The development of immunotherapy has revolutionized cancer care (1). These drugs block tumor-induced immunosuppression and help immune cells detect and attack cancer cells (1). Immunotherapy effectively prevents the progression of many types of cancer but has limited effect on colorectal cancer (CRC), suggesting that this type of cancer uses different strategies to evade immune recognition (2). CRC ranks third among the most common types of cancer and second among cancers leading to death worldwide (2). Identifying the mechanisms involved in CRC immune evasion can help the development of new therapies to improve clinical outcomes.
Using a combination of in vitro and in vivo experiments, Silva et al. showed that expression of branched N-glycans in CRC cells contributes to immune suppression and evasion (3). In addition, changing the glycosylation profile of cancer cells restored anti-tumor activity (3).
Keep reading to learn more about this study and check out other publication highlights on the blog.
Cancer cells have a different glycosylation signature, or glycocalyx, than healthy cells (4,5). Glycan-binding receptors on the surface of immune cells recognize the glycocalyx of tumor cells and, in response, trigger immune activation (6,7). Examples of these glycan-binding receptors include galectins, Siglecs, and C-type lectins—dendritic cell-specific intracellular adhesion molecular-3-grabbing nonintegrin (DC-SIGN) and mannose receptors (6,7). However, tumor cells change their glycocalyx to drive cancer progression (4,5). For example, overexpression of b1,6-GlcNAc-branched N-glycans in gastrointestinal cancer cells often associates with an invasive tumor phenotype and poor clinical prognosis (8–12).
Despite the known association between overexpression of branched N-glycans and tumor progression, additional research is needed to elucidate the precise mechanisms mediating this relationship. The long-term goal is to identify new therapeutic targets to treat CRC and other types of cancer.
In the first set of experiments, researchers used human CRC samples to confirm previous findings: Overexpression of branched N-glycans associates with tumor progression. First, Silva et al. used fluorescein-conjugated Phaseolus vulgaris leucoagglutinin and Galanthus nivalis to target the b1,6-GlcNAc arm of branched N-glycans and terminal a-1,3-mannose residues, respectively. Then, they used immunohistochemistry (IHC) to quantify the expression of FOX3P in Tregs (T-regulatory) cells and Tbet in CD4+ T lymphocytes. Data showed that as the stage of CRC advances, the expression of b1,6-GlcNAc-branched N-glycans increases and of mannosylated N-glycans decreases. In addition, changes in the cancer cell glycocalyx were associated with more FOX3P-expressing Tregs cells and fewer Tbet-expressing cells in the tumor environment.
Transcriptomic analysis confirmed that FOXP3 expression gradually increases as cellular phenotype goes from normal dysplasia to colon adenocarcinoma. Researchers also quantified the levels of MGAT5, the gene that encodes GnT-V, the enzyme responsible for the expression of b1,6-GlcNAc-branched N-glycans. Data showed that the expression of MGAT5 steadily increases as the stage of human CRC progresses and positively correlates with FOXP3 levels. Silva et al. also reported a strong positive correlation between FOXP3 and MGAT5 expression in large datasets from patients with colorectal cancer.
Next, researchers sought to establish the causal relationship between the overexpression of branched N-glycans and immunosuppression. They transfected a gastrointestinal cancer cell line with the GnT-V enzyme to overexpress b1,6-GlcNAc-branched N-glycans. Transfected and mock cells were co-cultured with CD4+ T lymphocytes and dendritic cells. Overexpression of branched N-glycans in transfected cancer cells suppressed the release of cytokines IFN-g, TNF-a, IL-6, and IL-8 by immune cells. In addition, expression of MHC-I in transfected cancer cells was higher in the intracellular compartment than on the cell surface, suggesting that the presence of branched N-glycans promotes the internalization of MHC-I.
Mock and transfected cells displayed opposite glycoprofile—mock cells had high levels of mannose-enriched glycans, and transfected cells overexpressed branched N-glycans. These differences in the glycocalyx likely impact the interaction and communication of cancer with immune cells. To investigate this hypothesis, researchers performed an assay to assess the binding between C-type lectin receptors on immune cells and glycans on mock and transfected cells. Mock cells displayed a high affinity for DC-SIGN binding, which decreased with overexpression of branched N-glycans in transfected cells. Overall, data suggest that DC-SIGN receptors on immune cells have a high affinity for mannose enriched-glycans on healthy and early-stage cancer cells. But as the tumor stage progresses, the overexpression of branched N-glycans masks the recognition of CRC cells by DC-SIGN, resulting in impaired immune activation.
Silva et al. also showed that modulation of N-glycosylation in tumor cells can impact tumor progression in preclinical models. Researchers used kifunensine (KF) to inhibit N-glycosylation in the MC38 murine colon adenocarcinoma cell line. As a result, the expression of high-mannose N-glycans increased and that of branched N-glycans decreased. Then, immunocompetent mice received subcutaneous inoculation of MC38 plus KF cells or MC38 plus vehicle cells. Inhibition of N-glycosylation in MC38 cells contributed to a significant delay in tumor growth.
As an additional proof of concept, researchers used CRISPR-Cas9 to silence the MGAT5 gene (encodes GnT-V enzyme) in MC38 cells, resulting in the generation of 3 different clones. Next, they used P. vulgaris leucoagglutinin, G. nivalis, and biotinylated Concanavalin A to analyze the glycoprofile of each clone and validate the functional knockout of the enzyme. The clone with the lowest expression of branched N-glycans was inoculated into immunocompetent mice. Results confirmed that low expression of branched N-glycans contributes to suppression of tumor growth—only 1 of 11 mice developed a very small tumor. In addition, the removal of branched N-glycans impacted the immune response. Inhibition of N-glycosylation associated with a higher number of immune cells and increased expression of cytokines. Data also revealed that inhibition of N-glycosylation enhances immune recognition of cancer cells but doesn’t impact the tumor’s capacity to grow. Inoculation of wild-type and MGAT5-/- MC38 cells in immunocompromised mice resulted in tumor growth, regardless of the absence of branched N-glycans. Therefore, immune recognition of high-mannose glycans in tumor cells is essential to suppress tumor growth.
In a final experiment, Silva et al. crossed over a preclinical model of intestinal tumorigenesis with MGAT5-/- mice. Deleting the MGAT5 gene resulted in fewer tumors with smaller sizes and more prolonged survival. The absence of branched N-glycan overexpression also resulted in increased infiltration of immune cells, decreased number of FOX3P Tregs and M2 pro-tumor macrophages, and enhanced release of pro-inflammatory cytokines. Similar findings in different preclinical models of CRC reinforced the overarching conclusion that removing branched N-glycans from the tumor glycocalyx reduces immune evasion.
Characterizing the mechanism of immune evasion in CRC has a significant translational impact on public health. CRC is the third most common type of cancer, and many patients don’t respond to currently available therapies (2). Data from Silva et al.’s study provide robust evidence that the glycosylation profile of cancer cells plays a major role in the immune evasion process. Modulation of glycosylation improved anti-tumor activity in in vitro and in vivo experiments. Continued research on this topic can contribute to identifying therapeutic targets and developing new interventions to treat CRC.
If you want to learn more about the applications of glycobiology in cancer research, check out the Examining Altered Glycobiology in Cancer eBook, and stay tuned to the blog for more glycobiology highlights.