Construction Of A Model For The Study Of Activated Fibroblasts Promoting Angiogenesis Of Oral Squamous Cell Carcinoma And Study On The Mechanism

Tumor microenvironment is the cornerstone of tumor survival and development,and its rich blood supply provides favorable conditions for tumor growth and metastasis.Tumor stromal cells are an important part of the tumor microenvironment.During tumor development,tumor stromal cells and tumor cells establish close connections through a series of mechanisms,and provide support for tumor angiogenesis.Among the numerous tumor stromal cells,cancer-associated fibroblasts(CAFs)are the most abundant.CAFs is a type of fibroblasts considered to be activated,tumor cells can induce the transformation of normal fibroblasts to CAFs,and CAFs can also promote the growth and metastasis of tumor cells.There are various ways for CAFs to promote tumor development.Existing studies have confirmed that CAFs are widely involved in tumor development,such as cell metabolism,migration and immune escape,and play an important role in tumor angiogenesis.Long non-coding RNA is an RNA longer than 200 nucleotides that does not encode or encodes a small amount of protein.Long noncoding RNAs have been shown to play an important role in angiogenesis in a variety of tumors including head and neck squamous cell carcinoma(HNSCC),however,there is no study involving the regulation of long non-coding RNAs in CAFs on HNSCC angiogenesis.HNSCC is one of the most common malignant tumors in the head and neck,among which oral squamous cell carcinoma(OSCC)accounts for the majority.OSCC has abundant blood supply and poor prognosis.Stromal cells play an important role in the establishment of the blood supply network of OSCC.In this series of studies,based on in vitro 3D models and single-cell data,we explored the role of activated fibroblasts in angiogenesis from a new perspective of "shield machine" and "locomotive",contributing to a comprehensive understanding of tumor angiogenesis.Finally,we conducted the study on the regulation of long non-coding RNAs on the proangiogenic phenotype of OSCC-derived CAFs for the first time,and preliminarily explored the role of long non-coding RNA FOXF1 adjacent noncoding developmental regulatory RNA(FENDRR)in OSCC-derived CAFs in the angiogenesis of OSCC.We found that FENDRR can regulate the pro-angiogenic ability of OSCC-derived CAFs through the PI3K/AKT pathway,providing a theoretical basis for subsequent studies.Part.Ⅰ: The Role of Activated Fibroblasts in AngiogenesisObjectives: To investigate the role of activated fibroblasts in angiogenesis.Methods: A 2D fibroblast-human umbilical vein endothelial cell(HUVECs)co-culture model(FHCC)was established to observe the interaction between fibroblasts and HUVECs.HNSCC transcriptome data from TCGA database and single-cell data of HNSCC from the CIBERSORTx were downlowed;CIBERSORTx was used to detect the proportion of fibroblasts and vascular endothelial cells(VECs),and further explore the relationship between the proportion of fibroblasts and VECs;Data of CAFs and tumor cells from HNSCC single-cell data were extracted for heterogeneity analysis;Cell Chat software was used for intercellular communication analysis;Monocle software was used for single-cell pseudotime analysis;Immunofluorescence was used to detect the expression of VECs marker gene CD31,pericyte marker gene actin alpha 2(ACTA2)and fibroblast marker gene fibronectin in normal tissues;3D fibroblastsHUVECs indirect co-culture(FHICC)model was established to observe the effect of fibroblasts on the formation of microvessels;Suspension spheroid-forming technology was used to establish fibroblasts organoid(F-organoid)and fibroblast-HUVECs organoid(FH-organoid)to explore the possible mechanism of fibroblasts affecting microvascular formation.Results: The results of the 2D FHCC model showed a close spatial distribution relationship between fibroblasts and HUVECs,and HUVECs are distributed along the area where fibroblasts are located.TCGA data combined with CIBERSORTx analysis showed that the proportion of fibroblasts was positively correlated with the proportion of VECs;The results of intercellular communication analysis showed that there was a close interaction between v CAF,m CAF and VECs;Single-cell heterogeneity analysis showed that marker genes of pericytes and fibroblasts were highly expressed in m CAF and v CAF,respectively,and platelet-derived growth factor A(PDGFA)and its receptor platelet-derived growth factor receptor A(PDGFRA)were highly expressed in m CAF and v CAF,respectively;single-cell pseudotime analysis showed that v CAF may differentiate into m CAF and VECs;The results of immunofluorescence confirmed that both fibroblasts and pericytes co-localized with VECs in normal tissues;The results of the 3D FHICC model showed that fibroblasts can form a porous structure in Matrigel,with CD31-positive cells attached to the pores;The results of the F-organoid showed that fibroblasts are able to form channels by breaking down the matrix as they grow outward,and they also form pores at the cytosolic site after death;The results of the FH-organoid showed that vascular endothelial cells form microvessels in the area where fibroblasts pass,and vascular endothelial cells grow out close to fibroblasts.Conclusion: Activated fibroblasts can promote angiogenesis by creating channels in the matrix to form porous framework and pulling vascular endothelial cells.Part.Ⅱ: Lnc RNA FENDRR in Cancer Associated Fibroblasts Regulates the Angiogenesis of Oral Squamous Cell Carcinoma through the PI3K/AKT PathwayObjectives: To explore the role of long non-coding RNA FENDRR in OSCC-derived CAFs in the progression of OSCC,and the molecular mechanism of FENDRR regulating angiogenesis in OSCC.Methods: Immunofluorescence and Western Blotting were used for the identification of primary CAFs and normal fibroblasts extracted from OSCC tissues;high-throughput data analysis of FENDRR expression in OSCC was conducted;The expression and distribution of FENDRR in OSCC-derived CAFs and tissues were detected by QRTPCR and in situ hybridization;FENDRR in CAFs was overexpressed by lentivirus technology,and the FENDRR-overexpressed CAFs was used to construct FH-organoid to detect the change of microvascular formation ability;Tube-forming assay and transwell migration assay were used to detect the tube formation and migration ability of HUVECs co-cultured with FENDRR-overexpressed CAFs;KEGG and GSVA were used to analyze the status of PI3K/AKT pathway in OSCC tissues;Sh RNA technology was used to knock down FENDRR in normal fibroblasts cells;Western Blotting was used to detect the expression of PI3K/AKT pathway-related proteins matrix metallopeptidase 2(MMP2)and matrix metallopeptidase 9(MMP9)in normal fibroblasts with FENDRR knockdown and FENDRR-overexpressed CAFs.After cocultured with FENDRR-overexpressed CAFs,the tube-forming ability of HUVECs was detected by tube formation assay;An in vivo co-culture model was established using nude mice,and OSCC cells were mixed with FENDRR-overexpressed CAFs and control group respectively and injected into nude mice subcutaneously to form transplanted tumors;4 weeks later,tumor volume and weight were calculated,and the microvessel density of transplanted tumors in both groups was detected by immunofluorescence.Results: The results of immunofluorescence and Western Blotting showed that the CAFs and normal fibroblasts extracted from tumor tissues were in line with their respective characteristics;Gene chip data showed that the expression of FENDRR was down-regulated in the tissues of patients with OSCC compared those with normal tissues;The results of the q RT-PCR experiments confirmed that compared with normal fibroblasts,FENDRR was stably low expressed in CAFs;The results of in situ hybridization showed that FENDRR was mainly expressed in the stroma,and the level of FENDRR in normal oral mucosal stroma was significantly higher than that in OSCC tissue;The results of co-culture of CAFs and HUVECs showed that the microvascularforming ability of the FH-organoid constructed with FENDRR-overexpressed CAFs was significantly inhibited.Tube-forming assay and transwell migration assay confirm that HUVECs co-cultured with FENDRR-overexpressed CAFs have reduced tube formation and migration ability;KEGG and GSVA analysis showed that the PI3K/AKT pathway was activated in OSCC;The results of Western Blotting showed that FENDRR could inhibit the activation of PI3K/AKT pathway and the expression of MMP2 and MMP9;After using SC79 to activate PI3K/AKT the pathway,the reduction in the proangiogenic effect of CAFs caused by overexpressing FENDRR was reversed;The results of in vivo experiments showed that the tumor volume,weight and microvessel density of nude mice in the group mixed with FENDRR-overexpressed CAFs were lower than those in the control group.Conclusion: Long non-coding RNA FENDRR can regulate the pro-angiogenic phenotype of oral squamous cell carcinoma-derived carcinoma-associated fibroblasts through the PI3K/AKT pathway.