Uveal Melanoma Cells Elicit Retinal Pericyte Phenotypical and Biochemical Changes in an in Vitro Model of Coculture.

Carmelina Daniela Anfuso,Alfio Distefano,Giovanni Giurdanella,Anna Longo,Mario Salmeri,Angela Maria Amorini,Cesarina Giallongo,Gabriella Lupo,Guido Zanghì

doi:10.3390/ijms21155557

Abstract

Vascular pericytes are an important cellular component in the tumor microenvironment, however, their role in supporting cancer invasion is poorly understood. We hypothesized that PDGF-BB could be involved in the transition of human retinal pericytes (HRPC) in cancer-activated fibroblasts (CAF), induced by the 92.1 uveal melanoma (UM) cell line. In our model system, HRPC were conditioned by co-culturing with 92.1UM for 6 days (cHRPC), in the presence or absence of imatinib, to block PDGF receptor-β (PDGFRβ). The effects of the treatments were tested by wound healing assay, proliferation assay, RT-PCR, high-content screening, Western blot analysis, and invasion assay. Results showed profound changes in cHRPC shape, with increased proliferation and motility, reduction of NG2 and increase of TGF-β1, α-SMA, vimentin, and FSP-1 protein levels, modulation of PDGF isoform mRNA levels, phospho-PDGFRβ, and PDGFRβ, as well as phospho-STAT3 increases. A reduction of IL-1β and IFNγ and an increase in TNFα, IL10, and TGF-β1, CXCL11, CCL18, and VEGF mRNA in cHRPC were found. Imatinib was effective in preventing all the 92.1UM-induced changes. Moreover, cHRPC elicited a significant increase of 92.1UM cell invasion and active MMP9 protein levels. Our data suggest that retinal microvascular pericytes could promote 92.1UM growth through the acquisition of the CAF phenotype.

Highlights

We first aimed to determine if (i) pericyte-cancer-activated fibroblasts (CAF) transition would occur in response to the interaction with 92.1UM uveal melanoma in an in vitro model of coculture
(ii) in order to test the role of PDGF-BB in 92.1UM-induced pericyte-CAF transition, pericytes were cocultured in the presence of 5 μM imatinib (c/IMAHRPC), a tyrosine kinase inhibitor (TKI) used as an inhibitor of PDGF receptors (PDGFR) signaling (Figure 1A)
Western blot analysis of the typical pericyte markers (α-SMA and neural/glial proteoglycan antigen 2 (NG2)) showed a significant reduction of the NG2 protein level of about 40% in cHRPC; this effect was eliminated by 5 μM of imatinib in coculture medium that restored the NG2 protein to the control levels (Figure 1C,D)

Summary

Introduction

The tumor microenvironment (TME) is constituted by both the extracellular matrix and multiple infiltrating cell components including smooth muscle cells, pericytes, endothelial cells, mesenchymal stem cells, a variety of immune cells and cancer-associated fibroblasts (CAFs) [1,2,3]. Pericytes are mainly described as “mural” cells, specialized in vascular homeostasis, and they participate in angiogenesis by regulating the remodeling and stabilization of the blood–brain and blood–retina barriers [8]. Vascular pericytes are considered an important source of CAFs, the most critical and abundant components of the tumor mesenchyme [5,10,11]

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Results

Discussion

Conclusion