"Colorectal cancer (CRC) is the third most common cause of cancer-related deaths worldwide1. According to molecular signatures, it can be classified into four Consensus Molecular Subtypes (CMS) with CMS4 tumours being associated with worst relapse-free and overall survival2. CMS4 tumours are rich in mesenchymal stromal cells (MSCs) and have increased angiogenesis and inflamed immune phenotype. Understanding the cellular interactions in the tumour microenvironment (TME) will be essential for developing novel cell therapies for treating CRC such as CAR-T and CAR-NK therapies. Much of the research in the past has focused on 2D monolayer cultures or in vivo studies, however, successful clinical translation of therapeutics derived from these methods has been sub-optimal. 3D models offer a promising tool to bridge the gap between in vitro and in vivo work3. We developed a CRC spheroid that incorporates stromal cells, for assessment of the impact on cell viability and cell proliferation, as well as migration and invasive potential.
HCT116 and HT29 colorectal cancer cell lines were incorporated into collagen type I hydrogels and cultured alongside human MSCs (hMSCs) for 14 days to mimic the high stromal CMS4 CRC. Using Alamar blue and Cyquant, we have shown that cell proliferation and metabolic activity significantly increased from day 1-14. Using flow cytometry, we found that CRC cell lines composed the majority of proliferating cells in the gels. Calcein AM and Propidium Iodide stain were used to assess cell viability. Using ImageJ, we found that increasing the ratio of hMSCs in the culture led to a trend in increase in percentage viability of cells and a decrease in the percentage of dead cells. Introduction of an increased ratio of hMSCs also significantly increased cell outgrowth from spheroids, indicating increased invasive potential. These results indicate that the presence of hMSCs in CRC TME could play a role in viability of the CRC cells as well as assisting the metastatic ability of these cells. To further advance this model, we will incorporate these spheroids into a microfluidic device containing HUVECs as a vasculature-like component mimicking the increased angiogenesis in CMS4 CRC TME. Incorporating immune cells such as NK cells will further increase our understanding of cellular interactions taking place in the CRC TME. Having appropriate 3D models to assess immune and stromal impacts in CRC will increase knowledge of cellular interactions in the TME thus assisting in discovering novel target therapeutics.
Funded by SFI 18/EPSRC-CDT/3583, EPSRC EP/S02347X/1.
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3. Reidy E, Leonard NA, Treacy O, Ryan AE. A 3D View of Colorectal Cancer Models in Predicting Therapeutic Responses and Resistance. Cancers (Basel). 2021;13(2):227. Published 2021 Jan 10. doi:10.3390/cancers13020227"