Carcinoma-associated Fibroblasts-derived Extracellular Vesicles Carrying IGF-1 Promote The Proliferation Of Oral Squamous Cell Carcinoma Cells

BackgroundOral squamous cell carcinoma(Oral squamous cell carcinoma,OSCC)refers to a type of malignant tumors that occur in the oral and maxillofacial region and are dominated by squamous cells proliferation,accounting for about 90% of the incidence of oral and maxillofacial malignancies.With the advancement of medical technology,the treatment of other malignant tumors has made significant progress,but OSCC is still high in morbidity and mortality due to its rapid growth,and its biological characteristics of metastasis and recurrence.This is directly related to the microenvironment.Tumor microenvironment(TME)is a complex microenvironment composed of parenchymal tumors and various surrounding cells,which play an important role in the malignant progression of tumors.Recent studies have found that cancer-associated fibroblasts(CAFs)are the key mesenchymal cells for TME to affect tumor progression.Extracellular vesicles(EVs)is a general term for a type of tiny vesicles secreted by cells that encapsulate the contents of maternal cells.In recent years,researchers have found that EVs can transfer the contents of their encapsulation to target cells,thereby affecting the cell surface.type.According to the origin,diameter and biological characteristics of EVs,they are divided into two categories: Exosomes(Exo)with diameters ranging from 40-200 nm and Microvesicles(MVs)with diameters ranging from 100-1000 nm.After cells secrete EVs to the outside of the cell,the sizes of Exo and MVs have an overlapping range.Therefore,based on the diameter of the EVs,the group named EVs with a diameter less than Exo as small EVs(small EVs,sEVs).The purpose of this study was to clarify the effect of sEVs secreted by OSCCderived CAFs on tumor cell proliferation,and to explore its main molecular mechanisms.Materials and methodsOSCC cell line uses UM-SCC6 and CAL-27 cells.Primary CAFs and normal fibroblasts(Normal fibroblasts,NF)were isolated from two fresh OSCC tissues and normal gingival tissue identified by immunofluorescence(Immunofluorescence,IF)and Western blot(WB).The OSCC cell lines UM-SCC6 and CAL-27 were induced to detect the proliferation ability of the OSCC cell lines UM-SCC6 and CAL-27 using conditioned medium(CM)of CAFs and NF.The sEVs in CAFs/NF CM were obtained by differential ultracentrifugation.Transmission electron microscopy(TEM)was used to identify the size and shape of sEVs and WB to identify their markers.After labeling CAF sEVs with PKH67,observe the uptake of CAF sEVs by tumor cells.Compare the effects of CAF CM(CAF sEVs-del CM)with sEVs removed and CAF CM on tumor cell proliferation,and detect the proliferation and clonal strength of tumor cells induced by CAFs/NF sEVs.WB detects the activation intensity of downstream signaling pathways after CAF sEVs act on tumor cells.According to the results of the protein chip in the laboratory,the expression of IGF-1 in CAFs,NF,UM-SCC6 and CAL-27 cells were detected from protein and m RNA levels,and IGF-1 in CAF sEVs-del CM and sEVs were further detected the level of expression.Transient transfection downregulated the expression of IGF-1 in CAFs.Detect the degree of down-regulation of IGF-1 in cells and sEVs.Collect the CAF CM that down-regulates IGF-1 to induce tumor cells and observe the effect on proliferation and cloning.Collect sEVs in CAF CM that down-regulate IGF-1 and then induce tumor cells to observe the effect on proliferation and cloning.WB detects the activation of downstream signaling pathways in CAF CM that down-regulates IGF-1 after sEVs process tumor cells.Results1.OSCC-derived CAFs can promote the proliferation of tumor cells.After the identification of the primary CAFs and NF we extracted,we found that pan-cytokeratin(pan-CK)was negative in both CAFs and NF,vimentin(Vimentin,Vim)and fibroblast-specific protein 1(Fibroblast specific protein 1,FSP1)is positive in both CAFs and NF,while fibroblast activation protein(FAP)and α-Smooth muscle actin(α-SMA)are only found in CAFs and NF.CAFs were positive.WB results showed that CAFs contained Vim,FSP-1,FAP and α-SMA,while NF contained only Vim and FSP-1,and basically did not contain FAP and α-SMA.We used CAF/NF CM to induce UMSCC6 and CAL-27 and found that the ability of CAF CM to promote tumor cell proliferation was significantly higher than that of NF CM group.2.CAF sEVs can promote the proliferation of tumor cells.CAFs/NF sEVs were extracted by differential ultracentrifugation,and TEM observation revealed that they had a cup-shaped structure.NTA(nanoparticle tracking analysis)results found that the particle size of sEVs is about 70 nm.WB detection revealed that both CAFs and CAF sEVs expressed positive markers of sEVs such as CD63,CD9 and HSP70,while CAF sEVs did not express Calnexin,a negative marker of sEVs.Uptake experiments show that CAF sEVs can be taken up by UM-SCC6 and CAL-27,and sEVs are located around the nucleus.After CAF CM group and CAF sEVs-del CM(soluble factor)group induced UM-SCC6 and CAL-27,CCK8 detected the proliferation ability and found that the proliferation ability of CAF CM group was enhanced.After CAFs/NF sEVs induced UM-SCC6 and CAL-27,the results of CCK8 and plate cloning experiments showed that the proliferation and cloning ability of CAF sEVs group was significantly higher than that of NF sEVs.WB detected the activation of downstream AKT and ERK1/2 pathways after CAFs/NF sEVs induced UM-SCC6 and CAL-27,and found that the activation intensity of P-AKT and P-ERK1/2 in CAF sEVs group was significantly higher than that in NF sEVs.3.CAF sEVs carry IGF-1 and are highly expressed.According to the early protein chip results of our research group,we found that IGF-1 is highly expressed in CAF sEVs.The q RT-PCR method was used to detect the level of IGF-1 m RNA in CAFs,NF,UM-SCC6 and CAL-27.The results showed that IGF-1 in CAFs was significantly higher than that in NF,UM-SCC6 and CAL-27.WB detection results showed that the level of IGF-1 in CAFs was higher than that of NF,UM-SCC6 and CAL-27;IGF-1 was basically not expressed in sEVs-del CM;IGF-1expression was significantly higher in CAF sEVs For NF,UM-SCC6 and CAL-27 sEVs.4.The IGF-1 carried by CAF sEVs promotes tumor cell proliferation through AKT and ERK1/2 signaling pathways.We used transient transfection technology to down-regulate the expression of IGF-1 in CAFs.q RT-PCR and WB were used to detect the expression level of IGF-1 in CAFs at m RNA and protein levels.IGF-1 in the si-groups Compared with the NC group,the expression decreased significantly.WB detected the expression of IGF-1 in CAF sEVs.The expression of IGF-1 in the si-groups was lower than that in the NC group.After down-regulating IGF-1,CAF CM induced UM-SCC6 and CAL-27,CCK8 and plate cloning experiments showed that the proliferation and Number of cloned balls of NC group were higher than si-groups.The CAF sEVs that down-regulated IGF-1 were collected to induce UM-SCC6 and CAL-27,CCK8 and plate cloning experiments.The results showed that the proliferation and Number of cloned balls of the NC group was higher than that of the si-groups.CAF sEVs with IGF-1 down-regulated were used to induce UM-SCC6 and CAL-27,and WB was used to detect the AKT and ERK1/2signaling pathways.The results showed that compared with the NC group,the expression levels of P-AKT and P-ERK1/2 in the si-groups decreased.Conclusions1.OSCC-derived CAFs and their secreted sEVs have a significant ability to promote tumor cell proliferation.2.The IGF-1 secreted by OSCC-derived CAFs can promote the proliferation of tumor cells,and the IGF-1 secreted by CAFs is mainly carried by sEVs.3.IGF-1 carried by OSCC-derived CAF sEVs is a key factor that promotes tumor cell proliferation,and promotes tumor cell proliferation through AKT and ERK1/2 pathways.