Engineering extracellular matrix dynamics linked to ovarian cancer chemoresistance

Introduction:: Dysregulation of the tumor extracellular matrix (ECM) composition and structure critically contributes to a cancer cell’s ability to resist treatment, ultimately leading to over 90% of deaths from cancer. The tumor ECM is a dynamic, non-cellular structure constructed from cellular secretions and it is biochemically and biomechanically distinct in its composition compared to normal tissue ECM. High-grade serous ovarian carcinoma (HGSOC) is the most common type of ovarian cancer and one of the deadliest forms of cancer in women. HGSOC patients are treated with neoadjuvant chemotherapy, but over 80% of patients develop resistance within 5 years. Highlighting its clinical significance, ECM remodeling signatures in HGSOC are associated with an increased risk of treatment failure and patient mortality. While the effects of ECM composition and structure in therapeutic response have been well explored, the ECM changes associated with chemotherapy remain elusive. While the analysis of primary tumor biopsies has provided preliminary evidence of ECM changes associated with chemotherapy treatment, it’s not feasible to study single-cell spatiotemporal ECM dynamics involved in chemoresistance. Therefore, there is a critical need for the development and functional characterization of cancer biomimetic tissue-engineered technologies to profile spatiotemporal ECM dynamics to chemotherapy on a single-cell basis. The overall objective of this investigation is to identify spatiotemporal ECM dynamics in response to chemotherapy on a single-cell basis using functionally characterized cancer tissue-engineered technologies. In the absence of these models, the development of effective anticancer therapies that mitigate chemotherapy resistance in HGSOC will likely remain difficult.


Materials and Methods:: A cancer biomimetic tissue-engineered technology called the three-dimensional extracellular matrix (3D-ECM) was developed through the chemical crosslinking of a hybrid hydrogel formed from ECM-based collagen I and fibrinogen ensuring viscoelastic and fibrillar microstructure, parameters which are critical for 3D mechanosignaling. 3D-ECM was characterized for collagen content by immunohistochemistry and for stiffness by atomic force microscopy. HGSOC cell lines (Kuramochi and SKOV3) were incubated with cancer-associated fibroblast (CAFs) and co-cultured for 14 days in the absence or presence of cisplatin (10uM), recapitulating standard of care treatment with platinum-based chemotherapy. Spatiotemporal evolution of newly synthesized or nascent ECM in response to chemotherapy was visualized and quantified using our 3D-ECM and bio-orthogonal chemistry. Specifically, a non-canonical amino acid (azidohomoalanine, AHA) is incorporated into all deposited nascent ECM components and fluorescently labeled via copper-free cycloaddition with dibenzocyclooctyne-amine (DBCO). Moreover, spatiotemporal evolution of cell-ECM interactions via mechanosignaling through ECM-dependent integrin activation were assessed by visualization and quantification of the phosphorylation of focal adhesion kinase (pFAK) in co-culture within the 3D-ECM in the absence or presence of cisplatin. Cell Profiler was used for image-based spatiotemporal ECM dynamics quantification at single cell resolution including nascent ECM and FAK expression using predefined and optimized pipelines, where at least 100 cells were profiled. See Figure A for a methodological schematic. High-throughput drug screening of cisplatin in combination with ECM targeting agents was performed in order to identify whether a platinum resistant cell line can be re-sensitized to platinum therapy. Cell viability in 3D-ECM cultures was analyzed using CellTiter-Glo®-3D-assay.


Results, Conclusions, and Discussions:: The 3D-ECM was optimized to recapitulate key HGSOC physical diversity parameters including high collagen type I content and the stiffness of the ovary (2.97±0.68 kPa) (Figure B). 3D-ECM co-cultures allow for cancer-stroma cell interactions (Figure B), which are fundamental for tumor recapitulation and response to treatment, and also indicate that both cell types are important contributors to ECM remodeling. Bio-orthogonal metabolic labeling revealed that nascent ECM deposition was significantly enhanced (5-fold) in response to cisplatin treatment (Figure C). Cisplatin-treated 3D-ECM hydrogels showed significantly higher nascent ECM deposition when compared to PBS hydrogel controls, as well as higher matrix thickness in both HGSOC cell lines. Moreover, pFAK expression per cell was significantly increased after cisplatin treatment when compared to the PBS control in 3D-ECM in both HGSOC cell lines (Figure D). Finally, we identified that ECM targeting in combination with cisplatin restores efficacy of cisplatin overcoming platinum resistance. While the platinum resistant cell line shows a 5-fold increase in the half-maximal inhibitory concentration (IC50) when treated with cisplatin alone, cisplatin in combination with an ECM targeting agent reveled total re-sensitization to the level of the parental platinum sensitive cell line (Figure E). Critically, these results suggest that chemotherapy itself induces changes in the ECM, and those ECM-derived changes further promote chemoresistance and are targetable. Further characterization by proteomics of the nascent ECM dynamics in response to chemotherapy is underway and this will allow us to identify the specific chemotherapy-induced ECM dysregulated components influencing chemotherapy resistance. In conclusion, our results present a functionally characterized biomimetic tissue-engineered technology enabling the spatiotemporal profile of ECM dynamics in response to chemotherapy on a single-cell basis. Moreover, our results are expected to have an important positive impact because they will provide strong-evidence for further development of therapeutics that possess potentiality and specificity towards the chemotherapy-induced ECM changes, defining the best therapeutic window of opportunity, and ultimately providing new opportunities for improving chemotherapy effectiveness and mitigating chemoresistance in ovarian cancer.


Acknowledgements (Optional): : This project was supported by NIH/NIGMS Center for Cancer Biology 5P20GM103548 and NIH/NCI R21CA259158.




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