N-glycosylation by Mgat5 imposes a targetable constraint on immune-mediated tumor clearance

The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of β1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.


Figure S1 :
Figure S1: Histologic and immunofluorescent analysis of T cell-inflamed EV and Mgat5 KO tumors.A, Weights of 2838c3 EV, Mgat5 KO-A, and Mgat5 KO-B subcutaneous tumors in C57BL/6 mice.Data represent mean ± SEM (n = 6-7 mice/group).Statistical analysis done using one-way ANOVA.B, Microscopic evaluation of 2838c3 EV and Mgat5 KO tumors fixed at twelve days post injection by H&E or immunofluorescence PHA-L, CD8, or CC3.

Figure S2 :
Figure S2: Growth of T cell-inflamed lines in low glucose and FBS.A, Relative cell viability by CellTiter Glo analysis of 2838c3 EV and Mgat5 KO cells grown in high glucose (25mM) or low glucose (2.5mM).Viability is normalized to high glucose condition.Statistical analysis by one-way ANOVA.Error bars are SD.B, Relative cell viability by CellTiter Glo analysis of 2838c3 EV and Mgat5 KO cells grown in 10% FBS (normal) or 1% FBS.Viability is normalized to 10% FBS condition.Statistical analysis by one-way ANOVA.Error bars are SD.

Figure S3 :Figure S4 :
Figure S3: Characterizing Mgat5 KO in multiple PDAC lines.A, qPCR analysis of Mgat5 RNA content in the 2838c3, 6499c4, 6694c2, and 6419c5 EV lines.Statistical analysis by one-way ANOVA.Error bars are SD.B, Histogram of PHA-L binding to 2838c3, 6499c4, 6694c2, 6419c5 EV lines, normalized to mode.C, Histogram of PHA-L binding to non-T cellinflamed (6694c2, 6419c5) EV and Mgat5 KO cell lines.D, Relative growth of non-T cellinflamed (6694c2, 6419c5) EV and Mgat5 KO cell lines in vitro.Statistical analysis done using two-way ANOVA with n = 3 replicates per data point.E, Weights of 6694c2 EV, Mgat5 KO-A, and Mgat5 KO-B subcutaneous tumors in C57BL/6 mice.Tumor weights were obtained from dissected tumors at day 22.Data represent mean ± SEM (n = 8-10 mice/group).Statistical analysis done using one-way ANOVA.

Figure S5 :Figure S6 :Figure S7 :
Figure S5: Immune involvement in Mgat5 KO tumor clearance.A, Confirmation of depletion of CD4+ T cells via flow of the spleen at experiment end.Statistics using one-way ANOVA for this and all depletion validation.B, Confirmation of depletion of CD8+ T cells via flow of the spleen at experiment end.C, Confirmation of depletion of NK cells via flow of the liver at experiment end.D, Subcutaneous tumor growth over time of 2838c3 EV under isotype control (n=6) or NK depletion (n=6), and Mgat5 KO-A tumors under isotype control (n=6) or NK depletion (n=7).Left, all four lines.Right, Mgat5 KO lines under isotype and NK cell depletion alone.Statistical analysis done using two-way ANOVA.Data represent mean ± SEM.E, Subcutaneous tumor growth over time of 2838c3 EV (n=10) and Mgat5 KO-A (n=10) tumors in MuMT mice lacking B cells.Data represent mean ± SEM.Statistical analysis using two-way ANOVA.

Figure S12 :
Figure S12: Therapeutic sensitivities of non-T cell-inflamed EV and Mgat5 KO tumors.A, MFI of PDL1 in 6694c2 EV and Mgat5 KO-A cells, with and without 100ng/mL IFNg preincubation for 24 hours.Statistical analysis done using one-way ANOVA.Error bars are SD.B, Normalized viability, as assessed by CellTiter Glo, of varying concentrations of swainsonine in 2838c3 WT after 72h of incubation.Error bars are SD.C, Flow cytometry for PHA-L of 2838c3 WT cells treated with and without 3µM swainsonine for 72 hours.