups.

Postoperative complication rates and the hospital LOS w ere similar. Prehab patients had better postoperative w alking capacity at both 4 w eeks (p = 0.01) and 8 w eeks (p < 0.01). At 8 w eeks, significantly more prehab patients w ere recovered compared w ith the control group (p < 0.01).

Significantly shorter LOS in patients receiving the intervention (P=0.029) and full functional recovery at 6 w eeks (100%, not significant from control). No differences in mortality and complications (P>0.05).

Compared w ith usual care, the supervised exercise demonstrated larger effects than usual care on physical activity level. Significant time effects w ere found for secondary outcomes: hand-grip strength, cardio-respiratory fitness (6MWT), and physical activity level (P<0.05)

Preoperative PA w as associated w ith a higher chance of feeling highly physically recovered 3 w eeks after surgery, compared to physical inactivity (P=0.038). No statistically significant associations w ere seen w ith LOS, mental recovery, re-admittances or w ith re-operations.

No differences betw een the groups in mean functional w alking capacity over the prehabilitation period or at postoperative follow ‐ up.

Respiratory muscle endurance increased in the preoperative period in the intervention group compared to the control group (P<0.01). There w as no significant difference in postoperative complications and length of hospital stay betw een the tw o groups.

Results

Primary: functional w alking Functional w alking capacity in the prehabilitation capacity (6MWT); secondary: self group increased compared to the rehabilitation reported physical activity, LOS, group (P=0.006). No significant difference in self complications rate, health-related reported physical activity, complication rates and QoL, anxiety, depression LOS betw een both groups (P>0.05). SD: standard deviation; IQR: Interquartile range; RCT: randomized controlled trial; prehab: prehabilitation; rehab: rehabilitation; LOS: length of stay; QoL: quality of life; 6MWT: 6-min w alk test; SF-36: Medical Outcomes Study 36-Item Short-Form Health Survey; PA: Physical activity

Design

First author

Tab le 1. Summary of the characteristics of the 8 studies included in this literature review.

operations) (P>0.05). Lin et al (15) evaluated the effects of supervised-exercise intervention with usual care in a nonrandomized controlled trial of patients with CRC undergoing chemotherapy. The supervised-exercise group received a combined aerobic and resistance exercise program for 12 weeks. Significant interactions between intervention and time were observed for the physical activity level and role functioning and pain subscales of QoL (P<0.05). Role functioning is an important part of health-related quality of life (17). The time main effects were significant for the secondary outcomes: hand-grip strength, cardiorespiratory fitness (measured by 6MWT), and physical activity level (P<0.05).

Bimodal prehabilitation: physical exercise & nutrition The evaluation of the impact of physical activity and nutrition on clinical outcome was performed in one study included in this review (12). Chia and colleagues (12) compared elderly patients who got two weeks prehabilitation and two to six weeks rehabilitation consisting of exercise and nutrition (n=57) compared with a group receiving standard care (n=60). These patients all received a major colorectal surgery. Patients receiving the intervention had a significant shorter LOS (P=0.029) and full functional recovery at 6 weeks (100%), however, this was not significantly different from the control group (95.7% ; P=0.157). There were no significant differences in complications or 30-day mortality (P>0.05).

Trimodal prehabilitation: exercise, nutrition & anxiety

physical

The effect of a trimodal intervention program on clinical outcome was assessed in three studies (8,13,14), where functional walking capacity measured by 6MWT was assessed as the primary outcome, and besides selfreported physical activity and LOS were evaluated. Patients were offered a home-

based intervention combining physical training sessions, nutrition counseling and anxietyreduction strategies. Physical training consisted of 3 training sessions per week of either 20 minutes of aerobic exercise and 20 minutes of resistance exercise (14), or 30 minutes (8) of aerobic and resistance exercise (8). In the study of Bousquet-Dion et al. (13), physical training consisted of 3-4 days per week 30 minutes of moderate intensity aerobic activity and 3-4 times per week resistance exercises + once per week supervised session. The nutritional component was similar in all three studies and consisted of supplementation of whey protein at 1.2 g/kg of body weight per day, and this supplement was preferably ingested within one hour after their training session. Li et al (8) compared the prehabilitation group with a control group that received no intervention, whereas the other two studies (13,14) compared the prehabilitation group with a control group that received the same intervention for 8 weeks post-surgery (rehabilitation). Two studies found an increased postoperative walking capacity in the prehabilitation group compared to the control group (P<0.05) (8,14), whereas one study did not observe any increase in walking capacity (P>0.05)(13). Neither one of the three studies showed a significant difference in hospital LOS or postoperative complication rate between the intervention and control groups (P>0.05). Two studies that evaluated health-related quality of life, measured with the 36-Item Short Form Survey, did not find any significant effects of intervention (P>0.05) (8,14).

Discussion This review aimed to assess the effect of prehabilitation on several parameters of clinical outcome in recovery of CRC patients after surgery. Results of this study show that all three assessed types of prehabilitation (unimodal, bimodal, trimodal) were capable of

improving clinical outcome in CRC patients after surgery. Trimodal prehabilitation, consisting of a combination of physical exercise, nutritional counseling, and anxiety reduction, was found to be the most promising for the optimal recovery after CRC surgery.

Unimodal prehabilitation A restricted amount of significant differences were found regarding to the effect of physical prehabilitation on clinical outcome after surgery, compared to the control group. It has been previously shown that 6MWT is strongly correlated with surgical outcome (19). Nevertheless, conflicting results were reported regarding functional walking capacity as measured by 6MWT (10,15). Exercise causes physiological stress, and this causes the cell to adapt to stress and therefore prepare for the stress from the surgery (20). However, two studies assessing LOS could not detect any significant differences in the high physical activity groups (9,11). It is suggested that outcomes might be improved if the frequency, intensity and intervention time are higher and longer, as suggested in a study investigating spinal surgery (18).

Bimodal prehabilitation A bimodal prehabilitation strategy comprising a combination of physical exercise and nutritional counseling seems an appropriate approach to target the muscular system. Other studies showed that CRC patients display increased muscle protein breakdown as a result of an inflammatory status (20). If muscle mass can be increased by prehabilitation with nutrition and physical activity it might prevent adverse postoperative outcomes (21). Furthermore, the intervention of a surgery causes chronic inflammation which possibly leads to more complications. Physical exercise and specific dietary changes can help to decrease the chance of complication by decreasing the inflammation and thereby increase the revalidation time (20). Results of

the bimodal prehabilitation study performed by Chia et al. show a significantly shorter LOS in the intervention group compared to the control group (P=0.029). However, no significant differences were found regarding full functional recovery, complication rate, or 30-day mortality (P>0.05). The effect of nutritional counseling might be greater if the doses of supplements are given more frequently and over a longer time period (21).

Trimodal prehabilitation To prevent the chance of sarcopenia and thereby the chance of complications, muscle mass must be improved. Muscle mass can increase by combining PA and nutrition (21). However, besides this, anxiety can also have a significant impact on complications after surgery and LOS. Mood distress is a common phenomenon in patients with cancer and also has a big impact on the quality of life. Psychological wellness can help to provide the optimal effect of exercise and nutritional interventions (20). Therefore, we can suggest that providing a trimodal prehabilitation to CRC patients can be the optimal intervention to improve clinical outcome after cancer treatment. We found that two studies assessing the effect of trimodal prehabilitation on surgery outcome found an increase in the postoperative walking capacity in the prehabilitation group compared to the control group (P<0.05) (8,14), whereas one study did not observe any increase in walking capacity (P>0.05)(13). Neither one of the three studies included in this review showed a significant difference in hospital LOS or postoperative complication rate between the intervention and control groups (P>0.05), whereas healthrelated quality of life, measured with the 36Item Short Form Survey, did not show any significant effects of intervention in two studies including this outcome parameter(P>0.05) (8,14).

Limitations A limited number of studies were published that used prehabilitation as an intervention in CRC patients. Therefore, this review was restricted to analysis and assessment of just eight studies. Moreover, most studies included in this review are performed with elderly people with an age of >60 years. However, one study does not meet this norm (15). This impedes comparison of the effect of intervention on patients with other studies. Moreover, the included studies had a relatively low number of subjects in each study arm (ranging from 17 to 60 patients), which impairs the power of the analysis of the studies. If there is a lack of power, it is not possible prove the real effect of the intervention. Furthermore, the studies used in this review were relatively heterogeneous regarding to the duration of the intervention, frequency of exercise, and nutrition and supplements recommended. This makes fair comparison between studies more difficult. Besides, the intervention was not in every study compared to standard care as a control, which makes it difficult to assess the

real effect of prehabilitation. Different study types were included in this review, ranging from randomized control trials, nonrandomized controlled trials, observational prospective cohort studies, to pilot studies. Furthermore, the chance of bias is high, since CRC patients often are of higher age and patients with specific diseases are excluded.

Conclusion The large heterogeneity of studies regarding prehabilitation limits the possibility of standard implementation into clinical use. Trimodal prehabilitation seems the most promising approach in decreasing postoperative complications and improving the postoperative fitness of patients after treatment. CRC patients with low levels of physical activity and poor dietary habits are most likely to improve their functional status with this approach. More homogeneity among studies is needed to provide more reliable evidence for the implementation of multimodal prehabilitation as standard preoperative care in CRC patients in the future.

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Predicts Adverse Postoperative Outcome After Colorectal Cancer Surgery. Ann Surg. 2015 Feb;261(2):345–52. Wong C-L, Lee HH-C, Chang S-C. Colorectal cancer rehabilitation review. J Cancer Res Pract. Elsevier; 2016 Jun 1;3(2):31–3. Le Roy B, Selvy M, Slim K. The concept of prehabilitation: What the surgeon needs to know? J Visc Surg. 2016 Apr;153(2):109–12. Li C, Carli F, Lee L, Charlebois P, Stein B, Liberman AS, et al. Impact of a trimodal prehabilitation program on functional recovery after colorectal cancer surgery: a pilot study. Surg Endosc. 2013 Apr 9;27(4):1072–82. Dronkers J, Lamberts H, Reutelingsperger I, Naber R, Dronkers-Landman C, Veldman A, et al. Preoperative therapeutic programme for elderly patients scheduled for elective

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Appendices Appendix A: Search strategy (((((("Exercise"[Mesh] OR exercise[tiab] OR Exercises[tiab] OR Physical Activity[tiab] OR Physical Activities[tiab] OR Physical Exercise*[tiab] OR Acute Exercise*[tiab] OR Aerobic Exercise*[tiab] OR Exercise Training*[tiab] OR physical intervention[tiab] OR resistance exercise*[tiab] OR exercise program[tiab])) OR ("diet therapy" [Subheading] OR nutritional support[tiab] OR nutrition counseling[tiab] OR nutritional

16.

17.

18.

19.

20.

21.

Exercise Program and Usual Care in Patients With Colorectal Cancer Undergoing Chemotherapy. Cancer Nurs. 2014;37(2):E21– 9. Eng JJ, Chu KS, Dawson AS, Kim CM, Hepburn KE. Functional walk tests in individuals with stroke: relation to perceived exertion and myocardial exertion. Stroke. 2002 Mar;33(3):756–61. Anatchkova MD, Bjorner JB. Health and role functioning: the use of focus groups in the development of an item bank. Qual Life Res. NIH Public Access; 2010 Feb;19(1):111–23. Nielsen PR, Jørgensen LD, Dahl B, Pedersen T, Tønnesen H. Prehabilitation and early rehabilitation after spinal surgery: randomized clinical trial. Clin Rehabil. 2010 Feb 26;24(2):137–48. Pecorelli N, Fiore JF, Gillis C, Awasthi R, MappinKasirer B, Niculiseanu P, et al. The six-minute walk test as a measure of postoperative recovery after colorectal resection: further examination of its measurement properties. Surg Endosc. 2016 Jun 27;30(6):2199–206. Minnella EM, Carli F. Prehabilitation and functional recovery for colorectal cancer patients. Eur J Surg Oncol. Elsevier; 2018 Jul 1;44(7):919–26. Looijaard SMLM, Slee-Valentijn MS, Otten RHJ, Maier AB. Physical and Nutritional Prehabilitation in Older Patients With Colorectal Carcinoma. J Geriatr Phys Ther. 2018;41(4):236–44.

counseling[tiab])) OR (Prehabilitation[tiab] OR prehab[tiab])) AND (colorectal cancer*[tiab] OR bowel cancer[tiab] OR colon cancer[tiab])) AND ("therapy" [Subheading] OR Radiotherapy[tiab] OR Chemotherapy[tiab] OR Surgery[tiab] OR surgical[tiab] OR Drug therapy[tiab] OR esophagectomy[tiab] OR oesophagectomy[tiab] OR pancreatectomy[tiab] OR colectomy[tiab])) AND ("Treatment Outcome"[Mesh] OR treatment tolerance[tiab] OR treatment completion[tiab] OR complication*[tiab] OR infection*[tiab] OR overall survival[tiab] OR LOS[tiab] OR 6MWT[tiab] OR physical functioning[tiab])

DIPEPTIDE DISTRESS: A BRIEF REVIEW OF ONCOGENIC PROPERTIES OF ASPARTAME. Manus van Dongen, Viktor Yurevych Radboud University Medical Centre/Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands

Over the last decades artificial sweeteners have become increasingly popular in the food industry. There is an increased demand for sugar replacements in many foods. One of such sugar replacements is aspartame. Though it is widely used today, it gained a reputation of being unhealthy in the public eye, partly caused by claims of its carcinogenicity. This article provides a brief review of carcinogenic potential of this sweetener.

Artificial sweeteners are used to reduce the caloric content of foods and beverages. The molecules used as artificial sweeteners are less energy rich, but still cause a sensation of sweetness. Aspartame is a synthetic dipeptide artificial sweetener discovered by James Schlatter in 1965 in a search for gastric ulcer drugs (Nakanishi, Kamikubo, & Matsuno, 1985). It is 200 times sweeter than glucose, allowing for its usage in small doses and thus making the product almost non-caloric (Handbook of pharmaceutical excipients, 2003). It was approved for commercial use in the United States in 1981 and the European Union in 1994 and has become widely used ever since in over 6000 food and hygiene products and about 500 pharmaceutical products, and is estimated to be consumed by over 200 million people worldwide (Aspartame Information Center, 2005). Aspartame is a methyl ester of L-aspartyl-Lphenylalanine (Figure 1) stable under dry conditions in temperatures from 30°C to 80°C (Nakanishi et al., 1985). Upon ingestion, it is metabolized by esterase and peptidase in the gut into 3 amino acid isolates, yielding 50% of phenylalanine, 40% of aspartic acid and 10% of methanol. Phenylalanine is metabolized in the liver, undergoing a conversion into L-tyrosine by phenylalanine hydroxylase, followed by conversion of L-tyrosine into L-DOPA or L-3,4dihydroxyphenylalanine by tyrosine hydroxylase. LDOPA is further converted into physiological catecholamines dopamine, noradrenaline and adrenaline by decarboxylase (Choudhary & Pretorius, 2017). Aspartic acid is also converted in the liver into L-lysine and L-methionine by aspartate kinase. At high concentrations, aspartic acid may cross the blood–brain barrier and bind to the Nmethyl-D-aspartate (NMDA) receptor, among other glutamate binding sites, resulting in an influx of calcium ions into cells. This causes increased firing of action potentials and higher rates of neuron depolarization can potentiate neurodegeneration (Humphries, Pretorius, & Naude, 2008). The enzyme responsible for metabolism of methanol (CH3OH) varies depending on the species. In primates, methanol is metabolized into formaldehyde (HCHO) in the liver by alcohol dehydrogenase. 10

Formaldehyde is then oxidized into formic acid (HCOOH) by formaldehyde dehydrogenase. An accumulation of folic acid could cause metabolic acidosis and tissue injury, potentially linked to low liver folate concentrations in humans (Johlin, Fortman, Nghiem, & Tephly, 1987).

Figure 1: Structure of aspartame and its metabolites (in color) (Taken from Choudhary & Pretorius, 2017). The safety of aspartame and its metabolites was a subject of frequent discussion. Its potential carcinogenicity too remains an important concern. The current knowledge on the safety of aspartame is mostly based on animal studies - and suggests that even the recommended safe dosages of aspartame might not be safe for human consumption (Choudhary & Pretorius, 2017).

Preclinical evidence Food additives and other food ingredients are required to be extensively screened before implementation. These tests include cellular assays such as the Ames test. This test is used to measure the mutagenicity of a compound. It is also required to do animal testing. These usually consist of rodent bioassays. A number of in vitro genotoxicity studies have been performed on aspartame, including bacterial, chromosomal aberration and DNA repair tests. From the combined results of the bacterial studies a conclusion can be drawn that aspartame is not a

bacterial mutagen. However, the possibility of induction of oxidative or cross-linking damage to the bacteria in these tests cannot be excluded. Apart from the Ames tests, the possible nitrosation of aspartame and its potential to form mutagens has also been investigated. Assays conducted on S. typhimurium showed that nitrosation of aspartame resulted in the formation of nitroso-species with mutagenic activity towards the bacteria. However, it was concluded that the nitrosation products of aspartame would not contribute significantly to the endogenous burden of nitrosation products when translated to a human setting (Rencuzogullari et al., 2004). Phenylalanine was also tested for mutagenicity using E. coli species, where a small increase in base-substitution mutations was reported, in contrast with the negative results reported for aspartame in S. typhimurium. Based on the available bacterial studies, it was concluded that aspartame is not a bacterial mutagen (Schmid, GĂśggelmann, & Bauchinger, 1986). However, currently available bacterial studies did not include mutagens able to act specifically on AT base-pairs, though such activity would be detected in mammalian cell tests for chromosome damage. There, chromosome damage was linked to formaldehyde, a secondary metabolite of aspartame and a potent DNA cross-linking agent. However, there is no evidence to suggest that these findings are translatable onto a human model, as the trials have been performed in mammalian cell cultures where chromosomal damage could be attributed to formations of small amounts of intracellular formaldehyde. Similarly, aspartame-derived formaldehyde was implicated in disrupting DNA repair through unscheduled DNA synthesis (UDS) in Syrian hamster embryo cells (Hamaguchi & Tsutsui, 2000), through the results obtained in that study may not be representative in vivo due to high metabolic activity of the chosen cell model. Thus far, no in vitro studies were able to clearly indicate the potential of aspartame to induce mutations in bacteria or contribute to chromosomal damage or primary DNA damage in mammalian cells. Several in vivo studies of carcinogenic capabilities of aspartame has been performed on rodents since the 1980s. A number of studies have looked for health effects in aspartame-fed lab animals, which often exceeded the maximum dose of 4,000 mg/kg per day approved for human consumption over their lifetimes. These studies have not found any health problems that are consistently linked with aspartame (Magnuson et al., 2007). Two large-scale studies published by a group of Italian researchers at the Ramazzini Institute are often cited as proof of the carcinogenic effects of aspartame. They suggested that very high doses of aspartame might increase the risk of some bloodrelated cancers (leukemias and lymphomas) in rats.

In those studies, Sprague-Dawley rats that were fed aspartame from 8 weeks of age throughout their lifetimes. The results demonstrated a statistically significant increase in incidence of leukemias and lymphomas in rats of both sexes, an increase in pelvic lesions in female rats and malignancies of peripheral nerves in males (Soffritti et al., 2006). A later study confirmed these findings, demonstrating further that the incidence of the mentioned malignancies increased upon prenatal exposure to aspartame (Soffritti, Belpoggi, Esposti, Falcioni, & Bua, 2008). A third subsequent study performed on Swiss mice that were exposed to aspartame from a prenatal stage demonstrated a statistically significant dose-related increase in incidences of liver and lung cancer (P<0.05) in male mice (Soffritti et al., 2010). Given such results, the authors concluded that aspartame can be considered a transspecies carcinogen that could likely be carcinogenic to humans consuming it in large quantities (Soffritti et al., 2014). However, an in-depth evaluation of the above studies by the European Food Safety Authority (EFSA) uncovered a number of potential flaws ranging from study design to the interpretation of results. With an increasing number of publications contradicting the results of the Ramazzini study, it can be concluded that there is no solid evidence this far proving that aspartame is carcinogenic for animals (Magnuson et al., 2007).

Epidemiological evidence The epidemiological data on aspartame is still limited and not consistent. One epidemiological study was conducted in nurses and health professionals (Schernhammer et al., 2012). The cohort of nurses was first started in 1976 and included 121,701 females aged between 30 and 55 years. The cohort with health professionals was started in 1986 and included 51,529 males aged between 40 and 75 years. Follow-up for disease incidence was every 2 years and dietary intake was reassessed every 4 years. The dietary questionnaires looked at frequency of consumption of around 130 foods. The assessment of artificial sweeteners was mostly done by looking at soft drink consumption. The different types of soft drinks were all assessed. From the consumption of diet soda the aspartame consumption could be calculated. The disease incidence assessed all cancer, but relations were found for blood cancers. The cohorts were followed over 22 years. With both cohorts of men and women pooled an elevated risk for leukemia was found (RR: 1.42; 95% CI: 1.00, 2.02) in participants who consumed â&#x2030;Ľone serving of diet soda per day. For non-Hodgkin lymphoma an increased risk was only found in males with consumption of â&#x2030;Ľone serving of diet soda per day 11

(RR: 1.31; 95% CI: 1.01, 1.72). For this male consumption group the risk for multiple myeloma was also increased (RR: 2.02; 95% CI: 1.20, 3.40). So these are some important and unexpected sex differences. Looking at the methodology of the study the male and female cohorts are entirely different groups. It is not one cohort of males and females. The difference in results might be influenced by these differences in groups. Both groups do work in the medical field, but there might still be different occupational influences. A possible explanation which is presented by the authors is that males have higher alcohol dehydrogenase type 1 enzymatic activity. From aspartame methanol can be formed by hydrolysis in the gut. This methanol can then be converted to formaldehyde, a class 1 carcinogen, by alcohol dehydrogenase (Humans, 2006). Mechanistically this explanation makes sense for the difference between sexes. Another limitation of this study is the inaccuracy for aspartame intake. In this study only diet soda was used as a source of aspartame, but there are of course many different sources. These sources are now missed. Since this study was started so early it has effectively looked at aspartame usage since it was first allowed to be used in food. Therefore, it assessed the lifetime exposure to aspartame of the participants in this study. Different types of cancers are not really mentioned in this study though the authors have published a different study using these cohorts to look at the risk for pancreatic cancer. In this study no association between diet soda consumption and elevated risk of pancreatic cancer was found. Unfortunately the risk for other cancers is not mentioned in both studies so it is unknown whether there is an association in this area. In an Italian case-control study the effect of artificial sweeteners on gastric, pancreatic and endometrial cancer was studied. Cases and controls were interviewed during their hospital stay with a questionnaire. This questionnaire determined the usual diet in the 2 years before diagnosis. The results showed no relation between aspartame consumption and any of the three cancers. Recall bias obviously plays a role here, so this could influence the results. Furthermore, the diet was only assessed for 2 years prior to diagnosis and this is a major limitation. These carcinogenic effects are often seen over long times and in this study the early diet of these cases is unknown. Another large study looked at hematopoietic and brain cancers (Lim et al., 2006). In this study 473,984 participants aged 50 to 71 years old filled in a questionnaire. These questionnaires were sent from 1995 to 1996. The cancer incidence in these participants was determined from registries up to December 31, 2000. The results showed no association between aspartame intake and hematopoietic and brain malignancies. In this study the time between the diet assessment and cancer incidence assessment was 12

quite short. This is especially because the participants were fairly old. Again we know nothing about diet during their youth. Because aspartame was only approved in 1981 the length of time wherein these participants could be exposed is limited. So in essence this study looks at later stage in life, short term exposure to aspartame. This could underestimate the risk. To summarize, there is clinical evidence supporting that aspartame could increase the risk of cancer, specifically hematopoietic cancer, but there is also evidence suggesting there is no relation.

Conclusion Since it was first approved for use in the United States, the safety of aspartame has been a subject of debate both in industry and in academia. To date, however, there has not been any evidence of aspartameâ&#x20AC;&#x2122;s carcinogenicity that could withstand scientific criticism. The sweetener was demonstrated not to be a mutagenic agent both in bacterial and mammalian cells, and the results of the largest studies linking aspartame consumption to cancer are contradicted by an ever increasing amount of findings proving the contrary. The epidemiological evidence around aspartame and cancer is still limited. Some studies contradict each other completely. Generally evidence seems to weigh more to side of no increased cancer risk, but no conclusions can be made. Many studies are limited by their short follow-up. The long-term effects of aspartame have not been studied sufficiently. Even though aspartame has been on the market for decades, there remains a need for centralized studies monitored and validated by independent agencies to put a stop to the debate and controversy. However, some regulatory bodies like the EFSA accept the conclusion that aspartame is a carcinogen based on the available evidence, seeing no further need to review opinion on the safety of this sweetener.

References Aspartame Information Center. (2005). Aspartame Information Center homepage. Retrieved from http://www.aspartame.org Choudhary, A. K., & Pretorius, E. (2017). Revisiting the safety of aspartame. Nutr Rev, 75(9), 718-730. doi:10.1093/nutrit/nux035 Hamaguchi, F., & Tsutsui, T. (2000). Assessment of genotoxicity of dental antiseptics: ability of phenol, guaiacol, p-phenolsulfonic acid, sodium hypochlorite, p-chlorophenol, m-cresol or formaldehyde to induce unscheduled DNA synthesis in cultured Syrian hamster embryo cells. Jpn J Pharmacol, 83(3), 273-276. Handbook of pharmaceutical excipients. (2003). Fourth edition / edited by Raymond C. Rowe, Paul J.

THE EPIGENETIC AND BIOCHEMICAL EFFECTS OF VITAMINS B AND D, AND CALCIUM ON PREVENTION OF COLORECTAL CANCER DEVELOPMENT Emre Dilmen S4594525 – Iris Teunissen van Manen S4579798 – Daphne Roelofs S4589785

ABSTRACT Global colorectal cancer (CRC) rates are expected to further increase over the next decade. It is suggested that this may be due to a more western lifestyle in low- and middle-income countries. Thus, lifestyle factors are considered important risk factors for developing CRC, one of such factors being diet. Vitamins and minerals derived from a healthy diet may have a protective effect on cancer development using biochemical and epigenetic mechanisms. This review elaborates on the epigenetic and biochemical effects vitamin B and D, and calcium have on the development of CRC. Based on these mechanisms, it can be recommended to ensure the daily intake of these vitamins and minerals to prevent development of CRC. However, it is not possible to give general recommendation due the heterogeneity in tumours, patients and healthy people. Dietary recommendation to decrease the risk of CRC development could therefore be improved by giving personalised advice based on information about a patient's medical history and genetic background.

INTRODUCTION In 2018, 14000 patients were diagnosed with colorectal cancer (CRC) in the Netherlands. With this incidence, CRC reaches the top three of most occurring cancers in both men and woman, making it 12% of all cancers. Five years after diagnosis, 65% of the patients are still alive, which is significantly more compared to twenty years ago, when the five-year survival was 50% (2). Whereas the incidence of CRC now seems to stabilise in the Netherlands, and has even been decreasing over the past few years, the global population of CRC patients is still rising and is expected to further increase by 60% in the next ten years. Especially in low- and middle-income countries, the incidence and mortality rates are increasing rapidly. This pattern of incidence correlates with the adaptation of a more western lifestyle and could therefore highlight the importance of lifestyle factors that can contribute to the development of CRC (3). In 2015, a paper by Tomasetti et al. was published that stated that environmental factors contribute for 35% to the development of cancer, leaving the other 65% due to “bad luck”, or random mutations during cell divisions (4). But what are the environmental contributors to CRC, and how

can the development of this cancer be prevented by adapting to a healthier lifestyle? First, many lifestyle factors that are generally considered to be risk factors for other cancer types, including CRC, are smoking, obesity, and physical inactivity. However, especially in CRC, diet is thought to have a significant effect on cancer development. Dietary risk factors include alcohol and red, processed meat, whereas the consumption of whole grains, fibre rich products, low-fat dairy and fruit and vegetables are believed to have a protective effect (1, 5). This raises the question about which components derived from these products are responsible for these protective effects, and by which mechanism they can prevent the development of CRC. A possible answer to this question may be the involvement of epigenetic regulators and other mechanisms that result in differential expression of tumour suppressor genes and oncogenes (6). Previous research has identified which components of a healthy diet are associated with a decreased risk of CRC. Among others, these include vitamins B and D, and calcium. The mechanism in which these vitamins and minerals contribute to cancer prevention has partly become evident from experimental data. However, results from clinical trials using oral supplementation are not always consistent (1). This review elaborates on the epigenetic and biochemical effects that vitamins B and D, and calcium have as potential CRC preventive substances. Finally, we aim to formulate dietary recommendations based on the mechanistic properties of these vitamins and minerals.

EPIGENETIC MECHANISMS Before going into depth about the epigenetic effects that certain vitamins have to inhibit cancer development, we will first explain mechanism to perform these epigenetic changes.

HISTONE MODIFICATIONS One of the most potent mechanisms that can introduce changes on the expression of oncogenes or tumour suppressor genes, is histone modification. Histones are the proteins that package the DNA strands into nucleosomes. The DNA is wound around eight histone

subgroup proteins, which together form a nucleosome. One method to modify histones, is by acetylation. Acetylation neutralizes the positive charges of lysine residues in the nucleosome, weakening the interaction between DNA and histone cluster. This weakened interaction makes DNA more accessible for the transcription of DNA to RNA and for proper DNA replication. Special lysine acetyltransferases and lysine deacetylases regulate this process with a high turnover for efficient polymerase activity at the weakly connected DNA strands (7, 8). These acetyltransferases can also acetylate other proteins, such as the cancer related proteins p53 and MYC. Therefore, mutations in the acetyltransferases may not only lead to effects in the histones, but also on these other cancer related proteins (9). A different way to alter the access to DNA, is by phosphorylating the histones. Since the phosphates in the DNA are negatively charged as well as the additional phosphates to the histones, the charges will result in repulsion of DNA and histones. A similar charge repulsion between histones and DNA is created when histones are ADP-ribosylated. This modification is more linked to giving access to proteins of DNA repair (7). The connection between these epigenetic mechanisms dies not only involve vitamins, but also include the role of fibre-rich products in cancer development. The dietary fibres are converted to butyrate in the colon by the microbiome. Subsequently, this butyrate on its turn acts as a histone deacetylase inhibitor. This results in the upregulation of tumour-repressor genes in colorectal cancer cells, which will lower the risks of further development of cancer (10).

DNA METHYLATION Besides histone modifications, another major epigenetic mechanism that involves direct chemical modification of the DNA is DNA methylation. DNA methylation occurs when an additional methyl group (CH3) is added to the DNA which can affect the function and expression of the gene. The methylation is carried out by a family of DNA methyltransferases (Dnmt) that transfers S-adenyl methionine (SAM), a methyl group donor, to the fifth carbon of a cytosine residue to form 5-methylcytosine (5mC). These methyl groups in the major groove of the DNA inhibit transcription and therefore the normal functioning (Figure 1). DNA methylation is essential in human embryonic development and plays important roles in key processes like X-chromosome inactivation, regulating tissue-specific gene expression and genomic imprinting. Over 98% of methylation in somatic cells occur on the site where a cytosine nucleotide precedes a guanidine nucleotide (the CpG site), whereas in embryonic stem cells, almost 25% of methylation occurs in non-CpG sites (11). Importantly, the dysregulation of DNA methylation also contributes to cancer development (12). The early stages of CRC are associated with occurrence of the epigenetic events, including methylation of cancer-related genes, such as APC, p16INK4a and TIMP3 (13). Furthermore, polymethylation of these genes can be caused by low levels of SAM, which is synthesized depending on the nutritional factors like intake of vitamins and minerals like folic acid and vitamin B12 (14). Since previous studies have shown that diet may influence epigenetic changes involved in cancer development, the role of vitamins and minerals as epigenetic regulators in cancer should be considered when treating patients.

DIETARY COMPONENTS TO PREVENT CRC

Figure 1: Methylation of DNA on a CpG site, facilitated by Dnmt proteins by transferring SAM, the methyl donor, to form 5mC. This modification can eventually affect the gene expression.

As described previously, the intake of food products that are associated with a lower risk of CRC include low-fat dairy, fruit and vegetables. Vitamins B and D, and calcium have been identified to be the components in these foods responsible for this association. Keeping in mind the aforementioned mechanisms to alter the expression of cancer-related genes, we will now further elaborate on the biochemical and epigenetic effect of vitamins B and D, and calcium as potential cancer-preventive substances.

VITAMIN B

important cofactors in this process (Figure 2). In addition, folate and folic acid are important at the start of this pathway leading to proper DNA methylation and DNA synthesis. When these processes cannot be executed properly due to a lack of vitamin B in the cycle, it may lead to CRC (figure 2).

Vitamin B consists of a subgroup of different substances which is collectively called the vitamin B complex. A wellknown vitamin B type is folate, or its synthetic form folic acid. Folate can mostly be found in green leafy plants, grains and citrus fruits. It is an important factor in DNA synthesis, where a deficiency in folate may lead to a misincorporation of an uracil nucleotide instead of a thymine. However, when much folate is available, many nucleotides will be available for possible pre-cancerous cells. Epigenetically, folate is a donor for methyl groups required for DNA methylation. Therefore, folate is associated with the methylation status of DNA (15). A study by Farkas et al. found that more CpG sites in the folate transporter gene RFC1 were methylated in CRC tissue compared to healthy tissue, leading to dysregulation of these proteins. This suggests that folate may play a role in the formation of CRC tumours (16).

Cohort studies and meta-analyses have shown that there is an increased risk in developing CRC when a vitamin B6 deficiency is identified. Therefore, vitamin B may help in preventing CRC by proper regulation of DNA methylation and synthesis (17). However, a contrary view on this has raised, stating that pre-existing malignant cells may benefit from the B vitamins, which could lead to more replication and more cancer cells. The CpG island methylator phenotype (CIMP) CRC seems to be related to a low intake of folate and other B vitamins as well, thus this deficient intake may result in aberrant CpG methylation and could thereby contribute to CRC (18). Moreover, in a special population with mutations in the gene encoding for methylenetetrahydrofolate reductase (MTHFR), which is important for the pathway for a methyl

The B vitamins play an important role in DNA methylation and DNA synthesis by providing the nucleotides and methyl groups. Especially vitamins B6 and B12 are

Figure 2: Description of the biochemical and epigenetic mechanism in which B-vitamins are involved in the prevention of the development of colorectal neoplasia (1).

donor for DNA methylation, shows more frequent this CIMP type of CRC (19). In summary, previous research has shown that folate and other B vitamins play an important role in the DNA methylation and DNA replication by being key players in the cycle to provide methyl groups. Therefore, vitamin B intake should be considered as an important component in a healthy diet that decreases the risk of developing CRC. However, this protective mechanism may work negatively on pre-existing tumours by enhancing proliferation and DNA replication by increasing vitamin B levels. Thus, for the healthy population it is recommended to take enough vitamin B complex via a healthy diet, but people being predisposed for CRC, are advised to not exceed the maximum daily intake of vitamin B complex.

CALCIUM Calcium ions are important electrolytes that are involved in many different biochemical processes in the human body. However, calcium is also one of the molecules that is believed to have a protective effect against the development of CRC. This may explain why a moderate intake of calcium-containing dairy products are associated with a lower risk of colorectal neoplasia (1). From epidemiological studies, it has become clear that a high intake of calcium is associated with a reduced risk of CRC, and that low calcium intake can increase this risk (20-22). However, the data from clinical trials in which patients were either given calcium supplements or a placebo could not confirm the protective effect of calcium (23). This may be explained by sufficient levels of calcium derived from a regular diet, or by relatively short follow-up periods. In addition, the effect of calcium may be dependent on the expression of calcium-sensing receptor (CASR). In some types of CRC, the expression of CASR is downregulated, hereby preventing the uptake of calcium. This might explain why patients that already have had CRC, increased intake of calcium does not significantly change the risk of recurrent neoplasms (24). The mechanism by which calcium could prevent cancer does not directly involve epigenetic modification but does eventually result in downregulation of proliferative genes and upregulation of genes involved in differentiation and apoptosis. There are two theories explaining this potential anti-cancer effect. First of all, from research conducted in the previous century, it has been concluded that calcium can bind to free fatty acids and bile in the lumen, hereby preventing damage and inflammation of the epithelial layer of the gut (1, 25). Secondly, calcium may have a more direct effect on proliferation by binding to CASR, which intracellularly 16

inhibits the protein kinase C-signalling pathway and decreases the proliferative response (26, 27). Despite this convincing experimental data, clinical data confirming the importance of dietary calcium to prevent CRC is still lacking. Thus, enough calcium intake may prevent CRC by its anti-inflammatory and anti-proliferative effects, which also explains the negative association between dairy intake and CRC. However, it should be noted that calcium supplementation may not be effective in preventing cancer development in patients with CASRnegative neoplasms.

VITAMIN D Evidence suggest that daily intake between 1000 and 2000 IU/day of vitamin D3 can reduce the risk for CRC (28). However, what are the mechanisms involved in this protective characteristic, and can they be translated to clinical practice? Vitamin D regulates epigenetic mechanisms through its ability to carry out acetylation reactions and methylation state of genes. The vitamin D receptor (VDR) recruits histone acetylases that acetylate chromatin and increases the expression of certain DNA demethylases (29). The first mechanism involves the most important genes in CRC development. These are the ones encoding for the mediators of the Wnt signalling, like DKK1 and Wnt5a. The Wnt signalling ensures homeostatic proliferation of colonic crypts. The hyperactivation of the Wnt pathway plays a major role in the development of colorectal tumour growth. The disturbance of this pathway, leading to the development of CRC due to dietary factors were described before. The regulatory genes DKK1 and Wnt5a are often silenced due to hypermethylation in case of CRC (30). Furthermore, in 2012, Rawson et al. established the relation between vitamin D and CRC by its effects on DNA methylation. A negative association was found between DKK1 methylation and overall vitamin D intake (p=0.001) and a negative association between Wnt5a methylation with overall vitamin D intake (p=0.05). Because DKK1 has tumour suppressive characteristics, this negative correlation can represent the link between vitamin D and protection for CRC (31). Second mechanism involves the responsiveness of the cell to vitamin D, which is determined by the level of VDR and the concentration of calcitriol in the nucleus of the cell. Calcitriol is the active metabolite of vitamin D which is known to promote epithelial differentiation of human colon carcinoma cells that express the vitamin D receptor and inhibit proliferation (31). Calcitriol is determinative in this process due to its ability to modulate gene expression. It has been reported that local production of a metabolite (1,25(OH)2D) by vitamin D intake can

regulate up to 200 genes (32). These protective and regulatory properties of vitamin D suggest that besides the molecular mechanisms, a clinical application in prevention of CRC might be feasible.

DISCUSSION CRC is a common type of cancer that makes up about 12% of all cancers. Although the 5-year survival rate is increasing, so is the global prevalence of the disease. Lifestyle factors such as smoking, consumption of alcohol and dietary preferences have been linked to this increase in prevalence. In this review, molecular mechanisms involved in development of CRC like histone modulation and DNA methylation are described that can be affected by dietary factors like vitamin and mineral intake. Evidence found suggests that supplemental intake of

vitamin D and B, and calcium can have protective properties against the development of CRC. However, these protective properties depend on other factors such as the heterogeneity of the tumour and the overall genetic makeup of the patient. This is confirmed by the aforementioned clinical trials that failed to confirm these effects in clinical settings. Therefore, it is difficult to provide an overall recommendation regarding these vitamins and minerals. A new perspective regarding this topic might be the upcoming emphasis on personalized medicine, which can also be applied here. In conclusion, unveiling the heterogeneity of these tumours and the patients can provide opportunities for tailored therapies and prevention methods involving drugs and supplements like vitamins and minerals.

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Microbes with a sense of tumor: A study to evaluate the effect of gut microbiota on immune checkpoint inhibition. Viktor Yurevych, Manus van Dongen Radboud University Medical Centre/Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands

Checkpoint inhibitors are a promising new type of immunotherapies for cancer. Unfortunately the treatment is not effective for all patients. Here a study design is presented to examine how the microbiome and its effect on the metabolome influence the response to anti-PD-L1 treatment. For this microbiome samples from several mouse models are examined. This should lead to bacterial species and metabolites which are involved in anti-PD-L1 response. Identifying new druggable targets could lead to adjuvants for anti-PD-L1 treatment, improving treatment efficacy.

Checkpoint inhibitors are a new and emerging type of immunotherapies used in the treatment of cancer. These are drugs which remove dumorinduced inhibition of immune response. Tumor cells activate pathways which protect them from an immune reaction and these pathways can be targeted. A checkpoint inhibitors block a inhibitory pathway leading to a restored immune reaction. Common targets of checkpoint inhibitors are PD-1, PD-L1 and CTLA4. The PD-1 (programmed cell death 1) protein is present on the surface of T-cells. Itâ&#x20AC;&#x2122;s activation leads to decreased cell activity. PD-1 is activated by PDL1, which can be expressed by tumor cells. By this mechanism a tumor is able to facilitate immune evasion. In order to decrease this effect an intervention could target PD-L1, biologicals which do this are atezolizumab, avelumab and durvalumab. It is also possible to block the binding of PD-L1 to PD-1 by targeting PD-1 itself. This is the mechanism of nivolumab, pembrolizumab and cemiplimab. CTLA-4 is another receptor on the T-cell related to immune response. CTLA-4 activation by antigen presenting cells leads to a reduced T-cell response. Ipilimumab targets CTLA-4. Overall, checkpoint inhibitors show promise as an effective treatment is certain cancers. However, a lot of patients donâ&#x20AC;&#x2122;t respond to these drugs. (Syn et al. 2017) It is important to identify which factors influence checkpoint inhibitor response so that patients can be screened beforehand. In this way useless treatments and needless healthcare spending can be avoided. Colorectal cancer (CRC) is the one of the leading causes of cancer-related death, and improving the outcome of the disease, especially in metastatic CRC (mCRC) patients, represents an unmet clinical need. (Bilgin et al. 2017) The relationship between tumor cells and host immune cells in the tumor microenvironment (TME) and TME-related immune response escape is a recognized hallmark of cancer. Owing to the

success of immunotherapy in treating various other forms of cancer, trials for mCRC patients are ongoing. (Passardi et al. 2017) However, despite its efficacy, a significant portion of patients does not respond to immunotherapy. (Bilgin et al. 2017) One of the factors which might influence checkpoint inhibitor response is the microbiome. It was demonstrated that gut microbiota has the potential to contribute to CRC development through altering intestinal biofilms, microenvironment, or modulating immune reactions. Bacterial biofilms are the highest order of bacterial structures present in the intestine, and normally act as the first line of defense against invading microbial pathogens, potentiating inflammatory immune responses or producing genotoxic compounds. Significant changes in colon biofilms are a characteristic feature of CRC. (Dejea et al. 2014) Analysis of stool samples of CRC patients showed significant differences in bacterial genera present in the gut microbiome and demonstrating an overall decrease in the diversity of gut microbiota with a parallel increase in Fusobacterium, Peptostreptococcus, Bacteroides, Eubacterium, Proteobacteria, Prevotella, and Clostridium genera in CRC patients. (Nakatsu et al. 2015) Additionally, evidence suggests that some species present within CRC-specific gut microbiome may contribute to tumor immune escape, either my suppressing T cell proliferation. (Nosho et al. 2016) As T cells are crucial for the success of any checkpoint inhibition therapy, it is important to understand the conditions within the CRC TME that may hinder therapy. It was demonstrated for oligonucleotide immunotherapy and chemotherapy it that the microbiome influences response by modulating the tumor microenvironment. (Iida et al. 2013) For ipilimumab, a checkpoint inhibitor targeting CTLA4, it has been shown that the treatment relies on the microbiome. (Vetizou et al. 2015)

Figure 1: Concept of the study

Here certain types of bacteria were shown to have a immunostimulatory effect. The same is seen when targeting PD-L1. (Sivan et al. 2015) The mechanism of this effect is still unknown and specific effect on the microenvironment is also not identified. Here, we propose a study design to show the effect the microbiome has on the tumor microenvironment and how this influences checkpoint inhibitor treatment response. Workplan As it is impossible to effectively recreate the gut microbiome in an in vitro system, mice models will be used for this study. A standard mouse model for studying CRC is the MC38 Xenograft model bearing MC38 colon adenocarcinomas. Sterile mice with xenografted MC38 tumors will be used as a positive control and tumor-free mice will be used as a negative control. Microbial diversity assessment Gut microbial diversity within tumor-free mice and mice bearing MC38 xenografts will be compared by performing 16S rRNA sequencing of stool samples and colon epithelial swabs. 16S rRNA sequencing provides a convenient way of analyzing the microbiota due to the bacterial speciesâ&#x20AC;&#x2122; specificity, allowing for accurate 20

taxonomic identification through comparison of sequencing data with existing 16S rRNA databases. However, this method does not allow for identifying new species within the mouse gut microbiome, which cannot be excluded. To address this issue, metagenomic shotgun sequencing will be performed to further validate 16S rRNA sequencing data and identify potential novel bacterial contributors to tumor immune escape. Analysis of gut microbiota on anti-PD-L1 treatment To elucidate the effect of gut microbiota on the effect of PD-L1 inhibition, survival rates of tumor-bearing MC38 xenograft mice and sterile mice will be compared upon receiving an injection of an anti-PD-L1 antibody (Nivolumab). Tumor volume will be monitored for 30 days post injection. The mice will be sacrificed 40 days after the nivolumab injection, after which the immune surrounding of the tumors will be assessed using fluorescence-activated cell sorting (FACS) with antibodies characteristic to tumor-infiltrating CD8+ T cells, dendritic cells and natural killer (NK) cells. The cellular composition of the tumor infiltrate will be compared between sterile tumor-bearing mice and MC38 xenograft mice to

elucidate an effect of gut microbiota on PD-L1 inhibition. Analysis of gut microbial metabolome in relation to PD-L1 inhibition To analyse the potential effect of gut microbial metabolites on PD-L1 inhibition, a metabolomic analysis of the resected tumors from sterile and MC38 xenograft mice will be performed using mass spectroscopy (MS). The metabolomes of MC38 xenograft mice and sterile tumor-bearing mice will be compared to identify any bacterial metabolites that could have an effect on the tumor microenvironment and immune escape. To validate the metabolomic findings, CD8+ T cells will be cultured with nivolumab in the presence of the identified metabolites that could be implicated in immune escape or PD-L1 inhibition, followed by an assessment of their activity through analysis of markers of CD8+ activation or anergy via FACS. Additionally, qPCR can be performed to further confirm T cell activation or anergy by expression profiling. A bioinformatic study can then be performed to identify the potential bacterial producers of the metabolites of interest, followed by co-culturing of CD8+ T cells with the identified bacterial species in the presence of nivolumab to validate the bioinformatic findings. Experimental Findings: The data gathered from the metabolomic analysis and its subsequent experimental validation would identify the exact bacterial species or a list of species specific to the CRC microbiome that are responsible for hampering with PD-L1 inhibition through production of specific metabolites. This would help identify new druggable targets to use as anti-PL-L1 adjuvation for improved treatment efficacy, which could be tested in a follow-up study.

References

Proc Natl Acad Sci U S A, 111: 18321-6. Iida, N., A. Dzutsev, C. A. Stewart, L. Smith, N. Bouladoux, R. A. Weingarten, D. A. Molina, R. Salcedo, T. Back, S. Cramer, R. M. Dai, H. Kiu, M. Cardone, S. Naik, A. K. Patri, E. Wang, F. M. Marincola, K. M. Frank, Y. Belkaid, G. Trinchieri, and R. S. Goldszmid. 2013. 'Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment', Science, 342: 967-70. Nakatsu, G., X. Li, H. Zhou, J. Sheng, S. H. Wong, W. K. Wu, S. C. Ng, H. Tsoi, Y. Dong, N. Zhang, Y. He, Q. Kang, L. Cao, K. Wang, J. Zhang, Q. Liang, J. Yu, and J. J. Sung. 2015. 'Gut mucosal microbiome across stages of colorectal carcinogenesis', Nat Commun, 6: 8727. Nosho, K., Y. Sukawa, Y. Adachi, M. Ito, K. Mitsuhashi, H. Kurihara, S. Kanno, I. Yamamoto, K. Ishigami, H. Igarashi, R. Maruyama, K. Imai, H. Yamamoto, and Y. Shinomura. 2016. 'Association of Fusobacterium nucleatum with immunity and molecular alterations in colorectal cancer', World J Gastroenterol, 22: 557-66. Passardi, A., M. Canale, M. Valgiusti, and P. Ulivi. 2017. 'Immune Checkpoints as a Target for Colorectal Cancer Treatment', Int J Mol Sci, 18. Sivan, A., L. Corrales, N. Hubert, J. B. Williams, K. AquinoMichaels, Z. M. Earley, F. W. Benyamin, Y. M. Lei, B. Jabri, M. L. Alegre, E. B. Chang, and T. F. Gajewski. 2015. 'Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy', Science, 350: 1084-9. Syn, N. L., M. W. L. Teng, T. S. K. Mok, and R. A. Soo. 2017. 'De-novo and acquired resistance to immune checkpoint targeting', Lancet Oncol, 18: e731e41. Vetizou, M., J. M. Pitt, R. Daillere, P. Lepage, N. Waldschmitt, C. Flament, S. Rusakiewicz, B. Routy, M. P. Roberti, C. P. Duong, V. PoirierColame, A. Roux, S. Becharef, S. Formenti, E. Golden, S. Cording, G. Eberl, A. Schlitzer, F. Ginhoux, S. Mani, T. Yamazaki, N. Jacquelot, D. P. Enot, M. Berard, J. Nigou, P. Opolon, A. Eggermont, P. L. Woerther, E. Chachaty, N. Chaput, C. Robert, C. Mateus, G. Kroemer, D. Raoult, I. G. Boneca, F. Carbonnel, M. Chamaillard, and L. Zitvogel. 2015. 'Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota', Science, 350: 1079-84.

Bilgin, B., M. A. Sendur, M. Bulent Akinci, D. Sener Dede, and B. Yalcin. 2017. 'Targeting the PD-1 pathway: a new hope for gastrointestinal cancers', Curr Med Res Opin, 33: 749-59. Dejea, C. M., E. C. Wick, E. M. Hechenbleikner, J. R. White, J. L. Mark Welch, B. J. Rossetti, S. N. Peterson, E. C. Snesrud, G. G. Borisy, M. Lazarev, E. Stein, J. Vadivelu, A. C. Roslani, A. A. Malik, J. W. Wanyiri, K. L. Goh, I. Thevambiga, K. Fu, F. Wan, N. Llosa, F. Housseau, K. Romans, X. Wu, F. M. McAllister, S. Wu, B. Vogelstein, K. W. Kinzler, D. M. Pardoll, and C. L. Sears. 2014. 'Microbiota organization is a distinct feature of proximal colorectal cancers',

The impact of bacterial toxins on the hallmarks of cancer A review Lieke Schaeken, s125623; Brechtje Vanderfeesten, s1025772; Marceline Sanders, s4018087. BMS69 Tumors of the Digestive Tract. MSc Biomedical Sciences, Radboud University.

Abstract

Introduction Cancer development is a multifactorial process

The “hallmarks of cancer” cover several

(1,2). The evolution of cancer is depending on

biological competences that logically define

the micro- and macro-environment (3–5).

how a normal cell might progress to a

Hanahan

tumorigenic state by multistep development

“Hallmarks of Cancer”, which cover six

within a complex “tumor microenvironment”.

biological competences that logically define

Recently, it has become well established that

how a normal cell might progress to a

microbiota

tumorigenic state by multistep development

modulating various aspects of host physiology.

within a complex “tumor microenvironment”

Several bacterial taxa belonging to the human

(6). They include sustaining proliferative

gut microbiome in a subset of the healthy

signaling,

population contain toxin-producing strains

resisting cell death, enabling replicative

which affect key host processes, such as

immortality,

cellular signaling, and some directly attack the

activating invasion and metastasis. These

genome. In this review, we will outline

hallmarks were later updated with the addition

information on the involvement of bacterial

of genome instability, generating the genetic

(protein) toxins in cancer development. We will

diversity that accelerates the acquisition of the

give an overview of a variety of toxins and their

hallmarks, and inflammation, which promotes

main actions that might be involved in

multiple hallmark functions. Other emerging

carcinogenesis by affecting several hallmarks

hallmarks have been added to the list,

of cancer and we will also briefly discuss the

including

clinical

metabolism and evading immune destruction

also

play

application

integral

these

developing anti-cancer therapies.

roles

toxins

and

Weinburg

evading

growth

inducing

proposed

suppressors,

angiogenesis,

reprogramming

the

and

energy

(3). In addition to cancer cells, tumors contain a range of recruited, supposedly normal cells that

contribute

the

acquisition

characteristic hallmarks by creating this tumor

development of cancer by modulating most, if

microenvironment. Therefore, nowadays, the

not all, established host factors that comprise

genetics, as well as the lifestyle and immune

the hallmarks of cancer.

and stromal cells, cytokines, proteases and hormones,

are

contributors

acknowledged to

cellular

major

malignant

transformation (3,4). Recently, it has become well established that microbiota also play integral roles in modulating various aspects of host physiology. The involvement of bacteria in carcinogenesis has been recognized starting from the initial link between Helicobacter pylori and cancer, when it was discovered that infection predisposed humans to gastric cancer (7). The host processes that become highly

Several bacterial taxa belonging to the human gut microbiome in a subset of the healthy population, contain toxin-producing strains (12). These toxins affect key host processes, such as cellular signaling, and some directly attack the genome. This can be accomplished by damaging DNA, either directly, by enzymatic attack,

indirectly,

provoking

inflammatory reaction that produces free radicals. Other bacterial toxins can affect DNA repair mechanisms (9).

dysregulated during carcinogenesis include

In this review, we will outline information on

cellular metabolism and immune function.

the involvement of bacterial (protein) toxins in

Disturbance of the microbiota disrupts these

cancer development. We will give an overview

homeostatic processes by production of

of a variety of toxins and their main actions that

virulence factors, and thereby advances

might be involved in carcinogenesis by

development of numerous diseases including

affecting several hallmarks of cancer (Figure 1).

inflammatory

and

The relevant hallmarks of cancer associated

colorectal cancer (CRC) (8). Among the

with microbial toxins that will be discussed

virulence factors are bacterial (protein) toxins,

comprise sustaining proliferative signaling,

and these have been the targets of a large

genomic instability and mutation, tumor

number of studies (9â&#x20AC;&#x201C;11), since a variety of

promoting inflammation and avoidance of

these products are somehow connected to the

immune destruction.

bowel

disease

(IBD)

Figure 1. Summary of microbial toxins affecting several hallmarks of cancer. Abbreviations: CagA, Cytotoxinassociated gene-A; BFT, Bacteroides fragilis toxin; AvrA, Avirulence protein A; VacA, Vacuolating cytotoxin A; CNF1, Cytotoxic necrotizing factor 1; CDT, Cytolethal distending toxin; SEs, Staphylococcal Enterotoxins.

Sustaining proliferative signaling

intercellular adhesion molecule, is a common

Conceivably,

fundamental

target of intestinal bacteria resulting in

characteristic of cancer cells involves their

epithelial proliferation. BFT promotes cleavage

ability

proliferation.

of E-cadherin, which enables the nuclear

Deregulation of growth-promoting signals by

translocation of Î˛-catenin and promotes

cancer cells cause them to evade their actual

transcription of proto-oncogene c-Myc (14).

the sustain

most chronic

destinies (3). The majority of protein toxins that influence tumor onset and progression interfere with pathways related to cellular proliferation (10).

including

Escherichia

coli,

Klebsiella

pneumonia, and other Enterobacteriaceae. These highly conserved 54-kb genomic pks

Bacteroides fragilis is a non-spore-forming

islands synthesize colibactins, which are

obligate anaerobe that is part of the normal

genotoxic

colonic microbiota. A toxin-producing strain of

producing (pks+) E. coli induce a senescence-

this

Enterotoxigenic

associated secretory phenotype (SASP), in

Bacteroides fragilis (ETBF), has been identified

which senescent cells - cells ceased to divide;

as enteropathogenic. ETBF secretes a heat-

often considered a barrier for proliferation -

labile protein now known to be of the family of

secrete growth factors that stimulate epithelial

bacteria,

termed

B. fragilis toxins (BFTs) (13). E-cadherin, an

Certain bacteria carry pks genomic islands,

substances

(15).

Colibactin-

proliferation and thereby enhance tumor

induction of vacuole formation, upon binding

growth (16).

of VacA to the epithelium (22). This leads to a

Fusobacterium nucleatum (F. nucleatum) is a Gram-negative anaerobe, and is often seen in disease conditions (17). F. nucleatum found in the colon is seen as a potential candidate for CRC susceptibility, in addition to the discovery that there is an enrichment of F. nucleatum in cancers of CRC patients (17â&#x20AC;&#x201C;19). FadA adhesin, a bacterial cell surface adhesion component, is likely the factor causing the carcinogenic effect of F. nucleatum. Patients with adenomas and adenocarcinomas have a 10-100 times higher FadA gene levels in the colon tissue, compared to normal individuals. FadA mediates its effect via similar mechanisms as seen in BFT. It binds host E-cadherin, thereby activating Î˛-catenin signaling, which in turn leads to increased expression of transcription factors, oncogenes and inflammatory genes, along with growth stimulation of CRC cells (19). Among

the

bacteria

most

commonly

the Helicobacter species, which are present in an estimated 50% of people worldwide (20). Helicobacter pylori is a microaerophilic Gramnegative bacterium and can be found in the gastric mucus of the human stomach. H. pylori is a risk factor for severe disease in the gastric area. The main toxins produced by H. pylori are gene-A

proinflammatory

response

and

increased cell proliferation. It is suggested that the opening of membrane pores causes a disruption of diverse pathways depending on cell

type

and the

local

environmental

conditions. The stomach and duodenum are the regions most commonly involved, with various forms of gastric cancer progressing from gastritis and peptic ulcers (20). On the other side, specific genotypes of the H. pylori virulence factor, CagA, represent main factors in gastric cancer, inducing altered intracellular signaling in epithelial cells (23). CagA can be injected into the host target cell and localizes to the inner side of the cell membrane (21), where it gets phosphorylated within motifs (24). A higher number of motif copies of the toxin that are involved accompanies increased epithelial cell cytoskeleton changes and an enhances a proinflammatory and carcinogenic

associated with the development of cancer are

Cytotoxin-associated

marked

(CagA)

and

Vacuolating Cytotoxin A (VacA) (21). The most significant effect of VacA on host cells is the formation of membrane pores and the

environment (25). The protein toxin Cytotoxic necrotizing factor 1 (CNF1) is produced by certain pathogenic E. coli strains. CNF1 permanently activates targets of the Rho GTPase family. The active site of CNF1 is at the carboxyl terminus and it promotes deamidation of glutamine the the Rho GTPase enzymes (26). Binding of CNF1 has multiple effects on the cell. First of all, it leads to increased cell proliferation and it diminishes the rate of senescence which can facilitate oncogenesis. Secondly, CNF1 causes the

activation of NF-κB, which results in an increase

mechanism behind the induction of these DSBs

in pro-inflammatory cytokines and increased

has been partly elucidated. Bossuet-Greif et al.

cell migration (27). Lastly, CNF1 is capable of

identified DNA interstrand cross-links (ICLs) as

protecting the cell against apoptotic stimuli by

primary mechanism by which colibactin

increasing the amount of anti-apoptotic

induces DNA damage. Such ICLs result in

proteins (28).

replication

Salmonella typhimurium is a Gram-negative pathogen. This pathogen secretes Avirulence

stress

and

favor

DSBs.

Accumulation of the ICLs favors aging in tissue and leads to genomic instability (32).

protein A (AvrA), a protein toxin that influences

The cytolethal distending toxin (CDT) is a

eukaryotic cell pathways. AvrA increases β-

holotoxin produced by certain gram-negative

catenin signaling, and thereby enhances

bacteria,

proliferation in intestinal epithelial cells (29), as

Campylobacter jejuni. CDT is made up of three

explained before.

subunits: CdtA, CdtB and CdtC. The subunits

such

Escherichia

coli

and

are encoded by three genes organized into one

Genomic instability & mutation Genomic instability is a characteristic of most

operon. The CdtA and CdtC subunits are

cancer cells (30). A number of bacterial toxins

responsible for binding to the host cell

are able to damage the DNA and cause

membrane, while CdtB is the enzymatic

genomic instability and mutations and, by

subunit (33). Since CDT functions by acting as a

means of this action, are thought to be related

DNase it needs access to the nuclear

to cancer development. Several well-studied

compartment of the host cell. Binding of CDT to

toxins have been described to cause genomic

the host cell leads to internalization and

instability and mutations.

subsequent translocation of the CdtB subunit to the nucleus (34). The active CdtB subunit has

Besides enhancing sustained proliferative signaling, colibactin has also been linked to cancer development by creating double-strand breaks (DSBs) in eukaryotic cells. This has been demonstrated

Cuevas-Ramos

and

colleagues who showed that colibactin genes are expressed in vivo and induce γH2AX foci, a sensitive marker of DSBs, in enterocytes (31). How the colibactin inflicts DNA damage, directly or indirectly, leading to DSBs was poorly understood. However recently, the

been shown to be functionally and structurally similar to mammalian deoxyribonuclease I (DNase I) (35,36). Low doses of CDT induce single strand breaks (SSBs), which are later converted into DSBs during the S-phase of the cell cycle (37). The DNA damage repair (DDR) cascade gets activated as a result of these SSBs and DSBs, leading to G2/M cell cycle arrest and/or G1/S transition and initiation of DNA repair (38). However, in some circumstances, the DDR is counteracted by the survival signals

which favors the proliferation of the cells, thus

quickly towards cancer development (42).

contributing to tumorigenesis.

Besides, inflammation allows the proliferation

Numerous studies have demonstrated that the VacA toxin that was mentioned before, induces ROS production and mitochondrial DNA mutations in gastric epithelial cells (39). This

of pathogenic opportunistic bacteria to the prejudice of symbionts (43,44), further shaping an environment predisposed to progression of cancer.

can be explained by the role of VacA on

Different toxins have been implemented in

autophagy. It has been reported that VacA

promoting inflammation in tumors. One of

disrupts autophagy within human gastric

these includes VacA. VacA may exert an effect

epithelial cells (40), leading to an increase in

on host cells by binding to a cell membrane

cellular ROS. These increases in cellular ROS

receptor and initiating a proinflammatory

concentrations can contribute to oxidative

response, by inducing the production of

DNA damage and subsequent molecular

proinflammatory cytokines TNF-Îą, IL-1Î˛, IL-6,

alterations

neoplastic

IL-10 and IL-13 (45). In addition, it has recently

transformations and cancer. However, other

been shown that VacA enhanced production

studies reported that VacA induces autophagy

and secretion of interleukin-8 (IL-8) (46).

that

lead

via its binding to LRP1 (41), which results in the accumulation of ROS and eventually leads to induction of autophagy. Therefore, regardless of its mechanism of action, VacA leads to increased cellular ROS and thereby contributes to the pro-tumorigenic environment of H. pylori.

a tumor-inflammation promoting factor. CagA contributes to the inflammatory response by initiating a signal transduction cascade, resulting in IL-8 production (46). CagA also can interact with the hepatocyte growth factor Met which activates a pathway leading to NF-kB

Tumor-promoting inflammation In addition to deregulated cellular proliferation and

Next to VacA, CagA has also been recognized as

genomic

instability,

deregulated

inflammatory response has been recognized as an important mechanism contributing to bacteria-induced cancer. Bacterial protein toxins act on immune cells in diverse ways, thus stimulating a persistent inflammatory state characterized by activation of specific immune cells and production of cytokines and metabolites. These stimuli can push cells more

activation (47). Staphylococcus aureus is a gram positive bacterium and is responsible for common diseases. S. Aureus secretes over 20 different staphylococcal enterotoxins (SEs), with SEA and SEB being the best characterised (48). SEs are considered superantigens and have the ability

trigger

T-cell

activation

and

proliferation. A link between SEs and immune dysregulation in cutaneous T-cell lymphoma

has been established (49). However, exact

immunomodulatory cell may serve as a

mechanisms concerning this link need to be

mechanism to circumvent host immune

further elucidated.

responses. Besides, VacA can block activation

As mentioned above, the microbial toxin CNF1 causes the activation of NF-ÎşB, which results in an increase in pro-inflammatory cytokines and increased cell migration, contributing to tumor-promoting inflammation (27).

of NFAT, a key transcription factor required for T cell activation (53,54). Sundrud et al. reported that VacA inhibited the proliferation of

primary

human

CD4+

cells

and

demonstrated that this inhibitory effect on proliferation is not attributable to VacA effects

Avoiding immune destruction

on NFAT activation of IL-2 expression (55).

Another additional hallmark of cancer involves

Furthermore, it has been reported that VacA

the

inhibits the proliferation of CD8+ T cells and B

ability

cancer

cells

evade

immunological destruction, in particular by T

cells (56).

and B lymphocytes, macrophages and natural killer cells (3). Recently, several bacterial toxins have been shown to disrupt immune functions

While

some

antitumor immunity, others are able to stimulate

(50).

resident microbiota inhibit antitumor

immunity.

Bifidobacterium is able to inhibit the avoidance Fusobacterium nucleatum excretes Fap2. Fap2

of the immune destruction. This bacteria

augments

enhances

the

avoidance

immune

cell

function

and

destruction by silencing the tumor-killing

subsequent

capabilities of cytotoxic immune cells. This is

cytotoxic T cells, which was found to be

accomplished via direct interaction with the

corresponding

immune inhibitory receptor TIGIT (T-cell

subcutaneous melanoma xenograft models in

immunoreceptor with immunoglobulin and

mice (57). However, it has not been elucidated

immunoreceptor

which toxin is causing this effect and if this also

tyrosine-based

inhibitory

tumor-killing with

capabilities

reduced

growth

of of

motif domains) (51).

applies to gastrointestinal cancers. Similarly,

In addition to its action to induce proliferation

Bacteroides

and initiating a proinflammatory response,

nontoxigenic

VacA secreted by H. pylori can pass through

augment antitumor cytotoxic T-cell immunity

tight junctions and inhibit T-cell activation and

and is associated with T-cell responses specific

proliferation,

for these bacteria (58).

possibly

contributing

suppression of the immune response (46). VacA might also induce apoptosis of infected macrophages (52). Elimination of this key

dendritic

thetaiotaomicron B.

fragilis

and

polysaccharides

Clinical application Elucidating the underlying mechanisms of the toxins that were mentioned in this review,

might be of great potential in developing anti-

caused by colibactin from E.coli and help to

cancer therapies. Several toxins are already

suppress DNA damage. Further research of this

being studied as potential target for clinical

method is required for testing efficacy and

application.

toxicity (59,60).

Targeting the interaction of the toxin with the

Staphylococcal enterotoxins might also be

host cell, by neutralizing the tumor-promoting

targeted

activity of the bacteria, is a suitable option to

antibodies.

use in new therapeutic strategies. This has

identifying targets for SEB, since it is relatively

already been performed in a murine xenograft

stable and highly resistant to denaturation

model with F. nucleatum, where the use of a

(61). Most of the experimental therapies that

synthetic peptide derived from the FadA-

target SEB are performed using monoclonal

binding site on E-cadherin led to a decreased

antibodies that directly bind to and neutralize

interaction between FadA and E-cadherin and

SEBâ&#x20AC;&#x2122;s interaction with host cell receptors,

eliminated FadA-induced CRC cell growth and

leading to a reduction in proinflammatory

oncogenic and inflammatory responses (19).

cytokine production and counteracting the

Possibly, this method can also be adopted in

toxic and lethal effects of the toxin (62,63).

targeting BFT, and thereby prevent the cleavage of E-cadherin by this toxin, since FadA displays a somewhat similar mechanism as BFT. Therefore, neutralization of the toxin might be an appropriate opportunity for preventing toxin-induced aberrant epithelial proliferation. Another approach using small molecules interferes at the level of synthesis of the microbial toxin. One example are small molecule inhibitors that prevent effects caused by colibactin produced by E.coli (59). These small molecule inhibitors comprise boron based compounds which are ligands of CIbP, a key enzyme involved in colibactin synthesis. Targeting ClbP blocks the deleterious effect of this toxin in vitro and leads to a significant decrease in tumor numbers in vivo. They

using Most

anti-toxin studies

neutralizing focussed

Further research has to be conducted to elucidate the potential of targeting other toxins and for identification of small molecules that can counteract the mechanisms of toxins. It must be taken into account that the reaction of individuals can differ and further safety issues must be solved.

Conclusion In this review we presented an overview of microbial toxins and the impact of these toxins on the hallmarks of cancer. We have shown that toxins can affect several hallmarks of cancer, signaling,

including avoiding

sustaining

proliferative

immune

destruction,

genome instability & mutation, and tumorpromoting inflammation. Toxins seem like a promising therapeutic target since they mostly

suppress proliferation and tumorigenesis

have a specific enzymatic activity on their molecular targets. Depending on the specific toxin(s)

involved

tumorigenesis,

personalized approach must be applied, since the response of the human body to bacterial toxins can vary per individual. Despite great advances

elucidating

the

underlying

mechanisms of tumorigenesis of the toxins, further research is essential to get a more comprehensive understanding of the complex interaction between microbiota and host. Only then, toxins can be a future target for therapy of cancer.

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THE ULTIMATE FUTURE ORGANOID MODEL: INCORPORATING THE NERVOUS AND IMMUNE SYSTEM IN AN INTESTINAL ORGANOID MODEL TO STUDY MICROBIOME-GUT INTERACTIONS IN THE CONTEXT OF COLORECTAL CANCER. Emre Dilmen S4594525

â&#x20AC;&#x201C;

Iris Teunissen van Manen S4579798

ABSTRACT Colorectal carcinoma is one of the most prevalent cancer worldwide. The microbiome is believed to be one of the important factors that is involved in the development of this cancer. Multiple models are available to study the interaction between the gut microbiome and the intestinal epithelial layer. Intestinal organoids derived from stem cells are an emerging method to resemble the in vivo situation and therefore facilitate research. Nowadays, these organoids are used to study the effects of the microbiome on the development of colorectal cancer. However, important interactions with the nervous and immune system are still missing in this model. This review provides an overview of the importance of the interactions between the microbiome and the immune and nervous system. Subsequently, a possible future direction of research in developing an organoid incorporating these components is proposed to consider the important interactions with the microbiome. BACKGROUND Colorectal carcinoma is one of the most commonly occurring cancers worldwide and is responsible for more than 700.000 deaths annually [1]. The etiology of this type of cancer has been extensively studied and involves pathogenic mutations in proto-oncogenes and tumour suppressor genes [2]. In 5-10% of the cases, germline mutations are present that predispose patients to develop colorectal cancer. The remaining of the patients have pathogenic mutations in cancerrelated genes that have been acquired later in life. These somatic mutations can be caused by random errors during DNA replication. However, DNA damage can be significantly enhanced by external factors, including toxic compounds derived from tobacco smoke, radiation and infections [3, 4]. It is believed that up to 20% of all cancer cases are initially driven by infections. Especially in colorectal

â&#x20AC;&#x201C;

Daphne Roelofs S4589785

cancer, the role of the infections in the development of cancer is broadly studied due to the inflammatory properties of the gut microbiome, which is composed of all microorganisms that reside in our intestines. The bacteria in the gut can stimulate cancer development via multiple mechanisms [5]. First of all, many bacteria can produce toxins that cause DNA damage in enterocytes, which can result in genome instability. Other metabolites promote oxidative stress, resulting in increased inflammation. In addition to this, pathogenic bacteria have also been found to influence intracellular protein pathways involved in proliferation and cell survival. However, whereas many pathogens have been associated with colorectal cancer in epidemiological studies, it is often difficult to confirm causality [4]. In contrast to viruses, little information is available about the mechanisms in which bacteria may stimulate oncogenesis. To confirm whether a certain bacteria or bacterial product can stimulate tumor development, experimental models are required [4]. These models conventionally consist of monolayers of intestinal epithelial cells, followed by experiments with germfree or gnotobiotic mice. 2D cultures can be used to provide evidence about some phenotypic characteristics of intestinal cells and their interaction with microorganisms. However, monolayer cultures are difficult to preserve and do not have all characteristics of intestinal cells in vivo. Similarly, animal models also encounter certain challenges. Gnotobiotic mouse are considered as low-throughput models and require time-consuming and costly facilities [6, 7]. An alternative to these aforementioned experimental models is the use of human organoids. Using stem cells derived from intestinal biopsies [8] or using induced pluripotent stem cells (iPSCs) [9], a gut-like structure can be cultured containing differentiated intestinal epithelial cells. Unlike standard 2D models, these 3D models do not only consist of enterocytes, but also contain goblet cells, enteroendocrine cells, and Paneth cells. Due to these characteristics, organoids are a

promising method to study the interaction between human intestinal cells and the microbiome in the context of cancer development. However, the technique is relatively new and can still be improved on multiple levels to facilitate future research on the role of bacteria in cancer development [4]. The ideal intestinal organoid for microbiome research does not only include intestinal cells, but should also take the role of immune cells and the nervous system into account. Previous research has already taken some steps in this direction [7, 10] but an organoid combining these components has not yet been published. This paper will review recent progresses made to improve intestinal organoids to model the effect of the gut microbiome on colorectal cancer. Based on this literature, we aim to recommend a direction for future research to develop a validated organoid model that enables the investigation of potential pathogenic microorganisms in cancer development. THE IMMUNE SYSTEM The immune system and immune cells show an important dynamic interaction. Both can influence each other via various mechanisms. One of such mechanisms, is via short-chain fatty acids (SCFA). These are products of the fermented dietary fibres, which is done by the bacteria of the microbiome. These SCFAs

can inhibit histone deacetylase, which will influence epigenetic. In addition, they can activate G-coupled proteins, which occurs in T-helper cells. Via this activation, these cells will produce more IL-10, which has anti-inflammatory effects [11]. Furthermore, immune cells indirectly indirect interaction via butyrate, a SCFA that can affect dendritic cells. In the presence of butyrate, dendritic cells will facilitate differentiation of naive T-cells towards antiinflammatory T-regulatory cells and inhibit differentiation to pro-inflammatory interferonproducing cells. [12]. Finally, butyrate influences macrophages as well. When macrophages are treated with butyrate, less proinflammatory mediators are detected [12]. This shows that the microbiome can have multiple indirect influences on the immune system and the production of cytokines via its metabolites, such as the SCFAs and butyrate (Figure 1, [12]). A more direct link has been shown via germ-free mice. These mice lack a microbiome and have shown deficiencies in immune functioning. The deficiencies include structural abnormalities in the spleen and the Peyers patches in the gut, but also include functional deficiencies in all immune cells. In addition, when these mice are colonized with commensal flora at early age, they will still develop a proper functioning immune system [13]. Moreover, the microbiome plays a key role in the initiation of the development of gut-

Figure 1: The interactions and pathways of short-chain fatty acids (SCFA) in the intestinal wall, leading to different effects on the immune system

associated lymphoid tissue [14]. These observations indicate the important role of the microbiome in the development of a healthy immune system. An additional connection between the microbiome and the immune system, has been found in the effectivity of checkpoint inhibitors. This rather new type of therapy to treat cancer consists of interfering with the checkpoint molecules on immune cells, which prevents inactivation by tumor cells. Tumors cells can express PD-L1 on their membrane, which can bind to PD1 on Tcells, and thereby inactivate the T-cell from recognizing the tumor cell. Via anti-PD-1 treatment, the T-cell cannot be inactivated and will kill the tumor cell. The same principle can be applied for CTLA-4 on T-cells and CD80 on antigen presenting cells. Anti-CTLA-4 treatment will ensure proper activation and maturation of T-cells. It has been shown that the efficacy of anti-CTLA-4 depends on the interaction with the microbiome. Germ-free mice did not respond to this type of treatment. In addition, the presence of certain gut bacteria, including B. fragilis, are specifically associated with successful anti-CTLA-4 treatment. This was hypothesised because recolonizing germ-free mice with this bacteria in various ways restored the response to this treatment [15]. A similar interaction has been found for anti-PD-1 treatment and the Bifido bacterium [16]. Additionally, the S. aureus can increase the level of PD1 expression of antigen presenting cells, which leads to fewer T-cells to attack tumor cells and more T-regulatory cells [17]. Therefore, S. aureus has a negative effect on tumor killing by the immune system. In summary, these indirect and direct interactions between the microbiome and immune cells show that the immune system is very important in proper functioning of the microbiome, and vice versa. Since it is known that there is a crucial interaction between the immune system and the microbiome, several studies have made gut organoids in a coculture

with immune cells. One example is an organoid containing macrophages. In this model, enteroids were seeded on one side of a permeable membrane and macrophages on the other side. This ex vivo mini-gut with two monolayers of different cells types could be used to innate immune responses to different factors [10]. Such a model may be used to study the effects of microbiotic toxins on the macrophages or enterocytes, and can be a valuable tool to study the interaction between macrophages and enterocytes. Furthermore, Nozaki et al. developed a method to coculture intraepithelial lymphocytes in vitro in an intestinal organoid [18]. These intraepithelial lymphocytes include different subsets of T-cells, which makes this model interesting to study interactions between these subsets and the enterocytes and the behaviour of the T-cells in different environments. A comparable approach has been used by Rogoz et al. They developed a 3D enteroid model which they also coculture with T-cells. However, they suggest that their method could also be adapted to use a coculture with other immune cells, like macrophages or dendritic cells [19]. Therefore, gut organoids with an immune compartment is expected to be suitable to study interactions between different immune cells and the epithelial layer. By addition of factors from the microbiome, the reaction to these factors can be studied and the interaction between those cells. THE NERVOUS SYSTEM It has already been established that the microbiota in the gut interacts locally with intestinal cells and the enteric nervous system (ENS). Experimental evidence derived from clinical trials suggest that the microbiota also interact directly with the central nervous system through neuroendocrine and metabolic pathways [20]. First of all, the microbiome has been associated with several neurological and behavioural diseases or

Table 1: several species of microbiomes and their products that can influence the development and functioning of the nervous system.

SPECIES

PRODUCED NEUROTRANSMITTER

Bifidobacterium

GABA

Lactobacillus

GABA

Escherichia

Norepinephrine, serotonin and dopamine

Streptococcus

serotonin

Enterococcus

Serotonin

Bacillus

Norepinephrine and dopamine

abnormalities of the central nervous system[21]. For example, it has been found that 70% of autism spectrum disorder (ASD) patients have gastrointestinal tract-related symptoms. Both metabolites of the microbiome (4-ethyl-phenylsulfate and 3-(3-hydroxy phenyl)-3-hydroxypropionic acid) and its composition were associated with ASD. For example, children with ASD had significantly higher concentrations of Faecalibacterium and Clostridium in the microbiome of the gut compared to children without ASD [22]. Similar relations between the dysregulation of the microbiome and anxiety and depression, schizophrenia and Alzheimer’s disease[23, 24]. These clinical studies have indicated an important link between the gut microbiome and the central nervous system. Bacteria can produce hormones and neurotransmitters and are highly responsive to these, which affects their growth and virulence. These produced neurotransmitters range from excitatory to inhibitory neurotransmitters (Table 1). Some of these compounds reach the brain through the blood are signal via the vagus nerve. This nerve innervates parasympathetic control of the digestive tract and other organs such as the heart and lungs. Bacteria can respond to some of these hormones and neurotransmitters in several ways. For example, norepinephrine was shown to induce growth in proteobacteria such as E. coli. In addition, the hormones produced by bacteria can affect the host as well. Hormones like cholecystokinin and glucagon-like peptide-1 target the human brain through the vagus nerve. Moreover, this nerve could be directly affected through SCFA produced by the microbiome [25]. Thus, the vagus nerve plays an important role in communication between the microbes and the CNS. However, it is not the only route of communication. Another route of communication between the microbiome and the nervous system is via the enteric nervous system (ENS). This A unique property of the gastrointestinal (GI) tract is the fact that it has a ‘’second brain’’, called the ENS. The ENS is one of the main divisions of the autonomic nervous system and consists of the neurons in the GI tract. It has several essential functions, including gastrointestinal motility, blood flow and epithelial barrier permeability and fluid exchange. The ENS is developed from neural crest cells (NCCs) that migrate to the foregut and proliferate to colonize the entire GI-tract [26]. If this migration or proliferation in the GI tract is disrupted, enteric

neuropathies can arise. These enteric neuropathies are also present in digestive diseases such as inflammatory bowel diseases (IBD), like Crohn’s disease. [7]. IBDs are known to increase the chance of cancers. Therefore, dysbiosis of the microbiome in the gut can indirectly be linked to the development of colorectal cancer via its influence on inflammation. A second interaction between the ENS and the microbiome can be found in early development. Similar to the microbial population in the gut, the development of the ENS continues after birth. It has been shown that germ-free mice have early postnatal structural and functional abnormalities of the ENS. Furthermore, the gut microbiome and its products influence the neural excitations in the ENS, thereby regulating the sensory to the CNS and the gut motility [27]. This indicates that the microbiome in the gut plays a significant role in the development and regulation of the nervous system. Therefore, the microbiome and its products are essential and should not be overseen in research to for example colorectal cancers. Organoids that include the nervous system can become a thorough research model and be the next best preclinical tool compared to animal studies. Some effort has been made towards developing an organoid containing nerve cells. Recently, Workman et al. published a method to incorporate the ENS into a human intestine organoid. To achieve this, human vagal NCCs were generated through manipulating signaling pathways of iPSCs with retinoic acid to express Hox genes. In order to incorporate the NCCs into the organoid, gut spheroids with the NCCs were mechanically added by centrifugation and allowed in three-dimensional growth conditions for 28 days. The incorporation of functioning ENS cells were verified by histological examination and protein, RNA and gene expression. After 6 to 10 weeks of growth, the organoids became vascularized and grew to 10 to 30 mm in diameter with mature intestinal tissues, functional intestinal stem cells and submucosal and myenteric layers of smooth muscle cells. The organoid had coordinated cell proliferation, migration and development of the ENS. Despite these similarities based on histology and function, the organoids with incorporated ENS did not fully resemble all characteristics of the human gut. For example, the organoid had smaller nerve bundles compared to an adult human intestine, which was similar in size to a human fetal gut. Similarly, the development of 39

submucosal plexus was retarded in the organoid and appeared similar to a human fetal gut as well [7]. Thus, although high similarities, the current protocol for differentiation from iPSCs to NCCs can be improved to fully assemble the in vivo situation. THE COMPLETE ORGANOID The interaction between the nervous system, immune system and microbiome in the gut shows the importance of a complete model to study the influence of the microbiome on colorectal cancer. Therefore, future studies should aim at developing a more complete organoid model to study this. This could be achieved by combining methods of previous discussed studies that developed organoids with either immune cells or a nervous system. Thereby, a combined organoid can be made with both components to study the effects of the microbiome on the intestines, taken into account the important interactions of the immune and nervous system. Firstly, this could be performed by adding toxins or other factors excreted by the microbiome to the complete organoid, and study the effects these have, depending on the outcome measures. Another approach may the incorporation of living bacteria to the co-culture. This will result in organoids containing at least four different components; intestinal epithelial cells, nerve cells, immune cells and bacteria. Previous studies have shown the possibility to culture bacteria in an organoid for an extended period of time [6]. It is now required to further investigate the exact characteristics the complete model should have in terms of cell types and culture conditions. If future development is successful, this model could be applied for multiple studies, including colorectal cancer. When studying the influence of the microbiome on developing colorectal cancer, possible read-outs for this model may include the effects on cell division or proliferation. This can include measuring the expression of certain genes or proteins after exposure to a specific bacteria or toxin. Furthermore, this complete model should represent all important interactions of the microbiome and thereby provide improved information about the mechanisms of developing colorectal cancer influenced by the microbiome.

DISCUSSION This review has highlighted the requirement of organoids to investigate the role of the gut microbiome and their metabolites on the development of colorectal cancer. To obtain validated information about the complex interaction between microorganisms and intestinal cell in the context of cancer development, the ideal organoid should not only consist of human enterocytes, but should take the role of the immune system and the nervous system in account. Multiple studies have already been performed that combined gut organoids made from either primary cells or iPSCs together with certain components of the nervous-or immune system [28]. However, it remains challenging to compose a model that contains all the previously mentioned compartments and also enables the organoid to be exposed to bacteria or toxins. To fill this gap, we recommend to direct future research towards identifying the possibilities of a complete model to study the interactions of the microbiome with enterocytes or other cells of the gut epithelial layer. It would be feasible to develop model including nervous and immune system compartments, since previous studies have shown functioning organoid models containing one of these important systems. Therefore, we expect it to be possible to combine methods and adapt methods to develop a model with both the nervous and immune system. The method to develop such a model could be by combining these organoids, which would include the addition of cells on a gut organoid under the right conditions. However, defining these optimised conditions is one of the challenges that have yet to be solved in future research. Once a complete organoid can be manufactured, the future perspective of microbiome research in the context of cancer development is promising. Besides facilitating fundamental research about the specific mechanisms in which certain bacteria or toxin contribute to oncogenesis, intestinal organoids may also be used in personalized medicine. Using stem cells derived from a patient's intestinal biopsies, a Personalised organoid can be designed that can be used for diagnostic purposes. Furthermore, combined with samples of a patientâ&#x20AC;&#x2122;s own microbiome, this model could be used to study drug responsiveness and facilitate the choice for a certain treatment [29]. This

approach may also profit from the use of iPSCs. Gut organoids derived from human pluripotent cells are currently still being optimised, but have the potential to surpass primary tissue-derived models due to their ability to differentiate into a complete epithelial gut layer with that is histologically very similar to the in vivo situation. In addition, cloning genetically modified cells is more convenient in iPSCs compared to primary tissue. This can be used to validate potential targets of microorganisms or their metabolites [28]. Finally, future possibilities for a validates organoids may include its application in high throughput screening for potential pathogenic bacteria and toxins. This approach was recently published for an organoid model composed of cells derived from mice colons. In this study, computer-controlled microinjections were used to design gut organoids with a hypoxic lumen in which microbiota could be investigated [6]. These examples show much potential for an ideal multicomponent intestinal organoid to facilitate research in both personalised healthcare, or high throughput pharmaceutical studies. This could subsequently

contribute to a reduction and replacement of animal models. In summary, researchers have started using organoids as a preclinical tool to mimic the in vivo situation as an improved alternative for monolayer cell cultures. One of the applications includes studying the effects of the microbiome in the development of colorectal cancer. However, this new emerging technique is still in development and lacks the complete resemblance of the in vivo situation. In case of gut organoids, two essential components are missing to date. These are the immune system and the nervous system. Incorporating the immune system was achieved by various groups. However, incorporation of the nervous system is a more novel method and requires optimization. Due to similar methods, we foresee a possibility of combining the two systems to better mimic the in vivo interactions and facilitate research towards unveiling the connection between colorectal cancer and the microbiome.

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Arnold, J.W., J. Roach, and M.A. Azcarate-Peril, Emerging Technologies for Gut Microbiome Research. Trends Microbiol, 2016. 24(11): p. 887-901.

NAB-PACLITAXEL TO IMPROVE THE OVERALL SURVIVAL IN PANCREATIC CANCER EMRE DILMEN S4594525

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ABSTRACT Pancreatic cancer is a type of cancer with a growing incidence and a poor prognosis. It is expected to be the second highest cause of death by cancer in the near future. Therefore, treatment of this type of cancer is important. However, due to the microenvironment driven by desmoplasia around the pancreatic tumour, the delivery of chemotherapy to the tumour is limited. As a result, the median overall survival of pancreatic cancer treated with conventional gemcitabine is still low at approximately 7 months. A new approach has been found to increase the intratumoral concentration of chemotherapeutics by binding them to albumin. Using this mechanism, nabpaclitaxel has increased access to the tumour environment, despite the high stroma content surrounding the tumour cells. When used in combination with the cytostatic drug gemcitabine, nab-paclitaxel seems to significantly increase the overall survival. This review provides an overview of the mechanism of nab-paclitaxel in combination with other chemotherapies and the clinical benefits that have been studied. In addition, future perspectives of this approach will be discussed.

INTRODUCTION The pancreas is an important organ located in the upper left abdomen behind the stomach and is surrounded by the small intestine, liver, and spleen. It is generally 12 to 18 centimetres long, shaped with a head and tail and weights around the 90 grams. The pancreas is an essential organ due its role in digestion with its exocrine function and regulating blood glucose with its endocrine function. 95% of the pancreas consists of exocrine tissue that secretes between 1,5 to 2 litres of an enzyme-rich fluid into the duodenum. This fluid contains salt, sodium bicarbonate, and enzymes. The main enzymes are proteases to break down proteins, lipases to break down lipids and amylases to degrade carbohydrates. To ensure that the pancreases does not digest itself (which can happen in case of e.g. pancreatitis), these enzymes are activated in the intestine. The endocrine cells of the pancreas form ‘’islands’’ of Langerhans are distributed throughout the pancreas. These cells secrete hormones that regulate blood glucose

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DAPHNE ROELOFS S4589785

levels. After a meal, when the blood glucose level elevates, these cells secrete insulin which stimulates the absorption of the glucose into the cells. Insulin also inhibits the glucose production by the liver and stimulates glucose storage by the muscle cells and the liver. In contrast, when there is a low concentration of glucose in the blood, the pancreas releases the hormone glucagon that stimulates the liver to increase the release of stored glucose and start the production of glucose from proteins (1). Pancreatic cancer represents a major burden on healthcare and the patients diagnosed with it. In 2015, the incidence of pancreatic cancer was estimated to be 367.000 and it was responsible for 359.000 deaths. In developed countries, this is the fourth highest cause of death due to cancer and it is expected to increase to be the second highest (2). Since 1990, an increasing trend in pancreatic cancer incidence can be observed in the Netherlands (Figure 1; (3)). The exocrine adenocarcinoma, or pancreatic ductal adenocarcinoma (PDAC), makes up the majority of the incidence, compared to the neuroendocrine carcinoma (3). The highest determinant risk for pancreatic disease is smoking, which increases the risk two to threefold. 15% to 30% of all cases are thought to be due to smoking. Age is another major risk factor, as most patients are diagnosed after 50 years, with a peak between 70 and 80 years (4). Other risks include heavy alcohol consumption, diabetes mellitus, and to some degree, genetic background. Genes associated with pancreatic cancer are BRCA2, BRCA1, CDKN2A, ATM, STK11, PRSS1, MLH1 and PALB2. Mutations in the KRAS pathway accounts for over 90% of all pancreatic cancer cases. Genes such as TP53, CDKN2A and SMAD4 play the most prominent role in this pathway. Genomic events such as hypermethylation, point mutations and homozygous deletion are all associated with the pathogenesis (5). As a result of these mutations, the epithelial neoplasia from which pancreatic cancer often arises is able to promote cell proliferation, migration and invasion through activating signalling pathways. This process is facilitated by signalling molecules like TGFα, IGF1, FGF, HGF and various tyrosine kinase receptors (6). The oncogene mutations in pancreatic cancer cells promote reprogramming of the cell’s metabolism, which enhances the cells ability to acquire and recycle nutrients. 43

Figure 1: The incidence of pancreatic cancer in the Netherlands (3)

In addition, progressor mutations cause differential interactions with surrounding cells and stimulates the production of extracellular matrix. One of the characteristics of pancreatic cancer is the dense collagenous stroma surrounding it, resulting in a hypoxic environment. This stroma is mostly made of collagen made by the cancer-associated fibroblasts activated by the pancreatic cancer cells. These cancer-associated fibroblasts also produce hyaluronic acid which accumulates, leading to increased interstitial pressure and a decreased vascular supply. This stiff matrix promotes tumour aggression due to upregulated glycolytic capacity, increased amino acid metabolism and angiogenesis, reduced chemotactic signals and the poor infiltration of immune cells. Combined, these effects result in an aggressive tumour environment (7). The window to diagnose pancreatic cancer is relatively large; it takes more than 10 years for a neoplastic cell in the pancreas to create a distant metastasis. However, the clinical symptoms are non-specific and occur when the cancer has reached later stages. These symptoms include unexpected weight loss, pain in the abdomen and diabetes mellitus signs (8). A CT scan can provide a diagnosis for the size and location of the tumour. Also, the surrounding tissues can be visualized for possible metastasis. The location of the tumour is very important in case of pancreatic cancer. If the cancer is located in the head of the pancreas, it can obstruct both the bile ducts and the pancreatic duct. A tumour in the tail will obstruct only the distal parts of the pancreatic duct. Early identification of these symptoms as signs of pancreatic 44

cancer could improve a patient's prognosis, but is often difficult in practice. To treat patients, a palliative approach can be chosen in case of metastasis and a severe disease progression. A resection is possible in fewer than 20% of the patients, of which 80% will relapse (5). Therapeutic interventions for mutations that can cause pancreatic cancer like KRAS, CDKN2A and TP53 are currently not available. In addition, therapeutic intervention with conventional cytostatic drugs is complicated by the dense collagenous stroma that prevents drugs delivery to the cancer cells. In 1997, the drug gemcitabine for the treatment of pancreatic cancer was approved by the FDA. Although other drugs have entered the market, gemcitabine is still considered a treatment option because it has relatively low side effects. This chemotherapeutic becomes activated once in the cell by obtaining three phosphates via enzymatic activity, forming the active state called gemcitabine triphosphate. Subsequently, gemcitabine triphosphate will be incorporated in the DNA by misinterpreting it as a nucleotide, leading to an irreversible DNA damage and therefore cell death (9). In 2012, gemcitabine combined with albuminbound paclitaxel (nab-paclitaxel) showed improved efficacy compared to gemcitabine alone, and this combination was therefore approved by the FDA. Gemcitabine/nabpaclitaxel treatment resulted in increased concentrations of gemcitabine in the tumour, hence the increased efficacy (10). This increased efficacy makes it an interesting approach to treat pancreatic cancer patients with nab-

paclitaxel/gemcitabine combination therapy, to increase the overall survival of patients. Therefore, this review provides an overview of the mechanism of nab-paclitaxel and its performance in the clinic. Furthermore, insights for the future to increase the efficacy and safety of this therapy will be provided.

endothelial layer and increased uptake of albumin by malignant cells may explain higher intratumoral concentrations of nab-paclitaxel compared to solventbased paclitaxel found in animal experiments (15).

SPARC AND NAB-PACLITAXEL IN PANCREATIC CANCER

NAB-PACLITAXEL’S MECHANISM OF ACTION WORKING MECHANISM AND DELIVERY

Before going into details about the use of nab-paclitaxel in pancreatic cancer and its performance in clinical trials in combination with other drugs, it is important to understand the working mechanism of this pharmaceutical. Paclitaxel is a cytostatic drug that inhibits cell division by depolymerization of microtubules. This process prevents cells from progressing to the G1 phase, which results in cell death. This characteristic is used to target fast-dividing tissues and reduce tumour size (11, 12). Due to its high solubility, paclitaxel was initially formulated as a solvent-based drug dissolved in ethanol and the formulation vehicle Cremophor EL. However, this formulation showed an unfavourable toxicity profile and could result in Cremophor EL-associated hypersensitivity reactions. To prevent the use of toxic solvents, a formulation of paclitaxel with human serum albumin (nabpaclitaxel) was developed. Albumin is a soluble protein that functions as a biomolecule carrier and can therefore dissolve hydrophobic drugs by forming complexes with these particles. These complexes can subsequently circulate through the blood and travel to the tumour (11). To reach tumour cells, the nab-paclitaxel complex has to cross the endothelial layer. This can be achieved in two ways. First of all, particles can reach tumour cells by paracellular transport. The enhanced permeation and retention (EPR) effect states that blood vessels surrounding tumours are typically leaky, which promotes the supply of macromolecules to the tumour, but also improves drug delivery (13). A second mechanism to cross the endothelial layer is by transcytosis, which is facilitated by the binding of albumin to its receptors on endothelial cells, including glycoprotein 60 (gp60). As a result of this receptor-ligand interaction, the cell membrane will invaginate the nabpaclitaxel particles in vesicles that will be released at the apical side in the interstitial space. This process was found to be significantly enhanced in nab-paclitaxel compared to the solvent-based (14-16). Furthermore, tumour cells are known to take up and metabolise albumin from their microenvironment to promote their growth and survival (17). The combination of enhanced transport through the

In addition to the aforementioned mechanisms that could explain increased intratumoral levels of nab-paclitaxel, another molecular mechanism for tumour accumulation of nab-paclitaxel has been proposed, which is specifically relevant for pancreatic cancer. One of the major components of the stroma surrounding pancreatic cancer cells is so-called “secreted protein acidic and rich in cysteine” (SPARC). This glycoprotein that can bind to albumin is found overexpressed in 80% of PDACs and is associated with tumour growth, metastasis, and a poor prognosis (14, 18, 19). Interestingly, in a clinical trial by Von Hoff et al. that studied nab-paclitaxel in pancreatic cancer, patients with a high SPARC expression in the stroma were found to have a significantly higher median overall survival compared to patients with low SPARC expression (20). This association was supported by animal studies that demonstrated decreased nab-paclitaxel levels in SPARC null tissues. It should be noted that additional experiments have indicated that SPARC can be saturated with nabpaclitaxel, and that no link has been found so far between SPARC and nab-paclitaxel uptake by tumour cells. However, the albumin-binding ability of SPARC and its increased abundance in PDAC stroma highlights the importance of this glycoprotein to increase intratumoral paclitaxel levels in pancreatic cancer (11, 14, 16).

NAB-PACLITAXEL AND GEMCITABINE

Both clinical and preclinical studies have indicated a synergistic effect of nab-paclitaxel in combination with gemcitabine compared to conventional monotherapy with gemcitabine. To explain these findings, several mechanisms have been proposed (11). First of all, a preclinical study with a murine model containing human pancreatic tumours found that the concentration of gemcitabine was higher in the tumour tissue when given in combination with nab-paclitaxel. In addition, it was observed that treatment with nab-paclitaxel resulted in a reduced amount of stroma surrounding the tumours. As decreased stromal mass could potentially improve access to tumour cells, this finding was used to explain the increased intratumoral concentrations of gemcitabine when co-administered with nab-paclitaxel (20). However,

the mechanism in which nab-paclitaxel reduces stromal components in not yet understood. A second mechanism that could explain increased concentration and efficacy of gemcitabine in pancreatic cancer when given in combination with nab-paclitaxel, is the proposed inhibitory effect of nab-paclitaxel on cytidine deaminase, the enzyme that metabolizes gemcitabine. The importance of this enzyme in pancreatic cancer was determined using a spontaneous mouse model that autonomously develops pancreatic cancer. It was shown that nab-paclitaxel could decrease cytidine deaminase protein levels by induction of reactive oxygen species (ROS) and increase the active metabolite of gemcitabine (21). Both potential pathways of interfering with the intratumoral concentration of gemcitabine indicate that nab-paclitaxel does not only have anti-cancer properties by itself, but can also increase the efficacy of co-administered cytostatic drugs.

MACROPHAGE ACTIVATIO N BY NAB-PACLITAXEL

A final mechanism of action that nab-paclitaxel may use to target pancreatic cancer, is by activating tumourassociated macrophages. This finding was recently proposed in a paper by Cullis et al. The researchers of this paper showed that nab-paclitaxel is taken up by macrophages via micropinocytosis and that this event is followed by polarisation towards M1 macrophages. Especially the macrophage population in the tumour tissue upregulated antigen-presenting and co-stimulatory molecules needed to activate T-cells in nab-paclitaxel treated mice. With these results, the authors proposed an additional tumour suppressive role of nab-paclitaxel via immune-stimulation (22). The observation that macrophages shift from the fibrogenesis-promoting M2 to the M1 subtype after internalisation of nab-paclitaxel, may also explain the depletion of stroma associated with nabpaclitaxel treatment in previous studies (20). Future research will have to investigate whether a combination of nab-paclitaxel with additional macrophage-activating drugs could improve the treatment in pancreas cancer, potentially by also using a nab-formulation for these compounds to increase their concentration in the tumour environment (22).

NAB-PACLITAXEL IN CLINICAL TRIALS PROOF OBTAINED FROM PREVIOUS TRIALS

Pre-clinical studies have shown promising results in the use of nab-paclitaxel monotherapy and its combination with 46

gemcitabine. In a mouse model for pancreatic cancer, nabpaclitaxel increased the intratumoral concentration of gemcitabine. As described previously, this is potentially related to the inhibitory effect of nab-paclitaxel on the gemcitabine-degrading enzyme cytidine deaminase activity (21). Because of this synergistic effect, many clinical trials have been performed towards this combination. Among these studies is the aforementioned phase I/II clinical trial by Von Hoff et al. (20). In this study, the researchers aimed to establish the most effective and safest concentration for administration using the optimal dosage scheme of this gemcitabine/nab-paclitaxel combination. Based on their results, the authors recommended dosage scheme of 1000 mg/m2 gemcitabine with 125 mg/m2 nab-paclitaxel, which should be administered weekly for 3 weeks, repeating every 4 weeks. This dosage scheme resulted in a 1-year survival of 48% and a median overall survival of 12.1 months, which is a significant difference compared to current treatment (20). In addition to the phase I/II trial, the same research group also performed a phase III trial to investigate the effects of gemcitabine/nab-paclitaxel combination therapy in an international multicentre trial (10). The 1-year survival rate of gemcitabine/nab-paclitaxel treated patients was significantly higher compared to just gemcitabine treated patients, 35% and 22% respectively. Furthermore, the median survival of 8.5 months in the intervention group was found to be higher than the 6.7 months in the control group. Besides this, nab-paclitaxel also influenced the median progression-free survival from 3.7 to 5.5 months (10). These two studies were the foundation for further research to prove the effectivity and safety of nabpaclitaxel. In a retrospective study towards a small group of pancreatic cancer patients who received gemcitabine/nabpaclitaxel as a first-line treatment, a median overall survival of 10 months and a median progression-free survival of 6.7 months was found (23). These survival rates are thus even higher compared to those observed in the study from Von Hoff et al. In addition, a study performed in Wales compared their observed survival rates with these of the founding study by Von Hoff et al. The authors stated that their small cohort showed a comparable survival, which included a median overall survival of 8.4 months for the group that received combination therapy (24). Besides the performance of nab-paclitaxel in clinical trials, the effectiveness of gemcitabine/nabpaclitaxel treatment in a clinical setting is also important. Recently, a paper has been published about a retrospective observational study performed in Sweden (25). This study compared the overall survival of patients with locally

advanced pancreatic cancer and patients with metastatic disease, both treated with the combination therapy. It was found that the overall survival was 17.1 and 9.4 months respectively. Even though these subgroups differ in survival, this difference was not significant and the patients with metastatic disease still had benefit from the combination therapy compared to a monotherapy. Furthermore, other differentiations in disease and treatment showed differences in survival, but this was still an improved survival compared to gemcitabine monotherapy (25). Thus, clinical trials and additional retrospective studies have shown the benefits of a nabpaclitaxel/gemcitabine combination therapy in increasing the overall survival and 1-year survival rates. Furthermore, the stage of the disease and if a patient received a previous treatment, still showed benefits for the patients.

CURRENTLY ONGOING CLINICAL TRIALS

Besides the published clinical trials and survival rates, different phase III studies are currently being executed to study the effects of nab-paclitaxel in different settings and combinations. One of such combinations is tested in a phase III trial with paclitaxel, gemcitabine and napabucasin (26). This last substance is known to have effects on different cancer pathways, however the exact target is yet unknown (27). Besides this current running trial to this combination, another phase III study is comparing a nabpaclitaxel/gemcitabine/napabucasin combination therapy to the nab-paclitaxel/gemcitabine combination therapy (28). These show that additional compounds can further enhance the beneficial effects of nab-paclitaxel on the treatment of pancreatic cancer. Additionally, the effects of nab-paclitaxel in specific patient populations is being studied currently, for example in patients with metastatic pancreatic cancer or as an adjuvant therapy after resection of the pancreas (29, 30). Thus, current running trials are focussing on improving and expanding the combination therapy with gemcitabine and nab-paclitaxel.

DISCUSSION This review showed the working mechanism of a rather new and promising drug delivery strategy for pancreatic cancer. Since this type of cancer is known for its impenetrable stroma, a new strategy is to bind chemotherapies to albumin. This nab-paclitaxel will be able to accumulate in the tumour tissue via multiple mechanisms and kill the tumour cells. Furthermore, various preclinical and clinical studies have indicated that nabpaclitaxel is able to improve the intratumoral

concentration of co-administered anti-cancer drugs. Clinically, it has shown that patients receiving nabpaclitaxel/gemcitabine treatment have an improved overall survival compared to patients receiving conventional gemcitabine monotherapy. Still, trials are being executed to show the benefits of nab-paclitaxel in treating pancreatic cancer. Directions of currently running studies are towards effectivity in specific patient groups. Nab-paclitaxel/gemcitabine combination therapy has shown several advantages. Mechanistically, nabpaclitaxel shows a better drug delivery to the tumour compared to non-bound paclitaxel or other chemotherapies for pancreatic cancer. Due to the high density stroma around these tumours, drug delivery is limited. To counter this, albumin-bound chemotherapies can be used to increase the intratumoral drug concentration via various ways. Furthermore, clinically, different clinical trials and retrospective studies have shown that the nab-paclitaxel/gemcitabine combination therapy results in a greater overall survival compared to a conventional gemcitabine monotherapy. Finally, the progression-free survival is increased by this combination therapy. A disadvantage of nab-paclitaxel as a treatment for pancreatic cancer may be that even though it showed to improve overall survival, the increase in survival is still rather low. In addition, several side effects have been reported for nab-paclitaxel, including several haematological side effects, fatigue and neuropathy (10). These adverse events are classified as grade 3 adverse events, being severe and undesirable. Given this rather low increase in survival and severe side effects, it is important to consider the quality of life in these extended months. However, the quality of life has not yet been studied in patients receiving a nab-paclitaxel/gemcitabine combination therapy. This is something to consider in future trial as the increase in survival may be not favourable for patients if the quality of life is lowered significantly. Some future directions may be towards the use of nab-paclitaxel as a neoadjuvant or adjuvant treatment for pancreas resection. Furthermore, the tumour specific SPARC may be used as a biomarker to predict the effectiveness of this treatment in a patients, to use a personalized approach. Lastly, new combinations and additional compounds are tested to further improve the tumour killing and thereby the survival of pancreatic cancer patients. One of such new combinations may be nabpaclitaxel/gemcitabine/napabucasin as explained earlier. Additionally, another combination may be with a TNFÎą inhibitor. Since TNFÎą levels have been reported to be 47

elevated in pancreatic cancers, treatment with anti-TNFα has been proposed. In preclinical studies, anti-TNFα therapy showed a reduction in desmoplasia, which could indicate an antitumor effect and improve drug delivery (31). However, these results seem contradictory to the previously described mechanism of ab-paclitaxel to improve cancer treatment by stimulating inflammation in the tumour environment. One possible explanation for this conflicting data could be the involvement of the tumourassociated fibroblasts, which may be activated by both M2 type macrophages and TNFα. A combination of stimulating macrophage polarisation from the fibrogenesis-promoting M2 type to the T-cell activating M1 type, and the inhibition of fibroblast activation by anti-TNFα could therefore result in a potential benefit for anti-cancer treatment in pancreatic cancer (22).

In summary, several mechanisms were described explaining the beneficial effect of nab-paclitaxel in combination with other drugs. Furthermore, the results from clinical trials have indicated the clinical potential of a nab-paclitaxel/gemcitabine combination therapy in pancreatic cancer. However, the prognosis for patients diagnosed with pancreatic cancer is currently still poor, indicating the requirement of improved pharmaceutical treatment. The results of the ongoing clinical trials may give a direction for future research to work towards this aim. The advancements in treatment of pancreatic cancer so far may improve the low overall survival rate of patients, but further research should be pursued to unveil new approaches.

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Cracking the stromal shell: A brief insight into metabolic reprogramming of cancer-associated fibroblasts in pancreatic cancer. Viktor Yurevych, Manus van Dongen Radboud University Medical Centre/Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands

Pancreatic cancer is a cancer with a poor prognosis. Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of dense fibrous stroma around the tumor, which contributes to immune escape, drug resistance and metastatic growth (Saison-Ridinger et al. 2017), contributing to a low 5-year survival rate of 5-8%. The current gold standard for PDAC treatment is surgery or combinational chemotherapy, although treatment often fails due to the hostile tumor microenvironment (TME). Cancerassociated fibroblasts (CAFs)(von Ahrens et al. 2017) are the main cellular component of the tumor stroma. They were demonstrated to engage in active signalling cross-talk with pancreatic tumors (Schnittert, Bansal, and Prakash 2019), which, combined with their production of tumor stroma, present a potent barrier to effective PDAC therapy. As such, CAFs represent a potent therapeutic target to potentiate PDAC treatment. This review briefly explores the metabolic aspects of CAF activity and glances at potent existing options for CAF-based PDAC treatments.

Reprogramming of CAFs in pancreatic cancer A transitioning from oxidative phosphorylation (OXPHOS) to glycolysis, also known as the ‘glycolytic switch’ is a common hallmark of aggressive cancers, and is known to occur in PDAC. However, CAFs also undergo metabolic reprogramming, through a multitude of fibroblast-specific and tumor-induced mechanisms. The major player in CAF reprogramming is Hypoxia-inducible factor 1-alpha (HIF1-ɑ), which, when overexpressed, causes a cell to undergo a glycolytic switch. Expression of HIF1-ɑ and NFκB the CAFs ceuses them to adopt a phenotype similar to that of the Warburg effect in tumors, also exhibiting aerobic glycolysis. (Sotgia et al. 2011; Pavlides et al. 2010) As such, CAFs secrete more lactate and pyruvate than normal fibroblasts. Reactive oxygen species (ROS) produced by the tumor also cause HIF1-ɑ expression in surrounding cells, albeit through a different mechanism: HIF1-ɑ induces autophagy in CAFs, which promotes degradation of caveolin-1, a component of membrane caveolae needed for autophagy. Caveolin is a marker of a metabolic connection between the tumor and CAFs, as its loss causes disruption of mitochondrial function through elevated levels of nitric oxide (NO). This further elevates the levels of ROS and contributes to HIF1-ɑ activation. Following the collapse of mitochondrial functioning and metabolic devolution from OXPHOS to glycolysis, the remaining mitochondria are removed through mitophagy. 50

From this point onwards, CAFs rely mostly on glycolysis to sustain themselves. As mentioned earlier, this leads to an overproduction of lactate - a process actively exploited by tumor cells for producing energy and new biomolecules through a ‘lactate shuttle’ process, where lactate is used as a precursor for gluconeogenesis, allowing tumor cells greater production of energy and biomolecules. A major side effect of lactate accumulation is the acidification of the tumor microenvironment. Low TME pH activates the metalloproteinase MMP-9, giving tumor cells greater freedom to undergo epithelial-to-mesenchymal transition (EMT) - an event often associated with formation of metastases and spread of disease. In addition to that, an acidic TME enhances the chemoresistance of tumor cells, rendering standard chemotherapy less effective. Until recently, another aspect of CAF metabolism was often overlooked: CAFs were demonstrated to produce active exosomes which, upon their intake by the tumor, trigger the metabolic reprogramming of the latter by inhibiting tumor OXPHOS and promoting glycolysis and glutaminolysis in tumor cells. Apart from that, the exosomes contain metabolites like lipids and amino acids, in addition to TCA cycle intermediates, allowing for nourishment of the tumor by readily providing it with nutrients and promoting tumor growth in a nutrient-deprived microenvironment. Although a switch to glycolysis promotes proliferation in tumor cells, it has an opposite effect on CAFs - their proliferation becomes significantly slower compared to normal pancreatic fibroblasts. This is connected to an

increase in CAF catabolism, wherein the biomolecules obtained through glycolysis are channelled into production of stromal components or metabolites for the tumor instead of driving proliferation. This phenomenon is further backed up by an extensive network of metabolic cross-talk between tumor cells and CAFs, where tumor cells were demonstrated to induce hypoxic-like conditions for CAFs to further boost their switch to glycolysis. (Pavlides et al. 2012) Through this, and the mechanisms described above, tumor cells are able to effectively ‘hijack’ CAFs and induce the latter to produce ketone bodies, fatty acids, glutamine and high-energy nutrients to sustain the tumor (Martinez-Outschoorn, Lisanti, and Sotgia 2014) while providing additional effects of an acidic microenvironment to promote tumor progression and metastasis (Kato et al. 2013) and a hypoxic microenvironment for enhancement of cancer invasiveness. (Eguchi et al. 2013) Pancreatic stellate cells (PSCs) are a major component of the stroma and occupy around half of its mass. This type of CAFs can switch between active and quiescent phenotypes, and in their active state were shown to overproduce ECM components like fibronectin and collagen, leading to increased hypoxia. Quiescent PSCs are normally involved in vitamin A storage, exocrine and endocrine secretion, phagocytosis and maintenance of normal pancreatic stroma, whereas activated PSCs overproduce stromal components and are involved in promoting angiogenesis through secreting factors such as VEGF. (Schnittert, Bansal, and Prakash 2019) PSCs are activated by macrophages, platelets or pancreatic acinar cells, or by endothelial cells due to inflammation, with cytokines and growth factors such as TGF-b, IL-1, IL-6, platelet-derived growth factor (PDGF) and VEGF. Once activated, PSCs can support their activated status through an autocrine loop, while also being stimulated by ROS and mechanical pressure. In the end, such metabolic symbiosis between the tumor and CAFs creates a ‘fibroblast addiction’, further potentiating CAFs as perspective targets for PDAC treatment. Activated PSCs are considered to be the most potent target, exhibiting a highly proliferating phenotype that secrete elevated levels of stromal components together with MMPs that remodel the stroma. The importance of activated PSCs in PDAC biology has become more evident in the past decade. PSCs affect PCCs signalling and vice versa: this heterotypic interaction further supports desmoplasia and promotes tumour growth, invasion, chemo- and radioresistance.

Genetic and metabolic reprogramming of PSCs as a therapeutic target Due to the limited benefits offered by conventional PDAC treatments, hampering the tumor-supportive functions of the pancreatic tumor stroma through PSC modulation is a promising approach for improving PDAC treatments. It has been demonstrated, however, that PSC depletion contributes to disease aggressiveness rather than offer any therapeutic benefits. Therefore, PSC reprogramming is the main approach currently in development, due an expanding mass of evidence in favor of inducing PSC quiescence as a possible therapeutic strategy. (Schnittert, Bansal, and Prakash 2019) In theory, doing so would not only affect the protumor functionality of PSCs, but would also reverse the tumorigenic TME, thus blocking its adverse effects on treatment and anti-tumor immunity. Several approaches targeting PSCs have been developed, showing various degrees of efficacy. One approach, aiming to target the SHH (hedgehog) pathway crucial for PSC proliferation, was shown to have an effect completely opposite of what was expected, contributing to tumor aggressiveness and mortality and leading to discontinuation of the associated clinical study. However, it also demonstrated that tumor stroma possesses some tumor-suppressive properties that cannot be ignored in treatment. Therefore, preservation of the stroma should also be taken to account in designing prospective PDAC treatments. However, the pro-tumor properties of PDACassociated stroma are overshadowed by its protumor characteristics. One of them is the mechanical stress induced by stromal components like hyaluronic acid and collagen, which can contribute to tumor growth through activation and deregulation of mechanosensing cellular pathways such as Hippo-YAP/TAZ. (Gruber et al. 2016) As such, using hyaluronidase was proposed as a treatment option for depleting the tumor stroma, though the proposed treatment also induced thromboembolism in patients. Matrix metalloproteinases (MMPs) were also explored as a possible druggable target for PDAC, as their overexpression in PSCs was shown to contribute to tumor cell migration and formation of metastases. (Tjomsland et al. 2016) However, MMP inhibitors have demonstrated little clinical efficacy when compared with standard PDAC chemotherapy. 51

Targeting p53 for PSC reprogramming P53 mutations have been previously reported to occur in cancer-associated pancreatic fibroblasts, though that finding has been challenged by several groups. (Saison-Ridinger et al. 2017) However, tumor-associated PCSs treated with Nultin-3, a compound that stabilizes the p53 protein and induces p53-mediated signalling, showed a significant decrease in proliferative capacity. PSCs treated with Nultin-3 fell into a reversible quiescent-like state caused by cell cycle arrest mediated by p53â&#x20AC;&#x2122;s downstream effectors. P53 was found to inhibit the gene activation signature of tumor-associated PSCs, which include cell adhesion molecules, inflammatory mediators, signalling molecules and lipid metabolism genes. Interestingly, the effects of p53 pathway activation were also applicable to animal models, as tumor-bearing mice treated with a synthetic version of Nultin-3 demonstrated decreased expression of tumor stromal components and tumor-supportive genes in PSCs. This opens up a possible avenue for therapy, as inducing PSC quiescence could likely make pancreatic tumors more susceptible for standard therapy through the removal of the stromal barrier. Inducing PSC quiescence using vitamin A Aside from p53 rescue, a different approach to induce PSC quiescence involves metabolic reprogramming of tumor-associated PSCs into their quiescent state using vitamin analogs. As the PSCs become activated during PDAC initiation, they lose the characteristic lipid droplets containing vitamin A, which often coincides with vitamin A deficiency in PDAC patients. Vitamin A was demonstrated to inhibit DNA synthesis in tumor-associated PSCs, reducing their proliferative capacity and initiating apoptosis in surrounding tumor cells. In studies using 3D organotypic models it was shown that ATRA, a vitamin A metabolite, could induce quiescence in PSCs. (Chronopoulos et al. 2016) This effect was shown to be through activation of the retinoic acid receptor Î˛, leading to downregulation of actomyosin. This impairs the mechanosensory function of PSCs, suppressing extracellular matrix remodelling. Overall this inhibits cancer cell invasion. Another study tried the combination of ATRA with gemcitabine and showed that ATRA leads to an increased effect of chemotherapy. (Carapuca et al. 2016) Unfortunately, in a small-scale clinical study the addition of a different retinoid (13-cis52

retinoic acid) to gemcitabine showed no added benefit. (Michael et al. 2007) Combination of this retinoid with interferon treatment also showed no benefit. Though the limited clinical data available is disappointing further research into different retinoids is warranted. Conclusion While the reprogramming of CAFs in PDAC occurs on many levels, metabolism is as important as tumor-driven genetic regulation in terms of its functional consequences. This is especially pronounced in PSCs, and the induction of their senescence is a valid option for prospective studies for PDAC treatments. Of the discussed options, P53-based therapies offer the best output, and could be explored as adjuvant therapeutic options to potentiate the effect of currently used therapies. References Carapuca, E. F., E. Gemenetzidis, C. Feig, T. E. Bapiro, M. D. Williams, A. S. Wilson, F. R. Delvecchio, P. Arumugam, R. P. Grose, N. R. Lemoine, F. M. Richards, and H. M. Kocher. 2016. 'Anti-stromal treatment together with chemotherapy targets multiple signalling pathways in pancreatic adenocarcinoma', J Pathol, 239: 286-96. Chronopoulos, A., B. Robinson, M. Sarper, E. Cortes, V. Auernheimer, D. Lachowski, S. Attwood, R. Garcia, S. Ghassemi, B. Fabry, and A. Del Rio Hernandez. 2016. 'ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion', Nat Commun, 7: 12630. Eguchi, D., N. Ikenaga, K. Ohuchida, S. Kozono, L. Cui, K. Fujiwara, M. Fujino, T. Ohtsuka, K. Mizumoto, and M. Tanaka. 2013. 'Hypoxia enhances the interaction between pancreatic stellate cells and cancer cells via increased secretion of connective tissue growth factor', J Surg Res, 181: 225-33. Gruber, R., R. Panayiotou, E. Nye, B. Spencer-Dene, G. Stamp, and A. Behrens. 2016. 'YAP1 and TAZ Control Pancreatic Cancer Initiation in Mice by Direct Upregulation of JAK-STAT3 Signaling', Gastroenterology, 151: 526-39. Kato, Y., S. Ozawa, C. Miyamoto, Y. Maehata, A. Suzuki, T. Maeda, and Y. Baba. 2013. 'Acidic extracellular microenvironment and cancer', Cancer Cell Int, 13: 89. Martinez-Outschoorn, U. E., M. P. Lisanti, and F. Sotgia. 2014. 'Catabolic cancer-associated fibroblasts transfer energy and biomass to anabolic cancer cells, fueling tumor growth', Semin Cancer Biol, 25: 47-60. Michael, A., M. Hill, A. Maraveyas, A. Dalgleish, and F. Lofts. 2007. '13-cis-Retinoic acid in combination with gemcitabine in the treatment of locally advanced and metastatic pancreatic cancer--report of a pilot phase II study', Clin Oncol (R Coll Radiol), 19: 150-3. Pavlides, S., A. Tsirigos, I. Vera, N. Flomenberg, P. G. Frank, M. C. Casimiro, C. Wang, P. Fortina, S. Addya, R. G. Pestell, U. E. Martinez-Outschoorn, F. Sotgia, and M. P. Lisanti. 2010. 'Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the "reverse Warburg effect": a transcriptional informatics analysis with validation', Cell Cycle, 9: 2201-19.

Pavlides, S., I. Vera, R. Gandara, S. Sneddon, R. G. Pestell, I. Mercier, U. E. Martinez-Outschoorn, D. WhitakerMenezes, A. Howell, F. Sotgia, and M. P. Lisanti. 2012. 'Warburg meets autophagy: cancer-associated fibroblasts accelerate tumor growth and metastasis via oxidative stress, mitophagy, and aerobic glycolysis', Antioxid Redox Signal, 16: 1264-84. Saison-Ridinger, M., K. E. DelGiorno, T. Zhang, A. Kraus, R. French, D. Jaquish, C. Tsui, G. Erikson, B. T. Spike, M. N. Shokhirev, C. Liddle, R. T. Yu, M. Downes, R. M. Evans, A. Saghatelian, A. M. Lowy, and G. M. Wahl. 2017. 'Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy', PLoS One, 12: e0189051. Schnittert, J., R. Bansal, and J. Prakash. 2019. 'Targeting Pancreatic Stellate Cells in Cancer', Trends Cancer, 5: 128-42. Sotgia, F., U. E. Martinez-Outschoorn, S. Pavlides, A. Howell, R. G. Pestell, and M. P. Lisanti. 2011. 'Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment', Breast Cancer Res, 13: 213. Tjomsland, V., E. Pomianowska, M. Aasrum, D. Sandnes, C. S. Verbeke, and I. P. Gladhaug. 2016. 'Profile of MMP and TIMP Expression in Human Pancreatic Stellate Cells: Regulation by IL-1alpha and TGFbeta and Implications for Migration of Pancreatic Cancer Cells', Neoplasia, 18: 447-56. von Ahrens, D., T. D. Bhagat, D. Nagrath, A. Maitra, and A. Verma. 2017. 'The role of stromal cancer-associated fibroblasts in pancreatic cancer', J Hematol Oncol, 10: 76.

Making the step into clinical trials: nanocarrier-modulated RNAi therapeutics for pancreatic ductal adenocarcinoma The transferability of nanocarrier-based RNAi therapeutics for pancreatic ductal adenocarcinoma Lieke Schaeken, s125623; Brechtje Vanderfeesten, s1025772; Marceline Sanders, s4018087. BMS69 Tumors of the Digestive Tract. MSc Biomedical Sciences, Radboud University, February 2019.

Recently, scientists from Tel Aviv University have developed a new sophisticated potential nanotherapeutic for pancreatic ductal adenocarcinoma (PDAC) which may improve therapeutic response (1). A combination of microRNA (miRNA) and small interfering RNA (siRNA) was delivered to PDAC tumors by means of an efficient nanocarrier. They made use of a miRNA-mimic to increase miR-34a together with siRNA to silence the serine/threonine Polo-like kinase 1 (PLK1) oncogene. A biodegradable amphiphilic polyglutamate amine polymeric nanocarrier (APA) was developed, and thereafter polyplexes of APA-miRNA-siRNA were systematically administered to orthotopically injected PDAC-bearing mice. It was observed that the polyplexes accumulated inside the tumors and showed no toxicity to normal cells. Ultimately, this resulted in enhanced antitumor effect due to inhibition of MYC oncogene, a common target of both miR-34a and PLK1 (1). Given that this particular nanoparticle shows potential to deliver small RNAs to tumors in a preclinical setting, this study might serve as a basis for developing synergistic RNA interference (RNAi) therapeutics for the treatment of PDAC. However, attention must be paid with regard to transferability of this preclinical approach to clinical studies. Future clinical translations of this type of therapeutic agent will depend on resolving questions

relevant to the toxicity and efficacy of this type of multicomponent establishment. An important aspect in the translation from preclinical to clinical studies that must be considered is the resemblance of the mouse tumor model to the human tumor. In this study, orthotopic transplantation was performed with different PDAC cell lines in severe combined immune deficiency (SCID) mice. These cell line-based xenografts are comparable to human PDAC tumors. However, limitations occur when translating this to humans. Xenografts select for the most aggressive cancer clones, causing them to grow as homogenous masses with limited stromal infiltration even though PDAC consists of a stromal microenvironment (2). The previously mentioned study by Gibori and colleagues assessed the immunogenic potential of the APA nanocarrier and found that IL-6 and TNF-Îą cytokine secretion - representing the innate immune response - were not induced, whereas complement activation of the immune response was not assessed (1). However, the use of immunocompromised murine models in cell line based xenografts constitutes another weakness regarding the translatability of preclinical studies, since development of the tumor occurs without the generally present selective pressure of an immune system on PDAC (3). Therefore, this may eventually result in a greater tumor burden (2).

The resemblance of pharmacokinetics and pharmacodynamics between animal and humans is also important when translating a preclinical study to clinical trials. Zuckerman et al. correlated preclinical animal data and human phase Ia/Ib clinical data of CALAA-01, a targeted polymer-based nanoparticle containing siRNA. CALAA-01 is used as an experimental therapeutic for cancer (4). They found that the evidence provided from animal studies for this nanoparticle therapeutic appear to correctly assess the action observed in the clinic. They suggest that scaling the dose from animal models to patients by body weight is most suitable for translation of the CALAA-01 nanoparticle, based on Cmax instead of area under the curve (AUC). Eliasof et al. looked at the correlation between animals and human for CRLX101, a polymeric nanoparticle which contains camptothecin (5). They show that the pharmacokinetics are similar in rats, dogs, mice and humans and scaling can be done for CRLX101 by body surface area. Distribution of the nanoparticle in the tumor seems similar in the human gastric tumor and the mouse xenograft. Although, there are still some interspecies differences to keep in mind, such as serum albumin binding and altered release of plasma components. Other important aspects when considering translatability involve safety and feasibility. The aforementioned nanoparticle that Gibori and colleagues developed showed no toxicity in vivo in mice and showed a solid safety profile after evaluation ex vivo (1). Passive targeting of siRNAs and miRNAs to tumors is facilitated by nano-sized polymeric carriers, which minimize non-specific targeting to healthy organs and reduce the number of small RNAs administered to reach the desired therapeutic effect. However, one of the obstacles include minimizing the potential offtarget effects. Gibori et al. observed

accumulation of a small amount of polyplexes in the spleen and kidneys. This obstacle could potentially be overcome by using other methods to deliver the RNAi-based therapeutics. A phase I/IIa clinical study by Golan and colleagues (6) utilized siRNA delivered from a biodegradable polymeric matrix implant. The implant was inserted into locally advanced pancreatic cancer (LAPC) tumors and regionally released a siRNA drug against KRAS directly into the inner core of the tumor for an extended duration of time. This delivery system appeared to be efficient, site-specific and safe. Such an approach could be of great potential in making the step into more effective and safe clinical studies with RNAi based therapeutics. Both monotherapies of siRNA against PLK1 and miR-34a have entered clinical trials. PLK1siRNA has been evaluated in early clinical trials (phase I) for cancer therapy through systemic administration. PLK-siRNA was delivered in lipid nanoparticles to patients with adrenocortical cancer. Safety and antitumor activity were assessed. Preliminary results show that PLK1-siRNA was generally well-tolerated by the majority of subjects, and 40% of subjects had a clinical benefit after therapy (7). Enrollment is currently continuing in the phase II cohort for further evaluation of PLK1-siRNA. A clinical study of liposome encapsulated miR-34a administered to patients with unresectable primary liver cancer or advanced or metastatic cancer with or without liver involvement or hematologic malignancies has been terminated since five patients experienced immune related adverse events (8). The combination of miRNA and siRNA delivered with an APA nanocarrier to PDAC tumors has yielded promising results in preclinical studies, but its efficacy and safety in patients still needs to be elucidated. As

stated above, there are many challenges encountered when making the step from preclinical to clinical studies. When these aspects are taken into consideration, we believe the efficacious translation of this dualtargeting combination to clinical studies is feasible.

References 1.

Gibori H, Eliyahu S, Krivitsky A, BenShushan D, Epshtein Y, Tiram G, et al. Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer. Nat Commun. 2018 Jan 2;9(1):16.

2. Krempley BD, Yu KH. Preclinical models of pancreatic ductal adenocarcinoma. Chinese Clinical Oncology. 2017 Jun 30;6(3). 3. Frese KK, Tuveson DA. Maximizing mouse cancer models. Nat Rev Cancer. 2007;7(9):654–8. 4. Zuckerman JE, Gritli I, Tolcher A, Heidel JD, Lim D, Morgan R, et al. Correlating animal and human phase Ia/Ib clinical data with CALAA-01, a targeted, polymerbased nanoparticle containing siRNA. Proc Natl Acad Sci U S A. 2014 Aug 5;111(31):11449–54. 5. Eliasof S, Lazarus D, Peters CG, Case RI, Cole RO, Hwang J, et al. Correlating preclinical animal studies and human clinical trials of a multifunctional, polymeric nanoparticle. Proc Natl Acad Sci U S A. 2013 Sep 10;110(37):15127–32. 6. Golan T, Khvalevsky EZ, Hubert A, Gabai RM, Hen N, Segal A, et al. RNAi therapy targeting KRAS in combination with chemotherapy for locally advanced pancreatic cancer patients. Oncotarget. 2015 Sep 15;6(27):24560–70.

7. Demeure MJ, Armaghany T, Ejadi S, Ramanathan RK, Elfiky A, Strosberg JR, et al. A phase I/II study of TKM-080301, a PLK1-targeted RNAi in patients with adrenocortical cancer (ACC). J Clin Oncol. 2016;34(15_suppl):2547–2547. 8. A Multicenter Phase I Study of MRX34, MicroRNA miR-RX34 Liposomal Injection - Full Text View - ClinicalTrials.gov. Available from: https://clinicaltrials.gov/ct2/show/NCT01 829971