# Engineered Nanoclusters to Selectively Reduce Mesenchymal and Epithelial Melanoma Cell Viability

## TL;DR

Calcium sulfide nanoclusters selectively reduce melanoma cell viability without harming normal cells, potentially offering a new treatment approach.

## Contribution

The study introduces CaS nanoclusters as a novel method to target both epithelial and mesenchymal melanoma phenotypes.

## Key findings

- CaS nanoclusters reduced melanoma viability by inducing intrinsic apoptosis with minimal impact on fibroblasts.
- Treated mesenchymal melanoma cells showed vinculin delocalization, while epithelial-like cells had increased focal adhesion points.
- Caspase 3 and 9 expression increased significantly in treated melanomas, supporting apoptosis induction.

## Abstract

The ability of melanoma cells to switch among different phenotypes facilitates metastasis and evasion of treatments. We use CaS nanoclusters to selectively reduce the viability of epithelial- and mesenchymal-like melanoma phenotypes with no significant effect on benign fibroblast viability. The nanoclusters reduce melanoma viability in melanomas with either phenotypes by intrinsic apoptosis as supported by fluoresccence imagng measurements of translocated cytochrome-c and the increase in caspase 3 and 9 average expression. Interestinly, vincluin was found to delocalize in the cytoplasm of treated melanomas with the mesenchymal-like phenotype. In melanomas with epithelial-like phenotypes, the number of focal adhession points at the cell membrane-extracellular matrix interphase is found to increase compared to the control. The results encourage future preclinical work with animal models.

Melanoma is the most common type of skin cancer. Melanomas are well known for their ability to metastasize to other organs, including the lungs, liver, brain, and bones. The ability of melanoma cells to switch among different phenotypes is a key mechanism that underscores their metastatic potential. The objective of this work is to report here on the effect of calcium sulfide (CaS) dispersions in melanoma cells. Melanomas with the epithelial- and mesenchymal-like phenotypes were observed during cell culture preparation. The dose-dependent viability was explored up to slightly less than 3% per volume of cell culture. The dispersion reduced the relative percentage of melanomas with the epithelial- and mesenchymal-like phenotypes to (57 ± 5) and (55 ± 5)%, respectively, at 24 h post treatment. In contrast, the viability of normal fibroblasts treated with the dispersion or melanoma cells treated with the reactants used to prepare the dispersion remained nearly constant, with a value range of (100.0 ± 0.2)% for the control and (97 ± 4)% and (93 ± 2)% for doses as high as 2 and 3% per volume of cell culture, respectively. Fluorescence imaging measurements were consistent with the release of cytochrome c from the mitochondria and its translocation to the cell nuclei. The average expression of caspases 3 and 9 was found to be 3 and 1.4 times higher than in the corresponding melanoma control, respectively, which was consistent with intrinsic apoptosis. The response of vinculin expression was slightly different in both cell phenotypes. Vinculin was found to delocalize in the cytoplasm of treated mesenchymal melanoma cells, with a slightly higher concentration at the end of the actin fibers. A statistically significant increase (p < 0.0001) in the number of focal adhesion points (FAP) at the edge of the cell membrane–external cellular matrix (ECM) interphase was observed in post-treated melanoma that exhibited the epithelial-like phenotype. The changes in vinculin expression and FAP and the reduced viability of the melanomas were consistent with regulation of proteins associated with programmed cell death. It is thus proposed that the sulfides produced from the reactions of the nanoclusters in the acidic environment facilitate the regulation of proteins required to initiate apoptosis, although other processes may also be involved. We conclude that CaS may be an adequate chemical to selectively reduce melanoma viability with little effect on benign fibroblasts.