Observing variable effects of Microbial Treatments in Different Cervical Cancer Cells
Jocelyn Mejia Mentor: Victoria Mgbemena Department if Biology Introduction: The American Type Culture Collection (ATCC) is a repository for different cell lines which are derived from primary tumors or metastatic sites. Each cell line is cataloged based on individual cell line characteristics, culture methods, and specifications. A comprehensive report detailing how each of the existing cervical cancer cell lines responds to the cervical commensal microbe, Lactobacillus acidophilus (BSL1), has yet to be compiled. Further, there is not a comprehensive profile of immune factors secreted by the cells under different conditions. ‘Lactobacillus Acidophilus is one of the more populous bacteria species found in the vaginal tract, comprising about 90% of the vaginal microbiome. This bacterium is important for maintaining the vaginal pH in an acidic range, thereby limiting the growth of other strains of bacteria. The cell lines C33-A, SiHa, DoTc2-4510, and Ca-Ski will be profiled in the presence and the absence of Lactobacillus. Our laboratory aims to study the growth requirements for each of the cell lines and assess the impact on gene expression. Cervical cancer is among one of the fourth most common cancers among women with approximately 570,000 cases worldwide and 311,000 deaths reported in 2018. [1]. Symptoms in the early stages are undetectable unless getting proper screening, leading to a misdiagnosis such as premenstrual symptoms. However, many gynecologists have help increase early detection of cervical cancer with persistent recommendations of pap-test exams during regular checkups. [2] The screening of cells from a pap-test is examined by a pathologist and is checked for abnormalities. Other test is done for further examination for the cervical cells, including colposcopy, loop electrosurgical excision procedure (LEEP) and cone biopsy to determine the stage and aggression of the carcinoma. [3] Early detection and proper diagnosis and treatment help reduce the risk of cancer-related death. The vaginal microbiome is made of many different types of cells and bacteria, including Lactobacillus acidophilus (BSL1). Research has shown that changes in the vaginal microbiome contribute to the formation of cervical cancer. In a healthy vagina, the balance of the microbiome is an important factor that allows for protection against sexually transmitted illnesses, such as Human papillomavirus (HPV) and multiple gynecological diseases, including cervical carcinomas. [4] A disturbance in the vaginal pH in an acidic range can lower one chance of fighting an infection such as HPV, which contributes to 70% of cervical cancer. [5] BSL1 is a major influence in Cell lines used to further understand the change and development in cervical cancer cells in our study will include C33-A, SiHa, DoTc2-4510, and Ca-Ski obtained from ATCC repository. Hypothesis: We hypothesize that we will observe cytokine expression changes in cells treated with Lactobacillus Acidophilus. Specific Aim 1: Investigate the effect of Lactobacillus Acidophilus on gene expression in cervical cancer cell lines. Specific Aim 2: Investigate the effect of Lactobacillus Acidophilus in the presence of different germline and somatic PALB2 mutations in cervical cell lines and cervical cancer cell lines. Specific Aim 3: Assess expression changes in a cell co-culture model of the microbiome for HPV positive and HPV negative cancer cell lines. Materials and methods: Cervical cancer lines will be grown in-vitro using cell growth medium and factors. Cells will be transfected stably with plasmid vectors made to encode various PALB2 mutations. Cells will be transfected with factors Sox2, Oct-4 and Nanog and supplemented with growth medium. Cells will then be treated with: 10ng/mL of Lactobacillus Acidophilus, then incubated for 24 hours in 37-degree Celsius incubator. Cell lysates will be collected and analyzed using microarray (Affymetrix Gene Chip). Later, PALB2 mutations will be expressed in cell lines and cells will be assayed for changes in gene expression. Finally, different cell lines will be co-cultured in a 2D model in the presence of an array of BSL-1 level microbes. We began by culturing Dotc2 4510, which do not heavily secrete the pro-inflammatory cytokine IL-6 [6], yet is modulated by STAT3, a transcription factor responsible for the production of cytokines and interferons. Results/Expected Results: Changes in the microbiome will yield observable effects on cell expression profiles and function. We observe differences in the expression level of PALB2 in treated, DMSO treated DoTC2 4510 and HeLa lysates. We also observed a difference in the secretion of factors including G-CSF, Serpin E1 and IL-1Ra. More G-CSF and Serpin E1 was secreted from DMSO, control treated cells than treated cells, and less IL1Ra was secreted from DMSO, control treated cells than treated cells. We later assayed for surface glycoprotein ICAM-1, which produces pro- inflammatory Page 141 factors. ICAM1 seemed to have increased expression in treated cells. of 3
