Development of a Stromal Microenvironment Experimental Model Containing Proto-Myofibroblast Like Cells and Analysis of Its Crosstalk with Melanoma Cells: A New Tool to Potentiate and Stabilize Tumor Suppressor Phenotype of Dermal Myofibroblasts.

Angelica Avagliano,Gaetano Calì,Stefania Masone,Alessandro Arcucci,Stefania Montagnani,Antonino Iaccarino,Maria Rosaria Ruocco,Mariorosario Masullo,Federica Aliotta,Rosarita Nasso,Claudio Bellevicine,Giuseppe Fiume,Gennaro Sanità

doi:10.3390/cells8111435

Abstract

Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression. The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages. In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids. In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts. The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of α-smooth muscle actin (α-SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts. Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts. The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium. An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity. Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids. Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts. Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.

Highlights

Melanoma is one of the most metastasizing and drug resistant solid tumors and is characterized by an impressive plasticity influenced by the interaction between cancer cells and the stromal microenvironment that regulates melanoma growth, progression and therapeutic response [1]
After 216 h of 3D culture, we deepened the phenotype study of cells from spheroids transferred to cell culture dishes and reverted to adhesion growth to compare the levels of activation, inflammation and mesenchymal markers with respect to myofibroblasts grown as monolayer and spheroids
We demonstrated that spheroids collected at 72 and 216 h show an impressive and comparable reduction of α-smooth muscle actin (α-SMA) levels with respect to the myofibroblast monolayer [26]

Summary

Introduction

Melanoma is one of the most metastasizing and drug resistant solid tumors and is characterized by an impressive plasticity influenced by the interaction between cancer cells and the stromal microenvironment that regulates melanoma growth, progression and therapeutic response [1]. Fibroblasts belong to a heterogeneous population of mesenchymal cells characterized by an exceptional phenotypic plasticity and the capability to secrete large amounts of soluble factors, extracellular matrix (ECM) components, and extracellular vesicles. These cells can regulate the physiological turnover of the ECM, but they can participate in wound healing and senescence [6]. In many fibro-contractive diseases and even in developing and specialized normal tissues, mechanical stress and specific factors can induce the expression of α-SMA and the differentiation of proto-myofibroblasts into myofibroblasts. Myofibroblasts represent activated fibroblasts, are characterized by a cytoskeleton with extensively developed stress fibers and α-SMA expression, and are devoted to contraction and ECM synthesis during wound remodeling and contraction [7,10,11]

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Discussion

Conclusion