470529 Systematic Modulation of the Physical Microenvironment for Characterization of Cancer Cell Lines and Primary Tissue
The tumor microenvironment includes a range of physical, chemical, and cellular components such as pressure, hypoxia, acidosis, the extracellular matrix (ECM), stromal, and immune cells. These factors can influence the gene and protein expression of the various types of cells that make up the tumor as well as influence the selection of cells that can thrive in that environment; however, typical cancer cell culture rarely uses hypoxia and pressure nor utilizes substrates similar to the native ECM. We designed a system study the influence of hypoxia, pressure, and native ECM conditions on cancer cell lines and primary cells, with the goal of creating a more relevant culture environment for translational studies.
Here, we comprehensively studied how hypoxia, pressure, and ECM composition and organization 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 which includes a custom bioreactor that allows us to control both 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 controlling ECM composition and organization (aligned or unaligned collagen at concentrations from 1-2.5 mg/ml +/- fibronectin 0.1-10 microgram/ml) as both 2D ECM coatings and 3D hydrogels. Cell lines studied included models for brain cancer (U-87, A172), pancreatic cancer (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 immunotherapy targets CTLA-4, PD-1, and PD-L1.
Additionally, we applied our ability to tune culturing conditions to represent physiological conditions in the body for the successful culture of fresh primary tissues from normal volunteers as well as cancer patients for downstream analysis studies focused on biomarker discovery and as a clinical tool for disease monitoring and treatment decision-making.
We identified both common and unique gene signatures across these different cells lines, with the hypoxic conditions activating HIF1 pathway activation having common across the cells studied whereas pressure results in more restricted signatures. Some cancer cell lines and PBMCs differentially express immunotherapeutic targets, at low oxygen and high pressure culturing conditions, resulting in reduced expression of key targets. Moreover, biomimetic ECM studies revealed modulation of ECM to represent a “tumor”-like ECM dramatically altered cell growth, morphology, and focal adhesion organization. In a 3D hydrogel context, a “tumor” like ECM and a hypoxic environment dramatically enhanced tumor cell invasion. Consistently, analysis of mRNA-seq data revealed alterations in gene expression profiles of immunotherapeutic and drug-target pathways involving CTLA-4 and AR signaling as well as EMT targets. In contrast, we observed increased CD47 and CD44 expression at low oxygen and high pressure culturing conditions in cancer cell lines and PBMCs. Thus, supporting the notion that these are possible drug targets for tumors that are characterized by low oxygen levels and high interstitial fluid pressure.