Targeting tumor O‐glycosylation modulates cancer–immune‐cell crosstalk and enhances anti‐PD‐1 immunotherapy in head and neck cancer

Cells in the tumor microenvironment (TME) communicate via membrane‐bound and secreted proteins, which are mostly glycosylated. Altered glycomes of malignant tumors influence behaviors of stromal cells. In this study, we showed that the loss of core‐1 β1,3‐galactosyltransferase (C1GALT1)‐mediated O‐glycosylation suppressed tumor growth in syngeneic head and neck cancer mouse models. O‐glycan truncation in tumor cells promoted the M1 polarization of macrophages, enhanced T‐cell‐mediated cytotoxicity, and reduced interleukin‐6 (IL‐6) levels in the secretome. Proteasomal degradation of IL‐6 was controlled by the O‐glycan at threonine 166. Both IL‐6/IL‐6R blockade and O‐glycan truncation in tumor cells induced similar pro‐inflammatory phenotypes in macrophages and cytotoxic T lymphocytes (CTLs). The combination of the O‐glycosylation inhibitor itraconazole and anti‐programmed cell death protein 1 (anti‐PD‐1) antibody effectively suppressed tumor growth in vivo. Collectively, our findings demonstrate that O‐glycosylation in tumor cells governs their crosstalk with macrophages and CTLs. Thus, targeting O‐glycosylation successfully reshapes the TME and consequently enhances the efficacy of anti‐PD‐1 therapy.

) were seeded into one well of a 96-well plate.Viability was measured at 72 h using alamarBlue™ assays.Data shown are representative of three independent experiments and are presented as mean ± SD. ***P < 0.001, analyzed using a two-tailed Student's t-test.(B) Apoptosis assay.Mock or C1galt1 KO SCA9 and MTC-Q1 cells (1 × 10 6 ) were detached and washed with PBS.After centrifugation, cell pellets were resuspended in 100 µl cocktail containing annexin V-FITC and propidium Iodide (PI) and incubated for 15 min, followed by flow cytometric analysis.Apoptotic cells shown are annexin V + cells, including early and late apoptosis.Data shown are representative of three independent experiments and are presented as mean ± SD. **P < 0.01 and ***P < 0.001, analyzed using a two-tailed Student's t-test.HA-tagged IL-6 was overexpressed in the C1GALT1 KO 293FT cells.IL-6 in the conditioned medium was purified using anti-HA agarose beads.Upper panel, the protein coverage map of IL-6.A HexNAc was identified on IL-6 at T166.Lower panel, tandem mass spectrum of the O-glycosylated peptide NLDAITT(166)PDPTTNASLLTK (red boxes).We analyzed two independent batches of IL-6 samples, and the O-glycosylation site was consistent between them. ) were seeded into one well of a 96-well plate on day 0. ITZ (10 μM for SCA9 cells and 1.25 μM for MTC-Q1 cells) was added at 24 h.Viability was measured at 72 h using alamarBlue™ assays.***P < 0.001, analyzed using a two-tailed Student's t-test.Data shown are representative of three independent experiments and presented as mean ± SD. (B) Blood samples (100 μl) were collected (n = 8 for each group) from the submandibular vein at 27 d.Complete blood counts were evaluated within 24 h of blood collection.WBC, white blood cell; NEUT, neutrophil; LYMP, lymphocytes.*P < 0.05 and **P < 0.01.This experiment was performed once.

Figure S1 .
Figure S1.Cell viability and apoptosis of Mock and C1galt1 KO cells.(A) Cell viability of Mock or C1galt1 knockout (KO) SCA9 and MTC-Q1 cells.Cells (3× 10 3 ) were seeded into one well of a 96-well plate.Viability was measured at 72 h using alamarBlue™ assays.Data shown are representative of three independent experiments and are presented as mean ± SD. ***P < 0.001, analyzed using a two-tailed Student's t-test.(B)

Figure S2 .Figure S3 .
Figure S2.Residual Cas9 expression check-up and tumor infiltrating leukocytes analysis.(A) Real-time RT-PCR analysis of Cas9 expression in Mock and C1GALT1 KO cells.Mock cells transiently transfected with Cas9 vector containing the sequence of sgRNA against C1GALT1 were used as positive controls.Data shown are representative of three independent experiments and presented as mean ± SD. (B) Numbers of tumor infiltrating leukocytes analyzed using immunohistochemistry (n = 5).CD4 + cells, CD8 + cells, and FoxP3 + cells were counted under high power fields of a microscope.Upper panels, numbers of positive cells presented by mean ± SEM.NS, not significant, analyzed using a two-tailed Student's t-test.Lower panels, representative images of CD4, CD8, or FoxP3 expression in Mock and C1galt1 KO tumors.Scale bars, 50 μm.

Figure S4 .
Figure S4.Functional maps of C1GALT1-regulated genes.SAS cells were transfected with nontargeting siRNA or siRNAs against C1GALT1.Total mRNA was analyzed using cDNA microarray analysis, and differentially expressed genes were further analyzed in this functional map.A node denotes the enriched GO term (P < 0.01) and an edge represents the gene overlap score between nodes > 0.5.Node color encodes the enriched P-value and node size is proportional to the number of genes which are associated with a given GO term.Groups of functionally related GO terms are manually identified and labelled with the appropriate terms.

Figure S7 .Figure S8 .Figure S9 .
Figure S7.Effect of mutant IL-6 on differentiation of THP-1 cells.Mock or C1GALT1 KO SAS cells were transfected with IL6/pcDNA3.1 (Mock IL-6) or mtIL6 T166A/pcDNA3.1 (mt IL-6).Control cells were not transfected.THP-1 cells were differentiated into macrophages by incubation with 5 ng/mL PMA for 24 h.Macrophages were then cocultured with SAS cells in transwells for an additional 24 h.The expression of TNF and IL1B was evaluated by real-time RT-PCR analysis.Data shown are representative of three independent experiments and presented as mean ± SD. **P < 0.01, analyzed using a two-tailed Student's t-test.