Review Article

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Tools to study and target the Siglec–sialic acid axis in cancer

Laboratory for Cancer Immunotherapy, Department of Biomedicine, and Medical Oncology, Department of Internal Medicine, University Hospital Basel, Switzerland

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Kunio Kawanishi

Kidney and Vascular Pathology, University of Tsukuba, Ibaraki, Japan

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Cijo George Vazhappilly

Department of Biotechnology, American University of Ras Al Khaimah (AURAK), UAE

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Rachel Matar

Department of Biotechnology, American University of Ras Al Khaimah (AURAK), UAE

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Maxime Merheb

Department of Biotechnology, American University of Ras Al Khaimah (AURAK), UAE

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Shoib Sarwar Siddiqui

Corresponding Author

shoib.siddiqui@aurak.ac.ae

orcid.org/0000-0002-7028-290X

Department of Biotechnology, American University of Ras Al Khaimah (AURAK), UAE

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Heinz Läubli

Laboratory for Cancer Immunotherapy, Department of Biomedicine, and Medical Oncology, Department of Internal Medicine, University Hospital Basel, Switzerland

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Kunio Kawanishi

Kidney and Vascular Pathology, University of Tsukuba, Ibaraki, Japan

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Cijo George Vazhappilly

Department of Biotechnology, American University of Ras Al Khaimah (AURAK), UAE

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Rachel Matar

Department of Biotechnology, American University of Ras Al Khaimah (AURAK), UAE

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Maxime Merheb

Department of Biotechnology, American University of Ras Al Khaimah (AURAK), UAE

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Shoib Sarwar Siddiqui

Corresponding Author

shoib.siddiqui@aurak.ac.ae

orcid.org/0000-0002-7028-290X

Department of Biotechnology, American University of Ras Al Khaimah (AURAK), UAE

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First published: 29 November 2020

https://doi.org/10.1111/febs.15647

Heinz Läubli and Kunio Kawanishi have contributed equally.

Abstract

Siglecs are widely expressed on leucocytes and bind to ubiquitously presented glycans containing sialic acids (sialoglycans). Most Siglecs carry an immunoreceptor tyrosine‐based inhibition motif (ITIM) and elicit an inhibitory intracellular signal upon ligand binding. A few Siglec receptors can, however, recruit immunoreceptor tyrosine‐based activation motif (ITAM)‐containing factors, which activate cells. The role of hypersialylation (the enhanced expression of sialoglycans) has recently been explored in cancer progression. Mechanistic studies have shown that hypersialylation on cancer cells can engage inhibitory Siglecs on the surface of immune cells and induce immunosuppression. These recent studies strongly suggest that the Siglec–sialic acid axis can act as a potential target for cancer immunotherapy. Moreover, the use of new tools and techniques is facilitating these studies. In this review, we summarise techniques used to study Siglecs, including different mouse models, monoclonal antibodies, Siglec fusion proteins, and sialoglycan arrays. Furthermore, we discuss the recent major developments in the study of Siglecs in cancer immunosuppression, tools, and techniques used in targeting the Siglec–sialic acid axis and the possibility of clinical intervention.

Abbreviations

ALL: acute lymphoblastic leukaemia
AML: acute myeloid leukaemia
CMAH: cytidine monophospho‐N‐acetylneuraminic acid hydroxylase
Dap12: DNAX‐activation protein of 12 kDa
FACS: fluorescence‐activated cell sorting
GBS: group B streptococcus
ITAM: immunoreceptor tyrosine‐based activation motif
ITIM: immunoreceptor tyrosine‐based inhibition motif
LGALS3BP: lectin galactoside‐binding soluble 3 binding protein
MAG: myelin associated glycoprotein
MAPK: mitogen‐activated protein kinase
MCA: 3‐methyl‐cholanthrene
MDSC: myeloid‐derived suppressor cells
NK: natural killer
PD1: programmed death 1
PD‐L1: programmed death‐ligand 1
SAM: sialic acid mimetic
SCID: severe combined immune deficiency
SEB: staphylococcal enterotoxin B
SHP‐1: Src homology region 2 domain‐containing phosphatase‐1
SHP‐2: Src homology region 2 domain‐containing phosphatase‐2
TAM: tumour‐associated macrophage
TNBC: triple‐negative breast cancer
TNF‐α: tumour necrosis factor‐α
WT: wild‐type

Introduction

Altered glycosylation is one of the hallmarks of cancer [[1-3]]. One type of glycosylation often altered in cancer is associated with the monosaccharide sialic acid (Sia) and changes in sialylation are commonly observed [[4-7]]. Sias are nine‐carbon atom monosaccharides that are present at the terminal end of the glycan chain [[8, 9]]. Sia‐containing glycans are ligands for Siglec receptors [[8, 9]]. Siglecs are cell surface receptors that are mostly inhibitory, and upon binding with sialoglycans, they generate inhibitory intracellular signals [[10, 11]]. Thus, enhanced expression of sialoglycans on the surface of cancer cells can be immunomodulatory, and an imbalance in Siglec signaling can induce acceleration or inhibition of immune reactions [[8, 10, 11]]. There are two types of Siglecs, based on the motif that they carry. The ones that carry ITIM are inhibitory, while the ones that carry positively charged residues in the transmembrane domain and interact with DNAX‐activation protein of 12 kDa (Dap12) are activating in nature [[10-12]]. Siglecs can also be divided into evolutionarily conserved Siglecs and rapidly evolving Siglecs (also called CD33‐related Siglecs). The members of evolutionary conserved Siglecs include Siglec‐1 (CD169), Siglec‐2 (CD22), Siglec‐4 (myelin‐associated glycoprotein/MAG), and Siglec‐15. Members of the rapidly evolving CD33‐related Siglecs include Siglec‐3 (CD33), Siglec‐5 to Siglec‐XII, Siglec‐14 and Siglec‐16 [[10, 13, 14]]. Due to rapid evolution of CD33‐related Siglecs, for most of the members, there are no true orthologues present between human and mice. Therefore, the human Siglecs are named with a number whereas murine Siglecs are often named with letters. However, Siglec‐G is a clear orthologue of Siglec‐10 and there is no equivalent of Siglec‐H in humans. The murine Siglecs have nine members: Siglec‐1, Siglec‐2, Siglec‐3, Siglec‐4, Siglec‐15, Siglec‐E, Siglec‐F, Siglec‐G and Siglec‐H [[13, 15]].

Siglecs are mostly expressed on the surface and endocytic machinery of immune cells, including innate immune cells [[10-12]]. The recent findings showed that one of the isoforms of CD33, also called a minor form (CD33m), is not on the cell surface, but instead expressed in the peroxisomes [[16]]. The reason for the expression of this alternatively spliced isoform in peroxisomes is not fully clear. Figure 1 demonstrates a detailed expression pattern for Siglecs in the immune system. Several reports found the expression of Siglecs in non‐immune cells as well. Siglec‐9, Siglec‐10, Siglec‐11 and Siglec‐16 are expressed in the female reproductive tract [[17]]; Siglec‐3, Siglec‐4 and Siglec‐11 are found in the brain [[15, 18-21]]; Siglec‐5/14 in the amniotic epithelium [[22]]; Siglec‐XII in the epithelial tissue [[23]]; Siglec‐6 in the human placental trophoblast [[24]]; and Siglec‐7 in pancreatic islets [[25]].

**Fig 1**
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Immune cell expression of Siglecs: The schematic shows the expression of different Siglecs in the immune cells in humans. The * shows the expression of Siglecs only in an activated state.

The Siglecs have an extracellular region comprised of immunoglobulin‐like (Ig‐like) domains, a transmembrane domain and an intracellular domain [[26, 27]]. The Ig‐like extracellular domain includes an outermost sialoglycan‐binding V‐set domain and the underlying Ig‐like domains, namely C2‐set domains [[26, 27]]. Exceptionally, Siglec‐XII carries two V‐set domains (none of them bind to Sia due to a mutation in critical arginine), while other Siglecs carry only one V‐set domain [[23, 26, 27]]. There are variable numbers of C2‐set domains in Siglecs. For example, CD33 carries only one C2‐set domain, while Siglec‐1 carries 16 C2‐set domains. The intracellular region of inhibitory Siglecs carries ITIM and ITIM‐like motifs, which become phosphorylated upon ligand binding. Upon phosphorylation, they recruit signaling proteins such as Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) and Src homology region 2 domain‐containing phosphatase‐2 (SHP‐2), which blocks the mitogen‐activated protein kinase (MAPK) pathway, inhibit calcium signaling, c‐KIT signaling, and phosphoinositide 3‐kinase (PI3K)‐signaling [[26-29]]. Thus, activation of inhibitory Siglecs leads to immunosuppression. The activating Siglecs carry a positively charged residue in the transmembrane domain, which interacts with Dap 12 carrying an ITAM to generate an activating intracellular signal. Activation of these Siglecs may lead to an immunoactivatory response [[26, 27]]. Some Siglecs also appear as paired receptors where they bind to the same ligands but generate opposite signals to the cells. For example, Siglec 5/14 and Siglec 11/16 are paired Siglec receptors [[22, 30]]. These paired Siglec receptors have recently been explored in cancer, bacterial infection and inflammation [[30-33]].

As previously noted, Sia is often present at the terminal end of the glycan chain where it is linked to underlying Gal in α2‐3 or α2‐6 linkages, to GalNAc or GlcNAc in α2‐6 linkage, or to Sia in α2‐8 linkage [[8, 9]]. Each of these three linkages also determines the binding preference of a specific Siglec. For example, Siglec‐1 and Siglec‐4 bind preferentially to 2‐3’‐linked Sia, Siglec‐2 binds to 2‐6’‐linked Sia and Siglec‐9 binds with both 2‐3’‐ and 2‐6’‐linked Sia [[6, 9, 26]]. In this review, we intended to focus on the evidence in the literature about how hypersialylation in cancer blunts anti‐tumour immune response by engaging Siglecs on the cell surface. Moreover, we discussed the new tools, and techniques such as different transgenic/humanised mouse models related to Siglecs, and how to target the Siglec–Sia axis to combat cancer growth.

Mechanism of hypersialylation in cancer and its role in cancer progression

Under the normal homeostatic state, there is no immune response mounted against self‐cells. This homeostatic state is maintained by many different immune regulators and inhibitory immune receptors, including the interaction of Siglecs with sialoglycans that keep the whole system in a quiescent state [[34-37]]. Cancer cells are known to get hypersialylated, meaning abundant and aberrant decoration of sialoglycans on the cell surface. In this case, the tumour‐associated immune cells, in particular innate immune cells, express different Siglecs which can mediate interaction with sialoglycans on cancer cells and lead to immunosuppression [[5]] and ultimately to cancer progression. This section will discuss how hypersialylation is induced and how hypersialylation leads to cancer progression and metastasis.

There is increasing evidence that sialoglycan levels are higher in cancer cells than in healthy cells and that hypersialylation can modulate cancer growth [[5, 6, 9, 38, 39]]. There are several mechanisms of enhanced sialylation in cancer versus normal tissue: (1) increased expression of the enzymes called sialyltransferase which add Sia to the glycan chain ; (2) downregulation of the enzymes called neuraminidases or sialidases which cleave Sia from the glycan chains; (3) reduction in the function of neuraminidase; (4) increased availability of the substrate required for Sia incorporation; (5) enhanced sialylation through the hexosamine pathway; and (6) enhanced expression of Sia transporters [[5, 9]].

Several studies have described an enhanced sialylation. The enzyme ST6‐Gal1 which adds α2‐6‐Sia is highly upregulated in breast cancer, leukaemia, gastric cancer, colorectal cancer, acute myeloid leukaemia, liver, brain, and cervix cancer [[40-46]]. This high expression of ST6‐Gal1 is responsible for evading TNF‐α‐induced apoptosis and correlates with a poor prognosis [[47, 48]]. The ST3Gal1 which adds α2‐3‐Sia on core‐1 O‐glycans are upregulated in colon, breast, and bladder cancer [[46-48]], and plays a pivotal role in blunting the anti‐cancer immune response [[49]]. The elevated levels of ST3Gal3, ST3Gal4, and ST3Gal6 upregulate the Sialyl‐Lewis^A and Sialyl‐Lewis^X in multiple tumour types and correlate with poor prognosis [[5, 50]]. Sialyl‐Lewis^A and Sialyl‐Lewis^X are specialised sialoglycan comprised of Neu5Acα2‐3Galβ1‐3[Fucα1‐4]GlcNAcβ and Neu5Acα2‐3Galβ1‐4[Fucα1‐3]GlcNAcβ, respectively. Another sialoglycan that is highly elevated in diverse cancer types is Sialyl‐Tn, which is produced by ST6GalNAc1. This upregulation of Sialyl‐Tn is shown in breast cancer, colon cancer, gastric cancer, and adenocarcinoma [[51-54]], and it is considered as an important tumour marker [[55]]. Finally, the polysialic upregulation in tumours takes place due to two main enzymes, ST8Sia2 and ST8Sia4, and this abundance of polysialic acid is seen in breast, astrocytoma, and chronic myeloid leukaemia [[56-58]]. This aberrant expression of polysialic acid is known to regulate the PI3K/Akt pathway and to negatively regulate the expression of pro‐inflammatory cytokines [[58]]. In addition, the gangliosides (Sia‐decorated glycosphingolipids) are highly upregulated on melanoma and ovarian cancer [[59, 60]]. The mechanistic study showed that GD3 (a type of gangliosides) reduces the Natural Killer (NK) cell activity, which is considered to be mediated by Siglec‐7 [[61]].

The mechanism of hypersialylation through the altered activity of neuraminidases is highlighted in other recent reviews, but here we pinpoint some recent work on neuraminidases and cancer [[62]]. There are four known neuraminidases in mammals which can cleave Sias from the glycan chain. These neuraminidases are Neu1, Neu2, Neu3, and Neu4 [[62]]. Neu1 upregulation has recently been shown in gastric cancer to reduce cell apoptosis, reduction in cell invasion, migration and proliferation. This effect of Neu1 is mediated through the Wnt/β‐catenin pathway [[63]]. In a recent study on MDA‐MB‐231 (breast cancer) and PC‐3 (prostate cancer) cells, Neu1 inhibition led to enhanced migration, while Neu2 and Neu3 inhibition led to a decrease in cell migration [[64]]. The expression of Neu2 is shown to be upregulated in prostate cancer [[65]], but interestingly it also induces apoptosis in the leukaemia cell line [[66]]. The overexpression of Neu3 is seen in melanoma, prostate, ovarian and glioblastoma cancers [[67-71]]. In glioblastoma, Neu3 overexpression increases the radioresistance capacity through the PI3K/Akt pathway [[71]]. Neu3 is shown to be highly upregulated in colorectal cancer (CRC), which triggers epidermal growth factor receptor (EGFR) activation without causing any effect on mRNA and protein expression of EGFR [[72]]. The desialylation of GM3 and GD1a by Neu3 is also shown to enhance the phosphorylation of ERK and AKT [[73]]. Moreover, Neu3 blocks apoptosis in cancer cell lines. Conversely, a knockdown of Neu3 activate apoptosis via reduction in BCL‐xL [[74]]. Neu4 can cleave off the Sia from Sialyl‐Lewis^A and Sialyl‐Lewis^X. It is known to be downregulated in colon cancer and thus facilitates cancer metastasis [[75]].

The other mechanism for hypersialylation in cancer is the increased availability of CMP‐Sia substrate to the cells. There is not a lot of information available for this mechanism due to the lack of sensitive methods to quantify intracellular CMP‐Sia acid levels [[5]]. Both the levels of CMP‐Sia and the underlying glycans can regulate cancer progression. A recent study shows that the intracellular Sia levels determine the different stages of breast cancer [[76]].

Enhanced sialylation could also be attributed to changes in the hexosamine pathway. Sialoglycan presentation could be altered by changes in the levels of UDP‐GlcNAc through the alteration in the hexosamine biosynthetic pathway [[77]]. Moreover, the transcription of sialyltransferase can be increased by augmented levels of UDP‐GlcNAc [[78]]. Breast cancer cells increase the levels of CMP‐Sia and sialoglycans by switching to the hexosamine metabolism pathway [[79]]. Increased acceptor sites for sialylation are created by increased levels of UDP‐GlcNAc altering the N‐glycosylation patterns [[80]]. Another mechanism of increased sialylation is the upregulation of the Sia transporter, such as Slc35a1, which mobilises the activated CMP‐Sia from the nucleus to the Golgi. A knockdown of this gene has shown reduced availability of substrate for the sialyltransferase [[81]].

Major developments in the study of Siglecs as cancer immunosuppressant

Function of Siglecs as immune checkpoints

The overexpression of Programmed Death‐ligand 1 (PD‐L1) has been highly explored in cancer, and the blocking of interactions with the inhibitory receptor Programmed Death 1 (PD‐1) on T cells has indeed revolutionised cancer therapy [[82, 83]]. However, the majority of patients do not respond or only partially respond to checkpoint inhibitors, which points toward other immunosuppressive mechanisms, including other immunomodulatory receptors. Several recent studies have uncovered the role of inhibitory Siglecs in immune evasion in cancer [[84-86]]. The most abundant Siglec on innate immune cells in mice is Siglec‐E. In mice deficient for Siglec‐E (Siglec‐E^‐/‐), it was observed that the immunosurveillance against the tumour was increased and carcinogen‐induced tumours with MCA therefore appeared later than in littermate control mice, and experimental metastasis was strongly reduced in Siglec‐E‐/‐ mice. However, in a transplantable syngeneic subcutaneous tumour model, tumours grew larger. Further studies demonstrated that this enhanced tumour growth was due to the polarisation of macrophages into an M2‐like phenotype in this mouse model [[84]]. In contrast, M2‐like polarisation of human monocyte‐derived macrophages by sialyl‐T‐containing mucin‐1 (ST‐MUC1) could be inhibited by a Siglec‐9 blocking antibody [[87]]. Additional studies are needed to elucidate the exact role of inhibitory Siglecs on tumour‐associated macrophages. In a model of hypersialylation of cancer cells, it was shown that hypersialylated cancer cells behave like ‘super‐self’, blunting the anti‐tumour immune response by binding to Siglec‐7 on NK cells [[85]]. It was also shown that there is enhanced Siglec‐7 and Siglec‐9 binding to histological tumour tissue sections [[84, 86]]. This immunoevasion phenotype was shown to be NK cell‐dependent [[86]].

In order to characterise the ligands of CD33‐related Siglecs, tumour extract was passed through an affinity chromatography column and lectin galactoside‐binding soluble 3 binding protein

(LGALS3BP/Mac‐2 binding protein) was identified. LGALS3BP bound efficiently to Siglec‐5, Siglec‐9 and Siglec‐10 in a Sia‐dependent manner and is suggested to have immunomodulatory roles in cancer progression [[88]]. In a recent study, Siglec‐1 and CCL8 were identified as prognostic markers associated with poor outcome in endometrial and breast cancer [[89]].

Wang et al. employed a surface proteome‐wide T cell activity array to identify immunosuppressive molecules. Through their array, they have identified Siglec‐15, which has limited expression in normal tissues and is mainly found on tumour cells and tumour‐associated macrophages. Using Siglec‐15 knock‐out mice and a blocking antibody against Siglec‐15, it was identified that such ablation increases the anti‐tumour immune responses. Thus, Siglec‐15 is identified as a major immune suppressor that can be targeted for normalisation cancer therapy [[90]].

Siglecs expressed on neutrophils, macrophages, NK cells, DCs, and T cells interact with the sialoglycans of cancer cells which can blunt the anti‐cancer response. Thus, Siglecs have emerged as new immune checkpoints for which inhibitors are under development. In a recent study, it was shown that CD24 acts like a ‘don’t eat me’ signal and are highly expressed on ovarian and breast cancer cells. The receptor Siglec‐10 was expressed on tumour‐associated macrophages which interact with CD24. A knockdown of CD24 or Siglec‐10, or the blockade of Siglec‐10‐CD24 axis led to a reduction in the phagocytosis of the CD24‐expressing tumour cells. [[91]].

T cell‐expressing Siglecs and mucins in cancer progression

It has also been shown that CD33‐related Siglecs―in particular, Siglec‐9―can be expressed on the T cells, and T cell‐expressed Siglec‐9 can mediate immunosuppression in the cancer microenvironment. In B16 melanoma cells, hypersialylation is associated with enhanced T regulatory/T effector cell balance and reduction in NK cell activity [[81]]. Thus, glycomimetic blocking of the inhibitory effects of sialyglycans led to a decrease in cancer metastasis in a B16‐OVA mouse model [[92, 93]]. Transmembrane Mucin‐1 (MUC1) proteins can get decorated with sialylated O‐linked Tn glycans, converting it into MUC1‐ST. MUC1‐ST has the ability to increase the expression of PD‐L1 in macrophages by inducing an M2 TAM‐like phenotype in these macrophages. In addition, this sialylated MUC1‐ST can engage Siglec‐9 and lead to MEK‐ERK pathway activation in a calcium channel‐dependent manner. Surprisingly, this Siglec‐9 engagement does not induce SHP1/2 activation [[87]]. A recent study utilises secreted protease of C1 esterase inhibitor (StcE), which is a bacterial protease that cleaves the mucin domain. Using this protease, it was demonstrated that Siglec‐7 selectively binds to sialomucins, while it failed to bind with Siglec‐9 [[94]]. Another tumour‐associated mucin, MUC2‐ST, is also sialylated and engages Siglec‐3, thus inducing apoptosis in monocyte‐derived dendritic cells [[95]]. This data therefore point toward the role of mucins in cancer progression, and their sialylation can mediate the engagement of Siglecs, thereby inducing immunosuppression. Figure 2 shows the immune cells with Siglec expression engaged by the sialoglycans, highly expressed on the surface of cancer cells.

**Fig 2**
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Engagement of Siglecs by Sialoglycans lead to immunosuppression: The schematic shows the engagement of Siglecs expressed on the surface of immune cells and sialoglycans highly expressed on cancer cells. These Siglecs are inhibitory Siglecs and upon binding with sialoglycans it signals through SHP1/SHP2 for the inhibition of the cells. This inhibitory signaling ultimately leads to immunosuppression in the tumour microenvironment. This Siglec–Sia axis is shown to be involved between tumour cells and multiple immune cells such as macrophages, neutrophils, T cells, NK cells and myeloid‐derived suppressor cells (MDSCs).

Therapeutic targeting of the Siglec–Sia axis in cancer

Siglec‐blocking antibodies

Siglec‐7‐blocking antibody

The clone Z176 of anti‐Siglec‐7 has been used to decipher the effect of NK cell‐mediated cytotoxicity on cancer cell lines. A treatment of Siglec‐7 blocking antibody has led to the enhanced killing of cancer cells [[86]]. Another clone of Siglec‐7 antibody S7.7 was used to prevent the interaction of Siglec‐7 present on NK cells, with sialoglycan decorated on Jurkat cells [[85]].

Siglec‐9‐blocking antibody

The anti‐Siglec‐9 (clone E10‐286) Fab fragments were used to determine the effects of NK cell cytotoxicity on cancer cell lines. It was found to enhance the cytotoxicity, but the effects were lower than that obtained with Siglec‐7‐blocking [[86]]. It is noteworthy that the clone E10‐286 does not block Siglec‐9‐Sia interactions [[84]]. Another clone, 191240, directly blocks binding of sialic acid to Siglec‐9 [[96]]. It has been used previously where NK cell cytotoxicity was deciphered on sialoglycan‐decorated cancer cells. As outlined above, the decoration of cancer cells with sialylated glycans prevents the activation of NK cells. To decipher which one of the Siglec‐7 and Siglec‐9 receptors is mediating this process of inactivation of NK, the blocking antibodies of Siglec‐7 and Siglec‐9 were used. The blocking antibody of Siglec‐7 was able to abrogate this inactivation of NK cells, whereas the Siglec‐9‐blocking did not have an effect. This pinpoints the role of Siglec‐7 in the observed phenotype [[85]]. In another study, the relevance of MUC1‐ST in cancer progression was determined by MUC1‐ST‐induced monocytes to express factors related to inflammation. This effect was reversed by the treatment with anti‐Siglec‐9 antibody clone 191240 [[87]]. The effect of blockage of Siglec‐9 on activation of the T cell was identified using a staphylococcal enterotoxin B (SEB) test. When the full IgG Siglec‐9 clones (191240 and E10‐286) were used, T cell activation was inhibited. However, a monovalent carbohydrate‐recognising domain‐blocking using the Fab fragment of clone 191240 failed to inhibit T cell activation. [[31]]. Staphylococcal enterotoxin B (SEB) is a superantigen used in a test (the SEB test) for immune checkpoint inhibitors, including antibodies blocking PD‐1 in vitro [[97]].

Siglec‐10 blocking antibody

The anti‐Siglec‐10 (clone 5G6) was used as a blocking antibody to inhibit the CD24‐Siglec‐10 axis, and robustly improved the phagocytosis of tumour cells by macrophages. Moreover, the CD24‐blocking antibody (clone SN3) led to the same phenotype, thus confirming the interaction between tumour‐expressing CD24 and TAMs‐expressing Siglec‐10 [[91]].

Siglec‐15‐blocking antibody

The relevance of Siglec‐15 in cancer progression was discussed above. In the same study, a blocking antibody α‐S15, clone 5G12, was generated for Siglec‐15 using hybridoma technology. The blocking antibody treatment was shown to reduce the load of the established tumour [[90]]. Clinical trials are ongoing and the first activity as a single substance has been reported [[98]].

Sialic acid mimetics

Sialic Acid Mimetics (SAMs) have been recently produced and used in multiple studies for targeting the Siglec–Sia acid axis. It is important to note that several research groups have designed small‐molecule chemical inhibitors of Siglec–Sia interaction, which could also be used for cargo delivery to the Siglec‐expressing cells. In addition, certain chemically synthesised molecules inhibit the process of sialylation such as Neu5Ac3Fax. We will discuss both of these types of molecules in this section. It was initially observed that certain modifications of Sia such as 9‐O‐acetylation could modulate the binding with Siglecs [[99, 100]]. Therefore, the idea was conceived to chemically modify the Sia ligands of Siglecs at different positions and use them to target the Siglec–Sia axis. With this notion, the chemical modification at different carbons of Sia was carried out and used as an inhibitor for Siglecs [[101]]. These SAMs can be aligned on nanoparticles, polymers and living cells―thus harnessing its potential as an immune mediator [[101]]. There has been extensive research on SAMs to target CD22, which binds α2‐6‐linked Sia. A nanoparticle formulation with SAMs targeting CD22 has been used to deliver cytotoxic agent doxorubicin in B‐cell lymphoma cells. The nanoparticle was decorated with CD22 ligand and loaded with doxorubicin, and upon binding with the receptor CD22 the cargo to the lymphoma cells was delivered in vivo in a xenograft model [[102]].

The fluorinated analogs of Sia lead to the prevention of a de novo synthesis of natural substrates. This ultimately shuts down the sialyltransferase activity, thus promoting remodeling of the glycan chain [[103, 104]]. In an elegant study by Bull et al., SAM Ac₅3F_axNeu5Ac prevented the expression of sialoglycans in vivo and reduced tumour growth in many tumour models. It was observed that the intratumoural injection of mimetic enhanced the infiltration of NK cells and CD8 + T cells while lowering the T regulatory cell infiltration. This led to enhanced immunosurveillance in the tumour microenvironment [[92]]. Of course, these approaches are limited in that they may lead to severe toxicity in the body. Moreover, it could expose the underlying galactose group, which can bind with galectins and might trigger immunological responses. It has been shown in a recent study that the CRISPR/Cas9‐mediated knockout of the CMAS gene (an important gene in the sialylation pathway) led to complete loss of sialic acid on the cell surface in the MC38 cancer cell line. Interestingly, it led to enhanced tumour growth upon implantation in mice as compared to the control cell line. The mechanistic study showed less infiltration of CD8 + T cells in the tumour microenvironment [[105]].

Use of sialidases (neuraminidases)

The use of sialidases dates back several decades when it was observed that the treatment of murine leukaemia cell L1210 increased their immunogenicity. A treatment with Vibrio cholerae neuraminidase led to the loss of oncogenicity of leukaemia cells in mice [[106-109]]. These approaches did not show the exact mechanism for the increase in immunogenicity of cancer cells upon sialidase treatment. Moreover, they were not specific. Therefore, a recent study streamlined this sialidase treatment by chemically coupling the Vibrio cholera sialidase with anti‐HER2 trastuzumab. This led to specific targeting of the sialidase to the HER2‐expressing breast cancer cells. The treatment of the sialidase anti‐HER2 trastuzumab complex increased the immunosurveillance and killing of breast cancer cells by NK cells [[110]]. Gray and colleagues have recently identified a bacterial sialidase from Salmonella typhimurium, which was used for tumour therapy in in vivo mouse models and showed potent activity [[111]]. In this study, an anti‐HER2 antibody‐sialidase conjugate was designed which can efficiently cleave off the sialic acid, specifically from breast cancer cells. Furthermore, in a syngeneic breast cancer mouse model, there was reduced migration and activation of immune cells, but cleaving sialic acids resulted in enhanced activation of immune cells. The mechanistic study showed the phenotype was dependent on the Siglec‐E [[111]].

siRNA/shRNA and CRISPR/Cas9 tools to study Siglec–Sia axis

In recent years, many studies have used siRNA, shRNA, or the CRISPR/Cas9 method to knockdown the genes important for the synthesis, metabolism and cleavage of Sia. We are highlighting these studies in this section. Using the CRISPR/Cas9 genome editing tool, knockdown of the Cmas gene (important for the activation of sialic acid) led to a reduction of lung metastasis in vivo. Moreover, it was shown that the metabolism of sialic acid is highly upregulated in metastatic breast cancer [[112]]. ST6‐Gal‐I is the enzyme responsible for the synthesis of α‐2,6‐linked sialic acid. A knockdown of ST6‐Gal‐I in cervical cancer cells have shown reduced tumour growth, enhanced apoptosis and inhibition of invasion in the xenograft model [[113]]. The siRNA‐mediated knockdown of sialidase Neu3 triggers apoptosis in cancer cells due to the reduction of Bcl‐xL and induction of mda7 and GM3 synthase gene transcription [[74]]. A knockdown of golgi‐localised CMP‐sialic acid transporter slc35a1 in B16 melanoma cells using shRNA led to the reduction of sialylation in cancer cells. This reduction of sialylation improved the effector T cell function and reduction of regulatory T cell. This improved effector T cell function was dependent upon the infiltration of NK cells [[81]]. The various different approaches for targeting the Siglec–Sia axis are depicted in Fig. 3.

**Fig 3**
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Approaches to target Siglec–Sia axis: A) The schematic shows the different approaches utilised to target the Siglec–Sia axis against cancer progression. These approaches include blocking antibodies which block the interaction of Siglec with sialoglycans, Siglec‐drug conjugates, Sialidase conjugated with anti‐tumour antigen‐directed antibody, gene knock down technology, and sialyltransferase inhibitors.

Tools and techniques used in Siglec‐related cancer studies

Different mouse models of Siglecs used in cancer studies

Siglec‐E knockout mouse model (Siglec‐E^‐/‐)

Siglec‐E^‐/‐ mice were generated by neomycin insertion in the Siglec‐E gene in an Sv/129 mouse background. To obtain a C57BL/6 background, the Siglec‐E‐/‐ were backcrossed with this background for > 15 generations [[114]]. Siglec‐E^‐/‐ led to enhanced immunosurveillance in myelomonocytic cells and thus delayed the appearance of cancer in mice [[84]]. Another version of a Siglec‐E^‐/‐ mouse (EKO) is created by genomic deletion of exon 4 to exon 6 of the Siglec‐E gene [[33]].

HS9‐LysM‐Cre mouse model

In this model, the line was made to carry Lox‐Stop‐Lox‐hSiglec‐9 and LysM‐Cre transgenes. Upon expression of Cre recombinase in the cell lineage that expresses LysM (i.e., myeloid lineage), the stop codon (along with selection markers) between the two Lox sites is removed, allowing the expression of Siglec‐9 only in myelomonocytic cell lineage. These mice were later bred in Siglec‐E^‐/‐ (explained above) to generate Siglec‐9 expression in a mouse model that does not have Siglec‐E [[84]]. This model was used by Läubli et al. and found to reverse the phenotypes of Siglec‐E^‐/‐ mice.

SigE16 mouse model

These knockin mice express a chimeric receptor which combines the extracellular domain of Siglec‐E and the transmembrane and intracellular region of human Siglec‐16. Therefore, in this mouse, the E16 receptor binds with Siglec‐E ligands but generates activating signals inside the cells. The tumour size was reduced in the SigE16 mice size as compared to mice expressing the WT Siglec‐E [[31]].

CD4‐Siglec‐9 mouse model

This line is similar in design to the ‘HS9‐LysM‐Cre’ (described above), and the authors crossed Lox‐Stop‐Lox‐hSiglec‐9 with the CD4‐Cre transgenic mouse, to express human Siglec‐9 in all T cells. As T cell development in thymus includes a stage in which the cells are both CD4‐ and CD8‐positive (at this stage CD4‐Cre is expressed and excises LSL cassette), Siglec‐9 is expressed in all mature T cells. When these mice were used, a significantly larger tumour size was obtained as compared to the wild‐type (WT) mice [[31]].

Siglec‐15 knockout mouse model

The conditional Siglec‐15 knockout mice were bred with CMV‐Cre or LysM‐Cre to create global all tissue knockout, or myelomonocytic cell knockout, respectively. As discussed above, these mice showed improved immunosurveillance against tumours [[90]].

huNSG mouse model

Since there was no Siglec‐7/‐9 mouse model available, NSG (NOD/LtSz‐SCID IL‐2Rγ^null) mice were reconstituted with human NK cell component. The gain of function in the mutant mouse model was used to decipher the role of Siglec‐7 and ‐9 in cancer immunosurveillance [[86]].

These knockout mice have proved to be very useful in deciphering the role of Siglecs in cancer progression. It is easier to understand the function of mouse Siglecs that are evolutionarily conserved, such as Siglec‐15. In addition, the functionally equivalent Siglecs between humans and mice, such as Siglec‐E (functionally equivalent Siglec‐9) have also shed light on the importance of such receptors in the cancer phenotype. Moreover, the newly created humanised mouse models such as Siglec‐E16, have been used recently to understand the effect of Siglecs in a more comprehensive manner. Besides these advantages, there is a lot of work that needs to be done. It will be highly interesting to use several other knockouts/knockin mouse models that have already been created. For example, the Siglec‐F knockout was created long ago but has never been used for cancer studies. The newly created humanised CD33 and Siglec‐8 mouse models have also never been used for cancer studies. Considering these Siglecs and their respective cell‐type expression could be important for the phenotype, it will be interesting to use them. Besides Siglec‐E16, none of the Siglec mouse models used for cancer studies tried to target the Siglec‐based intracellular signaling. A list of all available mouse models to study Siglec biology is provided in Table 1. It is noteworthy that most of these Siglecs are expressed on immune cells which may play a crucial role in cancer progression but have never been used for such studies. In the future, these models should be used to pinpoint the role of one or multiple Siglecs.

Table 1. List of available mouse models to study Siglec biology.

Siglec mouse models	Phenotype	Tumour study performed	Tumour phenotype	References
Siglec‐1/CD169 Knockout mouse	No developmental abnormalities, slight changes in T cell and B cell populations	Yes	Activation of tumour antigen‐associated CD8 (+) T cells.	[[140, 141]]
Siglec‐2/CD22 Knockout mouse	Reduction of subsets of marginal zone B cells	No	NA	[[142]]
Transgenic expression of human CD22 (hCD22) in B cells	Phenotype identical to mCD22‐expressing mice	No	NA	[[143]]
CD22 knockout on a pure C57/BL6 background	Activated B cells, higher calcium mobilisation, no autoimmunity	No	NA	[[144-146]]
CD22 knockout on 129 X C57/BL6 background	Autoimmunity	No	NA	[[147, 148]]
CD22‐R130E (Sia‐binding is lost)	Reduced calcium signaling	No	NA	[[28]]
CD22‐Y2,5,6F (Lost signaling)	Increased calcium signaling	No	NA	[[28]]
Siglec‐G R120E x CD22 R130E mice	No autoimmunity	No	NA	[[149]]
CD22 × Siglec‐G Double‐deficient mice	Autoimmunity	No	NA	[[150]]
CD33 knockout mice	No major abnormalities	No	NA	[[151]]
Human CD33‐expressing mice in microglial cells	Reduced cargo uptake	No	NA	[[152]]
Human CD33 expressed in mouse mast cells	Suppression of IgE‐mediated mast cell activation	No	NA	[[153]]
Siglec‐4a/myelin‐associated glycoprotein (MAG)	Delayed myelination onset	No	NA	[[154]]
Siglec‐E‐ KO1 (Neomycin insertion)	Negatively regulate recruitment of neutrophils to lungs	Yes	Enhanced immunosurveillance, polarisation of TAMs to M2 macrophages	[[84, 114]]
Siglec‐E‐KO2 (Cross of R126D X (Nes‐cre)1Wme/J (Bal1 cre)	Deciphered the mechanism for reduced recruitment of neutrophils to lungs	No	NA	[[155]]
EKO mice (Siglec‐E^‐/‐) (LoxP‐mediated deletion)	Increased platelet activation upon group B Streptococcus (GBS) infection	No	NA	[[33]]
Siglec‐E R126D mutant mouse (Ligand binding lost)	Little expression of protein at protein level	No	NA	[[114]]
Sig‐E16	Enhanced killing of tumour cells by the expression of activating receptor in immune cells	Yes	Enhanced killing of tumour cells by the expression of activating receptor in immune cells	[[31]]
Siglec‐F knockout Cross of R114A X ZP3‐Cre (LoxP‐mediated deletion)	Enhanced infiltration of eosinophils in lungs	No	NA	[[156]]
Siglec‐F R114A mouse model	Very little mRNA and protein expression	No	NA	[[156]]
Siglec‐F knockout Cross of R114D X Tg (Nes‐cre)1Wme/J (Bal1 cre) Cre‐mediated deletion	Enhanced allergen‐mediated eosinophilia	No	NA	[[157]]
Siglec‐G knockout mouse	Autoimmunity	Yes	Aged mice develop lymphoma	[[158, 159]]
Siglec‐H knockout mouse	Mild systemic autoimmunity	No	NA	[[160]]
Siglec‐8 in mast cell (Conditional knockin)	No difference in mast cell number	No	NA	[[161]]
Siglec‐11 mouse model (Humanised)	Impaired phagocytosis and reduced microglial toxicity	No	NA	[[162]]
Siglec‐15 knockout mouse	Used in cancer study, enhanced immunosurveillance	Yes	Increased immunosurveillance against tumour by activated TAMs	[[90]]
HS9‐LysM‐Cre in Siglec‐E^‐/‐ background	Immunosuppression	Yes	Reverse the phenotype observed in Siglec‐E^‐/‐	[[84]]
CD4‐Siglec‐9 mouse model	Immunosuppression	Yes	Enhanced tumour growth	[[31]]
Soluble‐Siglec‐9 expressing mouse (sSiglec‐9)	Protection from Group B Streptococcus (GBS) infection	No	NA	[[162]]

Siglec‐Fc fusion proteins

In most of the cases, Siglec‐Fc has been created by cloning and expressing the extracellular domains of Siglec genes with the Fc region of human IgG. Many Siglec‐Fc have been generated and used to find the ligands of Siglecs on different cells and tissues. Siglec‐8‐Fc and Siglec‐9‐Fc have been used to find their ligands in the human airways and airway cell line calu‐3 [[115, 116]]. Siglec‐Fcs have not only been instrumental in finding ligands on human and mouse cells but also bacterial pathogens. A binding assay of various Siglec‐Fcs with Campylobacter jejuni lipopolysaccharides showed binding of Siglec‐7‐Fc with sialylated ligands. Since Siglec‐7 is expressed by leucocytes, especially NK cells, this finding could have important implications for the pathogenesis of C. jejuni infections [[117]]. In a recent study, it was found that neutrophils remain quiescent in the bloodstream due to the interaction of Sia ligands expressed on red blood cells (RBCs). This study on the interaction of Siglec‐9 and Siglec‐9 ligands sheds light on why neutrophils only get activated when they pass through the bloodstream [[35]]. Siglec‐E‐Fc has been used in the characterisation of Siglec‐E antibodies [[118]]. The Siglec‐Fcs that have been used for cancer studies are as follows:

Siglec‐9‐Fc chimera

The Siglec‐9‐Fc chimera (extracellular Ig‐like domains of Siglec‐9 fused with the Fc portion of human IgG) was used to determine the Siglec ligands in tumour histological tissues. In addition, an R120K‐Siglec‐9‐Fc was used as a control chimera, which did not show appreciable binding on tumour tissue sections. Another chimera K131Q‐Siglec‐9‐Fc also showed reduced binding to sialoglycan ligands on the glycan array [[84]]. In a further study, Siglec‐9‐Fc was used to detect Siglec‐9 ligands on cancer cells by flow cytometry and tumour sections by immunohistochemistry [[86]].

Siglec‐7‐Fc chimera

Siglec‐7‐Fc was produced and used for the cancer cell lines and tumour sections. CD33‐Fc was used as a negative control for these assays [[86]]. The glycopolymers were decorated on the surface of Jurkat cells and probed with Siglec‐7‐Fc [[85]].

Siglec‐E‐Fc

3‐methyl‐cholanthrene (MCA)‐injected mice develop sarcoma, which was stained for sialoglycan ligands using Siglec‐E‐Fc [[84]].

In this section, we provided the list of chimeric Siglec‐Fcs that have been used to find the Siglec ligands in the tumour tissue. This cloning, purification and application of such Fc proteins is well established and has proved to be very handy for such studies. An ideal control which is usually kept for such studies is arginine mutant Siglec‐Fc protein, which should not bind to Siglec ligands in a sialic acid‐dependent manner. Moreover, for some of these studies, healthy tissue is also used as a control, which has been described to show less or minimal binding. As shown, the list is not extensive and only three Siglec‐Fc proteins have been used for cancer studies. Since there is Siglec‐Fc proteins cloned and prepared for almost all Siglec receptors, it needs to be scaled up to identify other Siglec ligands in tumour tissues. A disadvantage of such a method is the fact that Siglec‐Fc only provides the information that the ligand is present, but no other information is provided for the type of ligands.

Sialoglycan microarray

Unlike RNA and proteins, glycan biosynthesis is not template dependent because there is no genetic code that determines their synthesis and sequence. Sia is a complex monosaccharide that has variable underlying sugar chains that determine their overall binding with proteins and underlying function. Therefore, they are more complex to study than other biological molecules. A large pool of natural and chemically synthesised sialoglycans libraries have been utilised to be immobilised on the surface of the glass slide surface [[119]]. Their binding preference with proteins and other molecules is detected, and thus overall biology is deciphered. Glycan microarray has three important aspects: robot type printer, glycan chemistry for conjugation and optics for detection [[120]]. Recently, the sialoglycan microarray has been used to study the binding pattern of anti‐Neu5Gc IgG in human sera. Neu5Gc is a type of Sia that is absent in humans due to a mutation in the Cytidine monophospho‐N‐acetylneuraminic acid hydroxylase (CMAH) gene, but our evolutionary cousins (great apes) do carry this molecule [[120]]. The sialoglycan array has been extensively used in determining the binding pattern of Siglecs, identifying the biomarkers for cancer and other applications [[84, 121, 122]]. Previously, it has been shown that Siglec‐15 expressed by TAMs bind with a tumour expressing sialyl‐Tn (sTn). However, in a recent study using glycan microarray, Siglec‐15 binds to many sialylated glycans at high‐affinity, compared to sTn and other related structures[[123]]. However, in this study a number of other sialylated probes were identified as high‐affinity binders of Siglec‐15, such as α2,3‐sialyl lactose, α2,6‐sialyl lactose and α2,6‐sialyl glycans with lacto‐N‐hexaose and lacto‐N‐tetraose containing backbone [[121]].

Neu5Gc gets incorporated in human tissues, especially tumours, due to an intake in the form of red meat. This incorporation of cancer cells leads to an inflammatory cascade when attacked by Neu5Gc antibodies generated in vivo [[124]]. A recent study on the characterisation of Neu5Gc antibodies and the risk of colorectal cancer utilises the powerful tool of sialoglycan microarray. In this study, it was shown that there was no correlation between CRC risk and Neu5Gc antibody or individual Neu5Gc‐bearing epitope. However, when all the Neu5Gc epitopes were combined, there was a positive correlation between the antibodies and CRC risk [[125]]. Similarly, in many such studies where Neu5Gc antibodies were characterised as a biomarker for cancer and other immune disorders, sailoglycan microarray was used [[39, 126, 127]]. Recently the cell‐based glycan array has been established where Chinese hamster ovary (CHO) cells were used to create an array of glycoforms that serve as a platform. The glycan‐binding proteins, such as Siglecs, were used to probe the interaction with this array using the flow cytometry method. The high‐affinity ligands of Siglec‐15 were discovered using this cell‐based glycan [[128]].

Conclusion and future perspectives

Based on recent findings, we have begun to understand the relevance of Siglecs in immunosuppression and inflammation. New animal models and Siglec antibodies (as discussed above) have enabled pre‐clinical studies which will pave the way for clinical intervention. A Siglec‐15 targeting antibody has already been successfully tested in a first‐in‐human trial (NCT03665285). Recent studies have used SAMs and sialidase which resulted in glycans without the terminal Sia [[62, 101]]. These approaches are good, but the lack of Sia at the cap of the sugar chain also exposes underlying galactose residues, which can bind to galectins. These studies should therefore provoke further analyses, taking into account the multiple roles of galectins in cancer progression and anti‐cancer immunity. Aberrant sialylation in cancer cells might also affect many different signaling events besides Siglec engagement. It might prevent immunogenicity of the cancer cells due to a dense array of glycans making the immune cells inaccessible to the cancer cells. In addition, it might affect another pathway that involves selectins that are pivotal for cancer metastasis [[129]].

The Siglec–Sia axis can be targeted by blocking antibodies that will prevent this interaction, and some of these antibodies have shown strong effects [[130]]. There is a Siglec‐E blocking antibody characterised recently, and it can also be used to test the role of Siglec‐E in tumour growth and metastasis [[118]]. However, we have to keep in mind that Siglecs are redundant in their functions and many Siglecs are expressed by a single immune cell type [[27]]. Even if we target a single cell type, which itself will be a limiting factor, the approach might need to target multiple Siglecs. Another way would be to target multiple cell types and multiple Siglecs, but this approach would also have the flaw that it might lead to systemic inflammation and autoimmune disorders, although no strong autoimmune phenotype was observed for Siglec‐E^‐/‐ mice [[131]]. Thus, a better understanding is needed to efficiently target this emerging immune checkpoint. A lack of information on intracellular signaling of Siglecs is another limiting factor in the development of therapeutic targeting. In addition, more clinical, translational data are warranted before going forward with any of the Siglec–Sia axis targeting agents.

The synthesis of SAMs by click chemistry approach has been improved since the year 2002 [[101]]. The problem with this approach is again the high toxicity with systemic injection. Some of the recent studies have also utilised the Siglec receptor for redirecting T cells to tumours by a chimeric antigen receptor (CAR) approach [[132-134]]. More emphasis should be given to this newly emerging field. The strategy involving sialidases with an anti‐HER2 antibody has shown a lot of promise [[110]]. Moreover, this has been shown to work in breast cancer, which needs to be replicated in other cancer types. Conjugation of sialidase with other cell surface receptors which are highly enriched on cancer cells would possibly be ideal, such as the folate receptor or PD‐L1 [[111, 135]].

A variety of mouse models have been developed to understand the role of Siglecs in cancer progression (Table 1). But there are multiple Siglecs for which a true mouse homologue does not exist [[13, 15]]. Recreating the human scenario in a mouse is difficult. The recent approaches to make humanised mouse models have partially tackled this problem [[31]].

In the last few years, several studies have shown the expression of inhibitory Siglecs on tumour‐infiltrating T cells. Based on this, one can assume that even the levels of Siglecs might increase in innate immune cells as well. So a cumulative effect of hypersialylation and increased Siglec expression could induce a ‘super‐self’ of cancer cells, which can efficiently blunt the anti‐tumoural immune response.

Nanoparticles have been used recently in cases of sepsis. Siglec‐E ligands were decorating nanoparticles, which led to engagement of Siglec‐E and ultimately reduced a cytokine storm and sepsis phenotype in murine models [[136]].

For a number of Siglec receptors, it is known that they undergo endocytosis upon ligand binding or cross‐linking with antibodies. Some cancer types are known to express Siglec receptors as well. For these, the drug can be loaded onto antibodies and incubated with cancer cells, which would lead to the cells being killed or their endocytosis. The proof of concept has been shown for Siglec‐XII expressing in epithelial cells [[23]], but there are reports for Siglec expression on other cancer types [[137]]. A thorough study is warranted to identify the tumour‐expressing Siglecs.

One of the new aspects of Siglec biology is the intracellular pool of Siglecs [[16]]. In this case, Siglecs are expressed not only on the cell surface but also inside the cells. In the case of neutrophils and monocyte‐derived macrophages, they are expressed in the peroxisomes [[16]]. However, the intracellular pool of Siglecs in tumour cells and their relevance is largely unknown. An approach is to develop anti‐oxidants for anti‐cancer therapy [[137, 138]]. The role of peroxisomes is well known in anti‐oxidation and reduction of reactive oxygen species [[139]]. It would be interesting to study the intracellular pool of Siglecs, their function in peroxisomes and their overall ability to suppress inflammation. Moreover, the splicing machinery that leads to the formation of CD33m in the case of CD33 and possibly other Siglecs would also be interesting to study regarding the targeting of the splicing machinery in cancer progression.

Based on current research evidence, Siglecs can be considered as new immune checkpoints. However, the exact role of Siglec–Sia interactions requires further studies, in particular during early clinical development of targeting agents. However, early clinical applications are promising and further results will support the establishment of Siglec–Sia targeting agents as new cancer immunotherapy.

Author contributions

All authors have read and agreed with the current version of the manuscript. HL and SSS were involved in the conceptualisation of the manuscript; KK prepared all three figures and graphical abstract; HL, KK, CGV, RM, MM, and SSS wrote the original draft preparation, and reviewed and edited the manuscript; SSS supervised the manuscript.

Acknowledgements

We are very thankful to Andrea Verhagen for critically reading the manuscript and providing feedback. I would like to sincerely thank Mr. Shadi Yacoub Al Shidrawi, Shadma Iram and Dr. Anne Phan for proofreading the manuscript. We are grateful to the department of research and community services, the American University of Ras Al Khaimah (AURAK), UAE, for providing assistance with this work. This work was supported by the seed grant of AURAK AAS/003/19.

Conflict of Interest

H.L. received travel grants and consultant fees from Bristol Myers Squibb (BMS) and Merck, Sharp and Dohme (MSD). H.L. received research support from BMS and Palleon Pharmaceuticals. All other authors declare no conflict of interest with the content of the manuscript.

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Early View

Online Version of Record before inclusion in an issue

Tools to study and target the Siglec–sialic acid axis in cancer

Abstract

Abbreviations

Introduction

Mechanism of hypersialylation in cancer and its role in cancer progression

Major developments in the study of Siglecs as cancer immunosuppressant

Function of Siglecs as immune checkpoints

T cell‐expressing Siglecs and mucins in cancer progression

Therapeutic targeting of the Siglec–Sia axis in cancer

Siglec‐blocking antibodies

Siglec‐7‐blocking antibody

Siglec‐9‐blocking antibody

Siglec‐10 blocking antibody

Siglec‐15‐blocking antibody

Sialic acid mimetics

Use of sialidases (neuraminidases)

siRNA/shRNA and CRISPR/Cas9 tools to study Siglec–Sia axis

Tools and techniques used in Siglec‐related cancer studies

Different mouse models of Siglecs used in cancer studies

Siglec‐E knockout mouse model (Siglec‐E‐/‐)

HS9‐LysM‐Cre mouse model

SigE16 mouse model

CD4‐Siglec‐9 mouse model

Siglec‐15 knockout mouse model

huNSG mouse model

Siglec‐Fc fusion proteins

Siglec‐9‐Fc chimera

Siglec‐7‐Fc chimera

Siglec‐E‐Fc

Sialoglycan microarray

Conclusion and future perspectives

Author contributions

Acknowledgements

Conflict of Interest

References

Figures

References

Related

Information

Siglec‐E knockout mouse model (Siglec‐E^‐/‐)