Establishment Of Oral Squamous Cell Carcinoma Cell Line WU-TSC-1 And Heterogeneity Of Cancerassociated Fibroblasts

Part Ⅰ Establishment and identification of oral squamous cell carcinoma cell line WU-TSC-1ObjectiveWe aim to establish a new human oral squamous cell carcinoma（OSCC）cell line WU-TSC-1 and characterize it as a cell model for further experimental studies Materials and Methods1.9 specimens were collected after surgical resection from radiotherapy and chemotherapy treatment-naive patients with correctly diagnosed primary OSCC at Hospital of Stomatology Wuhan University.The primary cells were obtained by enzymatic digestion and block culture methods.2.Short tandem repeat（STR）analysis of genomic DNA was conducted to determine the cell type and whether there was cross-contamination of other cells.By phase contrast microscopy,cell morphology and growth characteristics were observed,bacterial and fungal contamination were also monitored,and mycoplasma contamination was detected by kits.3.CCK8（Cell Counting Kit-8）proliferation experiment was used to calculate the doubling time of cultured cells.Concanavalin A（Con A）was used to detect its proagglutination function.4.The expressions of HNSCC markers were detected by immunohistochemical staining.Chromosomal karyotype was analysed after Giemsa staining.5.The stemness of cells was detected by in vitro spheroidization assay.The tumorigenic and lymph node metastasis abilities of these cells in vivo were detected by orthotopic transplantation and cervical lymph node metastasis assay in immunodeficient mice.6.Immunohistochemical staining was used to deteck the expressions of PD-L1 in primary tumor,transplanted tumor and cultured cells.Results1.Hematoxylin-eosin staining（HE）of the primary tumor showed well-differentiated OSCC with intercellular bridges and lamellar keratinization（keratin beads）in the center of the cancer nest.Immunohistochemical staining showed positive nuclear staining for Ki-67（17%）,Cyclin D1（14%）and P53（39%）.P16 and HPV16/18 staining results were negative.2.By primary cell cultivation of the block from human OSCC,epithelioid cell adherence was observed in one of the specimens.Cells with polygonal or square morphology were acquired and the characterisrics of obtained cells which can be stably passaged in vitro were consistent with the primary foci.These cells were free of bacterial,fungal and mycoplasma contamination.The analysis of 19 loci and one sex-determining locus（Amelogenin）by STR proved the human origin and specificity of these cells in vitro,as well as no cross-contamination by other cells,and the cell was named WU-TSC-1.3.The CCK8 experiment showed that the doubling time of WU-TSC-1 was 51.15 hours.Con A agglutination experiment showed that few fibroblasts gathered when the concentration of Con A was 100μg/m L,and agglutination reaction was observed in the WU-TSC-1 group when the concentration of Con A was 12.5μg/m L.4.Immunohistochemical staining showed that keratin CK5/6（61%）expression of WU-TSC-1 was positive in cytoplasma,expressions of Cyclin D1（17%）and P53（98%）were positive in nuclear.Karyotype analysis showed non-diploid chromosomes with large differences in the number and morphology.5.The spheroidization rate of WU-TSC-1 cells in vitro was 0.7%.In situ tumor formation rate in nude mice was 100%,and the rate of cervical lymph node metastasis was 25%.6.Expressions of PD-L1 in primary tumor,transplanted tumor and cultured cells were negative（TPS<1%）.ConclusionWU-TSC-1 was a stable and specific HPV-negative tongue squamous cell carcinoma cell line with in vivo tumorigenic and metastatic abilities,and may be innate immune tolerant to anti-PD-1/PD-L1 immunotherapy.Part Ⅱ Effects of Cancer-associated fibroblasts on the function of oral squamous cell carcinoma in vitroObjectiveCancer-associated fibroblasts（CAFs）are one of the core cellular components in tumor microenvironment（TME）,CAFs have complex roles in the progression of OSCC.In this part,we will mainly explore the effects of CAFs on the proliferation and invasion ability of OSCC in vitro.Materials and Methods1.Two kinds of fibroblasts were isolated and cultivated from 9 pairs of OSCC and adjacent（>1cm）normal tissues by enzymatic digestion.Expressions and trends of fibroblasts markers α-smooth muscle actin（α-SMA）,Vimentin and Fibroblast activation protein（FAP）were analyzed in the extracted fibroblasts.Fibroblasts used in subsequent experiments were passages 2-8.2.CCK8 assay and colony formation experiment were used to detect the proliferation activity of CAFs and NFs and their effects on the proliferation of OSCC.3.CAFs and fibroblasts in adjacent normal tissue（Normal fibroblasts,NFs）were cocultured with OSCC cell lines CAL27 and WU-TSC-1 in Transwell chambers,respectively,the invasion ability of CAL27 and WU-TSC-1 cells was observed to verify whether CAFs regulated the invasion of OSCC cells.4.Exosomes in the conditioned medium of CAFs and NFs cells were isolated and used to coculture CAL27 and WU-TSC-1 cells and the invasion abilities of OSCC cells were detected.Results1.Fibroblasts derived from OSCC tissue and adjacent normal tissue are all spindleshaped and there was no significant difference in appearance.Immunofluorescence staining showed that both CAFs and NFs expressed α-SMA,FAP and Vimentin in cytoplasm.Western blotting results showed that,compared with corresponding NFs,the expression of α-SMA was up-regulated in all CAFs,which was 5.69±3.42 times that of NFs（P<0.001）,and there were no consistent difference in the expressions of FAP and Vimentin.2.The CCK8 assay showed that the proliferation activity of CAFs increased by 42% at 96 h compared with that of NFs（P<0.001）;The colony formation experiment showed that the number of OSCC clones induced by CAFs conditioned medium increased by 86% after 7 days compared with that of NFs（P<0.05）.3.Transwell experiments showed that the promoting effect of CAFs on CAL27 invasion was significantly lower than that of NFs,which decreased by 59%（P<0.01）.Experiments with WU-TSC-1 and its same patient-derived fibroblasts yielded consistent trends,the invasion promoting role of CAFs on WU-TSC-1 decreased by 36%（P<0.01）.4.BCA（Bicinchoninic Acid Assay）protein quantification method showed that the amount of exosomes secreted by NFs was 1.25 times that of CAFs（P<0.05）.Compared with exosomes derived from NFs,the effect of exosomes derived from CAFs on promoting OSCC invasion decreased by 58%（P<0.001）.ConclusionCAFs in OSCC had stronger ability to promote tumor proliferation and inhibit tumor invasion than NFs.Part Ⅲ Prediction of potential molecules of CAFs inhibiting invasion of OSCC based on highthroughput sequencingObjectiveTo preliminary analyze the differentially expressed genes between cancerassociated fibroblasts（CAFs）and normal fibroblasts adjacent to cancer（NFs）.Materials and Methods1.Transcriptome high-throughput sequencing（m RNA-sequence）was applied to detect differentially expressed m RNAs between CAFs and NFs,and cluster analysis was performed on differentially expressed m RNAs.2.BGI Dr.Tom system was used to carry out functional enrichment analysis and KEGG pathway enrichment analysis of differentially expressed genes to screen signal molecules and pathways regarding invasion for follow-up research.3.The expressions differences of some chemokines in CAFs and NFs were detected by RT-q PCR.Results1.The sequencing results showed that each sample produced an average of 1.16 G data.The Pearson correlation coefficient of all m RNAs expressions between NFs and CAFs samples showed that the correlation between 2 samples was 93.5%.The screening criteria were set as log2FC≥1 and P value<0.05.Compared with NFs,there were 386 m RNAs up-regulated and 542 m RNAs down-regulated in CAFs.2.GO functional enrichment analysis and KEGG pathway analysis showed that the up-regulated m RNAs in CAFs were mainly concentrated in the signaling pathways related to cell contraction and cytoskeleton regulation,such as vascular smooth muscle contraction and c GMP-PKG signaling pathway;Down-regulated m RNAs in CAFs were mainly enriched in cancer development and metabolism-related signaling pathways,such as cytokine-cytokine receptor interaction,PI3K-Akt signaling pathway.3.RT-q PCR showed that the m RNA expressions of chemokines CXCL-1 and CXCL-2 decreased by 81.69%±11.25% and 71.46%±29.33% in CAFs compared with NFs,respectively.ConclusionCAFs exhibited significant myofibroblast-related properties and may be involved in suppressing cell chemotaxis in tumor microenvironment.