Extracellular Vesicles Secreted By Oral Squamous Cell Carcinoma-derived Carcinoma-associated Fibroblasts Carry Lysyl Oxidase To Promote Pulmonary Fibrosis

Background:Oral squamous cell carcinoma(OSCC)is the most common epithelial malignant tumor in the oral and maxillofacial region,accounting for about 90% of oral malignancies.It often metastasizes to regional lymph nodes,and develops distant metastases at late stages.Lung is the common target organ for distant metastasis.Carcinoma-associated fibroblasts(CAFs)are one of the most important mesenchymal cells in the tumor microenvironment and play an important role in the regulation of tumor metastasis.Existing studies have confirmed that tumor-derived products can regulate metastasis,including various molecules secreted by tumors.Extracellular vesicles(EVs)mediates communication between cells and can also mediate information exchange between the primary tumor and distant organs.It is a general term for small vesicles with a lipid bilayer structure secreted by cells.Exosomes are EVs derived from the endosomal pathway.At present,we ofen use differential speed combined with ultra-high speed centrifugation to separate EVs.It is difficult to trace the subcellular origin of EVs.Therefore,the International Association of Extracellular Vesicles refers to EVs with a diameter of less than 200 nm as small EVs(sEVs).At present,studies have reported that sEVs derived from CAFs play an important role in tumorigenesis,development and metastasis,but sEVs derived from CAFs promote the fibrosis of metastatic target organs to further participate in the construction of the pre-metastasis microenvironment haven’t been reported.Objective:The purpose of this study is to clarify the effect of sEVs secreted by OSCC-derived CAFs on the lung and its molecular mechanism,and to explore ways to reverse the loss of lung tissue.Materials and Methods:Two samples of OSCC were collected clinically,and primary CAFs were isolated by trypsin combined with type I collagenase digestion.They were named CAF-S1 and CAF-S2.UM-SCC6 was used in this study as tumor cells.Then we culture cells and collect sEVs depleted cell-conditioned medium,and extract CAF sEVs and UM-SCC6 sEVs from the cell-conditioned medium by differential combined ultra-high-speed centrifugation.C57BL/6J mice were injected with sEVs from different cell through the tail vein,and lung tissues were harvested on the 3rd,7th,and 14 th day of the experimental cycle.Hematoxylin-eosin staining(HE)staining and Ashcroft score were used to investigate the effect of OSCC-derived CAF sEVs in promoting lung tissue fibrosis in vivo.The expression of LOX in lung tissue was determined by immunofluorescence staining.To inhibit s EV-promoted lung fibrosis,CAF sEVs were injected through the tail vein of C57BL/6J mice and the LOX inhibitor β-aminopropionitrile(BAPN)was injected intraperitoneally.On the 14 th day of the experiment cycle,lung tissues were collected and stained by HE and immunofluorescence to investigate the changes of lung fibrosis and the expression of LOX in lung tissues.The immunofluorescence staining method was used to determine the target cells of CAF sEVs.Tumor cell adhesion experiment was used to further illustrate the effect of CAFs sEVs on the adhesion of tumor cells after the target cells.Results:The results of CAF-S1,CAF-S2 immunofluorescence staining showed that Pan-CK was negative,indicating that CAF-S1 and CAF-S2 were non-epithelial cells,Vimentin and FSP-1 were positive,indicating that CAF-S1 and CAF-S2 were stromal cells,α-SMA and FAP were positive,indicating that both CAF-S1 and CAF-S2 expressed typical CAF cell biological markers.The conditioned medium from CAF-S1 and CAF-S2 was collected,and sEVs was extracted by differential combined ultra-high speed centrifugation.Western blot results show that sEVs expressed CD9,CD63,CD81,HSP70 and don’t express Calnexin.The transmission electron microscopy pictures show that sEVs have typical cup-shaped structure.C57BL/6J mice were divided into four experimental groups.Mice injected with PBS,UM-SCC6-sEVs,CAF-S1-sEVs and CAF-S2-sEVs,injecting PBS through tail vein were taken as Control group.The mice were sacrificed on the 3rd,7th and 14 th day,then we took the lung tissues for HE staining,and the degree of pulmonary fibrosis was investigated by Ashcroft score.The results showed that the degree of pulmonary fibrosis in the CAF-sEVs group was significantly higher than that in the Control group(* p < 0.05,* p < 0.01),but there was no significant difference between the UM-SCC6-sEVs group and the Control group(p > 0.05).After 7 days of injection,the degree of fibrosis in CAF sEVs group was higher than that in Control group and UM-SCC6-sEVs group,but there was no significant difference between UM-SCC6-sEVs group and Control group.Fourteen days after injection,the degree of pulmonary fibrosis in CAF-sEVs group was significantly higher than that in Control group,and higher than that in UM-SCC6-sEVs group(* p < 0.05,* p < 0.001).The degree of pulmonary fibrosis in UM-SCC6-sEVs group was higher than that in Control group(* p < 0.05).These results suggest that both UM-SCC6 sEVs and CAF sEVs can promote pulmonary fibrosis,but the ability of CAF sEVs to promote pulmonary fibrosis is higher than that of OSCC sEVs.The results of immunofluorescence staining showed that 14 days after injection,the expression of LOX in lung tissues of CAF sEVs group was higher than that of Control group and UM-SCC6 sEVs group(* p < 0.05,** p < 0.01,*** p < 0.001),and the expression of LOX in lung tissues of UM-SCC6 sEVs group was also higher than that of Control group(* p < 0.05).These results suggest that LOX may play a role in sEVs promoting pulmonary fibrosis.In order to further study the mechanism of CAF sEVs promoting OSCC pulmonary fibrosis,C57BL/6J mice of the same age were injected with the same amount of CAF sEVs,and intraperitoneal injected with LOX inhibitor BAPN.The results showed that after two weeks of experiment,the expression of LOX in lung tissues of mice decreased significantly,and the degree of pulmonary fibrosis also decreased,suggesting that LOX is the key factor of CAF sEVs promoting pulmonary fibrosis,which can be inhibited by BAPN.CAF sEVs were labeled with green fluorescent dye PKH67 and injected into C57BL/6J mice through tail vein.24 hours later,lung tissues were taken for immunofluorescence staining.It was found that CAF sEVs was caught by FSP-1 positive cells and part of the sEVs was co-located with type I collagen.We treated lung fibroblasts(LFs),with CAF-sEVs in vitro.After treating 40 min,we vaccinated UM-SCC6 cells into the treated LFs.The results showed that the number of tumor cells adhered to LFs after CAF-sEVs treatment was significantly higher than that in the blank control group(* p < 0.05,* p < 0.01,* p < 0.001).Conclusions:1.The LOX carried by CAF-sEVs from OSCC can promote lung fibrosis.2.BAPN can reduce the ability of CAF sEVs to promote pulmonary fibrosis by inhibiting LOX activity.3.CAF sEVs can target LFs and improve its ability to promote tumor cell adhesion.