Investigation of vasculogenic mimicry in melanoma tissue

Abstract

Melanoma is an invasive and aggressive form of skin cancer developing from melanocytes in the basal layer of the epidermis. Worldwide, the incidence of melanoma continues to increase, accounting for approximately 80% of all skin cancer-related deaths in 2020. In South Africa, the overall incidence of melanoma is approximately 2.7 per 100,000 people. In comparison, melanoma accounted for a similar proportion of approximately 80% of all skin cancer-related deaths in 2012, with an estimated 55,000 deaths globally. The rapid proliferation of melanocytes increases oxygen and nutrient requirements and results in increased vascularisation as a coping mechanism to meet the metabolic requirements of the tumour. Angiogenesis, the formation of new blood vessels from existing micro-vessels, is an important mechanism of vascularisation in melanoma. Various anti-angiogenic drugs such as bevacizumab have been employed to inhibit blood vessel formation in melanoma. However, resistance develops partly due to alternative pathways of vascularisation, mainly vasculogenic mimicry (VM). Vasculogenic mimicry is the process of forming highly patterned matrix and tubular channels lined with tumour cells, evidently known to conduct fluid movement. The process entails melanoma cell proliferation and their migration, and their subsequent coalescing into vessel-like structures. The formation of VM is regulated by multiple factors including, vascular cell adhesion molecule -1 (VCAM1), vascular endothelial growth factor-A (VEGF-A), and nodal. Therefore, the aim of this study was to investigate the vasculogenic mimicry in melanoma tissue by evaluating cell proliferation, migration, and the expression of molecular regulators of vasculogenic mimicry. For in vivo studies, immunohistochemistry (IHC) was utilised to determine the expression of the proteins VCAM1 and VEGF-A in tissue samples of melanoma patients. As challenges were encountered with optimising western blotting technique, further studies were undertaken using in vitro models . The endothelioma (sEnd.2) and melanoma (B16-F10) cell lines, employed in this study as in vitro models for benign and malignant skin tumours, were maintained in an incubator at 37 °C in a humidified atmosphere containing 5% CO2. Melanoma and endothelioma viability and growth patterns were studied in vitro using the crystal violet assay. Furthermore, cell morphology characteristics were studied using polarisation-optical interference contrast and light microscopy to augment cell growth data. Since vascular endothelial growth factor receptor-1 (VEGFR-1) is known to signal vasculogenic mimicry, western blot was employed to investigate the expression of the key receptor which promotes vasculogenic mimicry, VEGFR-1. The in vitro scratch migration assay and real-time, label-free xCELLigence technologies were used to study the effect of VEGFR-1 blockade on the migration of cells as well as cell invasion and migration respectively. In vivo studies showed that VCAM1 and VEGF-A in melanoma tissue were positively expressed intratumorally, suggesting their potential involvement in tumour growth and vasculogenic mimicry. This expression may contribute to melanoma progression and survival by promoting the formation of vessel-like structures, thereby facilitating nutrient and oxygen supply to the tumour cells. The results from in vitro studies demonstrated significant attenuation of melanoma cell growth. Morphology study results showed significant changes including cell rounding, membrane blebbing and reduction in cell density, which indicate cytoskeletal disruption and apoptosis. These observations, along with decreased cell density, suggest a reduction in cell proliferation and an increase in cell death, confirming the apoptotic effects of sunitinib malate. Western blot studies revealed that melanoma cells express VEGFR-1. Cell migration plays a critical role in vasculogenic mimicry by allowing tumour cells to create vessel-like structures that mimic blood vessels, promoting tumour growth, invasion, and the bypass of conventional angiogenesis, which in turn aids tumour progression and therapy resistance. In this regard, cell migration studies showed that VEGFR-1 blockade significantly decreased the rate of cell migration and invasion, highlighting the crucial role of VEGFR-1 in these processes and its potential as a therapeutic target. Therefore, taken together, these outcomes further support the evidence for the presence of vasculogenic mimicry in melanoma and thereby provide an opportunity for further investigation on the use of combination therapies to improve patient outcome.