TUMOR MICROENVIRONMENT-DERIVED PARACRINE SIGNALING MODULATES HOXB8 MAST CELL DIFFERENTIATION AND LIPID METABOLISM IN COLORECTAL CANCER

Colorectal cancer (CRC) is a major global public health challenge in part due to its heterogeneous tumor microenvironment (TME). Mast cells (MCs) are resident cells of connective tissues and mucosal epithelia whose differentiation is driven by tissue-specific stimuli. Within the TME, MCs are found in the stroma or adjacent to hypoxic regions. However, the role of MCs progenitors (MCps) recruited to the TME remains poorly understood. Here, we aimed to determine how the TME influences MCps differentiation and contribution to CRC progression.

Immortalized MCps (iMCps) were differentiated for 7 days in RPMI supplemented with IL-3 (1 ng/mL), or conditioned medium from murine colon carcinoma (CM-MC38) or fibroblast (CM-NIH/3T3) cells. Toluidine Blue and Alcian Blue–Safranin staining were used to assess mast cell differentiation profiles. Lipid droplet (LD) accumulation was evaluated by osmium tetroxide staining. To investigate the functional impact of iMCps and differentiated MCs, tumor spheroids were generated by co-culturing 500 MC38 tumor cells with 4,500 NIH/3T3 fibroblasts and 1,000 MCs in 1% agarose-coated 96-well round-bottom plates.

Our results indicate that CM-MC38-derived MCs (TiMCs) displayed safranin-positive granules and higher LD content compared with CM-NIH/3T3-derived MCs (FiMCs), which showed reduced PGD2 production upon PMA stimulation. The incorporation of iMCps into spheroids had no effect on overall growth, but increased migratory capacity of day 5 but not day 3 spheroids. FiMCs lead to a similar behavior, with effects seen also on day 3, whereas IL-3-differentiated MCs and TiMCs reduced spheroid size and growth rate without affecting migratory capacity.

These findings suggest that the differentiation stimulus programs the MCs effects on spheroid growth and migration. We further intend to elucidate the molecular mechanisms underlying these processes and to investigate whether lipid metabolism reprogramming in MCs correlates with CRC progression.