Fibroblasts Influence Metastatic Melanoma Cell Sensitivity to Combined BRAF and MEK Inhibition.

Delphine Morales,Ines Ferreira,Catherine Lok,Mikael Magnano,Laxsika Mahenthiran,Pascale Vigneron,Muriel Vayssade,Warda Hamitou

doi:10.3390/cancers13194761

Abstract

Simple SummaryPreclinical 3D in vitro coculture models are known to be more complex systems than monolayer cell culture and mimic the physiological environment more closely. Three-dimensional dermal equivalents provide a relevant environment for cutaneous metastatic melanoma cells and are capable of modulating a cancer cell’s response to drugs. We showed that a combined targeted therapy (vemurafenib and cobimetinib) efficiently inhibits cell proliferation and induces apoptosis, especially in the 3D coculture model. A cancer-associated fibroblast population isolated from a cutaneous melanoma was also sensitive to the treatment but with no detectable induction of apoptosis. To better understand the complex crosstalk between melanoma cells and their microenvironment, we compared the influence of conditioned media obtained from healthy or cancer-associated fibroblasts on the response of metastatic melanomas to the drugs. Our data indicate that normal fibroblast supernatants potentialize the therapy’s efficiency, whereas cancer-associated fibroblast secretomes favor melanoma cell survival.The sensitivity of melanoma cells to targeted therapy compounds depends on the tumor microenvironment. Three-dimensional (3D) in vitro coculture systems better reflect the native structural architecture of tissues and are ideal for investigating cellular interactions modulating cell sensitivity to drugs. Metastatic melanoma (MM) cells (SK-MEL-28 BRAF V600E mutant and SK-MEL-2 BRAF wt) were cultured as a monolayer (2D) or cocultured on 3D dermal equivalents (with fibroblasts) and treated with a BRAFi (vemurafenib) combined with a MEK inhibitor (MEKi, cobimetinib). The drug combination efficiently inhibited 2D and 3D MM cell proliferation and survival regardless of their BRAF status. Two-dimensional and three-dimensional cancer-associated fibroblasts (CAFs), isolated from a cutaneous MM biopsy, were also sensitive to the targeted therapy. Conditioned media obtained from healthy dermal fibroblasts or CAFs modulated the MM cell’s response differently to the treatment: while supernatants from healthy fibroblasts potentialized the efficiency of drugs on MM, those from CAFs tended to increase cell survival. Our data indicate that the secretory profiles of fibroblasts influence MM sensitivity to the combined vemurafenib and cobimetinib treatment and highlight the need for 3D in vitro cocultures representing the complex crosstalk between melanoma and CAFs during preclinical studies of drugs.

Highlights

The first-line therapy for patients with metastatic melanoma (MM) with a BRAF V600E mutation is a BRAF inhibitor (BRAFi) such as vemurafenib [1]
This model made it possible to observe the effective inhibition of BRAFi on the proliferation of MM cells in coculture within the 3D models: SK-MEL-2 cells were sensitive to BRAFi in the 3D coculture, while they were resistant in the 2D configuration, highlighting that paracrine signaling in coculture models and extracellular matrix components are key points for drug efficiency
For the SK-MEL-2 cells, data revealed no effect of BRAFi alone, but the drug combination was efficient for inhibiting cell proliferation with a MEK inhibitor (MEKi) dose-dependent effect: SK-MEL-2 metabolic activity was found to be 95% lower than for untreated cells

Summary

Introduction

The first-line therapy for patients with metastatic melanoma (MM) with a BRAF V600E mutation is a BRAF inhibitor (BRAFi) such as vemurafenib [1]. We recently developed a 3D human dermal equivalent (dermal fibroblasts embedded in a collagen gel) to mimic the cutaneous environment for MM cells (SK-MEL-28 BRAF V600E mutant and SK-MEL-2 BRAF wt, NRAS Q61R) This model made it possible to observe the effective inhibition of BRAFi on the proliferation of MM cells (wild-type or mutated BRAF V600E) in coculture within the 3D models: SK-MEL-2 cells were sensitive to BRAFi in the 3D coculture, while they were resistant in the 2D configuration, highlighting that paracrine signaling in coculture models and extracellular matrix components are key points for drug efficiency. This shows the value of studying MM sensitivity to targeted first-line therapy treatment, the combination BRAFi + MEKi

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