Colorectal cancer is the third most common cancer in Australia and affects thousands of Australians each year. Tumour initiation in the bowel is predominantly driven by mutations to the tumour suppressor gene Apc in the intestinal epithelium. Current models solely focus on the impact of aberrant Wnt signalling activation in epithelial cells and their acquired competitive advantage, but fail to evaluate the concomitant remodelling of the supportive microenvironment during intestinal tumour initiation.
Here we observed, using an animal model of intestinal tumour formation, that amplification and infiltration of a specific stromal subtype to close proximity to Apc mutant cells is required to support expansion of mutant cell clusters and tumour growth.
To investigate the crosstalk between mouse mutant cells and intestinal fibroblasts, we generated co-cultures of ApcWT or ApcKO organoids with tissue-derived primary colon fibroblasts and assessed their ability to support organoid formation. Co-culture of ApcKO organoids with fibroblasts was significantly more potent in driving organoid formation and growth than ApcKO organoids alone, or than ApcWT organoids in co-culture, suggesting that Apc mutant cells remodel fibroblasts into pro-oncogenic cells. We characterised these alterations via RNA sequencing and identified several stromal-specific ligands being up-regulated by fibroblasts in response to co-culture with ApcKO organoids, driving activation of multiple downstream signalling pathways in these organoids. Genetic ablation of these ligands in fibroblasts or inhibition of signalling activation in mutant organoids using small molecule inhibitors in co-culture assays blocked ApcKO organoid formation and expansion. Importantly, similar mechanisms were observed with human intestinal tumour organoid/fibroblast co-culture systems. The potential ligands identified in vitro were also validated in human tumour tissues compared to healthy colon tissues.
Finally, we characterised the mechanism responsible for the pro-oncogenic reprogramming of fibroblasts by mutant epithelial cells. Targeting this signal using a blocking antibody significantly decreased the production of pro-oncogenic ligands by fibroblasts.
To conclude, we showed that Apc mutation alone is not sufficient for polyp fixation and expansion. Concomitant reprogramming of fibroblasts by Apc mutant cells supports tumour initiation and growth. This work provides major new biological insights into colorectal carcinogenesis and may inform the development of new therapies for colorectal cancer patients.