CSPG4 expression in soft tissue sarcomas is associated with poor prognosis and low cytotoxic immune response

Soft-tissue sarcomas (STS) are rare and serious tumors of mesenchymal origin affecting children and adults [1]. These heterogeneous tumors encompass more than 100 distinct pathological subtypes associated with variable biological and clinical behaviors [2]. Despite the successful advances in surgical resection, radiotherapy, and chemotherapy, the outcome of patients with non-metastatic STS is still poor: the disease recurs in approximately 50% of patients, often with distant failure [3‐6]. In the metastatic setting, the prognosis remains dismal with a 5-year survival rate inferior to 25% [7]. Currently, two issues represent major challenges in the management of STS. The first one is the improvement of prognostic factors that will help to better define the role, if any, of adjuvant chemotherapy. Historically associated with pathological grade [8] and size, and depth [9], the prognostic characterization is being refined, thanks to the contribution of genomic signatures such as CINSARC [10] or the immunologic constant of rejection (ICR) signature [11]. Clinical trials assessing prospectively the clinical utility of CINSARC (NCT03805022, NCT04307277) are ongoing. The second issue relies on the identification of novel therapeutic targets. The limited development of new chemotherapy drugs and targeted therapies during the last decade has not modified the overall prognosis, and doxorubicin remains the backbone of systemic treatment.

Chondroitin sulfate proteoglycan 4 (CSPG4), also called neural-glial 2 (NG2), is a cell surface proteoglycan, overexpressed in certain human cancers, with low expression in normal tissues and oncogenic roles in tumor growth and metastatic dissemination [12] via the promotion of cell proliferation, cell survival and drug resistance, angiogenesis, cell migration and invasion [13]. Inhibition of CSPG4 by gene deletion or treatment with anti-CSPG4 antibodies inhibits tumor growth in xenografts from some malignancies [14, 15]. Since CSPG4 is expressed in the mesenchymal progenitor cells [16, 17] and pericytes [18] from which STS are supposed to originate, its activation could play a role in sarcoma progression. Driving oncogenic mutations in Ng2/Cspg4-expressing cells leads to the formation of sarcomas [18]. During the last years, CSPG4 was described as a potential target of cellular immunotherapy in cancers [12]. CSPG4 is also known to influence activation, maturation, proliferation, and migration of different immune cell subsets suggesting likely interaction with immunotherapy efficiency [12].

Recent data suggested that the immune system might positively impact the outcome of patients with STS [11, 19, 20]. Several clinical trials testing immunotherapy based on immune checkpoint inhibitors (ICI) have been launched [21], but the results were relatively disappointing and remain controversial [22]. Identification of efficacy predictive markers, such as the presence of tertiary lymphoid structures [23, 24], is crucial in this so heterogeneous group of tumors. Another immunotherapy type is adoptive cellular therapy in which the T-cells are redirected by tumor antigen-specific chimeric antigen receptors (CAR-Ts). This approach, very effective in B-cell cancers [25‐27], remains challenging in solid tumors [28]. One approach dedicated to improving the efficacy and safety of CAR-based therapies is the engineering of immune effectors different from αβT-lymphocytes, such as γδT-cells, natural killer (NK), NKT, or cytokine-induced killer (CIK) cells [29]. A recent study revealed the therapeutic potential of CSPG4-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CSPG4-CAR.CIKs) in STS [30]. In this study, the CSPG4-CAR.CIKs effectively targeted multiple STS pathological subtypes in vitro and in vivo. Antitumor activity against STS spheroids was associated with tumor recruitment, infiltration, and matrix penetration. In vivo, the CSPG4-CAR.CIKs delayed or reversed the tumor growth in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma).

Expression of CSPG4 is poorly known in STS. To our knowledge, only three studies [30‐32] in the literature analyzed its expression in clinical samples, including respectively 251, 55, and 108 cases, but only the two smallest ones searched for correlations with tumor clinical features. To fill this gap and given the potential relevance of CSPG4 as a target for immunotherapy, we analyzed its expression in 1,378 localized STS clinical samples. We searched for correlations between expression and clinicopathological data, including disease-free survival (DFS), but also the components of the tumor immune landscape.

Our analysis was based on gene expression of CSPG4 in a very large series of clinical samples. The strong correlation between mRNA and protein expression levels of CSPG4 that we evidenced in 343 cancer cell lines suggests that CSPG4 protein expression parallels observations made at the transcriptomic level. Such mRNA level analysis allowed not only to avoid the classical limitations of immunohistochemistry (availability of antibodies, standardization, positivity cut-off, interpretation subjectivity…), but also to work on a large series of clinical samples and to search for correlations with expression of biologically and clinically relevant immune signatures. We found heterogeneous expression of CSPG4 in clinical STS samples, as reported in the three previous studies on STS, all also performed at the transcriptional level [30‐32]. Benassi et al. profiled 55 samples [31], Cattaruzza et al. 108 samples including the 55 previous ones [32], and Leuci et al. analyzed 251 TCGA samples [30]. Benassi et al. focused their analysis on 55 deeply localized, >5-cm diameter and high-grade lesions [31] and did not find any significant correlation between the CSPG4 expression and clinicopathological variables. The same team [32] extended this series to a total of 108 cases and found higher expression in synovial sarcoma. Leuci et al did not search for eventual correlations [30]. In our present study, we found that expression was mainly associated with the pathological type, with higher expression in LMS and MFS and lower expression in UPS and LPS. Other significant correlations existed with age, tumor site, and CINSARC risk, the “CSPG4-high" samples being more frequently high-risk according to CINSARC than the “CSPG4-low” samples.

Our prognostic analysis included a large series of 610 patients informative for DFS, the largest prognostic study reported so far. In uni- and multivariate analyses, high CSGP4 expression was associated with a higher risk of DFS event, independently from other prognostic variables including pathological tumor size and grade and CINSARC. The 5-year DFS was 61% in the “CSPG4-low” subgroup versus 49% in the “CSPG4-high” group, representing a 49% increased risk of event in the “CSPG4-high” group. These results are consistent with the sole other prognostic study published in the literature [31]. In a series of 108 patients with STS deeply localized, >5-cm diameter and grade 2–3 [32], the authors found higher CSPG4 expression in the metastases when compared with paired primary lesions and when compared with normal lung and other tissues. They confirmed this result at the protein level using IHC based on a monoclonal antibody generated by their own team, with high expression on the surface of neoplastic cells and neovascular structures of primary and secondary tumor masses. Finally, they demonstrated in multivariate analysis the independent unfavorable prognostic value of high CSPG4 expression for MFS [32]. This prognostic impact in STS was investigated at the functional level. Using in vitro and in vivo models, the same team [32] showed that CSPG4 controlled the tumor progression (local growth, cell adhesion, and motility, and cell survival) by mediating the interaction of sarcoma cells with the host extracellular matrix, in particular with collagen 6 (Col VI) that accumulates in the peri- and intra-lesional stroma. In another study [44], Hsu et al. showed that the effects of CSPG4 on STS growth depended of the tumor developmental stage: in established murine and human STS, inhibition of CSPG4, using anti-CSPG4 antibody immunotherapy or gene deletion, decreased the cell proliferation and tumor size and increased apoptosis, whereas Ng2/Cspg4 deletion at the time of tumor initiation resulted in the opposite effect on tumor growth. The prognostic value of CSPG4 expression is likely tumor type-dependent. We recently showed the good-prognosis value of high CSPG4 expression in a series of 309 GIST [45], whereas higher expression was associated with poorer prognosis in melanoma [46], glioblastoma [47], breast cancer [48], head and neck squamous cell carcinomas [49], and hepatocellular carcinoma [50].

The transmembrane proteoglycan CSPG4 had been originally identified by Dr Ferrone‘s team as a highly immunogenic tumor antigen on the surface of melanoma cells and was named High Molecular Weight Melanoma-Associated Antigen [51]. This antigen was then characterized by the same team in several cancers: melanoma [46], TNBC [14], malignant mesothelioma [15], acute myeloid leukemia [52], chordoma [53], glioblastoma [54], and osteosarcoma [55]. It is now identified as a potential therapeutic target for immune therapy in different cancers, including anti-idiotypic antibodies in melanoma [56‐58], monoclonal antibodies in triple-negative breast cancer [14] and melanoma [59], antibody-drug conjugate in melanoma [60], and CAR-T cells in many cancers [61]. In sarcomas, Leuci et al. recently demonstrated in vitro and in vivo the anti-tumor activity of CSPG4-CAR.CIKs in STS pre-clinical samples. We thus searched for eventual correlations between CSPG4 expression and immune/stromal features. Several of them were differentially enriched between "CSPG4-high" and "CSPG4-low" STS. "CSPG4-low" tumors had higher scores for immune signatures suggesting higher infiltration by immune cells, higher lymphatic vasculature and higher anti-tumor immune response. They were also associated with the presence of TLS and other signatures indicative of better response to ICI treatment [23], despite signs of exhaustion. In line with this, "CSPG4-low" tumors showed higher iCAFs and apCAFs, which respectively secrete cytokines to attract and entrap lymphocytes to turn them into harmless cells. Altogether, these data suggested that "CSPG4-low” STS could be better candidates for immune therapy involving ICI, which will fully unleash pre-infiltrated CD8 T cells cytotoxic potential [45]. By contrast, the "CSPG4-high" tumors displayed immune profiles suggesting an immune desert or immune-excluded tumor microenvironment, lacking all kinds of immune cells required to mount an effective anti-tumor response, except for the NK^bright cells. This lack of immune infiltrated cells can be related to the presence of myofibroblasts and myCAFs, which are fibroblastic subsets endowed with contractile features, which affect the distribution of blood vasculature [62]. They also secrete a massive amount of matrix and prevent lymphocyte accessibility to tumor cells. For all these reasons, immune desert tumor microenvironments have been reported to have limited sensitivity to ICI. In the study reported by Leuci et al., the tumor elimination in vitro after treatment with CSPG4-CAR.CIKs was dependent on the expression level of tumor cells, suggesting that "CSPG4-high" STS should represent the most candidate population for such treatment [30]. But our present data suggest that a treatment based on CSPG4-CAR.CIKs infiltrating cells that will target "CSPG4-high" tumor cells could be interesting if some aspects responsible for the immune desert can be overcome first. Notably, immune desert can be induced by hypoxia. Hypoxia is a key determinant of tumor aggressiveness, therapy resistance and has a dampening effect on antitumor immune responses and immune cells recruitment. Hypoxia and lactic acidosis induce the functional suppression of NK cells. We found that both hypoxia and lactic acidosis were enhanced in "CSPG4-high" tumors. This observation is consistent with a certain overexpression of CSPG4 induced by the chronic hypoxia in vitro [63] and likely explains why only immunoregulatory NK^bright cells are present in "CSPG4-high" tumors. A strategy based on CSPG4-CAR.CIKs would thus require to overcome the functional suppression. Hypoxia results from an immature chaotic microvasculature within the tumor. Strategies that seek to normalize the tumor vasculature, such as tyrosine kinase inhibitors (TKIs) that target pro-angiogenic receptors, should help reduce hypoxia, enhance tumor’s perfusion and optimize therapy uptake. This would be a key point to improve the efficacy of CSPG4-CAR.CIKs in STS.

Supervised analysis of transcriptomics data between the “CSPG4-high” and “CSPG4-low” tumors confirmed the immune desert observed in the “CSPG4-high” tumors and their enrichment in genes related to cell migration, collagen and extra-cellular matrix, response to stress, growth and development. That might explain in part their poorer prognosis when compared with the “CSPG4-low” tumors. No significant difference was observed regarding the frequency of gene amplification or gene deletion between both tumor groups, notably for CSPG4, suggesting that the DNA copy number is not responsible for the CSPG4 differential expression. Similarly, no gene showed a significant difference in term of mutation frequency between the “CSPG4-high” and “CSPG4-low” tumors. By contrast, 84 sites were differentially methylated between both tumor groups, calling for further investigations in order to assess an eventual functional link with CSPG4.

To our knowledge, our study is the largest one describing CSPG4 expression in clinical cancer samples, here in STS. We show that expression of CSPG4 in STS samples is heterogeneous and associated independently with shorter DFS and with an immune landscape not favorable to anti-tumor cytotoxic response. The main strength of our study lies in the high number of rare tumor samples analysed. Others include its originality and analysis of correlations with tumor immune variables. Limitations include the retrospective nature and associated biases, analysis of bulk samples, and absence of analysis at the protein level. Obviously, analysis of larger clinical series and protein analysis are warranted. Yet, our results suggest the prognostic value of CSPG4 expression in STS and describe the immune microenvironment of tumors candidate to specific immune therapy such as CSPG4-CAR.CIKs. Whether patients with STS, and what type of patients, will benefit from such immunotherapy deserve rapid assessment in prospective clinical trials.