(F-221) Microvascularized Multiniche Tumor-On-a-Chip for Studying Ovarian Cancer Drug Resistance

Introduction:: Ovarian cancer (OC) has the highest incidence-to-mortality ratio of all cancers. It is considered a silent killer because of the late diagnosis due to unspecific symptoms in the early stage and recurrence and drug resistance in more than 80% of patients. There are many factors that influence drug resistance in OC including the tumor microenvironment (TME) composed not only of cancer cells, but also of stromal cancer-associated fibroblasts (CAFs), and endothelial cells, that participate in extracellular matrix (ECM) remodeling and secretion of cytokines and signaling molecules that can affect the cancer cell behavior and drug response. The ECM can act as a physical barrier and generate gradients of nutrients, oxygen, and drugs. Low levels of oxygen, or hypoxia, is a key hallmark of ovarian tumors and is also associated with an increased risk of treatment failure and patient mortality. Moreover, more than 95% of cancer drugs fail in clinical trials due to lack of efficacy clearly highlighting the inability of the current preclinical models to accurately predict therapeutic response. Therefore, there is a pressing need to develop physiologically relevant preclinical models recapitulating the physical properties influencing chemoresistance including the ECM remodeling and hypoxia. Our overall objective is to develop a microvascularized multiniche tumor-on-a-chip microfluidic device for mimicking the hypoxic OC-TME and ECM remodeling and studying the influence of CAFs, endothelial cells, and cell-cell interactions on drug resistance in OC. In the absence of these models, the development of effective anticancer therapies that mitigate chemoresistance in OC will likely remain difficult.


Materials and Methods:: SU-8 lithography was used to develop polydimethylsiloxane (PDMS) tumor-on-a-chip formed by five chambers (Figure A). Specifically, the PDMS device is designed with a central cancer chamber for seeding OC cancer cell lines (Kuramochi or SKOV-3), flanked by four lateral microchannels. The adjacent stromal chambers include normal healthy fibroblasts or cancer-associated fibroblasts (CAFs) in monoculture or co-culture with human vein endothelial cells (HUVECs) and the lateral circulation chambers contain culture media and drugs, all together recreating the “surrogate” tissue and vascular network around a tumor. The stromal and central cancer chambers are 3D fibrinogen hydrogel-based allowing cell-cell and cell-ECM interaction, and the circulation chamber contains circulating media. 300 μl reservoirs are implemented in the inlets to generate laminar flow in the device. The chips were evaluated for growth, cell migration, and vessel formation by confocal microscopy over 2 weeks, where the cell types were identified by membrane surface staining or antibody labeling (cancer-DiD-magenta, stroma-anti-CD90 antibody-green, and HUVECs-anti-CD31 antibody-red). Development of an oxygen gradient across the central chamber containing OC cells was confirmed using a hypoxia-sensing dye (Image-iT Hypoxia Reagent). ECM remodeling by collagen I deposition was evaluated by immunohistochemistry. Drug penetration and drug uptake was evaluated by confocal microscopy using the cytotoxic drug doxorubicin (DOX), which exhibits natural fluorescence properties (Ex/Em: 480/590). To study drug response, standard of care OC treatment with paclitaxel and carboplatin (PTX/Carbo) was administered and cell viability (NUCLEAR-ID viability kit) was assessed.


Results, Conclusions, and Discussions:: The tumor-on-a-chip microfluidic device allowed for stromal and cancer niche recapitulation, as well as establishment of vessel-like structures validating the microvascularized multiniche assembly (Figure B). The chip was designed to study cell-cell interactions and migration between the compartments, where OC cells were identified proliferating and moving towards stromal chambers on day 5 and 7 (Figure C) initiating metastasis-like migration. The device was optimized to recapitulate physiologically relevant hypoxic gradients from the circulation chamber towards the cancer chamber (Figure D), as well as high collagen type I content provided by the stromal compartment (Figure E). Doxorubicin penetration and uptake experiments confirmed the device’ capabilities to mimic in vivo tumor drug diffusion and cell uptake, where the drug penetrated and was uptaken faster in the stromal chamber compared to the cancer chamber, but all drug reached the central chamber and the cancer cells by 24 hours (Figure F). Moreover, we observed increased drug resistance (less red dead cells) to PTX/Carbo treatment when cancer cells are co-cultured with CAFs when compared to normal fibroblasts, validating the microvascularized multiniche tumor-on -a-chip to mimic OC chemoresistance mechanisms (Figure G). Critically, these results suggest that physical properties of the TME including ECM remodeling, hypoxia, drug penetration, as well as cell-cell interaction with cancer-associated fibroblasts and endothelial cells are key contributors to OC chemoresistance. Moreover, we demonstrated that a precise recreation of the cellular crosstalk in the TME, as well as spatial organization through compartmentalization, are fundamental to determining the effect of these physical and biological mechanisms on drug resistance. In conclusion, our results present a functionally characterized microvascularized multiniche tumor-on-a-chip device enabling recapitulation of the hypoxic OC-TME and ECM remodeling and investigation of chemotherapeutic response and drug resistance influenced by drug penetration, cancer-associated fibroblasts and endothelial cells in OC. Our results are expected to have an important positive impact because they will provide strong-evidence for providing new opportunities for improving chemotherapy effectiveness and mitigating chemoresistance in ovarian cancer.


Acknowledgements (Optional): : This project is supported by NIH/NCI R21CA259158.


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