N-glycans in non-malignant tumor microenvironment cells dampen CAR-T cell function in solid tumors

Background Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy in solid tumors, largely due to immunosuppressive mechanisms within the tumor microenvironment (TME). While tumor-associated glycans are known to protect malignant cells from immune attack, the contribution of N-glycans expressed by non-malignant TME populations to CAR-T cell dysfunction remains poorly defined. Methods We investigated the role of N-glycans in non-malignant TME populations, focusing on M2-like macrophages and hepatic stellate cells in liver metastasis of colorectal (CRC) and pancreatic cancer (PDAC). Using in vitro co-culture systems, transcriptomic analysis, and tumor-bearing humanized mouse models, we assessed how pharmacologic or genetic disruption of key nodes of the N-glycosylation pathway ( MGAT5 , MAN2A1 and ST6GAL1 ) in immune and stromal compartments shapes T-cell function. Results In patient samples, a branched N-glycan signature was associated with transcriptional programs characteristic of tumor-promoting macrophages and stromal cells, linking N-glycosylation to an immunosuppressive TME. Disruption of N-glycan synthesis in non-malignant TME cells reduced their immunosuppressive and tumor-supporting functions. Single-cell RNA sequencing of tumor-bearing humanized mice showed depletion of protumor IL1β + macrophages and diminished inhibitory macrophage-T cell interactions following N-glycosylation blockade. Selective MGAT5 disruption in immune and stromal compartments suppressed immunosuppressive programs and enhanced CAR-T cell antitumor activity independently of tumor cell glycosylation. Conclusions These findings show that N-glycans expressed by non-malignant TME cells restrain CAR-T cell responses in CRC and PDAC, highlighting MGAT5 -dependent branching as a potentially actionable axis and supporting a broader role for multiple nodes of the N-glycosylation pathway.