THE GUT MICROBIOME AND LUNG CANCER MAYA DUMAS

Major: Biology

Class of 2023

Introduction

Lung cancer is a prominent and life-threatening cancer that affects many people worldwide today. It causes many deaths and has no real cure.1 Lung cancer is a disease that is usually detected too late and progressed far enough to make treatment difficult. This makes the need to find biomarkers to detect early-stage lung cancer an important part of the pathway of fighting lung cancer. Treatment of lung cancer is typically done by chemotherapy, which reduces the size of tumor cells in the lung. However, chemotherapy is not a cure or always an effective solution. T-cells are the response of the body to tumors. Therapies allowing for increased production of tumor-fighting T-cells have shown to have positive results in the survival rates of lung cancer patients.2

The gut microbiome is comprised of gut bacteria that help influence our metabolic, endocrine, and immune systems.3 The balance of the microbiome or the relative abundances of certain microbes has become more important in recent studies. Probiotics containing strains like Lactobacillus are used by people to help aid in their digestive health. Many doctors will stress the importance of probiotics in one’s health as it relates to the health of the gut microbiome. The gut microbiome is not something that has been as fully emphasized as an important part of daily health to avoid cancer when compared to habits like not smoking, maintaining a healthy weight, and sun protection.

1 Siegel, R. L., Miller, K. D., & Jemal, A. (2017). Cancer Statistics, 2017. CA: a cancer journal for clinicians, 67(1), 7–30. https://doi.org/10.3322/caac.21387

2 Reuben, A., Zhang, J., Chiou, S. H., Gittelman, R. M., Li, J., Lee, W. C., Fujimoto, J., Behrens, C., Liu, X., Wang, F., Quek, K., Wang, C., Kheradmand, F., Chen, R., Chow, C. W., Lin, H., Bernatchez, C., Jalali, A., Hu, X., Wu, C. J., … Zhang, J. (2020). Comprehensive T cell repertoire characterization of non-small cell lung cancer. Nature communications, 11(1), 603. https://doi.org/10.1038/s41467-019-14273-0

3 Zheng, Y., Fang, Z., Xue, Y., Zhang, J., Zhu, J., Gao, R., Yao, S., Ye, Y., Wang, S., Lin, C., Chen, S., Huang, H., Hu, L., Jiang, G. N., Qin, H., Zhang, P., Chen, J., & Ji, H. (2020). Specific gut microbiome signature predicts the early-stage lung cancer. Gut microbes, 11(4), 1030–1042. https://doi.org/10.1080/19490976.2020.1737487

Recently, more studies have indicated that the maintenance of a healthy gut microbiome may have more important implications on the health of other systems. For example, the gut-lung axis is the pathway that has been more recently researched in its relationship to relative immunity. Different bacteria and microbes play an important role in a person’s health. The relationship between the lung and gut will be explained in this review as the “gut-lung axis” plays a role in the development and progression of lung cancer.

This review will evaluate the question of how the gut microbiota is related to lung cancer, as well as discuss why it has become important in advancing lung cancer research. The studies in this review aim to answer the questions of how gut microbiota can help predict and slow the progression of lung cancer. I decided that it would be best to group my sources by subcategories that separate the sources on whether they present information that is relevant to the relationship between the gut-lung axis, and the prediction and treatment of early-stage lung cancer. First, the topic of the relationship of sodium butyrate in the reduction of cancer cells will be explained. The importance of the gut-lung axis will be discussed to give context to why the microbiome plays a role in lung cancer. Next, studies focusing on the distinct alterations of the gut microbiome has allowed for the detection of lung cancer will be discussed. Finally, some of the ways a healthy microbiome has been shown to slow or stop the progression of lung cancer will be explored. By the end of this review, the mechanism of how specific gut bacteria can help aid in lung cancer treatment and how they may serve as biomarkers (predictors) will be answered.

Methods

The first article (Zheng et al. 2018) was found using the Taylor and Francis Online database using keywords gut microbiota and limiting search results to original articles within the last five years that excluded literature reviews. The Taylor and Francis online database contains thousands of peer-reviewed journals and articles. Most of the sources found about the microbiota as biomarkers for lung cancer and the use of probiotics in treatment were found through bibliography mining of that source. Other sources, like the study that focused on butyrate-producing bacteria and its relation to lung cancer (Gui et al. 2020), were found using the Pro Quest database. I also used the PubMed database using the same filters used in the ProQuest database search but using the keywords “gut microbiome and cancer”. I used the keywords “gut microbiota and lung cancer” and limited searches to within the last five years and included clinical trials and articles, while excluding literature reviews. Two relevant sources were found through the bibliography mining of that source as well. The previous study also helped identify

“sodium butyrate cancer apoptosis” as a relevant keyword, and using the same filters in ProQuest helped identify a study that focused on the mechanism of how those specific gut bacteria can reduce cancer cells (Salimi et al. 2017). While performing keyword searches, I found that there was a limited number of studies that linked lung cancer specifically to gut microbiota. Therefore, I expanded my research to include studies that included other types of cancer but included other valuable information that relates to the mechanism of gut bacteria and cancer-killing. I specifically wanted to include information that explained why and how healthy gut microbiota can slow the progression of cancer by using sources that are related to sodium butyrate and T-cells. Therefore, I included the study by Sun et al. (2020). Although it relates to colorectal cancer, I wanted to show the mechanism that T-cells are regulated by within the gut microbiome. In addition, I wanted to show what kinds of treatments are effective in maintaining a healthy gut microbiome and the effects on cancer cells, which is why I used sources that related chemotherapy effectiveness to sodium butyrate. The one source that is very distantly related to my specific topic focused on early lung inflammation and changes in the gut microbiota (Arrieta et al. 2015). However, I found this source to be important in defining the relationship of how alterations in the gut microbiome may lead to a higher risk of immune system inflammation.

RESULTS & DISCUSSION

I: Sodium Butyrate and its inhibition of cancer cells

Author/Year Purpose Methods Results

Salimi et al. 2017

To investigate the role of sodium butyrate in apoptosis in breast cancer cells.

The Cytotec effect of Nabu on breast cancer cells was determined using an MTT assay. Flow cytometry was used to detect apoptosis.

Nabu apoptosis was accompanied by reduced reactive oxygen formation and impairment of the mitochondria. In normal breast cells, there were no significant differences. The study focuses helps show the pathway that sodium butyrate affects cancer cells Sodium butyrate was found to be a histone deacetylase inhibitor. Shows the mechanism by which butyrate inhibits cancer cells.

This section will explore the answer to the question of how sodium butyrate can affect the size of cancer cells or aid in their reduction. A study conducted on breast cancer cells in 2017 found that sodium butyrate apoptosis, or cell death, was accompanied by reduced reactive oxygen formation and impairment of the mitochondria of breast cancer cells. Sodium butyrate is an SCFA that has shown important anti-cancer activity as a histone deacetylase inhibitor.4

A study by Salimi et al. (2017) found that apoptosis of cancer cells was caused by sodium butyrate-induced activation of caspases 3 and 8. However, Salimi et al. also points out that sodium butyrate does not promote cell death in healthy cells. This study was also consistent with previous studies5 using other types of cancer cells that indicated that sodium butyrate induces cancer cell apoptosis via oxidative stress through decreased reactive oxygen formation. Salimi et al. indicates from these findings that this pathway of cell cycle arrest is not exclusive to only one type of cancer.

The mechanism in which sodium butyrate can inhibit tumor growth via mitochondrial impairment and oxidative stress is important to consider when thinking of the microbiome. Many gut bacteria produce sodium butyrate, so gut bacteria may have the ability to kill cancer cells, as many produce sodium butyrate. Salimi et al. state that “the relevance of Sodium butyrate and cancer cell growth has yet to be investigated in many cancers, (pg 2).”4 Although this study focused on breast cancer cells, evidence of apoptosis due to sodium butyrate has been shown in other types of cancers. More recent studies that have explored this phenomenon in lung and other cancers will be explored in the next section.

Before exploring the ways that research into the gut microbiome has allowed for new advances in lung cancer treatment and detection, the relationship between lung cancer and the microbiome must be given context. Although it is clear there is a relationship between gut and lung, there is still a lot that is unknown. The gut-lung axis gives insight into what specific microbial changes are important to the immune system and why. This section will explore the gut lung axis and how it relates to lung immunity and lung cancer.

The methodology of the three studies that will be mentioned was similar in that they analyzed the fecal microbiomes of mice and/or humans. Jin et al., Arrieta et al., and Gui et al. (2020), all used qPCR and 16sRNA techniques to analyze the microbiota present and determine the abundance and phyla of gut bacteria present. Gui et al. used a small sample size and did not use testing with germ-free mice to validate the findings like Arrieta et al. and Jin et al. The testing of germ-free mice helps validate these findings, as they are genetically engineered to mimic lung cancer. All three studies also found Faecelibacterium to have an important role, which indicates a relationship

4 Salimi, V., Shahsavari, Z., Safizadeh, B. et al. Sodium butyrate promotes apoptosis in breast cancer cells through reactive oxygen species (ROS) formation and mitochondrial impairment. Lipids Health Dis 16, 208 (2017). https://doi.org/10.1186/ s12944-017-0593-4

5 Louis, M., Rosato, R.R., Brault, L., Osbild, S., Battaglia, E., Yang, X. ... Bagrel, D. (2004). The histone deacetylase inhibitor sodium butyrate induces breast cancer cell apoptosis through diverse cytotoxic actions including glutathione depletion and oxidative stress. International Journal of Oncology, 25, 1701-1711. https://doi.org/10.3892/ijo.25.6.1701

II: Sodium Butyrate and its inhibition of cancer cells

Author/ Year Purpose Methods

Gui et al. 2020

To investigate the association between butyrate-producing bacteria and non-small cell lung cancer.

This study used qualitative PCR to detect the expression of eight butyrate-producing bacteria in healthy adults and NSCLC. Data was collected by fecal samples.

Important Findings

Sodium butyrate can induce apoptosis of lung cancer cells and inhibit their growth. This also applies to lung cancer as it increases the expression of p glycoprotein in lung cancer cells.

Jin et al. 2019

To investigate how microbiota of the lung promote cancer development via T-cells.

Used a genetically engineered mouse that mimics lung cancer. To examine the microbiota in the mice 16 S rDNA qPCR analysis was done.

Shows that depletion of microbiota is linked to the development of lung cancer in mice. Microbiota can help shape lung immunity and protect against airway inflammation by T-cells.

Arrieta et al. 2015 between lung immunity and lung cancer. Arrieta et al. (2015) found that antibiotic usage contributed to decreased microbial diversity in the gut leading to decreased lung immunity, while Jin et al. (2019) found that antibiotic usage helped contribute to the reduction of lung microbiota diversity in the airway which led to a decrease in the progression of lung cancer.

To investigate early infants’ microbial metabolism to see how it affects the risk of asthma .

Four bacterial genera were analyzed by qPCR from fecal samples collected from very young infants. Mice were injected with the four bacterial taxa.

Infants at the risk of asthma had gut microbial dysbiosis during the first few months of life. The abundance of Faecfalibacterium was reduced, as well as the other three genera. The mouse model demonstrated that these bacteria have a role in reducing the risk of asthma.

Arrieta et al. examined the gut microbiome of infants during their first 100 days of life. Exposure to antibiotics in infants was found to be a significant indicator of a loss of microbe populations. The significant reductions of populations Faecalibacterium, Lachnospira, Veillonella, and Rothia were linked to a higher risk of asthma and compromised immune systems. However, Arrieta et al. was unable to determine whether the changes in the gut microbiome were preexisting and caused an inflammation or are a result of asthma.

The findings of Arrieta et al. provided a basis for a study performed in 2019. Jin et al. were able to further explore the relationship of causality between lung immunity and the role of microbes. However, this study focused on lung cancer cells and the development of T-cells, which are vital to fighting lung cancer.6 Additionally, Jin et al. used findings from airway microbiota rather than intestinal microbiota. Jin et al. discovered that “depleting microbiota or inhibiting γδ T-cells or their downstream effector molecules all effectively suppressed lung cancer development (p4)”.7 Jin et al. results indicate that the reduction of certain lung microbiota leads to an improvement in lung cancer status.

The findings of Jin et al. help indicate that there are two separate and different types of relationships between the microbiomes of the lung and gut that contribute to lung immunity. However, how the interactions of the lung and gut microbiota contribute to one another are still unclear. Future research is needed in describing the distinct lung microbiota signatures in lung cancer patients and how these compare with gut microbiota.

Gui et al. (2020) also found that Firmicutes like Facelibacterium and Clostridium, which produce sodium butyrate, were significantly reduced in lung cancer patients. Gui et al. examined the gut microbiota in fecal samples of healthy and lung cancer patients. According to Gui et al. his results confirm “a significant correlation between the gut microbiota and lung cancer (p.6)”.

All three studies do not establish a clear indication as to whether the reduction or enlargement of a certain population of microbe/s is responsible for lung immunity. The results of Jin et al., Gui et al. and Arrieta et al. are consistent with the idea that the phyla of lung microbiota and gut microbiota both play a role in the gut lung axis and affect cancer development. The next section will explore how these distinct changes in the gut microbiome have allowed for a new way for lung cancer to be detected.

One of the ways the research on the gut lung axis has been helpful in lung cancer research is its usage as biomarkers. Specifically, the abundance of certain phyla present only in lung cancer patients helps indicate its presence. Zheng et al. (2020) and Ren et al. (2017) characterized OTUs, or operational taxonomic units, which are used to compare markers of specific bacteria. Ren et al. investigated the relationship between gut microbiota and hepatocellular carcinoma, or liver cancer. Zhuang et al. (2019) further investigated the alterations that the gut microbiome undergoes during lung

6 Reuben, A., Zhang, J., Chiou, S. H., Gittelman, R. M., Li, J., Lee, W. C., Fujimoto, J., Behrens, C., Liu, X., Wang, F., Quek, K., Wang, C., Kheradmand, F., Chen, R., Chow, C. W., Lin, H., Bernatchez, C., Jalali, A., Hu, X., Wu, C. J., … Zhang, J. (2020). Comprehensive T cell repertoire characterization of non-small cell lung cancer. Nature communications, 11(1), 603. https://doi.org/10.1038/s41467-019-14273-0

7 Jin, C., Lagoudas, G. K., Zhao, C., Bullman, S., Bhutkar, A., Hu, B., Ameh, S., Sandel, D., Liang, X. S., Mazzilli, S., Whary, M. T., Meyerson, M., Germain, R., Blainey, P. C., Fox, J. G., & Jacks, T. (2019). Commensal Microbiota Promote Lung Cancer Development via γδ T Cells. Cell, 176(5), 998–1013.e16. https://doi.org/10.1016/j.cell.2018.12.040

III: The role of the gut microbiota as biomarkers for lung cancer

Author/ Year Purpose

Zheng et al. (2020)

To investigate specific gut microbiome in predicting early-stage lung cancer.

Methods

16 S rRNA gene sequencing was performed using fecal samples from lung cancer patients and healthy individuals. The abundance of four phyla of gut bacteria was examined.

Important Findings

Establishes that an overall specific gut microbial biomarker may be used to predict lung cancer.

Zhuang et al. 2019

To investigate the gut microbiota of lung cancer patients.

The gut microbiota of 30 LC patients and 30 controls were examined via 16 S rRNA and analyzed for diversity and biomarkers.

Identified reduced Actinobacteria and Bifidobacterium as potential biomarkers of lung cancer. The study also offers a very detailed table of the decline in certain functions of microbiota in LC patients which other studies have not covered.

Ren et al. 2017 cancer rather than liver cancer. This was elaborated on further by Zheng et al. (2020) who investigated whether gut microbe biomarkers like those found by Ren et. al could be identified in early-stage lung cancer. The results were consistent with the findings of Zhuang et al. (2019), in that there were unique gut microbe signatures present in lung cancer patients as compared to healthy ones. Zheng et al. and Ren et al. both used cross-validation from different regions to confirm and validate the accuracy of the OTU system, however only Zheng et al. also used patients with liver cirrhosis to measure differentiation. Zheng et al. only used 13 OUT makers, compared to the 30 used by Ren et al.

To investigate the gut microbiome of patients with hepatocellular carcinoma and evaluate which biomarkers may be used to detect HCC.

Fecal samples of patients in China were collected. The gut microbiome of these patients was analyzed, and microbial gut markers were characterized.

Early-stage cirrhosis patients had a decreased amount of Actinobacteria and Bifidobacterium. The study indicates that early cancer like HCC can be predicted by microbial gut markers. The first study to characterize the gut microbiome of patients with HCC.

Ren et al. used gut microbiota data to determine biomarkers for liver cancer. This study was the first to use cross-regional validation, by using samples from different regions of China. This helped confirm the accuracy of the OUT-based system and shows a potential way for early detection of liver cancer via microbial markers. The OTUs were able to differentiate and predict between healthy patients and patients with liver cirrhosis which is not the same as liver cancer.

Zhuang et al. and Zheng et al. found Actinobacteria and Bifidobacterium were reduced in lung cancer patients, which Ren et al. also found to be reduced in the patients with liver cancer. Therefore, it can be inferred that the similar results indicate a similar signature for even different cancer types. The similar signatures are given by many cancers, as they seem to all involve a general decrease in butyrate-producing bacteria which may make it harder for the OTU model to accurately differentiate between different types of cancer.

The importance of these certain phyla is that they increase SCFA production.8 As mentioned in the previous section, SCFAs or short chain fatty aid in lung immunity by controlling T- cells, which protect the airway from inflammation8. However, there is not just one microbe that contributes to cancer, as the overall composition of the microbiome is more important. Zheng et al. states that “No microbe was found to be specifically increased only in the non-metastatic patients when compared with the healthy controls and the metastatic patients (pg 6).”8 This is important when considering that the microbiome of a lung cancer patient is very distinct and has many different individual alterations.

The Effectiveness Of The Biomarker System

The predictive model with 13 operational taxonomic unit (OTU)-based biomarkers achieved high accuracy in lung cancer prediction. Zheng et al. (2020) narrowed down the 97 OTUs detected to 13.

Figure 1, taken from Zheng et al., illustrates that there is a clear separation between the healthy and lung cancer patients, which indicates the effectiveness of the OTUbased system implemented. Zheng et al. concluded that their 13 OTU marker-based system is more “accurate and reproducible, (p.8)9” because of the efficiency of the 16 S rRNA sequencing.

However, there are limitations to this technique as it is not able to identify the full taxa of the gut microbiome. One study in 2021, found that “16 S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun

Gut microbes, 11(4), 1030–1042. https://doi.org/10.1080/19490976.2020.1737487

Gut microbes, 11(4), 1030–1042. https://doi.org/10.1080/19490976.2020.1737487

III: The role of the gut microbiota as biomarkers for lung cancer

sequencing (pg.1)”10. This indicates that a more accurate depiction of phyla not as abundantly present would be more effectively done by shotgun sequencing. This means that the findings of Zheng et al. (2020) as well as all the other studies conducted using 16 S rRNA may have not found certain less abundant phyla that may also act as more accurate OTU-based markers.

In this section, I will explore how alterations to the gut microbiota via external methods have allowed for a change in the development of lung cancer. All studies mentioned also used 16 S rRNA sequencing for identification using fecal samples. Gui et al. (2015) and Chen et al. (2020) both treated mice with multiple antibiotics followed by probiotics to establish a healthy microbiome followed by observation via in vivo tumor growth. Both Gui et al. and Chen et al. indicate that the increased production of certain gut microbiota via probiotics helps lead to tumor size reduction and enhances the efficiency of chemotherapy. Gui et al. and Chen et al. also used germ-free mice to help confirm their results. Sun et al. (2020) used only patients with colorectal cancer and is the only study within this review that was performed in a successful human clinical trial. Sun et al. was able to show how a new integrative cancer therapy can be developed to help increase the production of certain beneficial antimalignancy bacteria.

IV: The Gut microbiome in new lung cancer therapies

Author/ Year Purpose Methods Results

Chen et al. 2020 To investigate the Akkermensia muciniphilia effect on tumor growth using Lewis lung cancer mice.

Cisplatin and AKK were combined to intervene in the mice, while in vivo imaging was. used to measure tumor size. Transcriptome sequencing was used to screen different expressed genes.

This study found that AKK is often decreased in lung cancer patients.

Sun et al. 2020 To investigate the effectiveness of Quixie capsules on the survival of patients with tumors from colorectal cancer.

Randomized double placebo-controlled clinical trial in China that used a Quixie capsule or control group. Stool samples were collected and analyzed via 16 S rRNA analysis and blood samples.

Gut microbiota was utilized as a way to control T-cells which help fight cancers Q capsule could enhance T-cells and increase the abundance of anti-cancer butyrateproducing bacteria.

Gui et al. 2015 To investigate the anticancer response in wellbalanced microbiota in a mouse model.

The microbiome of mice from fecal matter was measured by 16 S rRNA sequencing.

Found that microbiota influences metabolism, tissue development, and immunity of the human body. The study showed that mice treated with chemo and Lactobacillus bacteria had a smaller tumor size and greater survival rate.

Sun et al., Chen et al., and Gui et. al. had similar findings in that the production of sodium butyrate-producing bacteria helps aid in slowing the progression of cancer. This section will show the findings of studies that have indicated that the treatment via probiotics or the promotion of a healthy microbiome allows tumors to be reduced. In the future, more research is needed into how this mechanism can be used within humans.

In a study conducted by Gui et al. in 2015, germ-free mice were used to measure the anti-cancer response in well-balanced gut microbiota. Mice were treated with heavy amounts of antibiotics so that all gut bacteria would be killed. One group was given probiotics in addition to cisplatin (chemo). One group of mice was given only probiotics (Lactobacillus) so that only healthy microbiota would live within the gut. Mice were treated with Lewis Lung cancer cells and their tumor sizes were monitored. Besides the placebo group of mice, the other groups were injected with cisplatin. The one group that was given both probiotics and cisplatin had a significantly smaller tumor size, compared to the group that was given only cisplatin.11

This study was elaborated on further in 2020 by Chen et al., who studied the specific interaction of Akkermensia munichphilia (AKK) in patients with lung cancer. Chen et al. suggest that AKK combined with cisplatin may enhance immune regulation, as AKK was found to be reduced in lung cancer patients.12 Chen et al., in comparison to other studies, was more representative in identifying the relationship of how certain microbiota help reduce the size of lung cancer specifically. In addition to chemotherapy, other methods of reducing tumor formation have been researched.

The study by Sun et al. (2020) focuses on how the gut microbiota is influenced by the treatment of cancer by using a therapy involving Quixie capsules. Sun et al. found that QX treatment was positively related to Actinobacteria, which are SFCAproducing and immunity-enhancing bacteria.13 This result indicates that the Quixie capsule can modulate the production of SCFA-producing bacteria which inhibit metastasis of cancer cells. Zheng et al. also found that a reduction in SCFAs leads to a progression in lung cancer which means this technique could be applied to lung cancer patients as well. This similarity indicates that Quixie capsules may have a positive effect on patients with lung cancer as well. Overall, these studies all indicate that the promotion of a healthy, diverse microbiome may help lead to a better prognosis in lung cancer patients.

Conclusion

The unique microbial signatures of the gut allow for the identification and enhancement of treatment against lung cancer. The studies discussed in this review indicate the overall importance of the abundance of sodium butyrate-producing bacteria within the gut microbiome. Sodium butyrate can promote cell death of cancer cells by mechanisms that are not specific to one cancer. The amount of sodium butyrate-producing bacteria within the gut seems to have a relationship with the overall immunity of a person and their ability to fight cancer. Recent studies that have examined the lungs’ relationship to the gut show that changes in the microbiome

11 Gui, Q. F., Lu, H. F., Zhang, C. X., Xu, Z. R., & Yang, Y. H. (2015). Well-balanced commensal microbiota contributes to anti-cancer response in a lung cancer mouse model. Genetics and molecular research: GMR, 14(2), 5642–5651. https://doi. org/10.4238/2015.May.25.16

12 Chen, Z., Qian, X., Chen, S., Fu, X., Ma, G., & Zhang, A. (2020). 0RW1S34RfeSDcfkexd09rT2Akkermansia muciniphila1RW1S34RfeSDcfkexd09rT2 enhances the antitumor effect of cisplatin in lewis lung cancer mice. Journal of Immunology Research, 2020. https://doi.org/10.1155/2020/2969287

13 Sun, L., Yan, Y., Chen, D., & Yang, Y. (2020). Quxie Capsule Modulating Gut Microbiome and Its Association With T cell Regulation in Patients With Metastatic Colorectal Cancer: Result From a Randomized Controlled Clinical Trial. Integrative cancer therapies, 19, 1534735420969820.https://doi.org/10.1177/1534735420969820 specifically, a decrease in sodium butyrate-producing gut bacteria are connected to the progression of lung cancer. However, it is not clear whether these changes in gut microbiota are a direct result of lung cancer or due to interactions with lung microbiota. The studies discussed in this review establish that distinct and specific changes in the gut microbiome balance are in some way related to immunity and lung cancer, but not very clear on why. It is possible that the diversity of microbiota may only be a result of decreased immunity, which in turn leads to the progression of lung cancer. Defining these relationships is important in understanding how to change the diversity of gut microbiota to stop the development of lung cancer. This is useful to the medical and pharmaceutical industries as it will allow for treatments that involve the increase of butyrate-producing bacteria to be researched for the treatment of lung cancer. In the future, more products and research that focus more on these specific bacteria should be applied to lung cancer.

THE FUTURE USAGE OF 16 S r RNA SEQUENCING IN MICROBIOME STUDIES

Most of the studies mentioned in this review utilized 16 S rRNA sequencing, which is the primary way of analyzing microbes from human fecal samples. As previously mentioned, newer technologies must be developed to analyze more of the less abundant phyla that may act as biomarkers. The studies that involved measuring the reduction of tumor size in patients because of the infusion of certain microbiota were not conducted directly on humans and were performed in vivo or in germ-free mice. This indicates a limitation in whether certain changes in microbe populations apply to mice as well as humans. Additionally, the fecal samples from human patients also did not necessarily account for unhealthy diets and lifestyle habits. These habits change people’s microbiome, so certain microbial changes in humans may not necessarily be from lung cancer, but other factors. Some studies overcame this challenge by using phyla from human fecal samples and injecting them into germ-free mice who were treated with antibiotics and then probiotics. However, not all studies confirmed their results via further animal testing.

More research into the 13 OTU-based system differentiation against other immunocompromising diseases and other cancers should be done to help confirm its accuracy. Additionally, more OTU markers may be identified as shotgun, metagenomic sequencing. This would be important in the medical communities as a viable way to detect lung cancer in a noninvasive method that does not require biopsy.