VCOM Research Day Program Book 2023

Medical Student Research Biomedical

15 Tumor Infiltrating Immune Cells in Human Lung Adenocarcinoma

Ramu Anandakrishnan; Catherine Bassett; Shourik Dutta; Diego Flores; Trent Kite; Ellis Linder-Elkins; Yaswitha Mikkilineni; Rajvi Patel; Nina Pillai; Veda Prasad; Addison Shenk; David Valent; Tarana Zaman Corresponding author: tkite@vt.vcom.edu

Edward Via College of Osteopathic Medicine-Virginia Campus

Although considerable progress has been made in reducing mortality rate in cancer patients, it remains a leading cause of death, second only to heart disease. Clearly, there is an urgent need for more effective treatments. A hallmark of cancer is the tumor cell’s ability to avoid immune destruction. These mechanisms include mutations in tumor cells that allow them to evade detection by immune cells, inhibit immune cell recruitment/ infiltrations, induce immune cell apoptosis, and the production of factors that inhibit immune response. However, the potential effect of somatic mutations in tumor infiltrating immune cells have not been previously studied. In a relatively recently discovered phenomenon, clonal hematopoiesis (CH); mutations in specific genes can lead to the clonal proliferation of individual cells resulting in a significant clonal population with somatic mutations. Three possible mechanisms have been proposed for CH. The mutation in a hematopoietic stem or progenitor cell causes (1) increased self-renewal; (2)

increased number of self-renewal cycles required to become a committed progenitor, or (3) increased epigenetic or transcriptional heterogeneity leading to clonal selection of highly proliferative states. Our work centered on identification of potential immunosuppressive mutations in tumor infiltrating immune cell subsets via tissue digestion of primary human lung adenocarcinoma tissue provided by the biospecimen repository at Carilion Clinic. Tissue was obtained, digested, and isolated using fluorescence activated cell-sorting (FACS). DNA and RNA were subsequently isolated for PCR analysis. Using primer sets designed to test each of 9 potential somatic mutations previously identified via in-silico analysis we normalized expression of each gene based on comparative cycle threshold (Ct) values normalized to GADPH expression. Cell culture models of SC 9855(Human Monocyte/Macrophage) gene edited with CRISPR systems were developed to recapitulate the mutations of interest. Media sampling for key

inflammatory mediators (IL-6, TNF alpha) were used to analyze the effect of the mutations on immune cell activity. This project will further provide evidence to the theory of CH as well as provide a novel perspective on tumor microenvironment interactions in cancer. Identifying driver mutations in mutated immune cells could provide the basis for a novel therapeutic approach in tumors highly enriched with immune cells.

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