Evaluation of immune responses in a novel ovarian cancer immune-oncology three-dimensional cell culture model

Abstract

<h3>Objectives:</h3> We aimed to develop and validate a novel ovarian cancer 3D immune oncology co-culture model consisting of high grade ovarian cancer tumor spheroids cultured with immune cells. We evaluated outcomes including tumor spheroid viability, ability to visualize immune cell migration and invasion, cellular expression of immune factors, and cell death resulting from immune co-culture. <h3>Methods:</h3> Spheroids were grown in 96-well U-bottom microplates using SKOV3 and OVCAR8 cell lines. Spheroids were then co-cultured with human peripheral blood mononuclear cells (PBMCs) from gynecologic cancer patients in 96-well 5 µm Transwell plates to allow for immune cell migration and invasion into spheroids. Confocal microscopy of live, labeled cells was performed to visualize migration and spheroid invasion. Flow cytometry was performed to determine spheroid viability over time and expression of immune factors MHC1 and PD-L1 on spheroid cells. Cancer cell death resulting from immune cell co-culture was quantified by propidium iodide immunofluorescence and compared to cell death in spheroids in the absence of PBMC or in spheroids treated with carboplatin. <h3>Results:</h3> SKOV3 and OVCAR8 spheroids became highly compacted after 48-72 hours in culture and retained excellent viability over 1 week. Strong e-cadherin expression was visible in both cell types at 48 hours. Live imaging of spheroids co-cultured with PBMC revealed robust invasion of the spheroids, which increased with increasing PBMC numbers and with time. MHC1 and PD-L1 levels were measured on spheroids alone cultured over 72 hours. Robust expression of MHC1 was observed on SKOV3 cells but was almost absent on OVCAR8 cells. Conversely, prominent expression of PD-L1 was noted on OVCAR8 cells. Immune cell killing of cancer cells was increased compared to controls for both SKOV3 and OVCAR8. OVCAR8 cells demonstrated different dynamics of cell death than SKOV3, with cell death in OVCAR8 peaking at 72 hours and cell death in SKOV3 peaking at 48 hours. <h3>Conclusions:</h3> Our findings demonstrate feasibility and reproducibility of an ovarian cancer 3D immune oncology co-culture model. As compared to traditional 2D tissue culture, this new technique may offer a more physiologic representation of cancer, its microenvironment, and its interaction with the immune system, which may improve preclinical study of cancer and antineoplastic agents.