Recent advances in the field of cancer immunotherapy, involving specific targeting modalities like monoclonal antibodies or chimeric antigen receptor (CAR) T cell therapies, depend on the identification of appropriate surface antigens. The search for appropriate targets is especially important for certain malignancies such as triple-negative breast cancer (TNBC), which lack expression of the human epidermal growth factor receptor 2 (HER2/neu), estrogen or progesterone receptors, thus rendering them insensitive to available targeted therapies.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a highly glycosylated transmembrane protein, and a member of the chondroitin sulfate group of glycosaminoglycans (GAGs). CSPG4, also referred to as melanoma-associated chondroitin sulfate proteoglycan (MCSP), high-molecular-weight melanoma-associated antigen (HMW-MAA), or neuron-glial antigen 2 (NG2), was first associated with malignant melanoma and subsequently implicated in the pathology of other solid tumors of different origins, as well as of hematological cancers (1). It has been investigated as a potential immunotherapy target due to its restricted/low distribution in normal tissues and overexpression in certain tumors at different disease stages, and based on evidence for multiple roles in supporting tumor growth and dissemination. Together, long-emerging studies point to CSPG4 as a promising target for cancer therapies, including immunotherapies with monoclonal antibodies. In this review, we summarize reported functions of CSPG4 in cancer and we examine the development of ongoing immunotherapy strategies, most notably monoclonal antibodies that target CSPG4.

Chondroitin sulfate proteoglycan 4 is heterogeneously expressed on normal tissues such as mesenchymal stromal cells—adult progenitor cells, which have been suggested to lose its expression during terminal differentiation (2). Early immunofluorescence/immunohistochemisry data on the distribution of CSPG4 in normal tissues suggest it is expressed in nevi, epidermis and hair follicles but not detected in brain, thyroid, thymus, lung, liver, ureter, testis, spleen, ovary, or peripheral nerves (3). In another early study, immunohistochemical analysis of fetal and adult human tissues suggest CSPG4 distribution in the adrenal cortex, liver, choroid and small intestine in the fetus and in adult peripheral nerves, liver, salivary glands, bladder, lung bronchial glands and sebaceous glands (4). In more recent studies, expression of CSPG4 and its rat ortholog has been reported at low or moderate levels on neuronal glial cells, arteriolar pericytes, smooth muscle cells, macrophages, melanocytes, articular chondrocytes, and others (5–11). At the RNA level, CSPG4 expression has been recently reported in skin, trachea, veins, lung, heart, muscle, diaphragm, adipose tissue, uterus, prostate, thymus, spleen, bone marrow, and gastrointestinal tissue, but importantly at 6.6 times lower levels than in tumors (12).

Its physiological functions are not completely understood and multiple studies report specific roles in different tissues throughout development. In placenta formation, the expression of CSPG4 on extravillous trophoblasts has been implicated in their differentiation and migration (13). CSPG4 is also proclaimed to be involved in angiogenesis and vascularization. Using murine in vivo models, NG2 was shown to induce de novo vascularization of otherwise the avascular corneal tissue, suggesting an important role in angiogenesis (14). Further reports suggest the involvement of CSPG4 in glial and oligodendrocyte formation and neuronal network regulation, epithelial keratinocyte replenishment, and epidermal stem cell positioning and homeostasis (15, 16).

Although a full understanding of the physiological roles of CSPG4 is still required, all reports suggest it is ubiquitously involved in multiple tissue development and homeostasis processes, and its roles may be differentially modulated based on the nature of the local tissue microenvironment (17). The regulation of CSPG4 expression is reported to be strongly affected by inflammatory cytokines such as TNF-α, interleukin (IL)-1α, IFN-γ, and TGF-β and hypoxia-induced mechanisms involving hypoxia-inducible factors. Furthermore, CSPG4 expression was described to depend on epigenetic pathways, certain transcription factors and microRNAs (see Ampofo et al. for review).

Its functional versatility could be explained by its protein scaffold structural characteristics (Figure 1). CSPG4 is a type I single pass transmembrane protein which exists as a core glycoprotein and chondroitin sulfate-decorated proteoglycan (18). Studies with the rat ortholog state CSPG4 consists of a large extracellular portion, a transmembrane domain and a short intracellular portion (19). The extracellular portion comprises three distinct domains. Located furthest from the membrane, D1 is composed of two laminin G-type subdomains and is abundant in disulfide bonds, important for the stability of tertiary structure. This domain is potentially involved in the interactions with the extracellular matrix (20). The middle domain, D2, comprises 15 CSPG4 specific repeats containing several potential glycosylation and chondroitin sulfate binding sites. The CS decoration may confer different attributes, including interaction with integrins and metalloprotease activation (21, 22). It is presently unclear whether CSPG4 is characterized with different glycosylation/glycanation patterns in normal or cancerous tissues. The D2 domain has also been proposed to directly bind collagens (23, 24). Although CSPG4 has no reported enzymatic functions, murine ortholog studies suggest it may bind growth factors and present them to receptor tyrosine kinases (RTKs), thus acting as a RTK coreceptor (25, 26).

Domain D3 is the one proximal to the cellular membrane, and contains putative protease cleavage sites as well as carbohydrate decoration, suggesting potential interactions with lectins and integrins (27, 28). Proteolytic cleavage may also allow ectodomain shedding. In support, levels of soluble CSPG4 have been reported in the sera of healthy individuals and patients with melanoma (29). The presence of soluble CSPG4 within circulation has been proposed as a potential diagnostic biomarker to aid melanoma detection and classification at the vertical growth phase (29). Moreover, CSPG4 may undergo endocytic recycling mediated by the endocytic receptor Stonin1 (30). Thus, endocytosis and ectodomain shedding of CSPG4 may point to different mechanisms involved in the turnover of membrane-bound protein.

The intracellular portion of CSPG4 is characterized by the presence of a threonine- and a proline-rich motif and a PDZ [postsynaptic density protein 95 (PSD-95)—Drosophila disc large tumor suppressor (Dlg1)—Zona occludens 1 (ZO-1)] domain (31, 32). The threonine motifs serve as kinase phosphoacceptor sites for protein kinase C α (PKCα) and extracellular signal-regulated kinase 1/2 (ERK1/2) (33, 34). The proline-rich domain and the PDZ domain most likely function as protein scaffolds for other intracellular proteins (31). The structural characteristics of CSPG4 may confer possible functions as a signaling mediator molecule, connecting the extracellular matrix (ECM) with two main intracellular signaling cascades—the integrin-focal adhesion kinase (FAK) axis through integrin interactions and the mitogen-activated protein kinase (MAPK) pathway through activation of RTKs and ERK1/2 (33, 35, 36). These may bestow functional attributes that could encompass promotion of cellular survival, proliferation, and motility. Importantly, studies with NG2 knockout mice suggest that deletion of this antigen is not lethal (37).

Therefore, research studies into CSPG4 point to stem cell origin, multiple functions in tissue development, together with regulation of expression by inflammation, hypoxia, decreased methylation mechanisms, as well as specific protein scaffolding structure and reported roles in cell signaling. These features may also translate to contributions in cancer pathology in several tumor types.

Early reports associated overexpression of CSPG4 with malignant melanomas, and more recently its enhanced expression has been identified in other cancer types. Despite these developments, the exact involvement of CSPG4 in the etiology of cancer is widely unknown.

It remains unclear whether CSPG4 has a role in tumor initiation or its expression only accumulates in tumors as a secondary tumor-associated event. Overexpression of CSPG4 has not been reported to be a result of genetic aberrations such as gene amplifications or chromosome translocation, suggesting CSPG4 may not be a primary driver in the onset of cancer. Even though its expression is not connected to the onset of epithelial tumors or hematological cancers, it has been linked to the putative mesenchymal stem cell origin of sarcomas and the epithelial–mesenchymal transition in melanoma, thought to be important for malignant transformation (36, 38–40). Several reports point toward potential roles in cancer growth and dissemination. Expression of the CSPG4 rodent homolog has been shown to localize in the invasive front of the filopodia of oligodendrocytes, suggesting involvement in mediating tumor cell motility and cancer dissemination (38, 41). In support, immunohistochemistry data evaluating CSPG4 expression in human melanoma claim higher levels in metastatic lesions than in primary tumors, and CSPG4 mRNA expression was reported to be predictive of metastasis formation in soft tissue sarcoma patients (6, 42).

Furthermore, CSPG4 is believed to contribute to cancer growth and progression through promotion of angiogenesis. Using cell line and patient derived glioblastoma xenografts in nude mice, Wang et al. reported that NG2 RNA interference in vivo decreased tumor volume and vasculature (43). In addition, a retrospective study focused on germline polymorphisms related to the function of pericytes in colorectal cancer, identified a CSPG4 polymorphism to be predictive of lower progression-free survival in patients treated with the monoclonal antibody (mAb) bevacizumab—specific for the vascular endothelial growth factor (44). More evidence is required to clarify the exact mechanism through which CSPG4 promotes angiogenesis in cancer.

Disparate reports therefore point to potential contributions of CSPG4 in cancer growth, vascularization, dissemination, and metastasis (Figure 1). These may provide opportunities for therapeutic interventions targeting CSPG4 and multiple cancer-associated pathways this molecule may be involved in.

Several studies have investigated the interactions between the chondroitin sulfate chains of proteoglycans or their degradation products and components of the immune system.

It has been reported that CS could stimulate monocytes to secrete IL-1β and to induce B cell proliferation in vitro (72). The effect of CS on B cell proliferation was corroborated by Aoyama et al., who demonstrated that CS could enhance murine B cell proliferation in vitro through PKC translocation and activation of protein kinase B (PKB, Akt) kinase (73). A role of CS in the maturation of dendritic cells was suggested with human monocyte-derived dendritic cells cultured in vitro in the presence of CS, hyaluronic acid, components of the ECM and human granulocyte-macrophage colony-stimulating factor (GM-CSF). CS-stimulated cells could differentiate faster than when cultured with GM-CSF and IL-4 (74). Another study utilizing splenocytes from ovalbumin (OVA)-immunized mice cultured ex vivo, showed that CS addition to culture stimulated secretion of Th1-type cytokines including IFN-γ, IL-2, and IL-12 and suppressed the secretion of Th2-type cytokines (IL-5 and IL-10) (75). Moreover, injection of BALB/c mice with CS and other glycosaminoglycans (GAGs) was shown to induce autoimmune conditions like rheumatoid arthritis through recruitment of CD4⁺ T cells (76). In addition, treatment of murine NK cells with chondroitinase or a proteoglycan biosynthesis inhibitor resulted in substantial decrease in IFN-γ secretion through interaction with murine IL-12. On the other hand, CSPG low-molecular weight disaccharide fragments could control the inflammatory response in a mouse model of autoimmune encephalitis, in a rat model of inflammation-mediated neuropathology of the eye, and in a delayed-type hypersensitivity model in Balb/c mice through reduced migration and activation of inflammatory T cells (77).

With regards to the role of chondroitin sulfate proteoglycans (CSPGs), an early study reported these located inside the granules of human NK cells and their exocytosis during NK cell-mediated cytotoxicity of tumor cells (78). Furthermore, a study of primary cultured human macrophages, showed secreted CSPGs as metabolic products of macrophages and increased secreted CSPG4 following lipopolysaccharide (79) stimulation (80).

A more recent immune monitoring study identified that both healthy individuals and melanoma patients have circulating CSPG4-reactive CD4⁺ T cells. The study did not report a significant correlation between the T cell responses against a HLA-DR presented CSPG4 peptide, quantified through IFN-γ production, and the tumor burden of patients. The authors showed that a smaller proportion of the melanoma patients (11 out of 42) compared to healthy volunteers (11 out of 13) exhibited T cell reactivity to CSPG4 (81). Other in vivo studies demonstrated that LPS induced the expression of the murine CSPG4 ortholog by rat microglia cells. Further in vitro experiments showed that NG2 RNA silencing of LPS-treated microglia blocked the mRNA expression of nitric oxide synthase (Yajima et al.), and of proinflammatory cytokines including IL-1β and tumor necrosis factor α (TNF-α), but not of chemokines like monocyte chemoattractant protein 1 and stromal cell-derived factor 1 α (SDF-1α). Importantly, this study demonstrated that NG2 is not only expressed upon stimulation of the microglia, but it likely has a role in regulating expression of pro-inflammatory cytokines (82).

In summary, chondroitin sulfate proteoglycans (CSPGs), CS carbohydrate chains, as well as small molecular weight CS degradation products and CSPG4, each appear to influence the activation, maturation, proliferation and migration of different immune cell subsets. The definitive roles for CSPG4 in the immunology of cancer are however still widely unexplored. More research is required to clarify whether the interactions between CSPG4, its amino acid or carbohydrate moieties and different components of the immune system could be exploited to enhance patient response to CSPG4 targeted immunotherapy or to be counteracted to avoid potential negative immunomodulatory functions.

Since CSPG4 is found to be overexpressed in a number of malignancies and based on its low and restricted distribution in normal tissues, alongside emerging evidence for crucial roles in cancer growth and dissemination, much research has focused on the development of different therapeutic approaches, including monoclonal antibodies. Some of these antibody-based strategies focused on CSPG4 are discussed below.

BRAF inhibitors, now approved for the treatment of patients with melanomas bearing mutant forms of BRAF, are often only effective for a short time before cancer recurs, due to intrinsic and acquired pathway resistance (115). Therefore, alternative treatments and treatment combinations that may overcome resistance mechanisms are desirable (116). The anti-CSPG4 mAb 225.28 combined with a BRAF inhibitor exhibited synergistic antitumor effects and enhanced efficacy against BRAF^V600E mutant melanoma cells in vitro compared to either agent alone (51). Furthermore, the mAb was shown to delay the development of resistance by melanoma cells. More recently, Pucciarelli et al. showed that combining polyclonal anti-CSPG4 antibodies, induced by mimotope vaccination, with the BRAF inhibitor vemurafenib synergistically reduced the proliferation and migration of melanoma cells in vitro (117). These preliminary findings suggest that combining anti-CSPG4 antibodies with pathway inhibitors may enhance the restricted success of BRAF inhibitors in melanoma.

Cancer theranostic agents, combining both diagnostic and therapeutic treatment in one targeted molecule are an emerging modality. As with other antibody therapeutic applications, CSPG4 has been recognized as a promising candidate for theranostic applications, based on high expression by tumor cells and low expression by healthy cells. In an in vitro photo-immunotheranostic study, single-chain variable fragment (scFv) antibodies recognizing TNBC targets, including CSPG4, as well as epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM), were conjugated to a potent photosensitizing agent and were used to target TNBC cell lines and tumor biopsy samples (118). The conjugated scFvs demonstrated high quality imaging capacity, and triggered apoptosis of cancer cells via induction of reactive oxygen species. Moreover, combinatorial administration of all three conjugated scFv antibodies together, increased cytotoxic activity against breast cancer cells in vitro by up to 40% compared with treatment by each individual agent alone. Further findings are awaited to confirm and provide further efficacy insights on these encouraging outcomes in future in vivo studies.

Despite advances in immunotherapy such as the emergence of checkpoint inhibitors for melanoma, mAb, or CAR T cell strategies that specifically target melanoma cells are still lacking. Localized overexpression in several aggressive tumor types and in tumor vasculature, combined with low and restricted distribution in normal tissues, as well as evidence for important functions to support cancer growth, angiogenesis and dissemination, represent important attributes that identify CSPG4 as a promising target for therapeutic approaches, including monoclonal antibodies (Table 1). Importantly, in order to develop more successful therapeutics, a better understanding of the functions of CSPG4 in cancer and its interaction with the immune system and the tumor immune stroma are urgently needed.

While many treatment strategies centered on CSPG4 appear to have had success both in vitro and in vivo in rodent models, the next steps require in-depth studies with humanized or human antibodies, in disease-relevant and in clinically congruent models of cancer, including animal models engrafted with components of human immunity. These will permit mechanistic and efficacy evaluations in systems better able to recapitulate the patient setting.

An exciting prospect for targeting CSPG4 is the observed synergy between anti-CSPG4 monoclonal antibodies and BRAF inhibitors. In melanoma, a proportion of patients’ tumors have constitutively activated BRAF. Small molecule inhibitors recognizing mutant forms of BRAF have proved very effective. However, clinical responses are often short-lived due to the emergence of resistance. Combinatorial studies of monoclonal anti-CSPG4 antibodies with BRAF inhibitors have demonstrated enhanced effects and delayed the occurrence of resistance. Further understanding of the mechanisms that underpin the efficacy of these and other combinatory strategies may offer important clues that stand to improve current treatments.

Additionally, targeting CSPG4 may lead to targeted therapy for triple-negative breast cancer patients who do not benefit from therapies apart from standard chemotherapy. Therefore, further research and translation into clinical trials could be especially beneficial for the TNBC patient group.

As with all therapeutic approaches, the benefits of treatment must be balanced with the likelihood and severity of adverse effects. CSPG4 is expressed at low levels in some normal tissues; therefore, it is important to evaluate and mitigate any on-target, off-tumor toxic effects of CSPG4-specific targeted therapy. Encouragingly, a phase I clinical trial investigating anti-CSPG4 radioimmunotherapy with a mAb (9.2.27) conjugated to an α-particle-emitting radioisotope which was administered systemically in patients with melanoma reported no adverse events while some clinical benefits were observed (108). Furthermore, CSPG4-based immunotherapy strategies would benefit from the development of more effective methods of treatment delivery, such as hypobaric pressure skin delivery, which would limit potential off-target effects and reduce the cost of the therapy (120).

The emergence of novel antibody-based approaches offers fresh optimism that aggressive cancers, such as TNBC, glioma and head and neck carcinomas, which do not benefit from currently available therapies, but for which CSPG4 expression and its tumor-promoting functions have been reported, may become responsive to treatments based on this target. Therefore, renewed focus on CSPG4 may in future translate into significant benefits for patients with cancer.

KI, SK, and AT conceived and designed the study. KI, AC, SM, GC, SC, MG, MN searched and studied the literature. KI, MG, SK and AT wrote the manuscript. AC, SM, GC, MN, JS, ST, KE discussed and interpreted the literature findings and helped to edit the manuscript. SK supervised the study.

SK and JS are founders and shareholders of IGEM Therapeutics Ltd. All other authors declare no conflicts of interest.