Development of Methods to Accurately Quantify Cellular Viability and Multidrug-Resistant Phenotypes within 3D Paper-Based Tumor Models

During cancer progression, cells undergo genetic mutations and epigenetic alterations that, in coordination with influences from the tumor microenvironment, lead to more aggressive cellular phenotypes, which can survive within cytotoxic conditions. Not only can these cancer cells evade antineoplastic chemotherapies, but they adopt a highly invasive mesenchymal phenotype that facilitates their invasion into the surrounding healthy tissue and can lead to metastasis. Multidrug resistance is thought to be the cause of treatment failure for over 90% of cancer patients whose disease has progressed to metastasis. The tumor microenvironment consists of a complex network of vasculature, biotic and abiotic chemical gradients, stroma cells, and extracellular matrix that all play a role in cancer survival and progression. Of particular interest are the consumption-based chemical gradients, specifically oxygen, that form throughout poorly perfused tumors due to cancer cell proliferation outpacing vasculature growth. These gradients lead to the formation of distinct phenotypic regions throughout a tumor mass with well oxygenated proliferative cells adjacent to the vasculature and necrotic cells at the hypoxic tumor core. These cellular phenotypes have different sensitivities to chemotherapies, making tumor models that spatially replicate cellular phenotypes ideal platforms for studies into multidrug resistance. Paper-based cultures (PBCs) are a 3D culture platform that can model tumor cross-sections by stacking multiple layers of cell-laden paper scaffolds together. By limiting the exchange between the PBC stack and medium to a single side, consumption-based abiotic chemical gradients form across the stack. These structures can be easily sectioned into 40 μm thick slices by pealing each scaffold apart. This ability to simply unstack the culture provides a spatial resolution that is ideal for studying how discrete microenvironments throughout the tumor model effect cellular response. A key measurement when studying drug potency and efficacy is cell viability. Determining an accurate assessment of the cellular viability in response to chemotherapeutic dosing is vital not only for drug screens of novel compounds but also in-depth studies of multidrug resistance mechanisms. The majority of viability assays are designed for 2D monolayer cultures and utilizing them with 3D culture platforms requires additional considerations for proper assays selection as 3D cultures have added complexity, which can pose experimental challenges. Indirect viability assay measure cellular phenomena as a marker for viability and provide a bulk readout of the entire sample. In this work, three indirect, end-point viability assays, traditional used with 2D monolayer cultures, were characterized with 3D PBCs. The analytical figures of merit for each assay were determined from calibration curves and the effectiveness of each assay in quantifying small changes in cell number was assessed with dose-response curves. In addition, optical coherence tomography—a biomedical imaging technique that senses near-infrared light backscatter from tissues to identify viable cells—was adapted to PBCs and marks the first instance of in situ longitudinal measurements of a stacked PBC. Indirect viability assays are often sensitive to the cellular microenvironment that influences the cellular phenomena being measured. These environmental influences can alter the assay sensitivity and cause misleading results when chemical gradients are present throughout a sample, as is the case with a PBC tumor model. In this work, we evaluated the influence of the oxygen gradient throughout PBC tumor models on the assay performance of the indirect viability assays and optimized experimental protocols to circumvent this influence. In addition, we adapted flow cytometry, an environmentally independent, direct measurement of cell viability for PBCs. With flow cytometry we were able to determine the distribution of viable cells present throughout PBC tumor models and confirm that multidrug resistance occurs within the hypoxic regions of the tumor model. We are currently using flow cytometry to investigate the possible mechanisms of drug resistance that cells within the hypoxic regions of the tumor model are using to evade drug induced cell death. In summary, my dissertation work has focused on successfully adapting, optimizing, and validating viability assays with PBCs to study the invasive and multidrug-resistant phenotypes that form throughout invasion and tumor model cultures.Doctor of Philosoph