Evaluation of Radiation and Ammonium Lactate Effects on Hyaluronic Acid Expression as a Pro-cancerous Factor in Supernatant and Exosome Isolated from Supernatant of Primary Mouse Fibroblast Cell Culture

Abstract

Background: Previous studies show that aberrant synthesis of Hyaluronan accelerates tumor growth, angiogenesis, and metastasis. The fibroblasts are probably responsible for most of the hyaluronic acid ( HA) accumulation in tumor microenvironment after radiotherapy. Our goal is to investigate and compare radiation and lactate effects on HA levels in supernatant and exosome isolated from supernatant of primary mouse fibroblast cell culture. Methods: Fibroblast cells were prepared from skin of C57BL6 mouse. These cells were divided into three groups (no treatment, cells treated with 10 mM ammonium lactate, and irradiated cells). Then supernatant was harvested from FBS-free culture media after 48 h. Exosomes were purified by differential centrifugation (300 x g for 10 min, 2000 x g for 30 min, 16500 g for 30 min) and were pelleted by ultracentrifugation (150,000 x g for 180 min). Size of exosomes was determined using a Zetasizer. HA concentration measured using a HA ELISA Kit. Data were analyzed using one-way ANOVA. Results: There was a significant increase in HA-coated exosomes isolated from supernatants of irradiated cells compared to untreated cell and cells treated with 10 mM ammonium lactate (P < 0.001). As well, there was a significant increase in the HA concentration in the supernatants of cells treated with 10 mM ammonium lactate relative to untreated cells and irradiated cells (P < 0.05). Conclusions: It seems that routine radiation therapy leads to massive shedding of HA-coated exosomes by normal fibroblast cells and thus exosomes-HA may contribute to tumor promotion and induce of the premetastatic niche.