Avagliano, Angelica (2020) Development and analysis of a stromal microenvironment experimental model containing proto-myofibroblasts: a new tool to study the crosstalk between stromal and melanoma cells. [Tesi di dottorato]

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Solid tumours are characterized by tumour microenvironment, composed of both neoplastic cells and a variety of tissue resident and recruited non-cancerous stromal cells, secreted factors, extracellular matrix (ECM) proteins, blood and lymphatic tumour vessels. The stromal compartment of tumour microenvironment, consisting also of fibroblasts, can favour or impede tumour growth and thus it has been recognized as an active and essential component of all solid tumours. Melanoma is one of the most aggressive solid tumours, composed of not only cancer cells but also the supporting stroma, which includes fibroblasts, fibroblast aggregates and cancer associated fibroblasts (CAFs). These fibroblast compartments affect differently melanoma growth during its distinct stages. Therefore, in this work, we have in vitro developed an experimental system resembling a part of the stromal microenvironment, represented by inactivated fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, namely spheroids. We have generated from human primary cutaneous myofibroblasts, isolated from normal skin, proto-myofibroblasts, which represent the intermediate cell type during fibroblast to myofibroblast differentiation. In particular, we have obtained proto-myofibroblasts from myofibroblast spheroids transferred to cell culture dishes and reverted to adhesion growth after 216 h of 3D culture. Likewise cells found in vivo, the proto-myofibroblasts of our experimental system are characterized by very low levels of α-smooth muscle actin (α-SMA) and cyclooxygenase-2 (COX-2) proteins, a cytoskeleton structure with less developed stress fibers, and increased proliferative and migratory capabilities. The analysis of the crosstalk between A375 or A2058 melanoma cell lines and fibroblasts in different stages of activation is the remarkable novelty of this work. In particular, for the first time, our study showed the anti-tumour role exerted by proto-myofibroblasts in vitro. Furthermore, the secretome analysis of proto-myofibroblast- and melanoma cell-conditioned media suggests that the down-regulation of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumour activity. This study has also showed that the viability, outgrowth and migration of proto-myofibroblasts from spheroids are not influenced by melanoma cell-conditioned media. Furthermore, the conditioned medium of proto-myofibroblasts does not affect the viability of both BJ-5ta cells and myofibroblasts. In the light of this evidence, it is possible to assume that proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.

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