Abstract
The overexpression of chondroitin sulfate proteoglycan 4 (CSPG4) is associated with several tumor types, including malignant melanoma, squamous cell carcinoma, triple-negative breast carcinoma, oligodendrocytomas or gliomas. Due to its restricted distribution in normal tissues, CSPG4 has been considered a potential target for several antitumor approaches, including monoclonal antibody (mAb) therapies. The aim of the present study was to characterize the impact of the CSPG4-specific mAb clone 9.2.27 on its own or in combination with the commonly used BRAF-selective inhibitor, PLX4032, on different functions of melanoma cells to assess the potential synergistic effects. The BRAF V600-mutant human melanoma cell lines, M14 (CSPG4-negative) and WM164 (CSPG4-positive), were exposed to the CSPG4-specific 9.2.27 mAb and/or PLX4032. Cell viability and colony formation capacity were evaluated. A 3D-cell culture spheroid model was used to assess the invasive properties of the treated cells. In addition, flow cytometric analysis of apoptosis and cell cycle analyses were performed. Incubation of the WM164 cell line with CSPG4-specific 9.2.27 mAb decreased viability, colony formation ability and the invasive capacity of CSPG4-positive tumor cells, which was not the case for the CSPG4-negative M14 cell line. Combined treatment of the WM164 cells with 9.2.27 mAb plus PLX4032 did not exert any significant additional effect in comparison to treatment with PLX4032 alone in the clonogenic and invasion assays. M14 cell cycle distribution was not influenced by the CSPG4-specific 9.2.27 mAb. By contrast, the exposure of WM164 cells to the mAb resulted in an arrest of the cells in the S phase. Moreover, combined treatment of the WM164 cells led to a significantly increased accumulation of cells in the subG1 phase, combined with a decrease of cells in the G2/M phase. On the whole, findings of the present study indicate that the CSPG4-specific 9.2.27 mAb exerts an anti-invasive effect on CSPG4-positive melanoma spheroids, which is not enhanced by BRAF inhibition. These findings provide the basis for further investigations on the effects of anti-CSPG4-based treatments of CSPG4-positive tumors.
Highlights
The present study first verified the effects of the potent BRAF V600 inhibitor, PLX4032, as well as those of the chondroitin sulfate proteo‐ glycan 4 (CSPG4)‐specific 9.2.27 monoclonal antibody (mAb), and the combination of both on the viability of melanoma cell lines
When combined with PLX4032, the anti‐CSPG4 mAb contributed to a significant, additional inhibition of WM164 cell viability, as compared with the cells treated with BRAF inhibitor alone, decreasing the viability by 37.3±5.4% after 24 h and by 60.7±2.4% after 72 h (Fig. 1A)
Higher antibody concentrations were associted with a higher percentage of cells arrested in the S phase (Fig. S4). These results indicate that the CSPG4‐specific mAb can lead to cell cycle arrest in the S phase and that the combination with the BRAF inhibitor PLX4032 may lead to an increased cell death of CSPG4‐postitive cells
Summary
Chondroitin sulfate proteoglycan 4 (CSPG4), known as high molecular weight‐melanoma associated antigen (HMW‐MAA) or melanoma chondroitin sulfate proteoglycan (MCSP) was first characterized on human melanoma cells 40 years ago [1]. CSPG4 is a single‐pass type I transmembrane protein expressed either as an 280‐kDa N‐linked glycoprotein or as a 450‐kDa chondroitin sulfate proteoglycan [2]. CSPG4 was originally associated only with melanoma progres‐ sion due to its widespread expression in the majority (≥70%) of these tumors [3], it was later detected in other hemato‐ logical and solid neoplastic conditions, including several types of leukemia [4], head and neck squamous‐cell carcinomas [5], triple‐negative breast carcinoma (TNBC) [6], gliomas [7], pancreatic tumors [8], soft‐tissue sarcomas [9] and malignant mesothelioma [10]. Only a limited number of anti‐CSPG4 monoclonal antibodies (mAbs) targeting CSPG4‐positive tumors has been described, including 9.2.27 mAb, 225.28 mAb and TP41.2 mAb [6,10,15]
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