Abstract 5122: Characterization of the effects of hypoxia and hydrostatic pressure on the expression of immunotherapeutic targets on cell lines used for translational research
Authors: Bruce A. Adams, James Lim, Luke Cassereau, Tianna Chow, Susan Bernstein
Journal: Cancer Research (2016)

Abstract INTRODUCTION The characterization of the tumor microenvironment includes mechanotransduction, hypoxia, acidosis, and tissue remodeling. These factors can influence the gene and protein expression of various cell types that make up the tumor as well as influence the selection of cells that can thrive in a given microenvironment; however, typical cancer cell culture rarely uses hypoxia and pressure nor utilizes substrates similar to the native extracellular matrix (ECM). We designed a system to study the influence of hypoxia, pressure, and native ECM conditions on cancer cell lines and primary cells, with the goal of creating culturing environments relevant for translational studies involving key immunotherapeutic targets. METHODS First, we studied how hypoxia with or without pressure influences cell biology and gene expression, using transcriptome profiling across a range of physiologically-relevant culturing conditions to mimic various tumor microenvironments found within the body. To do this, we utilized Xcell's primary cell culture platform that allows for the precise control of oxygen concentration (0.1%-20% O2) and hydrostatic pressure levels (26 to 260 mmHg / 0.5 psig to 5 psig). Additionally, we studied the influence of biomimetic substrates by ECM composition and organization (aligned or unaligned collagen at concentrations from 1-2.5 mg/ml +/- fibronectin 0.1-10 microgram/ml). Cell lines studied include models for brain (U-87, A172), pancreatic (PANC10.05), and prostate cancer (DU-145, PC-3, 22Rv1, LNCaP). We performed high-resolution immunofluorescence imaging and western blot protein expression analysis of key targets, including immunotherapeutic targets CTLA-4, PD-1, and PD-L1. Finally, we applied these culturing conditions to characterize primary tissues obtained from cancer patients for studies focused on biomarker discovery, patient monitoring and treatment decision-making. CONCLUSIONS We identified both common and unique gene expression signatures across different cells lines, with hypoxic conditions activating HIF1 signaling, whereas hydrostatic pressure resulted in restricted signatures of high clinical value. Cancer cell lines and PBMCs differentially express immunotherapeutic targets, at low oxygen and high pressure culturing conditions, resulting in reduced expression of key drug targets. Analysis of mRNA-seq data revealed alterations in gene expression profiles of immunotherapeutic and drug-target pathways involving CTLA-4 and AR signaling. In contrast, we observed increased CD47 and CD44 expression at low oxygen and high pressure culturing conditions in cancer cell lines and immune cells. Therefore, these results support the presence of physiologically-relevant drug targets in tumors and immune cells characterized by low oxygen and high interstitial fluid pressure. Citation Format: Bruce A. Adams, James Lim, Luke Cassereau, Tianna Chow, Susan Bernstein. Characterization of the effects of hypoxia and hydrostatic pressure on the expression of immunotherapeutic targets on cell lines used for translational research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5122.