Abstract A27: Engineering a 3D melanoma microenvironment and identifying novel therapeutic targets

Abstract

Abstract Resistance to targeted therapies remains an obstacle in curing cancers. A reason for this is testing of therapeutic agents in 2D culture conditions that do not recapitulate growth conditions of tumors in vivo. A critical component for defining the cell survival in vivo is the extracellular matrix (ECM). The ECM not only modulates cell-matrix and cell-cell interactions, but also determines drug accessibility to the tumor tissue. Many labs thus evaluate drug perturbations in 3D cell culture platforms using Matrigel and collagen scaffolds. A caveat with these models is the simplistic use of only basement membrane proteins or individual ECM proteins that, again, do not efficiently mimic the tumor microenvironment (TME). Although in vivo mouse models can be a better alternative, the time to conduct experiments, the associated cost, and inability to recapitulate patient TME remain a crucial challenge. Thus, we aim to regenerate an in vitro melanoma TME by engineering a melanoma extracellular matrix. We aim to do this by first identifying the patient melanoma ECM. On conducting proteomics for ECM proteins on patient tissues we identified collagen VI to be the most dominant ECM protein. However, prior work from other labs reports collagen I as the most abundant protein. This information was used to construct a platform through which we can recapitulate the melanoma ECM. We did this by generating cell-derived matrices (CDM) using human foreskin fibroblasts. On comparing the CDM from HFF1 to patient ECM, we could detect ~80 of ~120 core ECM proteins. Upon performing proteomic studies on CDMs, we again identified collagen VI as the dominant ECM protein. We also tested CDM deposition with cancer-associated fibroblasts, yielding similar results. This brought us to the question of whether collagen VI plays a role in drug resistance in melanoma. To address the role of cell-derived matrices and collagen VI, we created collagen VI KO fibroblasts through CRISPR/Cas9, which were then used to generate these matrices and will be used to evaluate its effect on drug treatments. While building this in vitro melanoma TME, we understood the need for a 3D assay to have a robust readout on effect of drugs in 3D compared to 2D. Although many viability and proliferation assays are used by various labs, these assays do not look at the effect of treatments at a single-cell resolution and neither distinguishes if the drug is cytostatic or cytotoxic. Towards that goal, we established a 3D assay by culturing melanoma cells in collagen and tested for Raf-Mek combination therapies. We made comparisons of cells either cultured in 3D as single cells or spheroids. Imaging of these platforms was performed on epifluorescence and light-sheet microscopes. We tested the platform for both viability and proliferation and the images were analyzed through individual Z planes of the 3D image. While we observed differences in viability and proliferation in 2D vs. 3D upon drug treatments, there was no difference in 3D single cells vs. spheroids. Citation Format: Vasanth Siruvallur Murali, Justin Cillay, Erik Welf, Gaudenz Danuser, Murat Can Cobanoglu. Engineering a 3D melanoma microenvironment and identifying novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A27.