Capturing the heterogeneity of the pancreatic ductal adenocarcinoma tumor microenvironment: novel triple co-culture spheroids for drug screening and angiogenic evaluation

Abstract

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) poses a significant health threat with poor response to current treatment options. The desmoplastic reaction, characteristic of PDAC, hinders therapeutic efficacy and emphasizes the need for novel in vitro models to study the complex tumor microenvironment and increase translatability. Three-dimensional in vitro co-culture models with clinically relevant numbers of cancer-associated fibroblasts and endothelial cells are still lacking and lead to failure of clinical trials and low improvement of patient survival.Methods MiaPaCa-2 and BxPC-3 cancer cell lines, RLT-PSC and hPSC21 pancreatic stellate cell lines and the endothelial cell line HMEC-1 were seeded in ultra-low-attachment round-bottomed plates to form triple co-culture spheroids. A growth assay including all cell lines was performed to evaluate if DMEM or MCDB131 is most ideal for spheroid formation and culturing. Multi-color flow cytometry was used to quantify cell populations after three days of spheroid formation to optimize the seeding ratios. Drug response profiles of mono-culture and triple co-culture spheroids were made using a cell viability assay. Finally, a tube formation assay with spheroid-conditioned medium was performed to showcase the potential of our model for angiogenic studies.Results We developed a panel of high-throughput triple co-culture spheroid models of pancreatic cancer cells, pancreatic stellate cells and endothelial cells. We were able to capture different facets of PDAC heterogeneity in scope of the tumor microenvironment using two different cancer and stellate cell lines, and one endothelial cell line. Importantly, drug responses varied between mono-culture and triple co-culture spheroids, underlining the impact of the tumor microenvironment, spatial arrangement, and spheroid density on therapeutic outcomes. Gemcitabine and paclitaxel treatments revealed different drug response profiles depending on the combination of BxPC-3 or MiaPaCa-2 with RLT-PSC or hPSC21 in a triple co-culture environment. A tube formation assay showcased the potential of our models to assess angiogenesis, providing a quantitative understanding of a treatment-induced response.Conclusions Our study brings sophisticated high-throughput in vitro models that are easy to reproduce and provide valuable insights into PDAC research to improve translatability and preclinical screening efficacy. In addition, our triple co-culture spheroids are cheap and include the heterogeneity of the PDAC tumor microenvironment.