Bioengineered Cancer-Fibroblast Interactions in Patient-Derived Colorectal Cancer Organoid Models

Abstract

The development of patient-derived organoid (PDO) technologies has greatly expanded the toolbox for drug discovery and personalized drug screening for several cancer types. While PDO models more closely represent the molecular characteristics and heterogeneity of patient tumors than traditional immortalized cancer cell lines, they are inherently limited in their ability to reflect the tumor microenvironment in vitro as they comprise exclusively of epithelial cells. The lack of stromal cells in PDO models, such as cancer-associated fibroblasts (CAFs), poses a major problem as these tumor microenvironmental components contribute to the various hallmarks of cancer and response to therapy. Particularly for colorectal cancer, CAFs comprise the majority of the tumor microenvironment and play important roles in cancer progression and drug resistance. In this study, we addressed this problem by establishing in vitro conditions that robustly enable the co-culture of CRC PDO with patient-derived CAFs. We report the development of an engineered tumor microenvironment consisting of CRC PDO encapsulated within a well-defined three-dimensional (3D) hyaluronan-gelatin hydrogel and co-cultured with patient-derived CAFs. Basement membrane extracts conventionally used for PDO culture exhibit batch-to-batch variability. Considering that the CRC extracellular matrix is high in hyaluronan and collagen I, and that hyaluronan-based matrices have been shown to be conducive for the culture of various human cancers, we hypothesized that hyaluronan-gelatin hydrogels may serve as a suitable alternative 3D matrix to support the co-culture of CRC PDO and CAFs. Through RNA- and whole-exome sequencing, we first show that these hydrogels are capable of maintaining key molecular characteristics of the original patient tumors in CRC PDO but not support the culture of CAFs. Further, based on our findings that CRC PDO culture medium poorly supports CAF viability, we developed a co-culture strategy that maintains the viability of both CRC PDO and CAFs. We found that in the absence of growth factors added to the co-culture, CAFs were able to maintain the proliferation of the cultured CRC PDO in the hydrogels and restore distinct biological pathways absent in the PDO culture alone but present in patient tissues. Lastly, we demonstrate that these CRC PDO-CAFs models are suitable for evaluating standard-of-care drugs, making them potentially very useful for realizing personalized cancer medicine.