Lung Tropism Of Extracellular Vesicles Derived From Salivary Adenoid Cystic Carcinoma Cells

Objectives: Salivary adenoid cystic carcinoma(SACC)is one of the most frequent malignancy in the oral and maxillofacial region.The lung is the most common organ in distant metastasis,accounting for about 74.5%.In recent years,it is widely concerned that extracellular vesicles(EVs)carry a variety of bioactive substances,such as proteins,nucleic acids and lipids,which can play a role in a variety of physiological and pathological processes,especially in tumor progression.The purpose of this study is to explore whether SACC EVs show different distribution in different organs;to verify whether SACC EVs can carry chemokine receptor CXCR4;to test whether CXCL12 /CXCR4 biological axis plays a role in the process of organ tropism of SACC EVs and its related mechanism.Materials and Methods: SACC-LM/83 derived small extracellular vesicles(sEVs)were extracted from SACC-LM/83 cell conditioned medium(CM)by differential centrifugation combined with ultracentrifugation.Under transmission electron microscope(TEM),the typical cup or disc-shaped and bilayer membrane structure of sEVs was observed.Using nanoparticle tracking analysis to detect the particle size distribution of SACC-LM/83 sEVs.WB was used to identify the protein expression of SACC-LM/83 sEVs,including CD63,CD9,HSP70 and CALNEXIN.Then,we selected 5-6 week-old C57BL/6 female mice to explore the distribution of SACCLM/83 sEVs in different organs.To determine the injection dose of sEVs in each mouse,BCA assay was used to detect the concentration of sEVs at first.sEVs was labeled with PKH67,and then injected into mice via tail vein.The control group was injected with PKH67 diluted with PBS.The mice were killed at 6h and 24 h,respectively.The lung,liver,spleen,kidney and brain tissues were harvested,fixed with 4% paraformaldehyde,dehydrated with 30% sucrose solution,and embedded with OCT.The fluorescence images were taken by an inverted fluorescence microscope IX71.Data were analyzed by Image Pro Plus software and quantified by Graph Pad Prism 7.0 software.We used a series of experiments to explore the expression of chemokine receptor CXCR4 in SACC-LM/83 cells and SACC-LM/83 derived sEVs,for instance,immunofluorescence staining,WB,flow cytometry and ELISA etc.Subsequently,we carried out the inhibition experiment in vivo.The PKH67 labeled sEVs was incubated with CXCR4 inhibitor AMD3100 at 4 ℃ overnight,and then injected into mice via tail vein to explore the effect of AMD3100 on the distribution of SACC-LM/83 derived sEVs in different organs.Results: 1.Differential centrifugation combined with ultracentrifugation was used to separate SACC CM to obtain sEVs.TEM showed that the morphology was typical cupliked,disc-shaped,with double-layered membrane structure.The particle size distribution of SACC-LM/83 sEVs is mostly between 50-100 nm by using nanoparticle tracking analysis.WB results showed that compared with SACC-LM/83 cells,sEVs expressed typical biomarkers CD63,CD9 and HSP70,while the CALNEXIN expression was negative.2.After PKH67 or SACC-LM/83 sEVs were injected into mice for 6 hours,sEVs distribution in different organs was examined.Compared with PKH67,SACC-LM sEVs mainly distributed in lungs and liver,lessly in spleen and kidney.No SACC-LM sEVs were detected in brain.And there was no significant difference in the distributions of SACC-83 sEVs in lung,liver and spleen.After PKH67 or SACC-LM/83 sEVs were injected into mice for 24 hours,the amount of SACC-LM sEVs in lungs was significantly higher than that in liver,spleen and other organs with a significant difference.There was no significant difference in the distributions of SACC-83 sEVs in lung,liver and spleen.Compared with PKH67 and SACC-83 sEVs,SACC-LM sEVs showed obvious lung tropism.The difference was statistically significant.3.Immunofluorescence staining,western blot,flow cytometry and ELISA examinations showed that SACC-LM/83 cells and sEVs express CXCR4.Compared with SACC-83 cells,SACC-LM sEVs carry high levels of CXCR4.The difference was statistically significant.4.Inhibitor experiments in mice showed that the amount of SACC-LM sEVs with AMD3100 treatment in lung tissue was significantly reduced,compared with the SACC-LM sEV group.This result suggests that SACC-LM sEVs lung tropism is significantly inhibited by CXCR4 inhibitor AMD3100.Conclusions:(1)sEVs derived from high lung metastasis potential SACC cells has obvious lung tropism in vivo;(2)SACC-LM/83-derived sEVs express the chemokine receptor CXCR4;(3)The CXCL12/CXCR4 axis may play a role in the lung tropism of SACC-LM sEVs.