Automation and High Throughput Technologies

Podium

3D Paper-Based Tumor Models to Characterize the Relationship Between the Tissue Microenvironment and Multidrug Resistant Phenotypes

• 

  Julie McIntosh

  Graduate Research Assistant
  UNC Chapel Hill

Multidrug resistance arises when a small population of cells in a tumor undergo changes in cellular phenotype that allow them to evade a particular treatment. We are developing a novel 3D paper-based culture screening platform that can stratify cells within tumor-like structures based on either environmental or phenotypic differences. Unlike other 3D culture platforms, we are able to rapidly separate (within seconds) discrete cell populations based on the location.

Our platform involves stacking cell- or extracellular matrix (ECM)-laden paper scaffolds together to create a 3D tissue-like structure, which models the tumor microenvironment with defined gradients of oxygen, waste, and nutrients. The stacks are disassembled by peeling the paper scaffolds apart to isolate each discrete sub-population for downstream analyses of cell viability, protein and transcript levels, or drug metabolism. Our system is also highly customizable by: i) altering the size, number, or shape of available seeding areas by wax printing seeding boundaries onto each scaffold; ii) manipulating gradients by changing seeded cell density, stack thickness, or limiting exchange with culture medium; or iii) enabling either monoculture and co-culture setups, which allows the user to simulate a wide variety of in vivo conditions based on the research of interest.

Here, we utilize this novel platform to investigate the phenotypic characteristics of chemotherapeutic resistant sub-populations within each culture. With a tumor cross-section model, we utilize flow cytometry to quantify the differences between cells at the drug resistant necrotic core of a tumor and cells at the proliferative outer shell. Specifically, we quantify the phenotypes that lead to passive resistance (e.g., decreased metabolic activity) or active resistance (expression of ATP-consuming drug efflux pumps). With an invasion assay setup, we investigate the relationship between drug resistance and the epithelial to mesenchymal transition (EMT) by isolating cells based on their level of invasiveness; a well-documented, observable phenotypic change that results in mesenchymal cells being more invasive than epithelial cells. The isolated sub-populations are analyzed for changes in i) drug response with dose-response curves, and ii) epithelial and mesenchymal specific protein expression levels with enzyme-linked immunosorbent assays (ELISAs) as a means to connect drug resistance to epithelial or mesenchymal phenotypes. This platform is an inexpensive customizable screening tool, which can be utilized to study specific subsets of cellular populations for advancements in both targeted chemotherapeutic development and understanding of the heterogeneity of tumor populations.