ABSTRACT: Adoptive transfer of CAR T cells demonstrated impressive results against B-cell malignancies, but still limited efficacy against solid tumors. In this context, multiple challenges need to be overcome, including poor tumor recognition and strong immunosuppression within the tumor microenvironment (TME). Our Unit has recently reported that pharmacological inhibition of N-glycan synthesis in cancer cells increases CAR T cell efficacy by improving tumor recognition and preventing T cell exhaustion. In this project, we investigated the role of N-glycosylation blockade on TME cells in the context of colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC)-derived liver metastases, exploiting CEA-specific CAR T cell therapy. Here, we evaluate the impact of N-glycosylation blockade on TME cells (both M2-like macrophages, M2-M and Hepatic stellate cells, HepSCs), analyzing phenotypic and transcriptional profile and performing in vitro functional assays. In vitro studies revealed that N-glycosylation inhibition abolishes the ability of both TME cells to restrain T cell proliferation and increases the elimination of cancer cell lines and patient-derived tumor organoids (PDOs from CRC-liver metastases and primary PDAC). Interestingly, these effects were associated with profound phenotypic and transcriptional changes in M2-M and HepSCs. Moreover, to evaluate the effect of N-glycosylation inhibition on TME cells in vivo, we exploited immunodeficient mice reconstituted with a human immune system (huSGM3), engrafted intra-liver with tumor cells and treated with CEA CAR T cells. Importantly, using these mice we observed that N-glycosylation inhibition, only with Tunicamycin, improves CEA CAR T cells fitness, increasing their antitumor activity in terms of survival. This effect is associated with the reduction of IL1B+ TAMs cluster and the absence of inhibitory interactions between TAMs and T cells in scRNAseq analysis. However, the combination of CAR with 2DG failed to enhance CEA CAR T cells efficacy. Indeed, by blocking also glycolysis it dampens lymphoid effector functions tuning down the immune response. Overall, these data suggest that blocking N-glycosylation can help overcome multiple barriers that currently limit CAR T cell efficacy in solid tumors, acting not only on tumor cells, but also on immunosuppressive tumor microenvironment cells.
