Regulation of urokinase receptor (uPAR) cleavage by cancer associated fibroblasts (CAFs) in oral cancer

Abstract

Oral squamous cell carcinoma (OSCC) is one of the frequently diagnosed type of oral cancers and is a leading cause of cancer associated mortality and morbidity worldwide. Cancer associated fibroblasts (CAFs) are activated fibroblasts that are found in association with cancer cells. CAFs are the most abundant stromal cells in the tumor microenvironment (TME). In the TME, cell interactions mediated by different soluble factors released from both stromal cells and cancer cells play a crucial role in tumor progression and metastasis. Of these interactions, high expression of the serine protease urokinase (uPA) and its receptor (uPAR) and the uPA inhibitor (PAI-1) have all been associated in triggering invasion and metastasis. The aim of this project was to study CAFs role in regulation of uPAR cleavage. We also aimed to identify soluble factors involved in cleavage regulation. Artificial CAFs were made in vitro by activating 3T3 cells (fibroblasts) using transforming growth factor β1 (TGF-β1) and conditioned media (CM) from AT84 cells (CM-uPAR and CM-EV). CMs from activated fibroblasts were harvested and used to treat OSCC cells overexpressing uPAR. For this, we optimized a culture medium that enabled us to activate fibroblasts in a controlled manner by supplementing the basal medium (RPMI) with low FBS (0.5%) and ITS (1%). Flp-In 3T3 cells treated with TGF-β1 showed high expression α-SMA and had elongated shape, which is a characteristic morphology of activated fibroblasts. However, Flp-In 3T3 cells treated with CM-uPAR and CM-EV from AT84 cells did not show activated phenotype. AT84-uPAR cells treated with CM prepared from TGF-β1 treated Flp-In 3T3 cells (CM-Flp¬+) revealed significantly (p=0.0005) higher full-length uPAR compared with CM-Flp÷ treated cells. Gel-zymography analysis of CM-Flp+ also exhibited the presence of high PAI-1 and matrix metalloproteinase 2 (MMP2). Thus, the detection of full-length uPAR might be due to the high PAI-1 expression that has a function of scavenging and inhibiting uPA activity as evidenced by low level of uPA in the CM-Flp+. Immunohistochemistry was also used to study the relative CAFs infiltration in different uPAR expressing mouse tongue tumor sections. The immunoratio analysis revealed high CAFs infiltration in high uPAR expressing tongue tumor sections. Together, these findings suggest the regulatory interplay between CAFs and uPAR expression in OSCC. These findings, however, warrant further investigation using more functional assays that illustrate this interplay between CAFs and uPAR.