P0144 Unveiling the role of the MFSD2A protein in contributing to the resolution of colitis-associated cancer inflammation
Authors: Cagliani S , Massimino L , Facoetti A , Spanò S , Maddipati K , Errico C , Nicolò S , Colasante G , Danese S , Ungaro F 
Published: January 2025

Abstract

Background
The intrinsic connection between inflammation and tumor promotion is well-established in colorectal cancer (CRC), where chronic inflammation, like ulcerative colitis (UC), plays a significant role in intestinal carcinogenesis (1). UC patients are at higher risk of colitis-associated cancer, emphasizing the inflammation-cancer connection. Previous studies from our laboratory revealed that UC patients with active inflammation have impaired production of inflammation-resolving DHA-derived metabolites due to a defect in the endothelial Major Facilitator Superfamily Domain containing 2A (MFSD2A) (2). Given the MFSD2A’s role in reducing colonic inflammation, we hypothesize it also counteracts cancer-associated inflammation.

Methods
Human Intestinal Microvascular Endothelial Cells (HIMEC) isolated from CRC and healthy samples were transduced with lentiviruses carrying either the GFP-tagged MFSD2A encoding sequence (MFSD2A-OE), or the relative GFP control, or the MFSD2A-targeting shRNAs, or the scramble as control, then analyzed by transcriptomics and lipidomics. A colon-adenocarcinoma cell line (Caco-2) was cocultured with either MFSD2A-OE or GFP CRC HIMEC to evaluate their proliferation rate. An orthotopic model of CRC was performed by intrarectally injecting CD1 nude mice with a cell mixture of Caco-2 and CRC HIMEC overexpressing either MFSD2A or GFP as control. Collected tumors were analyzed by flow cytometry and lipidomics (3).

Results
CRC HIMEC displayed a pro-resolving signature at the lipidomic level, consistently with higher MFSD2A expression as compared to the healthy. However, gene expression analysis shows a pro-inflammatory profile in CRC HIMEC, suggesting that MFSD2A levels alone are insufficient to induce a fully pro-resolving phenotype. We performed loss of function experiments to assess whether the pro-resolving profile in CRC HIMEC depends on MFSD2A. MFSD2A silencing reduces pro-resolving functions, confirming its role in balancing pro-inflammatory and pro-resolving signals in CRC regarding lipid production. Caco-2 co-cultured with MFSD2A-OE CRC HIMEC displayed a decrease in their proliferation rate after 48 and 72 hours of co-culture, indicating that endothelial MFSD2A overexpression reduces tumor cell proliferation. By comparison with animals injected with Caco-2 and GFP HIMEC, mice injected with a Caco-2/MFSD2A-OE CRC HIMEC mixture developed tumors with reduced weight and size, and increased pro-resolving lipid release. Additionally, flow cytometry showed increased M2 macrophage polarization, indicating a shift toward a pro-resolving immune response.

Conclusion
These data suggested that MFSD2A contrasts cancer-associated inflammation, ensuring the correct balance between pro-inflammatory and pro-resolving milieu.

References
1.Ungaro F, D'Alessio S, Danese S. The Role of Pro-Resolving Lipid Mediators in Colorectal Cancer-Associated Inflammation: Implications for Therapeutic Strategies. Cancers (Basel). 2020;12(8):2060. Published 2020 Jul 26. doi:10.3390/cancers12082060
2.Ungaro F, Tacconi C, Massimino L, et al. MFSD2A Promotes Endothelial Generation of Inflammation-Resolving Lipid Mediators and Reduces Colitis in Mice. Gastroenterology. 2017;153(5):1363-1377.e6. doi:10.1053/j.gastro.2017.07.048
3.Massimino L, Bulbarelli A, Corsetto PA, et al. LSEA Evaluation of Lipid Mediators of Inflammation in Lung and Cortex of Mice Exposed to Diesel Air Pollution. Biomedicines. 2022;10(3):712. Published 2022 Mar 19. doi:10.3390/biomedicines10030712