Introduction:: Colorectal cancer is the third most common cancer. Interactions between tumor cells and the tumor microenvironment (TME) promote cancer progression and therapeutic resistance [1]. Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells in the TME. CAFs secrete various soluble factors to biochemically interact with cancer cells and physically remodel the TME through deposition and degradation of the extracellular matrix [2]. Physiological tumor models are critical to understand specific disease mechanisms and identify effective treatments. We aimed to establish an organotypic model of primary colorectal tumor cells and CAFs for mechanistic studies and testing efficacy of drugs toward realizing personalized cancer medicine.
Materials and Methods:: CN0375 colorectal cancer cells derived from a patient was obtained through the NIH/NCI PDMR. Primary colorectal CAFs were obtained from Neuromics. Single cancer and CAF cells were seeded in a 1:2 ratio in 25 µl of growth factor reduced, phenol red-free Matrigel in each well of a 96-well plate. The Matrigel was polymerized for 30 minutes at 37 °C. Then, 200 µl of F12 medium containing a rock inhibitor (Y-27632) was added. The culture medium was replaced every two days and the morphology and metabolic activity of tumor organoids with and without CAFs were measured daily. To characterize the organotypic model, immunofluorescence was performed using vimentin as a fibroblast marker, actin as a marker for apical side of organoids, and Hoechst for nuclei. Images were captured with a confocal microscope and composite images were made in ImageJ. To identify specific mechanisms of interactions between CAFs and organoids, we used a phosho-RTK array to detect activities of 49 different receptor tyrosine kinases (RTK) in CRC cells stimulated with conditioned medium of CAFs. Then, qPCR and western blot were performed to pinpoint mechanisms of interactions of cancer cells and CAFs in the organotypic model. Drug studies with oncogenic pathway inhibitors were performed and analyzed.
Results, Conclusions, and Discussions:: Co-culture of cancer cells with CAF led to significantly larger organoids (Fig 1a). We quantified the number of organoids and their size. Results showed that there were a greater number of organoids under co-culture with CAFs. For example, more than 50 organoids of 250 µm diameter were presents in co-culture with CAFs than in monoculture of cancer cells (Fig 1b). This suggests that CAFs promote growth and proliferation of organoids of primary cancer cells. From immunofluorescence staining and 3D confocal images of organoids, we confirmed that CAFs surrounded the organoids (Fig 1c), closely resembling spatial distribution of cells in solid tumors, where stromal cells surround masses of cancer cells. Using a phospho-RTK array, we found that while EGFR, MSP (Marcophage stimulating protein), and Ephrin (Eph) were active in cancer cells at a basal level, CAFs predominantly activated MET on cancer cells by 1.6-fold (Fig 1d). An immunoassay to determine prominent soluble factors of CAFs showed only hepatocyte growth factor (HGF), which is the MET ligand, was abundantly present at a concentration of 6.7 ng/ml (Fig 1e). This suggest that CAFs promote organoid proliferation through HGF-MET signaling in cancer cells. We studied effects of CAFs on the response of organoids to MEK inhibition and found that CAFs shifted up the dose response curve (Fig 1f). Interactions of CAFs and cancer cells promoted resistance to MEK inhibition, suggesting that targeting of CAFs-cancer cells signaling is a potential therapeutic approach. We successfully formed an organotypic model of primary tumor cells, CAFs, and the extracellular matrix. This study offers a humanized tumor model for mechanistic studies of drug resistance and personalized cancer medicine.
Acknowledgements (Optional): : NIH grant CA216413.
References (Optional): : 1. Jin, M.-Z. and W.-L. Jin, The updated landscape of tumor microenvironment and drug repurposing. Signal Transduction and Targeted Therapy, 2020. 5(1): p. 166.
2. Winkler, J., et al., Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nature Communications, 2020. 11(1): p. 5120.
