Abstract A87: Myristoylated alanine rich C-kinase substrate (MARCKS) expression in lung cancer cells influences immune cell populations in tumor microenvironment in murine models

Abstract

Abstract Purpose: To determine if MARCKS expression in lung cancer cells alters the recruitment of immune cells to the tumor microenvironment while influencing outcome in in vivo models. Background: Lung cancer is the leading cause of cancer related deaths in the United States. There is increasing support that aberrant immune cell recruitment and activation in the tumor microenvironment leads to pro-tumor behavior. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) is an intracellular protein that has been described to alter cellular proliferation as well as be involved in lung epithelial cytokine secretion and immune cell migration. Depending on the type of cancer, MARCKS has either pro-tumor or anti-tumor properties. Currently, it is unknown whether MARCKS expression is beneficial or detrimental in lung cancer. We wanted to investigate if MARCKS expression was able to delay tumor burden as well as influence the immune cell populations being recruited to the tumor microenvironment. Methods: The C57BL/6 murine lung cancer cell line Lewis Lung Carcinoma (LLC) was engineered with lentiviral particles to over-express MARCKS (LLC-MARCKS). Western blot confirmed over-expression of MARCKS. Tumors were implanted into the flank of C57BL/6 mice and survival data was collected comparing LLC-MARCKS over-expression tumors versus empty lentiviral plasmid containing LLC tumors (LLC-Ctrl). Survival data was graphed by Kaplan-Meier and statistics calculated with log-rank survival test. To analyze immune cells in the tumor microenvironment, orthotopic lung nodules were established by tail-vein injections. Three weeks after tail-vein injections, lungs were resected from LLC-MARCKS and LLC-Ctrl tumors bearing mice for flow cytometry analysis. Differences in immune cells between groups were calculated by student's t-test. Results: Mice with LLC-MARCKS tumors had an increase in median survival time compared to LLC-Ctrl tumors (31 days vs. 26 days) and a trend toward increase survival (p=0.058). While collecting lungs, it was grossly apparent that mice with LLC-Ctrl tumors had greater tumor burden and signs of disease, such as bloody exudates, compared to mice with LLC-MARCKS tumors. H & E staining confirmed that LLC-Ctrl tumors had higher tumor burden then LLC-MARCKS tumors. Flow cytometry identified a decrease in F4/80+ macrophages (p<0.05) and neutrophils (p<0.05) in LLC-MARCKS cells compared to LLC-Ctrl. There was no difference in MDSC, DC subpopulations, CD4 T-cells, CD8 T-cells, B-cells, or T-regs. Conclusion: MARCKS expression in the murine lung cancer LLC cell line prolonged survival while also decreasing F4/80+ macrophages and neutrophils in the tumor microenvironment. Citation Format: Timothy D. Rohrbach, Travis D. Hull, John S. Jarboe, Nicholas Eustace, Yong Wang, Jessy S. Deshane, Christopher D. Willey. Myristoylated alanine rich C-kinase substrate (MARCKS) expression in lung cancer cells influences immune cell populations in tumor microenvironment in murine models. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A87.