3D models of tissue organ culture to study the role of the immune system and postbiotics in Colorectal cancer

Being one of the most prevalent forms of cancer, colorectal cancer (CRC) represents a significant public health concern. Mast cells (MCs) are immune cells involve in the maintenance of intestinal homeostasis as well as in orchestrating inflammation. The link between persistent inflammation and tumour transformation is an established knowledge and MCs are increasingly recognised as potential crucial players in tumor progression. CRC is characterized by an intimate interaction with microbiota which, as microbiota-derived metabolites like short chain fatty acids (SCFAs), can exert immunomodulatory effects. However, the relations between MCs and SCFAs during CRC pathogenesis still remained undescribed. Therefore, in this work, triangle interactions between MCs, tumoral colonic epithelial cells and SCFAs has been object of interest. Mice-derived tumoral organoids (TOs), as in vitro model of CRC, and mice-derived MCs were used to perform co-cultures, which were characterized, evaluating different parameters of interest. Secondly, the effects of SCFAs on MCs’ functionality and on MC-TO interactions were investigated. TOs appeared able to induce a phenotype switching of MCs, by increasing in their expression of the MC connective tissue marker mcpt4. Additionally, TO was shown to induce MC activation by increasing their expression of CD107a and production of TNF-α and incubation of MCs with the colture media (cm) of TOs led MC to release TNF-α. A reduction in TOs proliferation in presence of MCs was observed and also the GFP signal intensity of a murine GFP-expressing colon cancer cell line (GFP-MC38) was reduced by MCs. A drop in the expressional level of Lgr5, ChgA, and Lyz-1 intestinal markers was measured in TOs co-cultured with MC and TNF-α was identified as a key mediator of these MC-mediated alterations. Indeed, the proliferation of TOs co-cultured with TNF-α-deficient MCs was not reduced as in the case of co-culture with the WT ones. Additionally, also GFP signal intensity of GFP-MC38 cells was not altered with TNF-α -/- MCs or WT MCs together with a TNF-α blocking antibody. By blocking TNF-α in MC-TO co-cultures, the effect exerted by MCs on Lgr5, ChgA and Lyz-1 expressions was abolished. IL-33, a cytokine particularly express in CRC, was identified as a possible tumour mediator that could activate MC to produce TNF-α. TOs produce high amount of IL-33 and IL-33 stimulation as well as incubation with TO cm induced MC to produce TNF-α. By blocking ST2, the receptor for IL-33, in MCs incubated with TO cm, there was a reduction in their production of TNF-α. Additionally, a restoration of Lgr5, ChgA and Lyz-1 expression levels was observed in MC-TO co-cultures when the ST2/IL33 axis was blocked, suggesting the relevancy of IL-33 in modulating the MCs/TOs crosstalk. As SCFAs, acetate, butyrate, propionate and valerate were tested, and the most interesting results were observed with butyrate (NaB). MCs treated with NaB reduced their mcpt4 expression and the TNF-α release after IL-33 stimulation. NaB treatment of MC also inhibited IgE-mediated MC degranulation and TNF-α production. The increased mcpt4 expression by MCs co-cultured with TOs was reduced by NaB pretreatment of MCs, but it was not able to revert also the MC mediated alteration on TOs’ Lgr5 and ChgA expression. In conclusion, TOs have been shown to alter and to activate MCs, probably also via the IL-33/ST2 axis leading to increased release of TNF-α which in turn was responsible for the observed effects on organoids. Butyrate appeared to exert an inhibitory function on both IgE- and IL-33-mediated MC activation, by also reducing the subsequent release of TNF-α. However, it appeared that NaB did not exert a modulatory action on MC-TO interaction with regard to Lgr5 and ChgA expression.