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

Nasrin Zare, Amirhosein Kefayat, Shaghayegh Haghjooy Javanmard


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 × g for 10 min, 2000 × g for 30 min, 16500 g for 30 min) and were pelleted
by ultracentrifugation (150,000 × 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.


Exosomes; hyaluronic acid; radiation

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