| Background:Gastric cancer(GC)is a highly prevalent malignant tumor in the digestive system worldwide,ranking fifth in incidence rate and fourth in mortality among malignant tumors,according to the latest global cancer burden data(Globocan 2020).In 1965,Lauren divided GC into intestinal type,diffuse type,and mixed type based on its histological structure and biological characteristics.Intestinal GC is the most important histopathological subtype of GC,and its development is a multistaged and orderly process following the Correa model,which progresses from normal mucosa to chronic non-atrophic gastritis(NAG),chronic atrophic gastritis(CAG),intestinal metaplasia(IM),gastric dysplasia(GD),and ultimately to GC.Investigating the biological characteristics of epithelial cells at different stages of intestinal GC progression is of great significance for revealing the pathogenesis,prevention,early diagnosis,and treatment of intestinal GC.Currently,with the development of single-cell sequencing technology and bioinformatics analysis methods,the heterogeneity of epithelial cells between GC and non-cancer groups at the single-cell level has been reported.However,there is no report on the differentiation and origin of epithelial cells at different stages of intestinal GC at the single-cell level.Moreover,the complex microenvironment formed by a large number of mesenchymal cells densely arranged between epithelial cells in tumor tissue plays a key role in tumor proliferation,metastasis,and drug sensitivity through interactions with epithelial cells.To date,no studies have explored the transcriptome characteristics of microenvironmental stromal cells at different stages of the development of intestinal GC at the single-cell level.Fibroblasts are the main component of microenvironment cells and play a key role in the carcinogenesis of various solid tumors,including GC.Among them,cancer-associated fibroblasts(CAF)are activated fibroblasts associated with cancer cells that promote the occurrence and development of tumors through interaction with malignant epithelial cells via a large number of secreted proteins.Collagen Triple Helix Repeat Containing Protein 1(CTHRC1)is a secreted extracellular matrix protein that participates in the occurrence and development of various types of cancer.However,at present,the role and mechanism of high CTHRC1 expression in Fibroblasts in GC are still unclear.Therefore,this study aims to use single-cell transcriptome sequencing data to construct the map of epithelial cell differentiation and microenvironment cells at different stages of intestinal GC.We will describe the origin and differentiation characteristics of epithelial cells,the dynamic changes of mesenchyme cells,transcription regulation,and the interaction between epithelial and interstitial cells at different stages of intestinal GC.Moreover,we will use human tissue specimens to verify the expression of key regulatory molecules by immunofluorescence in situ.Finally,we will conduct additional in vivo and in vitro experiments to explore the role and mechanism of high CTHRC1 expression in Fibroblasts in the occurrence and development of GC at the tissue and cell levels.This study will provide new insights into the occurrence of intestinal GC and new methods and approaches for the early diagnosis,prevention,and treatment of GC and various gastric diseases.Purposes:1.Based on single cell sequencing,construct the differentiation map of epithelial cells at different stages of intestinal GC progression,and clarify the origin,differentiation and transcriptional regulation mechanism of epithelial cells at NAG,CAG,IM and GC stages2.Based on single cell sequencing construct the transcriptome characteristics of TME cells at different stages of intestinal gastric cancer and identify the characteristics and dynamic change trend of TME cell transcriptome at each stage3.To explore the effect of high CTHRC1 expression in Fibroblasts on the function of gastric cancer cells and its mechanismMaterial and methods:Part I:Based on single cell sequencing construction of epithelial tissue differentiation map at different stages of intestinal type gastric cancer1.Single cell sequencing data acquisition and analysisSingle cell sequencing data set(GSE134520)downloaded from GEO database included 12 sequencing data from 8 patients,including 3 cases of NAG,3 cases of CAG,6 cases of IM and 1 case of GC.The data was processed using R software package(version 3.0.1)for quality control,cell grouping,and cell type identification.Harmony R package was used for eliminating batch effect.We performed Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis to investigate different cell functions and signaling pathways.Monocle 2 was utilized to analyze the origin and differentiation of various epithelial cells.We calculated active transcription factors using SCENIC(version 1.1.0)and performed copy number variation(CNV)analysis of cells using Infer CNV.2.Immunofluorescence staining verification(1)Collection of 20 cases of early intestinal-type gastric cancer with submucosal dissection treated in the Department of Gastroenterology,the First Hospital of China Medical University,between June 2020 and December 2021,including cancerous,adjacent intestinal metaplasia,and normal tissues embedded in paraffin.The cohort consists of 13 male and 7 female patients with an average age of 58±8 years.The histopathological diagnosis was based on the latest Sydney System and WHO Classification of Digestive System Tumor(4th Edition,2010).Ethics approval for this study was obtained from the Ethics Committee of the First Hospital of China Medical University,and all participants provided informed consent prior to inclusion in the study.(2)Experimental method:immunofluorescencePart II:Based on single cell sequencing construction of microenvironment cell dynamic transcriptome atlas at different stages of intestinal gastric cancer1.Single cell sequencing data acquisition and analysisThis study comprised 15 sequencing datasets obtained from 11 patients,encompassing 3 cases of NAG,3 cases of CAG,6 cases of IM,and 3 cases of intestinal GC.The data were sourced from two public databases,with two cases of gastric cancer retrieved from the Genotype and Phenotype database(db Ga P,phs001818.v2)and the remaining cases from the GEO database(GSE134520).The analysis method employed was identical to that employed in the first part.2.Immunofluorescence staining verificationThe source and detection method of human tissue specimens are the same as those in Part IPart III:Effects and potential mechanism of high CTHRC1 expression in Fibroblasts on biological function of gastric cancer cells1.Research object1.1 Paraffin-embedded tissue specimens were collected from 111 patients who underwent distal gastrectomy at the Anorectal Surgery Department of the First Hospital of China Medical University from June 2012 to June 2020.Patients who received preoperative radiotherapy and chemotherapy were excluded.The study population consisted of 77 males and 34 females,with an average age of 61±11 years.Cancerous tissues and matched normal mucosal tissues from gastric cancer patients were obtained for paraffin embedding and pathological section preparation.The histopathological diagnosis followed the latest Sydney System and WHO Classification of Digestive System Tumor(4th Edition,2010),and TNM classification was determined according to the NCCN clinical practice guidelines(2nd edition,2018).Ethics approval for this study was obtained from the Ethics Committee of the First Hospital of China Medical University,and each participant provided written informed consent.1.2 Cells:AGS and HGC27 cell lines were purchased from American Type Culture Collection(ATCC).Cancer-associated fibroblasts(CAFs)were purchased from human gastric cancer tissues were provided by Meisen CTCC2.Material and method:2.1 Detection of CTHRC1 expression in GC tissue and its clinical significanceThis study utilized immunohistochemistry to detect CTHRC1 expression in both GC and para-carcinoma tissues.To determine any differences in CTHRC1 expression between the two tissue types,the Mann-Whitney U nonparametric test was employed.Additionally,the correlation between CTHRC1 expression and the clinicopathological characteristics of GC was analyzed using the Chi-square test.Finally,to examine the relationship between CTHRC1 expression and the prognosis of GC patients,the study utilized the Kaplan-Meier method,as well as univariate and multivariate Cox regression analyses.2.2 Effect of CTHRC1 secreted by fibroblasts on the biological function of gastric cancer cells2.2.1 Detection of CTHRC1 secreted by CAF.2.2.1.1 Detection of the subcellular localization of CTHRC1 in gastric cancer tissue by double-color immunohistochemistry.double-color immunohistochemistry staining was simultaneously performed with the anti-CTHRC1 antibody(green color)and anti-SMA antibody(red color),to observe whether the expression of CTHRC1 in fibroblasts.2.2.1.2 Construction and identification of CTHRC1 stably transformed fibroblast cell lineAfter virus transfection,stable cell strains were screened 72 hours later with puromycin at a concentration of 1μg/m L.The transfection efficiency was confirmed using an inverted microscope.Concurrently,overexpression at the m RNA level was confirmed using RT-q PCR,while overexpression at the intracellular protein level was verified using Western Blot.To further verify that CAF could secrete CTHRC1 protein,ELISA was performed on the supernatant of the cell culture.2.2.2 Effect of CTHRC1 on biological function of gastric cancer cells(1)Ed U was used to detect the effect of CTHRC1 on the proliferation of gastric cancer cells(2)The effect of CTHRC1 on the migration of gastric cancer cells was detected by scratch and Transwell.(3)The effect of CTHRC1 on vascular mimicry of gastric cancer cells was detected by three-dimensional gels.2.3 Study on the mechanism of CTHRC1 affecting the biological behavior of gastric cancer cells.2.3.1 Enrichment analysis of CTHRC1 pathway in gastric cancerThe present study utilized data from the TCGA database to investigate the genes associated with CTHRC1 using the R package"edger."Furthermore,a pathway enrichment analysis was performed to identify the most pertinent pathway related to CTHRC1.2.3.2 Mechanism of CTHRC1 promoting malignant biological function of GC cellsDetect the change of biological function of gastric cancer cells stimulated by CTHRC1,and detect the expression of key protein of PI3K-AKT pathway by WB test.At the same time,we added inhibitors of the above pathways to observe whether the malignant biological behavior of tumor caused by CTHRC1 was reversed.ResultPart I:Based on single cell sequencing construction of epithelial tissue differentiation map at different stages of intestinal type gastric cancer1.A total of 33 epithelial cell subclusters were obtained using single-cell sequencing data,including pit mucous cell(Pit mucous cell,PMC),gland mucous cell(Gland mucous cell,GMC),progenitor cells(Progenitor cell,PC),neuroendocrine cells(Neuroendocrine cell),intestinal stem cells(Stem cell),Undifferentiated cell,goblet cells(Goblet cell),enterocytes(Enterocytes,Ent),and adenocarcinoma(Adenocarcinoma).The above cell showed dynamic changes in different stages of intestinal gastric cancer.2.In NAG stage,PC,as the origin cell,differentiated into PMC and GMC cells through undifferentiated cells;These processes may be regulated by transcription factors TAGLN2 and TAF7,respectively.3.In CAG stage,the differentiation relationship of epithelial cells is similar to that of NAG stage,but the ability of PC cells to differentiate into GMC cells is significantly enhanced,which may be regulated by the transcription factors SPDEF and KLF10.4.In IM stage,PC and GMC eventually differentiate into terminal mature enterocytes cells through different cluster intestinal stem cells,but the differentiation ability of PC is significantly higher than that of GMC.The active transcription factors regulating the above two differentiation pathways are significantly different.TTR~+cells are the enteroendocrine progenitors,which mainly differentiate into goblet cells.5.In early stage of intestinal type gastric cancer,the cancer epithelial cells are mainly differentiated from PC cells.At this stage,PC cells specifically express KIAA0101 and PRAP1,and the transcription factor SIRT6 plays an important role in the above differentiation process.Part II:Based on single cell sequencing construction of microenvironment cell atlas at different stages of intestinal gastric cancer1.A total of 15 mesenchymal cell subclusters were obtained using single-cell sequencing data,mainly including T cells,B cells,macrophages,mast cells,fibroblasts,endothelial cells,and pericytes.The above cells showed significant dynamic changes in different stages of intestinal type gastric cancer,in which the proportion of T cells and macrophages increased significantly,especially in GC stage,B cells and endothelial cells decreased significantly.2.CD8~+T cells are mainly divided into five subclusters(CD8~+T C1-C5).During the dynamic progress of the disease,the composition of C1 and C3 clusters gradually decreased,but the proportion of C2 and C4 clusters gradually increased.The results of immune characteristic analysis showed that during the dynamic change of gastric diseases,some CD8~+T cells were exhausted to produce immunosuppression,while the other part dominated the activation of immune response to resist tumor cells.Treg cells are mainly divided into two subclusters(Tre C1 and Tre C2),most of which originate from GC stage.These Treg cells highly express multiple immune checkpoints(TIGIT,VSIR,HAVCR2,CD48,TMIGD2,CD80,CD44)and co-stimulatory molecules(TNFRSF9,TNFRSF4,TNFRSF18,CD27,CD70 and ICOS).NKT cells are divided into two subclusters(C1 and C2),most of which come from tumor tissue.Some immune checkpoints(TIGIT,HAVCR2,TMIGD2,CD48,CD44)and co-stimulatory molecules(TNFRSF9,TNFRSF18,TNFSF14)are expressed in the C2 cluster.3.B cells are divided into four cell subclusters(C1-C4),most of which originate from the inflammatory stage.C1 and C3 clusters gradually increased in CAG stage,and then decreased with disease progression.C2 cluster fluctuates during the development of the disease.C4 subgroup only appears in GC stage.The results of differentiation trajectory analysis showed that C2 cluster was the start of B cell differentiation.4.Macrophages include four subclusters(Mac C1-Mac C4).From CAG to GC,the proportion of Mac C1 and Mac C3 shows a significant downward trend,while Mac C2shows an upward trend,while Mac C4 only exists in GC stage.Different subpopulations of macrophages have different immune characteristics,and the above cell subclesters cannot be distinguished according to the expression of M1/M2 marker gene.5.Mast cells are divided into two subclusters(C1 and C2).During the dynamic change of the whole disease,Mas C1 cluster was gradually replaced by Mas C2,and Mas C2 specific high expression genes SLC18A2 and HDC.6.Fibroblasts are divided into five subclusters(Fib C1-Fib C5),which have significant heterogeneity.Human gastric tissue specimens were used to verify the marker genes of the different subclusters and their distribution in different disease stages.Among them,Fib C3 cluster only exists in GC tissues with high expression of CTHRC1.Trajectory analysis shows that Fib C2 cluster is the start of differentiation,Fib C3 at the end of differentiation,and a large number of transcription factors are activated in Fib C3cluster.7.Endothelial cells are divided into four subclusters(End C1-C4).In the process of tumorigenesis,End C1 cluster gradually decreases and End C2 cluster slightly increases.End C3 cluster mainly appear in the IM phase,while End C4 cluster only appears in GC stage.End C4 highly expresses a variety of immune cytokines.Trajectory analysis showed that End C1 cells had the lowest pseudotime value,whereas C3 and C4 clusters appeared at each end of the differentiation trajectory8.There are three subcluster of pericytes,and C1 cluster has the highest proportion in each disease stage,and its with slight changes.9.The results of cell-cell interaction showed that macrophages,fibroblasts and endothelial cells had the strongest interaction with enterocytes and cancer cells in all mesenchymal cells.Fibroblasts,as the most heterogeneous cell type,had the strongest effect on epithelial cells in all disease stages.Part III:Effects and potential mechanism of high CTHRC1 expression in Fibroblasts on biological function of gastric cancer cells1.Expression characteristics and clinical significance of CTHRC1 in GC tissueCTHRC1 is expressed in gastric cancer stromal fibroblasts,and its expression level is higher than that in adjacent normal tissues.The expression of CTHRC1 was correlated with tumor size(P=0.007),TNM stage(P=0.015),lymph node metastasis(P=0.017),invasive paratumor(P=0.01),tumor implantation metastasis(P=0.003),growth mode(P=0.039),depth of invasion(P=0.002),peripheral nerve invasion(P<0.001),peripheral lymphocyte infiltration(P=0.046),and VEGF expression(P=0.001)in gastric cancer.The expression of CTHRC1 is closely related to the poor prognosis of patients with gastric cancer(P=0.001,HR=4.101,95%CI=2.01-8.369).The results of Cox regression multivariable analysis showed that the expression of CTHRC1 in the stroma and TNM stage can be independent prognostic factors of patients with gastric cancer(P=0.004,HR=2.969,95%CI=1.419-6.212).2.Effect of CTHRC1 on the biological function of GC cells2.2.1 The results of two-color immunofluorescence and ELSA showed that CAF secreted CTHRC1 in gastric cancer.2.2.1.1 The co-expression of CTHRC1 and fibroblast marker gene(α-SMA)in GC paraffin section was detected by two-color immunofluorescence.The results showed that CTHRC1 was mainly expressed in the fibroblasts of gastric cancer.2.2.1.2 The successfully constructed lentiviral vector CTHRC1 was transfected into fibroblast cells.RT-q PCR,Western Blot and ELISA were used to detect the expression of CTHRC1 in fibroblasts and their culture supernatants,the experimental group was significantly higher than the control group(P<0.05).2.2.2 CTHRC1 recombinant protein GC cells and GC cells cultured in conditioned medium showed that CTHRC1 could promote the proliferation,migration and vascular mimicry of gastric cancer cells(P<0.05).3.Mechanism of CTHRC1 affecting the biological function of GC cells3.1 The enrichment results of CTHRC1 related pathway showed that it was related to extracellular matrix interaction,cell adhesion,proteoglycan in cancer,PI3K-AKT pathway,TGF-beta pathway,etc.The first three pathways indicate that CTHRC1 plays an important role in extracellular matrix remodeling.Further,we used in vitro cytology experiment to detect the effect of CTHRC1 on the above key pathway proteins by WB.3.2 CTHRC1 mainly affects the biological function of GC cells by activating PI3K-AKT pathwayThe recombinant protein of CTHRC1 was used to stimulate AGS and HGC27 cells,and the expression of PI3K-AKT key molecular protein was detected by Western Blot.The results of WB experiment showed that compared with the control group,the expression of p-AKT and p-PI3K in GC cells stimulated by recombinant protein increased,suggesting that CTHRC1 could activate PI3K-AKT pathway.3.3 CTHRC1 promotes malignant biological behavior of cancer cells and is blocked by PI3K inhibitorsTo investigate whether CTHRC1 promotes the malignant biological behavior of cancer cells through the PI3K-AKT pathway,conditional culture medium and gastric cancer cell lines(HGC27,AGS)were co-cultured,while different concentrations of PI3K inhibitors(10,30μmol/ml)were added.WB was used to detect the expression of key proteins in the PI3K-AKT pathway in gastric cancer cells.In addition,EDU,scratch,and three-dimensional culture were performed to detect changes in gastric cancer cell proliferation,migration,and vascular mimicry and other biological behaviors of cancer cells.The results showed that PI3K inhibitors significantly inhibited the activation of the above pathway,and reversed the CTHRC1-promoted malignant biological behavior of gastric cancer cells.Conclusion:1.Utilizing single-cell sequencing data,we successfully constructed a differentiation map of epithelial cells in various stages of intestinal GC.Specifically,we found that PC cells in both NAG and CAG stages serve as the origin of epithelial cells,which eventually differentiate into PMC and GMC cells via undifferentiated cells.During the IM stage,PC or GMC cells differentiate into mature enterocytes via distinct intestinal stem cell subclusters.TTR~+cells,identified as enteroendocrine progenitors,primarily differentiate into goblet cells.Notably,we observed that cancer epithelial cells primarily differentiate from PC cells.The differentiation process of epithelial tissue during different stages of intestinal-type GC is regulated by specific transcription factors.2.The construction of a microenvironment cell atlas for different stages of intestinal gastric cancer using single cell sequencing has provided insights into the heterogeneity of fibroblasts in particular.The Fib C2 cluster,characterized by the expression of ABCA8,is predominant in the intestinal metaplasia stage and interacts with absorption cell TFRC via B2M,suggesting a potential role in promoting changes in lipid and iron metabolism in epithelial cells.Targeting metabolic changes at the intestinal metaplasia stage may therefore represent a novel therapeutic strategy.Conversely,the Fib C3 cluster is specific to the cancer stage and exhibits high expression of matrix remodeling genes such as CTHRC1,highlighting its close association with gastric cancer onset and progression.Additionally,the C4 clusters of CD8~+T and Mac C2 cells significantly increase during the gastric cancer stage and exhibit high expression of multiple immune checkpoints,pointing towards their potential therapeutic value in cancer treatment.3.The expression of CTHRC1 is higher in stromal cells of gastric cancer and is positively correlated with various clinicopathological parameters,as well as poor prognosis in patients.Additionally,results from Cox regression multivariable analysis indicate that CTHRC1 expression in the stroma is an independent prognostic factor for patients with gastric cancer.CTHRC1 has been shown to activate the PI3K-AKT pathway,promoting the proliferation,migration,and vascular mimicry of malignant gastric cancer cells. |
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