Engineering an ex vivo high grade serous ovarian carcinoma 3D culture model for precision-based drug screening

Introduction:: 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. In HGSOC tumors, both hypoxia and the dysregulation of the extracellular matrix (ECM) composition and structure critically contribute to a cancer cell’s ability to promote growth and resist treatment. Both in vivo and in vitro studies have demonstrated how tumor cell-ECM interactions affect tumor progression and therapy response. However, high cost and species-dependent differences between humans and mouse models or mouse-derived materials, make it challenging to isolate mechanistic links between hypoxia, cell-ECM interactions, and treatment response. Tumoroids (“tumor-like-organoids”) have shown to retain both histological and genetic features of original tumors and are feasible for in vitro drug sensitivity assays, recapitulating clinical responses of the matched patients, but often lacking physiologically relevant physical characteristics. Therefore, there is a critical need for the development and functional characterization of technologies for precision-based drug screening accounting for physiologically relevant tumor characteristics. The overall objective of this investigation is to provide a translationally relevant 100% patient-derived 3D culture platform with controlled physiologically relevant physical properties to perform clinically relevant analysis of HGSOC treatment responses. In the absence of these models, the prediction of the best therapeutic regimen for each patient will likely remain difficult and the improvement of survival rates and reduction of healthcare costs unachievable.


Materials and Methods:: A precision-based tissue-engineered technology called the HGSOC patient-derived fibrin (HGSOC-PDF) was developed through the crosslinking of plasma fibrinogen to fibrin and contains patient-derived tumoroids within the human patient-derived plasma 3D matrix, vascularized with human umbilical vein endothelial cells (HUVECs) (Figure A). HGSOC-PDF was fully characterized and compared to the matched parental tumors at histological level (immunofluorescence of cytokeratin and PAX8), biochemical preservation by cytokine array, and physiologically relevant physical properties including oxygen content by oxygen microsensor, ECM composition, and stiffness by atomic force microscopy. Cell-ECM interactions via mechanosignaling through phosphorylation of focal adhesion kinase (pFAK) and translocation to the nuclei of Yes-Associated Protein (YAP) in the absence or presence of cisplatin (10uM) was evaluated in PDFs recapitulating physoxia/ECM^low compared to hypoxic/ECM^high. Cell Profiler was used for image-based quantification at single cell resolution using predefined and optimized pipelines, where at least 300 cells were profiled. 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. Primary biospecimens were categorized as sensitive and resistant by Response Evaluation Criteria In Solid Tumors or RECIST score (defined by pathology, radiology, and/or physician decision of follow-up response >6 months to standard of care treatment). Precision-based drug screens were performed in order to establish a predictive score able to distinguish sensitive from resistant patients using several read-outs including proliferation, apoptosis, viability and immune status.


Results, Conclusions, and Discussions:: HGSOC-PDFs recapitulated structural complexity, biochemical composition, and morphological features of HGSOC tumors. Cytokeratin (epithelial marker, red) and PAX8 (fallopian tube and HGSC marker, green) revealed the preservation of the native epithelium and tumor heterogeneity (Figure B). HUVECs plated on top of the PDF matrix formed tube-like structures after 24h (Figure C). The HGSOC-PDF model was engineered to recapitulate physiological oxygen levels of the healthy physoxic ovarian follicle tissue compared to the hypoxic ovarian tumor tissue. PDFs were profiled and showed that while incubation at 21% O₂ captures 6.7kPa oxygen content, 1.5% O₂ incubation recapitulates 0.77kPa inside HGSOC-PDFs (Figure D). The ECM production at 1.5% O₂ incubation revealed increased deposition of collagen I and laminin compared to the ECM composition at 21% O₂ incubation (Figure E). Additionally, increased stiffness in the matrix correlated with the increased ECM deposition in 1.5% O₂ incubation compared to 21% O₂ (Figure F). Using this model, we identified platinum resistance mechanisms via adhesion FAK and mediators of ECM mechanical YAP signaling, which were aggravated in hypoxia/ECM^high conditions. Both pFAK expression and nuclear to cytoplasmic ratio (nuc:cyto) of YAP were significantly increased in hypoxia/ECM^high compared to physoxic PDFs, and cisplatin treatment enhances this expression (Figure G&H). We also identified that ECM targeting in combination with cisplatin restores efficacy of cisplatin overcoming platinum resistance (Figure I). Finally, precision-based drug screens using HGSOC-PDFs allowed us to distinguish sensitive from resistant patients (Figure J). Critically, these results indicate that the PDFs are a unique in vitro model that allows for the recapitulation of physiologically relevant physical properties, making the PDFs a well-suited platform for precision-based prediction of therapeutic efficacy in HGSOC. In conclusion, our results present a reproducible and clinically translatable preclinical model assessing effective treatment options by predicting therapeutic efficacy and avoiding treatment with drugs that the tumor will be resistant to. Moreover, our results are expected to have an important positive impact because they will provide a valuable tool in predicting each individual patients’ response to therapy and permit a much more in-depth and clinically relevant analysis of HGSOC treatment responses than is currently possible.


Acknowledgements (Optional): : This project is supported by NIH/NIGMS DaCCoTA Ready to Go Award under U54GM128729 and NIH/NCI R21CA259158.


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