The impact of cancer related fibroblasts-derived exosomes on promoting the invasion and metastasis of osteosarcoma

Abstract

Objective To investigate the impact of cancer related fibroblasts-derived exosomes on promoting invasion and metastasis of osteosarcoma. Methods The osteosarcoma cell line Saos-2 was used to isolate cancer-associated fibroblasts (CAFs). Exosomes extract from the supernatants of CAFs and the morphology of exosomes was identified by electron microscopy. The expression of exosome-specific marker protein β-actin, CD63 were detected by Western blot. Cell scratches and Transwell chamber experiments were used to observe the effects of exosomes on the migration and invasion of Saos-2 cells. Twelve female BALB/c-nu nude mice of 5 weeks old were randomly divided into exosomes and control groups, with 6 in each group. The two groups were subcutaneously implanted by subcutaneous inoculation of human osteosarcoma Saos-2 cells and Matrigel mixed suspension to establish a nude mouse osteosarcoma model. Tail vein injection was performed after modeling. The exosome group was injected with a concentration of 0.1 μg/μL of exosome suspension through the tail vein, 30 μL per nude mouse, once a week, for a total of 6 injections. Mice in the control group were injected with normal saline at the same dose. After the first injection, the tumor formation of the nude mice was observed daily. After one week of tumor implantation, the nude mice had a subcutaneous lesion on the right side. The longest diameter and the largest vertical orthogonal diameter of the two groups were measured, and the tumor volume was calculated, and then measured once a week for a total of 6 weeks. After the last measurement was completed, euthanasia was performed, and the lump was completely removed and weighed. The lung tissue of nude mice was taken for HE staining to observe the lung metastasis of the two groups. Results Under microscope, the CAFs showed long fusiform shape and disordered arrangement. Immunohistochemical staining showed that the expression of α-SMA protein in CAFs was positive, suggesting that CAFs were successfully extracted. Exosomes were observed by Electron microscopy as the round and elliptical membranous vesicular structure of 70-100 nm. The results of Western blot showed that CD63 protein was highly expressed in exosomes, and β-actin protein expression was weak, suggesting successful isolation of CAFs derived from exosomes. The scratch test and invasion test showed that the migration distance, migration rate and number of invasive cells in exosomes group were (0.19±0.02) mm, 0.81%±0.11%, and (53.67±5.69), respectively, which were higher than those in control group (0.81±0.11) mm, 0.32%±0.11%, and (31.67±8.08), with statistically significant differences (t=6.209, 6.209, and 3.856, all P values 0.05). The third to sixth measurement of tumor volume were higher in the exosomes than in the control group (t=4.697, 4.825, 3.516, 2.706, all P values<0.05). After six measurements, the nude mice were sacrificed and the tumors were removed. The tumor mass of the exosomes group (1.62±0.29) g was greater than that of the control group (1.00±0.43) g, and the difference was statistically significant (t=2.929, P<0.05). HE staining of lung tissue sections showed that lung metastasis occurred in both nude mice. The number of lung tissue metastases in the Exo group was 6.63(3, 17), which was significantly higher than that in the control group 1.17(0, 3). The difference was statistically significant (W=36.000, P<0.01). Conclusions CAFs-derived exosomes can promote the migration and invasion of osteosarcoma cell line Saos-2, and facilitate the osteosarcoma growth and lung metastasis. Key words: Osteosarcoma; Exosomes; Cancer-associated fibroblasts; Neoplasm metastasis; Tumor microenvironment