N-glycans in non-malignant tumor microenvironment cells dampen CAR-T cell function in solid tumors
Camilla Sirini, Chiara Balestrieri, Beatrice Greco, Barbara Camisa, Laura Falcone, Gaia Castaldi, Martina Spiga, Eliana Ruggiero, Rita El Khoury, Elena Tiziano, Oronza Antonietta Botrugno, Giulio Giovannoni, Chia-wei Lin, Vigneshwaran Venkatesan, Samuele Ferrari, Marta Angiola Moresco, Alice Bergamini, Giovanni Tonon, Fabio Ciceri, Chiara Bonini, Monica CasucciBackground
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.