Abstract 4253: Modeling the effects of inflammatory stress on human intestinal epithelial cells in 3D enteroid co-culture system

The molecular pathogenesis of colitis-associated colorectal cancer (CAC) has been suggested to involve oxidative stress-induced DNA damage, resulting in mutations of tumor-suppressor genes and activation of pro-oncogenic pathways. However, the exact relationship between inflammation and colorectal cancer is not well understood due to the lack of a human model system that not only recapitulates in vivo growth/differentiation patterns, but also can be easily manipulated. In order to gain insight into the mechanism of colitis-associated colorectal cancer, we explored human intestinal enteroids as an ex vivo model to study the interaction of intestinal epithelial cells with immune cells under inflammatory conditions. Human intestinal crypts were isolated from adult intestinal tissue and grown in Matrigel culture to form enteroids. To model inflammation, we incorporated MHC-mismatched human peripheral blood mononuclear cells (PBMCs) into the Matrigel. TH1 immune response was induced by a cocktail of cytokines including IL-2, IL-7 and IL-15. A TH1 response was confirmed by the production of pro-inflammatory cytokines INFγ and TNFα over the course of 6-day treatment with the cytokine cocktail. Several cytokines related to TH1 and TH2 responses were induced. Interestingly, co-culture with human intestinal enteroids significantly enhanced the level of pro-inflammatory cytokines INFγ, IL-6 and TNFα, anti-inflammatory cytokine IL-10, as well as TH2 cytokine IL-13. Immunophenotyping of PBMCs shows that both CD4+ T cells (10-30%) and CD8+ T cells (1-10%) were activated upon TH1 induction. In addition, the percentage of activated CD8+ T cells was much higher in co-culture than in PBMC alone controls. Moreover, the cytokine cocktail dramatically induced the production of INFγ by purified CD4+ or CD8+ T cells but only when in co-culture with enteroids and not when cultured alone. In the enteroids, the TH1 response significantly inhibited cell proliferation and increased the apoptosis of epithelial cells when in co-culture only. Increased γH2AX (DNA damage marker) and impaired integrity of intestinal enteroids were also observed when in co-culture with the TH1 response. Cell-cell contact between enteroids and immune cells is required for these effects. Moreover, the TH1 response increases the mRNA levels of GRP78 (master regulator of ER stress) and Nrf2 (regulator of oxidative stress); decreases the mRNA level of Lgr5 [marker of active intestinal stem cells (ISCs)], but has no obvious effect on Bmi1 (marker of quiescent ISCs) in co-culture. Taken together, our data provide insight into the interaction of intestinal epithelial cells with immune cells at the molecular and cellular levels and establish this approach as a viable model for exploring the mechanism of CAC. Future studies will examine specifically for oxidative DNA damage and the effects of specific tumor suppressor inactivation on epithelial cell responses. Citation Format: Fang Wang, Dorottya Laczko, Mary Ann Crissey, Gary Falk, Bradley Johnson, Anil Rustgi, John Lynch. Modeling the effects of inflammatory stress on human intestinal epithelial cells in 3D enteroid co-culture system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4253.