CSPG4P12 polymorphism served as a susceptibility marker for esophageal cancer in Chinese population.

BACKGROUND: Chondroitin sulfate proteoglycan 4 pseudogene 12 (CSPG4P12) has been implicated in the pathogenesis of various cancers. This study aimed to evaluate the association of the CSPG4P12 polymorphism with esophageal squamous cell carcinoma (ESCA) risk and to explore the biological impact of CSPG4P12 expression on ESCA cell behavior. METHODS: A case-control study was conducted involving 480 ESCA patients and 480 healthy controls to assess the association between the rs8040855 polymorphism and ESCA risk. The CSPG4P12 rs8040855 genotype was identified using the TaqMan-MGB probe method. Logistic regression model was used to evaluate the association of CSPG4P12 SNP with the risk of ESCA by calculating the odds ratios (OR) and 95% confidence intervals (95%CI ). The effects of CSPG4P12 overexpression on cell proliferation, migration, and invasion were examined in ESCA cell lines. Co-expressed genes were identified via the CBioportal database, with pathway enrichment analyzed using SangerBox. The binding score of CSPG4P12 to P53 was calculated using RNA protein interaction prediction (RPISeq). Additionally, Western Blot analysis was performed to investigate the impact of CSPG4P12 overexpression on the P53/PI3K/AKT signaling pathway. RESULTS: The presence of at least one rs8040855 G allele was associated with a reduced susceptibility to ESCA compared to the CC genotype (OR = 0.51, 95%CI = 0.28-0.93, P = 0.03). Stratification analysis revealed that the CSPG4P12 rs8040855 C allele significantly decreased the risk of ESCA among younger individuals (≤ 57 years) and non-drinkers (OR = 0.31, 95%CI = 0.12-0.77, P = 0.01; OR = 0.42, 95%CI=0.20-0.87, P = 0.02, respectively). CSPG4P12 expression was found to be downregulated in ESCA tissues compared to adjacent normal tissues. Overexpression of CSPG4P12 in ESCA cells inhibited their proliferation, migration, and invasion capabilities. Furthermore, Western Blot analysis indicated that CSPG4P12 overexpression led to a reduction in PI3K and p-AKT protein expression levels. P53 silencing rescues the inhibitory effect of CSPG4P12 on p-AKT. CONCLUSION: The CSPG4P12 rs8040855 variant is associated with reduced ESCA risk and the overexpression of CSPG4P12 inhibited the migration and invasion of ESCA cells by P53/PI3K/AKT pathway. These findings suggest that CSPG4P12 may serve as a novel biomarker for ESCA susceptibility and a potential target for therapeutic intervention.

Pseudogene CSPG4P12 inhibits colorectal cancer progression by attenuating epithelial-mesenchymal transition

Colorectal cancer is one of the most common malignant cancers. Pseudogenes have been identified as oncogenes or tumor suppressor genes in the development of various cancers. However, the function of pseudogene CSPG4P12 in colorectal cancer remains unclear. Therefore, the aim of this study was to investigate the potential role of CSPG4P12 in colorectal cancer and explore the possible underlying mechanism. The difference of CSPG4P12 expression between colorectal cancer tissues and adjacent normal tissues was analyzed using the online Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database. Cell viability and colony formation assays were conducted to evaluate cell viability. Transwell and wound healing assays were performed to assess cell migration and invasion capacities. Western blot was used to measure the expression levels of epithelial-mesenchymal transition-related proteins. Colorectal cancer tissues had lower CSPG4P12 expression than adjacent normal tissues. The overexpression of CSPG4P12 inhibited cell proliferation, invasion, and migration in colorectal cancer cells. Overexpressed CSPG4P12 promoted the expression of E-cadherin, whereas it inhibited the expression of vimentin, N-cadherin, and MMP9. These findings suggested that CSPG4P12 inhibits colorectal cancer development and may serve as a new potential target for colorectal cancer.

MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

Metastatic melanoma poses significant challenges as a highly lethal disease. Despite the success of molecular targeting using BRAFV600E inhibitors (BRAFis) and immunotherapy, the emergence of early recurrence remains an issue and there is the need for novel therapeutic approaches. This study aimed at creating a targeted delivery system for the oncosuppressor microRNA 126 (miR126) and testing its effectiveness in combination with a phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) inhibitor for treating metastatic melanoma resistant to BRAFis. To achieve this, we synthesized chitosan nanoparticles containing a chemically modified miR126 sequence. These nanoparticles were further functionalized with an antibody specific to the chondroitin sulfate proteoglycan 4 (CSPG4) melanoma marker. After evaluation in vitro, the efficacy of this treatment was evaluated through an in vivo experiment using mice bearing resistant human melanoma. The co-administration of miR126 and the PI3K/AKT inhibitor in these experiments significantly reduced tumor growth and inhibited the formation of liver and lung metastases. These results provide evidence for a strategy to target an oncosuppressive nucleic acid sequence to tumor cells while simultaneously protecting it from plasma degradation. The system described in this study exhibits encouraging potential for the effective treatment of therapy-resistant metastatic melanoma while also presenting a prospective approach for other forms of cancer.

Exogenous recombinant N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) inhibits progression of B16F10 cutaneous melanomas and modulates cell signaling.

In the syngeneic, subcutaneous B16F10 mouse model of malignant melanoma, treatment with exogenous ARSB markedly reduced tumor size and extended survival. In vivo experiments showed that local treatment with exogenous N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) led to reduced tumor growth over time (p < 0.0001) and improved the probability of survival up to 21 days (p = 0.0391). Tumor tissue from the treated mice had lower chondroitin 4-sulfate (C4S) content and lower sulfotransferase activity. The free galectin-3 declined, and the SHP2 activity increased, due to altered binding with chondroitin 4-sulfate. These changes induced effects on transcription, which were mediated by Sp1, phospho-ERK1/2, and phospho-p38 MAPK. Reduced mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4), carbohydrate sulfotransferase 15 (N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase), and matrix metalloproteinases 2 and 9 resulted. Experiments in the human melanoma cell line A375 demonstrated similar responses to exogenous ARSB as in the tumors, and inverse effects followed ARSB siRNA. ARSB, which removes the 4-sulfate group at the non-reducing end of C4S, acts as a tumor suppressor, and treatment with exogenous ARSB impacts on vital cell signaling and reduces the expression of critical genes associated with melanoma progression.

CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma.

BACKGROUND: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40-60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. METHODS: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. RESULTS: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. CONCLUSIONS: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

NG2/CSPG4 attenuates motility in mandibular fibrochondrocytes under serum starvation conditions.

The migration of mandibular fibrochondrocytes is important for the development of the mandible, the homeostasis of the mandibular cartilage, and for the capacity of the tissue to respond to injury. Mandibular fibrochondrocytes have to overcome formidable obstacles during migration including a dense and heterogeneous three-dimensional matrix. Guiding the direction of cell migration and commitment to a migratory phenotype in this microenvironment necessitates a multivalent response to chemotactic and extracellular matrix-mediated stimuli. One of the key matrix components in the cartilage of the temporomandibular joint is type VI collagen. Neuron/glial antigen 2 (NG2/CSPG4) is a transmembrane proteoglycan that binds with collagen VI and has been implicated in a wide range of cell behaviors including cell migration, motility, adhesion, and proliferation. While NG2/CSPG4 has been shown to be a key regulator of mandibular cartilage homeostasis, its role in the migration of mandibular fibrochondrocytes during normal and cell stress conditions has yet to be resolved. Here, we address this gap in knowledge by characterizing NG2/CSPG4-dependent migration in mandibular fibrochondrocytes using primary mandibular fibrochondrocytes isolated from control and full length NG2/CSPG4 knockout mice, in primary mandibular fibrochondrocytes isolated from NG2|DsRed reporter mice and in an immortalized mandibular fibrochondrocyte cell line with a mutated NG2/CSPG4 ectodomain. All three cells demonstrate similar results, with loss of the full length or truncated NG2/CSPG4 increasing the rate of cell migration in serum starvation/cell stress conditions. These findings clearly implicate NG2/CSPG4 as a key molecule in the regulation of cell migration in mandibular fibrochondrocytes in normal and cell stress conditions, underscoring the role of NG2/CSPG4 as a mechanosensitive signaling hub in the mandibular cartilage.

Prognostic significance and immunoinfiltration analysis of genes associated with epithelial-mesenchymal transition and energy metabolism in bladder urothelial carcinoma.

BACKGROUND: Epithelial-mesenchymal transition (EMT) and aberrant energy metabolism are pivotal biological processes in tumor progression, significantly impacting tumor prognosis. However, the relationship between EMT, energy metabolism, and the immune microenvironment in bladder urothelial carcinoma (BLCA) remains inadequately understood. METHODS: Bladder cancer samples from The Cancer Genome Atlas were categorized into two groups via clustering analysis to elucidate disparities in expression, prognostic significance, and immune infiltration of genes associated with EMT and energy metabolism between these groups. Key genes associated with EMT and energy metabolism in BLCA were identified through Cox multifactorial regression analysis, immune infiltration analysis, etc. Subsequently, their prognostic significance in BLCA was validated. RESULTS: Cluster analysis revealed significant differences in the expression of genes associated with EMT and energy metabolism between the two groups. Group 2 exhibited significantly improved overall survival and progression-free survival compared to Group 1. Chondroitin sulfate proteoglycan 4 (CSPG4) emerged as the most critical gene associated with EMT, energy metabolism, prognosis, and immune infiltration in BLCA. Immunohistochemical assays demonstrated differential expression of CSPG4 in bladder tumors and normal bladder tissues, with high CSPG4 expression correlating with a poorer BLCA prognosis. Furthermore, CSPG4 exhibited an association with the immune checkpoint molecule programmed death-1 (PD1) in BLCA. CONCLUSIONS: EMT and energy metabolism exert pivotal influences on the immune microenvironment in BLCA. CSPG4 holds promise as a prognostic biomarker for patients with BLCA, offering valuable insights into potential immunotherapeutic strategies for this patient population.

Utilizing induced neural stem cell-based delivery of a cytokine cocktail to enhance chimeric antigen receptor-modified T-cell therapy for brain cancer.

Chimeric antigen receptor (CAR)-modified T-cell therapy has shown enormous clinical promise against blood cancers, yet efficacy against solid tumors remains a challenge. Here, we investigated the potential of a new combination cell therapy, where tumor-homing induced neural stem cells (iNSCs) are used to enhance CAR-T-cell therapy and achieve efficacious suppression of brain tumors. Using in vitro and in vivo migration assays, we found iNSC-secreted RANTES/IL-15 increased CAR-T-cell migration sixfold and expansion threefold, resulting in greater antitumor activity in a glioblastoma (GBM) tumor model. Furthermore, multimodal imaging showed iNSC delivery of RANTES/IL-15 in combination with intravenous administration of CAR-T cells reduced established orthotopic GBM xenografts 2538-fold within the first week, followed by durable tumor remission through 60 days post-treatment. By contrast, CAR-T-cell therapy alone only partially controlled tumor growth, with a median survival of only 19 days. Together, these studies demonstrate the potential of combined cell therapy platforms to improve the efficacy of CAR-T-cell therapy for brain tumors.

Targeting the melanoma-associated antigen CSPG4 with HLA-C*07:01-restricted T-cell receptors.

Intorduction: Chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen, is expressed in melanoma but also other tumor entities and constitutes an attractive target for immunotherapeutic approaches. While recent preclinical reports focused on anti-CSPG4 chimeric antigen receptors (CAR), we here explore T-cell receptor (TCR)-based approaches targeting CSPG4. Methods: The TCRs of two CSPG4-reactive T-cell clones (11C/73 and 2C/165) restricted by the highly prevalent HLA-C*07:01 allele were isolated and the respective αßTCR pairs were retrovirally expressed in CRISPR/Cas9-edited TCR-knockout T cells for functional testing. We also combined alpha and beta TCR chains derived from 11C/73 and 2C/165 in a cross-over fashion to assess for hemichain dominance. CSPG4+ melanoma, glioblastoma and lung cancer cell lines were identified and, if negative, retrovirally transduced with HLA-C*07:01. Results: Functional tests confirmed specific recognition of CSPG4+HLA-C*07:01+ target cells by the αßTCR retrieved from the parental T-cell clones and in part also by the cross-over TCR construct 2Cα-11Cß. Despite high surface expression, the 11Cα-2Cß combination, however, was not functional. Discussion: Collectively, 11C/73- and 2C/165-expressing T cells specifically and efficiently recognized CSPG4+HLA-C*07:01+ cancer cells which warrants further preclinical and clinical evaluation of these TCRs.

Immunohistochemical detection of the chondroitin sulfate proteoglycan 4 protein in primary and metastatic melanoma.

Treatment of malignant melanoma, the most aggressive form of skin cancer, continues to be a major challenge for clinicians. New targeted therapies with kinase inhibitors or drugs which modify the immune response are often accompanied by the development of resistance or severe side effects. In this context, chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, could be a potential target for alternative therapeutic approaches. The aim of the present study was to identify differences in the levels of CSPG4 protein expression in primary and metastatic melanomas as well as to analyze correlations between CSPG4 expression and histopathological data and patient characteristics. A total of 189 melanoma tissue samples from Lower Austria, including primary melanomas and melanoma metastases, were immunohistochemically stained for the expression of CSPG4 and statistical analyses were performed. A total of 65.6% of melanoma tissue samples stained positive for the expression of CSPG4. Primary nodular and primary superficial spreading melanomas demonstrated a significantly higher number of positively stained tissue samples for CSPG4 compared with primary lentigo maligna melanomas. No significant differences in the expression of CSPG4 were demonstrated between primary melanomas and melanoma metastases. The present study supports the advancement of the understanding of CSPG4 tissue expression patterns in melanoma patients and provides additional information for further investigation of CSPG4 as a potential therapeutic target.

CHST11-modified chondroitin 4-sulfate as a potential therapeutic target for glioblastoma.

Aberrant chondroitin sulfate (CS) accumulation in glioblastoma (GBM) tissue has been documented, but the role of excessive CS in GBM progression and whether it can be a druggable target are largely unknown. The aim of this study is to clarify the biological functions of CHST11 in GBM cells, and evaluate therapeutic effects of blocking CHST11-derived chondroitin 4-sulfate (C4S). We investigated the expression of CHST11 in glioma tissue by immunohistochemistry, and analyzed CHST11 associated genes using public RNA sequencing datasets. The effects of CHST11 on aggressive cell behaviors have been studied in vitro and in vivo. We demonstrated that CHST11 is frequently overexpressed in GBM tissue, promoting GBM cell mobility and modulating C4S on GBM cells. We further discovered that CSPG4 is positively correlated with CHST11, and CSPG4 involved in CHST11-mediated cell invasiveness. In addition, GBM patients with high expression of CHST11 and CSPG4 have a significantly shorter survival time. We examined the effects of treating C4S-specific binding peptide (C4Sp) as a therapeutic agent in vitro and in vivo. C4Sp treatment attenuated GBM cell invasiveness and, notably, improved survival rate of orthotopic glioma cell transplant mice. Our results propose a possible mechanism of CHST11 in regulating GBM malignancy and highlight a novel strategy for targeting aberrant chondroitin sulfate in GBM cells.

CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate.

PURPOSE: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products. METHODS: Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy. RESULTS: After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines. CONCLUSION: This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC.

Low-density lipoprotein receptor-related protein-1 (LRP1) in the glial lineage modulates neuronal excitability.

The low-density lipoprotein related protein receptor 1 (LRP1), also known as CD91 or α-Macroglobulin-receptor, is a transmembrane receptor that interacts with more than 40 known ligands. It plays an important biological role as receptor of morphogens, extracellular matrix molecules, cytokines, proteases, protease inhibitors and pathogens. In the CNS, it has primarily been studied as a receptor and clearance agent of pathogenic factors such as Aß-peptide and, lately, Tau protein that is relevant for tissue homeostasis and protection against neurodegenerative processes. Recently, it was found that LRP1 expresses the Lewis-X (Lex) carbohydrate motif and is expressed in the neural stem cell compartment. The removal of Lrp1 from the cortical radial glia compartment generates a strong phenotype with severe motor deficits, seizures and a reduced life span. The present review discusses approaches that have been taken to address the neurodevelopmental significance of LRP1 by creating novel, lineage-specific constitutive or conditional knockout mouse lines. Deficits in the stem cell compartment may be at the root of severe CNS pathologies.

A chimeric human/dog-DNA vaccine against CSPG4 induces immunity with therapeutic potential in comparative preclinical models of osteosarcoma.

The high mortality rate of osteosarcoma (OSA) patients highlights the requirement of alternative strategies. The young age of patients, as well as the rarity and aggressiveness of the disease, limits opportunities for the robust testing of novel therapies, suggesting the need for valuable preclinical systems. Having previously shown the overexpression of the chondroitin sulfate proteoglycan (CSPG)4 in OSA, herein the functional consequences of its downmodulation in human OSA cells were evaluated in vitro, with a significant impairment of cell proliferation, migration, and osteosphere generation. The potential of a chimeric human/dog (HuDo)-CSPG4 DNA vaccine was explored in translational comparative OSA models, including human xenograft mouse models and canine patients affected by spontaneous OSA. The adoptive transfer of HuDo-CSPG4 vaccine-induced CD8+ T cells and sera in immunodeficient human OSA-bearing mice delayed tumor growth and metastasis development. HuDo-CSPG4 vaccination resulted safe and effective in inducing anti-CSPG4 immunity in OSA-affected dogs, which displayed prolonged survival as compared to controls. Finally, HuDo-CSPG4 was also able to induce a cytotoxic response in a human surrogate setting in vitro. On the basis of these results and the high predictive value of spontaneous OSA in dogs, this study paves the way for a possible translation of this approach to humans.

Cspg4high microglia contribute to microgliosis during neurodegeneration.

Microglia play a critical role in the pathogenic process of neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD). Upon pathological stimulation, microglia are converted from a surveillant to an overactivated phenotype. However, the molecular characters of proliferating microglia and their contributions to the pathogenesis of neurodegeneration remain unclear. Here, we identify chondroitin sulfate proteoglycan 4 (Cspg4, also known as neural/glial antigen 2)-expressing microglia as a specific subset of microglia with proliferative capability during neurodegeneration. We found that the percentage of Cspg4+ microglia was increased in mouse models of PD. The transcriptomic analysis of Cspg4+ microglia revealed that the subcluster Cspg4high microglia displayed a unique transcriptomic signature, which was characterized by the enrichment of orthologous cell cycle genes and a lower expression of genes responsible for neuroinflammation and phagocytosis. Their gene signatures were also distinct from that of known disease-associated microglia. The proliferation of quiescent Cspg4high microglia was evoked by pathological α-synuclein. Following the transplantation in the adult brain with the depletion of endogenous microglia, Cspg4high microglia grafts showed higher survival rates than their Cspg4- counterparts. Consistently, Cspg4high microglia were detected in the brain of AD patients and displayed the expansion in animal models of AD. These findings suggest that Cspg4high microglia are one of the origins of microgliosis during neurodegeneration and may open up a avenue for the treatment of neurodegenerative diseases.

VT68.2: An Antibody to Chondroitin Sulfate Proteoglycan 4 (CSPG4) Displays Reactivity against a Tumor-Associated Carbohydrate Antigen.

The anti-CSPG4 monoclonal antibodies (mAbs) have shown anti-tumor activity and therapeutic potential for treating breast cancer. In addition, CSPG4 is a dominant tumor-associated antigen that is also involved in normal-tissue development in humans. Therefore, the potential for off-tumor activity remains a serious concern when targeting CSPG4 therapeutically. Previous work suggested that glycans contribute to the binding of specific anti-CSPG4 antibodies to tumor cells, but the specificity and importance of this contribution are unknown. In this study, the reactivity of anti-CSPG4 mAbs was characterized with a peptide mimetic of carbohydrate antigens expressed in breast cancer. ELISA, flow cytometry, and microarray assays were used to screen mAbs for their ability to bind to carbohydrate-mimicking peptides (CMPs), cancer cells, and glycans. The mAb VT68.2 displayed a distinctly strong binding to a CMP (P10s) and bound to triple-negative breast cancer cells. In addition, VT68.2 showed a higher affinity for N-linked glycans that contain terminal fucose and fucosylated lactosamines. The functional assays demonstrated that VT68.2 inhibited cancer cell migration. These results define the glycoform reactivity of an anti-CSPG4 antibody and may lead to the development of less toxic therapeutic approaches that target tumor-specific glyco-peptides.

Trophoblast-specific knockdown of CSPG4 expression causes pregnancy complications with poor placentation in mice.

The multifunctional molecule chondroitin sulfate proteoglycan 4 (CSPG4/NG2) plays key roles in organogenesis and tumorigenesis. However, its roles in placentation remain unclear. In this study, CSPG4 expression in human and mouse placentas was investigated through immunohistochemistry (IHC), qPCR and western blotting. The theoretical structure and function of CSPG4 were assessed using bioinformatic tools, and the functions of CSPG4 in fetal and placental development were investigated using a mouse model established by trophoblast-specific CSPG4 knockdown and a trophoblast cell line with CSPG4 knockout by lentivirus infection. The results showed that CSPG4 was mainly located in trophoblasts in both human placentas and mouse placentas, with a higher level in preeclampsia (PE) placentas than in healthy control placentas. Furthermore, there was a trend of increasing expression in mouse placentas during pregnancy. The 3D structure of CSPG4 was visualized using an M model composed of two chains, and the structure implied that CSPG4 was a multifunctional molecule containing multiple pockets with multiligand binding sites and enzyme active sites. Trophoblast-specific CSPG4 knockdown caused frequent fetal loss, and viable fetal development was restricted by poor placentation, with mice placentas having reduced weight and width. The proliferation and invasion of CSPG4-knockout trophoblasts were significantly inhibited, and as such, the molecular signaling of AKT and ERK phosphorylation was inhibited, and the expression of MMP2 and MMP9 was reduced. In summary, CSPG4 deficiency inhibited trophoblast proliferation and invasion, which was associated with AKT, ERK and MMP signaling. CSPG4 deficiency also caused pregnancy complications with poor placentation in mice.

Improving Osteosarcoma Treatment: Comparative Oncology in Action.

Osteosarcoma (OSA) is the most common pediatric malignant bone tumor. Although surgery together with neoadjuvant/adjuvant chemotherapy has improved survival for localized OSA, most patients develop recurrent/metastatic disease with a dismally poor outcome. Therapeutic options have not improved for these OSA patients in recent decades. As OSA is a rare and "orphan" tumor, with no distinct targetable driver antigens, the development of new efficient therapies is still an unmet and challenging clinical need. Appropriate animal models are therefore critical for advancement in the field. Despite the undoubted relevance of pre-clinical mouse models in cancer research, they present some intrinsic limitations that may be responsible for the low translational success of novel therapies from the pre-clinical setting to the clinic. From this context emerges the concept of comparative oncology, which has spurred the study of pet dogs as a uniquely valuable model of spontaneous OSA that develops in an immune-competent system with high biological and clinical similarities to corresponding human tumors, including in its metastatic behavior and resistance to conventional therapies. For these reasons, the translational power of studies conducted on OSA-bearing dogs has seen increasing recognition. The most recent and relevant veterinary investigations of novel combinatorial approaches, with a focus on immune-based strategies, that can most likely benefit both canine and human OSA patients have been summarized in this commentary.

Decitabine-Mediated Upregulation of CSPG4 in Ovarian Carcinoma Cells Enables Targeting by CSPG4-Specific CAR-T Cells.

The addition of CAR-T cells to the armamentarium of immunotherapy revigorated the field of oncology by inducing long-lasting remissions in patients with relapsing/refractory hematological malignancies. Nevertheless, in the lion's share of patients diagnosed with solid tumors, CAR-T-cell therapy so far failed to demonstrate satisfactory anti-tumor activity. A crucial cause of resistance against the antigen-specific attack of CAR-T cells is predicated on the primary or secondary absence of suitable target antigens. Thus, the necessity to create a broad repertoire of different target antigens is vital. We aimed to evaluate the potential of the well-established melanoma antigen chondroitin sulfate proteoglycan 4 (CSPG4) as an inducible antigen in ovarian cancer cells, using CSPG4-negative SKOV-3 ovarian cancer cells as a model. Based on the hypomethylating activity of the FDA-approved drug decitabine, we refined a protocol to upregulate CSPG4 in the majority of decitabine-treated SKOV-3 cells. CSPG4-specific CAR-T cells generated by mRNA-electroporation showed CSPG4-directed cytokine secretion and cytotoxicity towards decitabine-treated SKOV-3. Another ovarian cancer cell line (Caov-3) and the neoplastic cell line 293T behaved similar. In aggregate, we generated proof-of-concept data paving the way for the further exploration of CSPG4 as an inducible antigen for CAR-T cells in ovarian cancer.

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

BACKGROUND: Soft tissue sarcomas (STS) are heterogeneous and pro-metastatic tumors. Identification of accurate prognostic factors and novel therapeutic targets are crucial. CSPG4 is a cell surface proteoglycan with oncogenic functions. It recently emerged as a potential target for immunotherapy, including cell therapy based on CSPG4-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CSPG4-CAR.CIKs) in STS. However, expression of CSPG4 is poorly known in STS so far. METHODS: We analyzed CSPG4 gene expression in 1378 localized STS clinical samples, and searched for correlations with clinicopathological data, including disease-free survival (DFS), and with tumor immune features. RESULTS: CSPG4 expression was heterogeneous across samples. High expression was associated with younger patients' age, more frequent undifferentiated pleomorphic sarcoma and myxofibrosarcoma pathological subtypes, more frequent internal trunk tumor site, and more CINSARC high-risk samples. No correlation existed with pathological tumor size and grade, and tumor depth. Patients with high CSPG4 expression displayed 49% (95% CI 42-57) 5-year DFS versus 61% (95% CI 56-68) in patients with low expression (p = 3.17E-03), representing a 49% increased risk of event in the "CSPG4-high" group (HR = 1.49, 95% CI 1.14-1.94). This unfavorable prognostic value persisted in multivariate analysis, independently from other variables. There were significant differences in immune variables between "CSPG4-high" and "CSPG4-low" tumors. The "CSPG4-low" tumors displayed profiles suggesting higher anti-tumor cytotoxic immune response and higher potential vulnerability to immune checkpoint inhibitors (ICI). By contrast, the "CSPG4-high" tumors displayed profiles implying an immune-excluded tumor microenvironment, potentially induced by hypoxia, resulting from an immature chaotic microvasculature, and/or the presence of contractile myofibroblasts. CONCLUSIONS: Patients with "CSPG4-high" STS, theoretically candidate for CAR.CIKs, display shorter DFS and an immune environment unfavorable to vulnerability to CAR.CIKs, which could be improved by combining anti-angiogenic drugs able to normalize the tumor vasculature. By contrast, "CSPG4-low" STS are better candidates for immune therapy involving ICI.