Preclinical 3D bioprinted model of ovarian cancer tumor microenvironment to test miRNA-based personalized therapies

Epithelial Ovarian cancer (EOC) is the most lethal malignancy in women. Despite de-bulking surgery, chemotherapy and radiotherapy almost 90% of EOC patients will relapse and succumb. One of the main causes of drug resistance and metastasis is the tumor microenvironment (TME) consisting of cancer-associated fibroblasts (CAFs), endothelial cells (ECs), anergic immune cells, adipocyte stem cells (ASC) and other components1,2. Mounting evidence suggest that microRNAs (miRNAs) play critical roles in shaping the TME by inhibiting gene transcription in cancer cells3. We have previously shown that miR-200c was able to contrast the induction of the immune checkpoint PD-L1, c-myc and β-catenin oncogenes expression by combinatorial therapies in EOC cell lines and biopsies4. Another study showed that miR-200c inhibited epithelial-to- mesenchymal transition (EMT), by targeting ZEB15. Recently, ASCs were discovered as key players in EMT of OC1,6. Since miR-200c targets multiple genes and de-regulates different molecular pathways, more physiologically relevant human preclinical models are required to unravel its role in TME. In this work, we 3D printed a biomimetic human 3D printed-OCTME model where miR-200c transfected tumor cells interact closely with immune cells and ASCs. The proposed model conveys unprecedent physiological features to better evaluate the effects of miR-200c on i) anti- tumor T-cell response, ii) ASC immunomodulation and iii) EMT in OC cells. For the 3D TME model, a microfluidic-based extrusion bioprinter was used to deposit two GelMa-based bioinks containing miR-200c-transfected SKOV3 cells and ASCs isolated from healthy donor. The microfluidic spatial control on the material deposition results in the fabrication of cylindrical constructs with radial cell concentration gradient (i.e. tumor cells in the core, ASCs in the shell). After 3 days, PBMCs, isolated from healthy donors, were added on top of the constructs and let for 3 hours to penetrate the gel. The same bioprinting conditions were also used for the parental cell line transfected with the empty vector. Confocal microscopy of cell-labeled constructs at day 5 revealed the formation of a TME exhibiting the physiological architecture found in vivo. ASCs cells surrounded the tumor site enabling the interaction with tumor cells. T-cells traveled across the stromal tissue and reached the tumor site, demonstrating their successful inclusion in the structure. Cells viability and functionality were addressed by live/ dead assay and immunostaining of activated T-cells and SKOV3. The proposed study aims to develop a 3D TME in vitro preclinical model to better understand the interactions between tumor cells, immune cells and ASCs in the presence/absence of miR-200c. Our technology is a proof-of-concept assay toward a precision stratification of EOC patients before undergoing miRNA-based and drug therapies, to test the therapeutic effectiveness and avoid side effects of these treatments.