TME: Sialoglycans and Siglecs

TME: Sialoglycans and Siglecs.  Siglecs are widely expressed in the mammalian immune system. Recent studies have provided strong evidence that tumor-derived sialoglycans can effectively trigger these receptors, thereby regulating the function of different immune cell subtypes in TME.

Preface

The tumor creates a local tumor microenvironment (TME), which is composed of different cell types, extracellular matrix components, and soluble factors that support tumor growth and progression. TME usually has a high degree of immunosuppressive effect, preventing immune cells from clearing cancer cells, thereby negatively affecting cancer immunotherapy. Therefore, overcoming immunosuppressive TME is essential for the development of effective cancer immunotherapy.

Tumor cells express abnormal amounts of sialic acid on the cell surface. Sialic acid is a family of negatively charged sugar molecules that cover glycoproteins and glycolipids with glycan chains. The sialoglycans on tumor cells can participate in tumor cell-extracellular matrix interactions and tumor cell-cell interactions. It can form a protective area to protect tumor cells from immune recognition.

In fact, the negatively charged sialic acid part of the cap-shaped sialoglycan is believed to be responsible for this masking effect. The removal of sialic acid from the surface of TA3 mouse cancer cells with sialidase inhibited their growth when transplanted into immune-competent mice.

Human vaccination trials on melanoma and breast cancer cells treated with autologous sialidase show that removal of sialic acid can cause a strong anti-tumor immune response. Studies in the past few decades have shown that the role of sialic acid glycans in tumor immune evasion far exceeds the masking role of antigens, and sialic acid is an effective immunomodulatory sugar.

Many of the strong immunomodulatory properties of sialic acid glycans are thought to be specific interactions with sialic acid-binding immunoglobulin-like lectins (Siglecs). Tumor cells with abnormal expression of sialic acid glycans can interact with the Siglec family to regulate immune cell functions in TME.

TME seems to enhance the abnormal sialic acid secretion of tumor cells and also stimulate the expression of Siglec infiltrating immune cells. These data suggest that the dysregulation of the siaglycan-Siglec axis in cancer contributes to the formation of immunosuppressive TME, which is an obstacle for certain cancers to escape immunotherapy.

The immunosuppressive effect of sialoglycans

The link between tumor sialic acid glycans and immune regulation is awaiting progress in our understanding of tumor immunology and TME, as well as the development of tools to interfere with the abnormal expression of sialic acid glycans.

Existing studies have proved that intratumoral injection of Ac53FaxNeu5Ac (a selective sialic glycan biosynthesis inhibitor) can block the expression of sialic glycans in mouse B16-F10 melanoma and 9464D neuroblastoma tumors. Inhibit tumor growth and significantly change the immune cell composition of TME. Compared with tumors treated with phosphate buffered saline (PBS), the removal of sialic acid in TME resulted in an increase in the number of natural killer (NK) cells, CD4+ and CD8+ T cells, and the number of regulatory T cells (Treg) and myeloid cells Reduced, resulting in an effective immune response.

Some researchers have coupled bacterial sialidase with an anti-human epidermal growth factor receptor (HER) 2 antibody (trastuzumab) to selectively remove sialic acid from the surface of human HER2+ breast cancer cells. Compared with purely anti-HER2 or HER2-negative human breast cancer cells, human peripheral blood NK cells lyse these antibody-sialidase-treated cells more efficiently through antibody-dependent cytotoxicity (ADCC).

In another study, the researchers silenced the sialic acid transporter SLC35A1 gene. Compared with the control group B16 cells, the gene silenced mouse B16 melanoma cells showed lower expression of sialic acid and reduced growth rate, and the tumor contained more TME. The effector immune cells (NK cells, CD8+T cells).

In addition, in the mouse MC38 colorectal cancer model, after knocking out the GNE gene, the key enzyme in sialic acid biosynthesis, compared with the control tumor, the expression of sialic acid was reduced, tumor growth was delayed, CD4+ T cells, CD8+ Increased recruitment of T cells and dendritic cells (dc).

In addition, tumor-associated sialic acid glycans may also perform different functions according to tumor stages. For example, in established tumors (sialic acid blockers) before or after mice (KO cells) are inoculated with tumors, the reduction of sialic acid may have different effects on tumor growth and TME components.

From another perspective, the removal of sialic acid will expose potential glycans, and these glycans can be recognized or sensed, thereby affecting cell-cell communication. For example, exposure of galactose residues on mouse or human tumor cells will cause the galactose residues to be recognized by galectin. The human galectin family consists of 15 members, which have a variety of biological functions, including tumor immune evasion, suppression of effector T cell function, or expansion of regulatory immune cells in TME.

In general, the above studies support that tumor sialoglycans may have strong immunosuppressive properties and are at least partly involved in the formation of immunosuppressive TME. In addition, the interaction between tumor sialic acid glycans and TME may be two-way. There is evidence that factors in TME, such as cytokines or hypoxia, can enhance the expression of siaglycan on tumor cells, thereby promoting tumor growth and immunosuppression.

Siglecs can inhibit the function of cytotoxic effector cells in TME

Since the discovery of sialic acid recognition receptors, the Siglec receptor family has been considered as the main target of tumor sialic acid glycans for regulating many immune functions of tumor infiltrating immune cells. However, only recent studies have provided direct evidence that the sialogan-Siglec interaction does have a strong immunomodulatory effect on effector immune cells, such as NK cells and cytotoxic CD8+ T cells.

In particular, the sialic acid glycan ligands of human Siglec-7 and Siglec-9 have been found in some human cancers, including colorectal cancer, melanoma, and glioblastoma. It is worth noting that human peripheral blood NK cells express Siglec-7 and Siglec-9. Siglec-7 is a pan-marker of human NK cells, while Siglec-9 is expressed on a subtype of CD56dim-CD16+ NK cells.

The interaction of Siglec-7 and Siglec-9 with human blood NK cells and sialic acid glycans on human cancer cells inhibited their ability to dissolve these cancer cells, and treatment with sialidase or Siglec blocking antibodies increased their killing ability .

By integrating human Siglec-7-bound sialic acid glycan polymer into the membrane of human Jurkat T lymphocytes, the strong inhibitory effect of sialic acid-Siglec-7 interaction on the cytotoxicity of human NK cells has also been confirmed .

This immunosuppressive effect involves the phosphorylation of the tyrosine-based immunoreceptor inhibitory motif (ITIM) in human Siglec-7 and the recruitment of SHP-1, which dephosphorylates downstream activated immune targets. Anti-Siglec antibody blocking the siaglycan-Siglec-7/9 interaction improves the killing effect of human NK cells on human cancer cells in vitro. These main effects of Siglec-7 and Siglec-9 on NK cell-mediated cytotoxicity in vitro indicate that the use of targeting antibody-sialidase conjugates or sugar mimics to locally remove sialic acid from tumor cells can be used to promote NK cell-mediated tumor cell clearance in vivo, although this requires reliable testing.

Human T lymphocytes, including CD8+ T cells, were initially reported to express no or only a small amount of Siglecs. However, it has now been demonstrated that Siglecs expresses Siglec in large quantities on CD8+ T cells isolated from patients with non-small cell lung cancer (NSCLC), colorectal cancer, ovarian cancer, and melanoma, with Siglec-9 expressing the highest.

In particular, CD8+ effector memory T cells isolated from non-small cell lung cancer tumors expressed a large amount of Siglec-9 after expansion in vitro, but CD8+ effector memory T cells were detected in the peripheral blood of cancer patients and healthy blood donors The low expression. These findings indicate that the expression of Siglec-9 is induced by factors that have yet to be determined in TME.

Therefore, it is speculated that the up-regulation of the immunosuppressive signal on CD8+ T cells in TME may help reduce the CD8+ T cell response, thereby facilitating tumor immune evasion.

Similar to human NK cells, the sialoglycan-Siglec-9 interaction can lead to ITIM phosphorylation and SHP-1 recruitment of human CD8+ T cells cultured in vitro, thereby inhibiting T cell receptor (TCR) signaling and cytotoxicity. In addition, the expression of Siglec-9 on infiltrating CD8+ T cells in NSCLC patients is associated with reduced survival, and Siglec-9 polymorphisms have been shown to be associated with the risk of cancer.

Tumor infiltrating Siglec-9+CD8+ T cells isolated from patients with non-small cell lung cancer also express other inhibitory receptors, such as PD-1, TIM-3 and LAG-3. Other recent studies have shown that human and mouse T cells express Siglec-10 (CD4+ T cells) and Siglec-G and -E (CD3+ T cells), respectively, but their role in TME has not been studied.

A new concept is that the factors in TME may induce the expression of Siglecs on CD8+ T cell subsets and other immune cell subsets. Identifying these factors and conditions in TME should be the focus of future research.

It is worth noting that only inhibitory Siglec has been identified on human effector CD4+ T cells, CD8+ T cells and NK cells, mainly Siglec-7 and Siglec-9, but no activating Siglecs. This indicates that Siglecs on lymphocytes may serve as “checkpoints” of glycoimmunity, and tumor cells use it to suppress effector immune cells.

Sialoglycans and Siglecs can regulate DC cell function

Recently, the tumor sialogan-Siglec interaction has been proposed to “brake” tumor immunity by regulating the function of DC cells. In fact, DC cells are the main antigen presenting cells (APC) in the immune system, which can cause the activation of antigen-specific effector T cells.

They also interact with other innate lymphocytes and play an important role in the activation of NK cells by producing type I interferons in mice and humans. DC cells express a variety of inhibitory Siglec family members, including Siglec-3, -5, -7, and -9 (human DC cells) and Siglec-E, -G, and -H (mouse DC cells), which are considered to be critical Processes such as DC maturation, antigen presentation and the ability to activate tumor-specific CD8+ T cells.

For example, compared with wild-type mice, SiglecG-deficient mice (C57BL/6J SiglecG–/−) exhibited enhanced cross-priming of CD8+DC, partly due to the increased formation of MHC-like peptide complexes, thereby improving the Control of subcutaneous B16-F10 melanoma growth. In addition, the OVA antigen (6′-sialic acid-N-acetyllactosamine) in which mouse spleen CD11c+DC is modified by sialic acid glycans makes OT-II CD4+ T cells prefer Tregs rather than effector T cells. In co-culture experiments with DC cells from SiglecE–/–C57BL/6J mice, no dendritic cells rich in sialic acid OVA were observed to induce Treg activation, which indicates that Siglec-E is involved in this process.

These studies show that certain tumors can use the sialic acid-Siglec interaction to regulate human and mouse DC functions at multiple levels, thereby negatively affecting the induction of CD4+ and CD8+ T cell responses. In addition to inhibitory Siglecs, Siglec-15 has also been found on human DC, which may increase the level of regulation of Siglecs on DC. Therefore, analyzing the expression and function of other activating and inhibiting Siglec receptors on different DC subgroups inside and outside TME may have a deeper understanding of the DC behavior of TME.

Sialoglycans and Siglecs can regulate myeloid cells in TME

Myeloid cells are highly plastic cells that can exhibit tumor or anti-tumor functions, depending on their environment. Myeloid cells in TME are mainly composed of immunosuppressive and primary macrophages, neutrophils and myeloid-derived suppressor cells (MDSCs), which can limit effective anti-tumor immunity. Siglecs are widely expressed in human bone marrow cell subpopulations. For example, Siglec-3/CD33 is used as a common marker for human MDSCs. However, the role of Siglecs in myeloid cell biology is still unknown.

The effect of Siglec-9 and tumor sialoglycan on colorectal cancer cells can induce SHP-1 recruitment and inhibit the killing of tumor cells by neutrophils in vitro; further in vivo data show that compared to WT mice, Siglec- The occurrence and metastasis of MC38 tumors decreased in E-/- mice. Although the immunosuppressive effect of Siglec-9 on neutrophils has been established, the effect of Siglec-9 on the polarization and function of monocytes/macrophages remains to be studied; this is true for both human and mouse Siglec-E.

Other reports have shown that human monocytes and macrophages obtained from blood are recognized by Siglec-9 for O-glycosylation carrying sialic acid T antigen, a glycoprotein that is frequently overexpressed in many epithelial human cancers Mucin 1, which produces protocarcinoma factor. However, three recent studies have shown that human Siglec-9 does not bind to mucin-type O-glycans, and prefers N-linked glycans on glycoproteins. Therefore, the relationship between Siglec-9 and mucin on myeloid cells remains to be elucidated.

Recent studies have shown that Siglec-10, Siglec-1 and Siglec-15 regulate the activity of tumor-associated macrophages (TAMs) in mouse and human tumor TMEs. Siglec-10 is expressed by a large proportion of human TAMs in the TME of ovarian cancer and breast cancer patients. Its ligand, the glycoprotein CD24, is up-regulated on many human tumors, such as ovaries and breasts, and can act as a “don’t eat me signal” for macrophages, similar to the interaction of CD47 with SIRPa. A recent study showed that compared with the control group, human cancer cells lack CD24, human macrophages lack Siglec-10 or CD24, or use monoclonal antibodies to block Siglec-10 or CD24, which can enhance human macrophages. Phagocytosis and tumor cell clearance rate. Treatment of established human MCF-7 tumor-bearing mice with anti-CD24 monoclonal antibodies showed the therapeutic potential of interfering with the CD24–Siglec-10 pathway. Compared with IgG-treated control mice, tumor growth was reduced.

Recently, it was found that in human breast cancer, Siglec-1 (CD169) is highly upregulated on TAMs. In addition to breast cancer, Siglec-1+ macrophages have also been found in human colorectal cancer and hepatocellular carcinoma. It is worth noting that in breast cancer, Siglec-1 is part of the new TAM gene expression profile composed of MHC-II, Fc receptors, TLRs, and other transmembrane receptors, and is related to the shorter disease of breast cancer patients. Specific survival time is related. Siglec-1 does not contain known intracellular signal motifs, but is related to the uptake of sialylated antigens or viral particles. Whether Siglec-1 also has an important function in breast cancer TAMs remains to be studied.

Human Siglec-15 is expressed by TAMs and can recognize the sialyl-Tn antigen, which is commonly expressed in human cancers. H157 cells expressing sialyl-Tn were co-cultured with human Siglec-15+ macrophages, and the cancer-promoting factor TGF-β was induced through the DAP12–Syk pathway. Recently, 6500 membrane proteins were screened through high-throughput analysis of Jurkat T cell activation, and Siglec-15 protein was identified as a potential immunosuppressive protein.

The degree to which Siglec-15 reduces Jurkat T cell activation is similar to the known T cell inhibitory molecular apoptosis ligand (PD-L) 1/2 and Fas ligand. In addition, in vitro stimulated by anti-CD3 antibodies, Siglec-15 inhibits the proliferation of human CD8+ and CD4+ T cells. Subsequent in vivo experiments using Siglec-15 KO mice (C57BL/6 Siglec15-/-) and Siglec-15 blocking antibodies showed that Siglec-15 present on TAMs inhibited antigen-specific CD8+ T cell responses and promoted Immunosuppressive TME.

Immunohistochemical analysis of human NSCLC tumor tissue arrays showed that Siglec-15 was expressed on CD68+ macrophages, mesenchymal cells and tumor cells themselves. In human bladder cancer, the expression of Siglec-15mRNA is negatively correlated with some immune marker genes (such as CD3E, IFNG, GZMA or GZMB). In addition, the protein sequence comparison shows that the immunoglobulin variable and constant regions of Siglec-15 are homologous to the B7 protein family; therefore, whether the binding of Siglec-15 to CD8+ T cells depends on sialic acid and whether it is related to B7 family members The same immunomodulatory function independent of sialic acid remains to be studied.

In summary, these studies indicate that Siglec-1, Siglec-10 or Siglec-15 may play a role in the formation of different TMEs. Human Siglec-10 may inhibit the immune response, and blocking the interaction with sialic acid glycans may help improve anti-tumor activity. However, inhibitory Siglec, such as Siglec-10, also recognize sialic acid glycans on healthy cells in the body as “self” and participate in immune regulation.

Similarly, the expression of human Siglec-1 is associated with positive and negative tumor parameters; for example, Siglec-1+ macrophages in breast and colorectal cancer patients are associated with tumor progression, while in hepatocellular carcinoma patients, they are better The clinical prognosis is relevant. For Siglec-15, the pro-inflammatory function of humans and mice has also been confirmed. In addition, Siglec-15 can regulate osteoclast function and bone resorption of the same species.

Therefore, studying the effect of systemic Siglec receptor blockade therapy not only on tumor immune function, but also has important significance for assessing the risk of adverse reactions or autoimmune reactions.

Summary

Siglecs are widely expressed in the mammalian immune system. Recent studies have provided strong evidence that tumor-derived sialoglycans can effectively trigger these receptors, thereby regulating the function of different immune cell subtypes in TME.

Sialylglycan-Siglec interaction shows a major inhibitory effect on effector lymphocytes, and a stronger regulatory effect on bone marrow cells (such as monocytes/macrophages, bone marrow mesenchymal stem cells and dendritic cells) .

Sialylglycan-Siglec interactions may have different functional results, depending on the suppression or activation of Siglec family members, the immune cell subsets they express, tumor types, species, and the background provided by other activation or suppression signals in TME.

Since the factors in TME can regulate the expression of sialic acid glycans in cancer cells and the expression of Siglec on immune cell subsets, it is necessary to clarify the role of these factors and regulatory mechanisms in different tumor types to understand the inhibition and activation of sialic acid polymerization. The full effect of the sugar-Siglec interaction.
This will be necessary for the final design of targeted and effective immunotherapy for specific cancers.

Proof-of-concept animal studies have shown the therapeutic potential of targeting the sialoglycan-Siglec axis. The understanding of the different functional consequences of the sialoglycan-Siglec interaction in TME will help to perfect these new treatment strategies and pave the way for their clinical applications.

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