Anti-angiogenic cancer therapies possess immune-stimulatory properties by counteracting pro-angiogenic molecular mechanisms. We report that tumor endothelial cells ubiquitously overexpress and secrete the intermediate filament protein vimentin through type III unconventional secretion mechanisms. Extracellular vimentin is pro-angiogenic and functionally mimics VEGF action, while concomitantly acting as inhibitor of leukocyte-endothelial interactions. Antibody targeting of extracellular vimentin shows inhibition of angiogenesis in vitro and in vivo. Effective and safe inhibition of angiogenesis and tumor growth in several preclinical and clinical studies is demonstrated using a vaccination strategy against extracellular vimentin. Targeting vimentin induces a pro-inflammatory condition in the tumor, exemplified by induction of the endothelial adhesion molecule ICAM1, suppression of PD-L1, and altered immune cell profiles. Our findings show that extracellular vimentin contributes to immune suppression and functions as a vascular immune checkpoint molecule. Targeting of extracellular vimentin presents therefore an anti-angiogenic immunotherapy strategy against cancer.

Poor immunogenicity of critical epitopes can hamper vaccine efficacy. To boost immune recognition of non- or low-immunogenic antigens, we developed a vaccine platform based on the conjugation of a target protein to a chimeric designer peptide (CDP) of bacterial origin. Here, we exploited this immune Boost (iBoost) technology to enhance the immune response against the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. Despite its fundamental role during viral infection, RBD is only moderately immunogenic. Immunization studies in mice showed that the conjugation of CDP to RBD induced superior immune responses compared to RBD alone. CDP-RBD elicited cross-reactive antibodies against the variants of concern Delta and Omicron. Furthermore, hamsters vaccinated with CDP-RBD developed potent neutralizing antibody responses and were fully protected from lung lesion formation upon challenge with SARS-CoV-2. In sum, we show that the iBoost conjugate vaccine technology provides a valuable tool for both quantitatively and qualitatively enhancing anti-viral immunity.

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-Tnhigh). RNA sequencing and subsequent GO term enrichment analysis of our Tnhigh 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-Tnhigh 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.

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.

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.

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.

Interferon α (IFNα) is a prompt and efficient orchestrator of host defense against nucleic acids but upon chronicity becomes a potent mediator of autoimmunity. Sustained IFNα signaling is linked to pathogenesis of systemic lupus erythematosus (SLE), an incurable autoimmune disease characterized by aberrant self-DNA sensing that culminates in anti-DNA autoantibody-mediated pathology. IFNα instructs monocytes differentiation into autoinflammatory dendritic cells (DCs) than potentiates the survival and expansion of autoreactive lymphocytes, but the molecular mechanism bridging sterile IFNα-danger alarm with adaptive response against self-DNA remains elusive. Herein, we demonstrate IFNα-mediated deregulation of mitochondrial metabolism and impairment of autophagic degradation, leading to cytosolic accumulation of mtDNA that is sensed via stimulator of interferon genes (STING) to promote induction of autoinflammatory DCs. Identification of mtDNA as a cell-autonomous enhancer of IFNα signaling underlines the significance of efficient mitochondrial recycling in the maintenance of peripheral tolerance. Antioxidant treatment and metabolic rescue of autolysosomal degradation emerge as drug targets in SLE and other IFNα-related pathologies.

The tumor microenvironment is immune suppressive, allowing tumor cells to escape from immune attack. By relieving this immune suppression, immune cells might be able to effectively eliminate the tumor. Therefore, a detailed understanding on how tumor cells induce an immunosuppressive tumor microenvironment is required. Glycosylation is the most abundant post-translational modification of proteins. It is highly diverse on a cellular level and strongly affected by oncogenesis. Tumor cells generally display an aberrant glycosylation profile and this is now well accepted as a new hallmark of cancer. Overexpression of glycan structures containing sialic acids (Sia) is frequently observed on tumors. Glycan structures can be recognized by lectin receptors expressed on immune cells, whereby sialic acids specifically bind to the Siglec receptor family. The majority of Siglec receptors has an immunoreceptor tyrosine-based inhibition motif and therefore, sialylated glycan structures are known to suppress ongoing immune responses. Hence, we hypothesize that tumor cells overexpress sialylated glycan structures in order to communicate with the immune system and to dampen immune attack. We have previously demonstrated that a genetically engineered Sialow glycovariant of the mouse melanoma B16 model displayed delayed growth in vivo compared to wild-type B16 Siahigh tumors (1). Moreover, higher frequencies of IFNγ-producing effector T-cells were detected in B16 Sialow tumors. This was paralleled by a 50% reduction in FoxP3+CD4+ regulatory T-cell frequencies in Sialow tumors (1), together indicating that tumor-associated sialic acids indeed play, indeed, a crucial role in suppressing the anti-tumor immune response. To check whether the inhibitory effect of sialic acids on the effector T cell response is tumor type specific, we investigated the role of sialic acids in a mouse colorectal cancer model (MC38). With the use of Crispr-Cas9 we successfully knocked out the N-acylneuraminate cytidylyltransferase (CMAS) gene, which is responsible for the sialylation pathway and thus the generation of sialylated glycans on the cell surface. In contrast to the B16 Sialow tumors, the Sianeg MC38 CMAS KO tumors displayed an increased tumor growth in vivo compared to the Siahigh MC38 Mock tumors. Moreover, less viable leukocytes could be recovered from the Sianeg MC38 CMAS KO tumors compared to the Siahigh MC38 Mock tumors. With the use of mass spectrometry, we analyzed the complete glycosylation profile of our Sianeg MC38 CMAS KO and Siahigh MC38 Mock cells and observed, in addition to the absence of sialylation, an increase in LacNAc (Galβ1-4GlcNAc) structures on the Sianeg MC38 CMAS KO cells. This glycan structure is a ligand for carbohydrate-binding Galectin receptors. Galectins are well known for their potential to induce leukocyte apoptosis after binding specific glycoproteins on the leukocyte surface. Using qPCR we observed higher mRNA levels of Galectin-1 in our MC38 CMAS KO cells. The enhanced production of Galectin-1 and the elevated expression of LacNAc together suggest that MC38 CMAS KO cells are able to recruit Galectin-1, thereby facilitating the enhanced immune suppression in these tumors. Currently, we are confirming the role of Galectins in our MC38 CMAS KO tumors. In conclusion, tumor cells alter their glycosylation profile and based on our data, we propose that tumor cells use these glycan structures as a mechanism to influence antitumor immunity and that this phenomenon might be tumor-type specific. Their immune inhibitory nature and their tumor-specific expression identifies tumor-associated glycans as potent candidates for future immune checkpoint blockade therapy. Reference: 1. Perdicchio M. et al. Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells. Oncotarget 2016;7(8):8771-8. Citation Format: Lenneke A.M. Cornelissen, Athanasios Blanas, Joost C. Van der Horst, Laura Kruijssen, Yvette Van Kooyk, Sandra J. Van Vliet. Bittersweet symphony: How tumor-associated glycan structures orchestrate immune evasion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B63.

Aberrant fucosylation in cancer cells is considered as a signature of malignant cell transformation and it is associated with tumor progression, metastasis and resistance to chemotherapy. Specifically, in colorectal cancer cells, increased levels of the fucosylated Lewisx antigen are attributed to the deregulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase 9 (FUT9). However, the lack of experimental models closely mimicking cancer-specific regulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding the substrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectal cancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the well-known murine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9 enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novo gene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes in MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen on the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4 and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expression stability of the induced enzymes. Moreover, Lewisx expression was found to influence core-fucosylation, sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. In conclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression is a promising method of transcriptional gene activation with broad application possibilities in glycobiology and oncology research.

[This corrects the article on p. 39 in vol. 8, PMID: 29527514.].