Aberrant sialylation functions as an important modulator of all steps of malignant transformation. Therefore, targeting sialylation regulators, such as sialyltransferases and neuraminidases, is a potential strategy for treating cancer. Here, we found that elevated α2,3-sialyltransferase III (St3gal3) was associated with dismal prognosis in high-grade serous ovarian carcinoma (HGSC). St3gal3 knockdown antagonized subcutaneous tumor growth in immunocompetent, but not immunodeficient mice, with enhanced accumulation of functional CD8+ T cells and antitumor immune gene signatures. St3gal3 knockdown inhibited intraperitoneal tumor growth and repolarized tumor-associated macrophages from a protumorigenic M2-like to a tumor-suppressive M1-like phenotype. In vitro, St3gal3 knockdown tumor cells guided bone marrow–derived macrophages (BMDM) toward the M1-like phenotype under both direct contact and distant Transwell coculture conditions. Depletion of macrophages rescued the suppressed tumor growth induced by St3gal3 knockdown and completely suppressed infiltration of functional CD8+ T cells that rely on macrophage-derived CXCL10. St3gal3 engendered an immunosuppressive HGSC microenvironment characterized by an abundance of pro-tumorigenic macrophages and reduced cytotoxic T-cell infiltration. In vivo, St3gal3 knockdown improved effectiveness of dual immune checkpoint blockade (ICB) with αPD-1 and αCTLA4 antibodies. Preclinical inhibition of sialylation with ambroxol resulted in decreased tumor growth and prolonged the survival of tumor-bearing mice, which was enhanced by the addition of dual ICB. These findings indicate that altered sialylation induced by St3gal3 upregulation promotes a tumor-suppressive microenvironment in HGSC and targeting α2,3-sialylation may reprogram the immunosuppressive tumor microenvironment and improve the efficacy of immunotherapy. Significance: Blocking sialylation augments antitumor immunity and enhances response to immune checkpoint blockade therapy, highlighting a potential therapeutic approach for treating patients with high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSC), with a modest response to immune checkpoint blockade (ICB) targeting PD-1/PD-L1 monotherapy, is densely infiltrated by M2-polarized tumor-associated macrophages (TAMs) and regulatory T (Treg) cells. The complement C5a/C5aR1 axis contributes to the programming of the immunosuppressive phenotype of TAMs in solid tumors and represents a promising immunomodulatory target for treating HGSCs. Here, we aimed to identify the relevance of C5aR1 in prognosis, immune microenvironment, and immunotherapy response in HGSCs. The expression and relationship of C5aR1 with tumor-infiltrating immune cells were assessed by immunohistochemistry and flow cytometry in the training cohort (n = 120) and fresh HGSC tissues (n = 36). Transcriptomic analyses of the xenografts delineated the mechanisms driving the immunomodulatory activity of PMX53, an orally bioavailable C5aR1 inhibitor. Therapeutic relevance was confirmed in ex vivo tumor cultures and The Cancer Genome Atlas (TCGA) datasets. C5aR1 expression independently predicted dismal prognosis and was linked to the immunoevasive subtype of HGSC, characterized by increased infiltration of pro-tumor cells (Treg cells, M2-polarized macrophages, and neutrophils) and impaired CD8⁺T functions. PMX53 antagonized subcutaneous tumor growth, modulated immunosuppressive mechanisms and synergized with aPD-1 in several tumor types. Single-cell RNA-seq analysis revealed predominant C5aR1 expression in TAMs, with an immunosuppressive-related expression signature in C5aR1⁺TAMs. Furthermore, the combination of C5aR1 and PD-L1 was associated with specific molecular characteristics and matched clinical response annotations. Therefore, the abundance of C5aR1 could predict an inferior prognosis in HGSCs, and incorporating PD-L1 may serve as a novel predictive biomarker to guide therapeutic options.

Background: The potent immunosuppressive properties of sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on myeloid cells and lymphocytes provide a strong rationale for serving as a therapeutic target. However, the expression profile and critical role of Siglec-9 in high-grade serous ovarian cancer (HGSC) remain obscure. This study aimed to elucidate the prognostic significance of Siglec-9 expression and its predictive value for immunotherapy in HGSC. Methods: Study enrolled two cohorts, consisting of 120 tumor microarray specimens of HGSC for immunohistochemistry (IHC) and 40 fresh tumor specimens for flow cytometry (FCM). Expression profile of Siglec-9 in immune cells was analyzed by both bioinformatics analysis and FCM. Role of Siglec-9 was studied to identify that Siglec-9⁺TAMs linked with an immunosuppressive phenotype by IHC and FCM, and block Siglec-9 was sensitive to immunotherapy by ex vivo and in vitro assays. Results: Siglec-9 is predominantly expressed on tumor-associated macrophages (TAMs). High Siglec-9⁺TAMs were associated with inferior overall survival (OS). Both tumor-conditioned medium (TCM) and tumor ascites induced enrichment of Siglec-9⁺TAMs with protumorigenic phenotypes. Siglec-9⁺TAMs were associated with immunosuppressive tumor microenvironment (TME) characterized by exhausted CD8⁺T cells and increased immune checkpoint expression. Blockade of Siglec-9 suppressed phosphorylation of the inhibitory phosphatase SHP-1 and repolarized TAMs to antitumorigenic phenotype and retrieved cytotoxic activity of CD8⁺T cells in vitro and ex vivo. Responders toward antiprogrammed death receptor-1 (anti-PD-1) therapy present more Siglec-9⁺TAMs than non-responders. Furthermore, blockade Siglec-9 synergized with anti-PD-1 antibody to enhance the cytotoxic activity of CD8⁺T cells in tissues with higher Siglec-9⁺TAMs. Conclusions: Siglec-9⁺TAMs may serve as an independent prognostic of poor survival but a predictive biomarker for anti-PD-1/antiprogrammed death ligand-1 immunotherapy in HGSC. In addition, the potential of immunosuppressive Siglec-9⁺TAMs as a therapeutic target is worth further exploration.

Background: Tumor‐associated neutrophils (TANs) play a critical role in modulating immune responses and exhibit significant heterogeneity. Our previous study demonstrated that jagged canonical Notch ligand 2 (JAG2) ⁺ TANs were associated with an immunosuppressive microenvironment in high‐grade serous ovarian cancer (HGSOC), but the underlying mechanism remains unclear. This study aimed to elucidate the role of JAG2 ⁺ TANs in tumor immunosuppressive microenvironment in HGSOC. Methods: HGSOC samples were collected, with 274 samples constituting two independent cohorts (training and validation cohorts) and an additional 30 samples utilized to establish patient‐derived tumor organoids (PDTOs). We characterized the number and phenotype of JAG2 ⁺ TANs by multiplex immunohistochemistry, flow cytometry, and single‐cell RNA sequencing (scRNA‐seq). We investigated the biological functions of JAG2 in immune evasion using in vitro co‐culture systems, flow cytometry, tumor‐bearing mouse models, and PDTOs. Results: JAG2 ⁺ TANs expressed elevated levels of immunosuppressive molecules, including programmed cell death ligand 1 and CD14, and had independent prognostic value for the overall survival of patients with HGSOC. scRNA‐seq analysis revealed that JAG2 ⁺ TANs exhibited a terminally mature phenotype. The infiltration of JAG2 ⁺ TANs was positively correlated with the abundance of effector regulatory T cells (eTregs). Interaction with JAG2 ⁺ TANs skewed CD4 ⁺ T cells towards an eTreg phenotype, a process that was suppressed by the Notch inhibitor LY3039478 and induced by recombinant Jagged2. Furthermore, we demonstrated that JAG2 ⁺ TANs enhanced Notch signaling activation, ultimately promoting recombination signal binding protein for immunoglobulin kappa J region (RBPJ)‐induced differentiation of naïve CD4 ⁺ T cells into eTregs. Clinically, JAG2 ⁺ TANs could serve as a biomarker for assessing immunotherapy resistance in various solid tumors. Pharmacological targeting of Notch signaling with LY3039478 or JAG2 neutralization antibodies enhanced the efficacy of programmed cell death protein 1 (PD‐1) monoclonal antibodies (mAbs) in both xenograft and PDTO models. Conclusions: The emergence of JAG2 ⁺ TANs is crucial for the differentiation of eTregs, which triggers immune evasion and resistance to anti‐PD‐1 therapy. Inhibiting Notch signaling with LY3039478 or JAG2 neutralization antibodies may overcome this anti‐PD‐1 resistance in HGSOC.

Qiu J, et al. J Gynecol Oncol. 2023 Jun;34:e64. https://doi.org/10.3802/jgo.2023.34.e64