Expression of the tumor-associated glycan Tn antigen (αGalNAc-Ser/Thr) has been correlated to poor prognosis and metastasis in multiple cancer types. However, the exact mechanisms exerted by Tn antigen to support tumor growth are still lacking. One emerging hallmark of cancer is evasion of immune destruction. Although tumor cells often exploit the glycosylation machinery to interact with the immune system, the contribution of Tn antigen to an immunosuppressive tumor microenvironment has scarcely been studied. Here, we explored how Tn antigen influences the tumor immune cell composition in a colorectal cancer (CRC) mouse model. CRISPR/Cas9-mediated knock out of the C1galt1c1 gene resulted in elevated Tn antigen levels on the cell surface of the CRC cell line MC38 (MC38-Tn high). RNA sequencing and subsequent GO term enrichment analysis of our Tn high glycovariant not only revealed differences in MAPK signaling and cell migration, but also in antigen processing and presentation as well as in cytotoxic T cell responses. Indeed, MC38-Tn high tumors displayed increased tumor growth in vivo, which was correlated with an altered tumor immune cell infiltration, characterized by reduced levels of cytotoxic CD8 + T cells and enhanced accumulation of myeloid-derived suppressor cells. Interestingly, no systemic differences in T cell subsets were observed. Together, our data demonstrate for the first time that Tn antigen expression in the CRC tumor microenvironment affects the tumor-associated immune cell repertoire.

No abstract

Many tumors display alterations in the biosynthetic pathways of glycosylation, resulting in increased expression of specific tumor-associated glycan structures. Expression of these altered glycan structures is associated with metastasis and poor prognosis. Antigen presenting cells can recognize tumor-associated glycan structures, including the truncated O-glycan Tn antigen, via specific glycan receptors. Tn antigen-mediated activation of the C-type lectin MGL on dendritic cells induces regulatory T cells via the enhanced secretion of IL-10. Although these findings indicate that MGL engagement by glycan ligands can modulate immune responses, the impact of MGL ligation on dendritic cells is still not completely understood. Therefore, we employed RNA sequencing, GO term enrichment and pathway analysis on human monocyte-derived dendritic cells stimulated with two different MGL glycan ligands. Our analyses revealed a reduced expression of genes coding for key enzymes involved in the glycolysis pathway, TCA cycle, and oxidative phosphorylation. In concordance with this, extracellular flux analysis confirmed the decrease in glycolytic activity upon MGL triggering in human dendritic cells. To our knowledge, we are the first to report a diminished glycolytic activity of human dendritic cells upon C-type lectin stimulation. Overall, our findings highlight the impact of tumor-associated glycans on dendritic cell biology and metabolism and will increase our understanding on how glycans can shape immunity.

Sialylated glycan structures are known for their immunomodulatory capacities and their contribution to tumor immune evasion. However, the role of aberrant sialylation in colorectal cancer and the consequences of complete tumor desialylation on anti‐tumor immunity remain unstudied. Here, we report that CRISPR/Cas9‐mediated knock out of the CMAS gene, encoding a key enzyme in the sialylation pathway, in the mouse colorectal cancer MC38 cell line completely abrogated cell surface expression of sialic acids (MC38‐Sianull) and, unexpectedly, significantly increased in vivo tumor growth compared to the control MC38‐MOCK cells. This enhanced tumor growth of MC38‐Sianull cells could be attributed to decreased CD8+ T cell frequencies in the tumor microenvironment only, as immune cell frequencies in tumor‐draining lymph nodes remained unaffected. In addition, MC38‐Sianull cells were able to induce CD8+ T cell apoptosis in an antigen‐independent manner. Moreover, low CMAS gene expression correlated with reduced recurrence‐free survival in a human colorectal cancer cohort, supporting the clinical relevance of our work. Together, these results demonstrate for the first time a detrimental effect of complete tumor desialylation on colorectal cancer tumor growth, which greatly impacts the design of novel cancer therapeutics aimed at altering the tumor glycosylation profile.

Activation of antigen-presenting dendritic cells (DCs) and the complement system are essential early events in the immune defense against invading pathogens. Recently, we and others demonstrated immunological crosstalk between signaling from receptors recognizing complement activation products and PAMPs on DCs. This affects DC effector function, as demonstrated by the finding that C5a prevents induction of pro-inflammatory cytokines by toll-like receptor (TLR) ligands in human monocyte-derived DCs (moDCs). Here, we demonstrate that this regulatory crosstalk is specifically important in 6-sulfo LacNAc dendritic cells (slanDCs), the most pro-inflammatory DC subset found in human. C5aR and TLR signaling show profound interference in the ERK/p38/CREB1 signaling pathways. C5aR signaling accelerates TLR-induced CREB1 phosphorylation both in moDC and slanDC. This is key in the regulatory effect of C5a on pro-inflammatory DC maturation by mediating induction of IL-10, which subsequently inhibits pro-inflammatory cytokine production via negative feedback signaling. Importantly, the regulatory effect of C5a affects T-cell immunity by decreasing Th1 and cytotoxic CD8 T-cell responses. The finding that the pro-inflammatory effector function of slanDC can be down modulated by activation products of the complement system highlights the existence of intricate regulatory interactions between various arms of the immune system. Intensive immune monitoring of patients suffering from complement-mediated diseases or patients receiving complement modulating compounds can give more inside in the contribution of complement receptor and TLR crosstalk in APCs in disease.

Cancer stem cells (CSCs) are located in dedicated niches, where they remain inert to chemotherapeutic drugs and drive metastasis. Although plasticity in the CSC pool is well appreciated, the molecular mechanisms implicated in the regulation of cancer stemness are still elusive. Here, we define a fucosylation-dependent reprogramming of colon cancer cells towards a stem cell-like phenotype and function. De novo transcriptional activation of Fut9 in the murine colon adenocarcinoma cell line, MC38, followed by RNA seq-based regulon analysis, revealed major gene regulatory networks related to stemness. Lewisx, Sox2, ALDH and CD44 expression, tumorsphere formation, resistance to 5-FU treatment and in vivo tumor growth were increased in FUT9-expressing MC38 cells compared to the control cells. Likewise, human CRC cell lines highly expressing FUT9 displayed phenotypic features of CSCs, which were significantly impaired upon FUT9 knock-out. Finally, in primary CRC FUT9+ tumor cells pathways related to cancer stemness were enriched, providing a clinically meaningful annotation of the complicity of FUT9 in stemness regulation and may open new avenues for therapeutic intervention.

Crosstalk between complement component 5a receptors (C5aRs) and TLRs in dendritic cells (DCs) occurs upon pathogen invasion; however, studies on C5aR and TLR crosstalk mainly focused on the modulating effect of C5a on TLR-induced cytokine production. To elucidate the breadth of C5aR and TLR4 crosstalk, the effect of simultaneous treatment with C5a and LPS was investigated in human monocyte-derived DCs (moDCs) 2 h after stimulation using whole transcriptome sequencing analysis. Although the effect of C5a on hallmark genes defining TLR4-induced DC maturation was limited at this time point, RNA sequencing analysis revealed a great variety of novel C5a targets, of which many interfere with TLR4-mediated immune activation. Analysis of functional relationships among these genes uncovered induction of a central immune regulatory network upon C5aR and TLR4 crosstalk, involving the transcription factors forkhead box (FOX)O1 and FOXO3 and the signaling molecules serum- and glucocorticoid-inducible kinase (SGK1), ribosomal S6 kinase 2 (RSK2), and PI3Kβ. C5aR and TLR crosstalk, furthermore, yielded down-regulation of mainly proinflammatory network branches, including IL-12B, IL-2Rα (IL-2RA), and jagged 1 (JAG1) and cooperative induction of predominantly anti-inflammatory network branches, including sphingosine kinase 1 (SPHK1), β2 adrenergic receptor (ADRB2), gastric inhibitory polypeptide receptor (GIPR), and four-and-a-half Lin11, Isl-1, and Mec-3 domains protein 2 (FHL2). Together, these data point toward induction of generalized immune regulation of DC function. Motif enrichment analysis indicate a prominent role for basic leucine zipper (bZIP) and IFN regulatory factor 4 (IRF4) transcription factors upon C5aR and TLR4 crosstalk. Additionally, differences were observed in the modulating capacity of C5a on DCs in the absence or presence of a pathogen (TLR stimulus). Our findings shed new light on the depth and complexity of C5aR and TLR4 crosstalk and provide new foci of research for future studies.

No abstract