Effects of vascular endothelial growth factor receptor-1 inhibition on vasculogenic mimicry and the metabolic profile of breast cancer cells in vitro

Abstract

Female breast cancer is the leading diagnosed cancer globally and the fifth leading cause of cancer mortality worldwide. The ability of breast cancer cells to form vessel-like structures through vasculogenic mimicry (VM) contributes to cancer progression. Vasculogenic mimicry provides a route for the transportation of blood and nutrients, which sustains the growth and survival of breast tumours. Thus, in patients with breast cancer, VM is associated with high tumour grade, metastasis, and poor prognosis. The steps involved in VM include the proliferation and migration of cancer cells, their invasion of the extracellular matrix, and finally, the formation of tube-like structures. The vascular endothelial growth factor receptor-1 (VEGFR-1) signalling pathway is involved in VM, and targeting VEGFR-1 might have clinical relevance and warrants consideration when designing targeted therapies for breast cancer.

Vascular endothelial growth factor receptor-1 is also associated with the metabolic adaptation of cancer cells, as observed in cancer patients who show a correlation between vascular endothelial growth factor A/VEGFR-1 expression and serum lactic acid levels. Indeed, VEGFR-1 promotes the Warburg effect associated with an enhanced acidic environment, which also induces the degradation and remodelling of the extracellular microenvironment. Therefore, the aim of the study was to investigate the effects of VEGFR-1 inhibition on the steps associated with VM, namely, cell growth, migration, invasion, and metabolism in breast cancer cells in vitro using VEGFR-1 inhibitors (ZM 306416 and sunitinib malate). Human breast cancer cell lines, MCF-10A, MCF-7, and MDA-MB 231 cells were maintained in an incubator at a temperature of 370C and in a humidified atmosphere containing 5% CO2. The effect of VEGFR-1 inhibition on cell viability was measured using the crystal violet assay on MCF-10A (a non-cancerous breast cell line), and breast cancer cell lines, MCF-7 and MDA-MB-231. For subsequent experiments, MDA-MB-231 cells were used as a model of investigation because profound effects (being highly responsive to the drugs of investigation) were observed with this cell line. Light microscopy was employed to study cell morphology. The effect of VEGFR-1 inhibition on cell migration and invasion was assessed using the scratch assay and the Boyden chamber, respectively. The optimisation of liquid chromatography with the tandem mass spectrometry method for the simultaneous assay of metabolites in cell culture preparations was determined. Lastly, the effects of treatment on the metabolic profile of breast cells were assessed using liquid chromatography with tandem mass spectrometry, enzyme-linked immunosorbent assay, and a pH meter/electrode. The results demonstrated that sunitinib malate had great efficacy and potency than ZM 306416, as sunitinib malate is a multi-kinase inhibitor. Overall, inhibiting VEGFR-1 reduces cell growth and alters breast cancer cell morphology. In addition, inhibiting the VEGFR-1 signalling pathway attenuates migration and invasion possibly by reducing ATP formation and the extracellular fluid acidity. Optimisation of mass spectrometry in terms of achieving mass ratio and ionisation mode of analytes (glucose-6-phosphate, fructose-6-phosphate, pyruvate, lactate, and glutamate) was achieved, however the optimisation of liquid chromatography was challenging, although it was discovered that analytes of interest in this study should be analysed using the Luna NH2 column to achieve retention and high separation. This study has formed the basis for further investigation of VEGFR-1 targeting in reducing VM and altering metabolic patterns in breast cancer to improve the treatment of this disease.