Exploring The Biological Mechanism Of CCR7 Based On The Tumor-stroma Crosstalk Networks In Breast Cancer Microenvironment

Tumor microenvironment consists of tumor cells and tumor stroma.Tumor stroma is composed of extracellular stroma and many stromal cells.Tumor cells and stromal cells autocrine or paracrine abundant cytokines,establishing the communication interaction network between tumor and stroma by interacting with its receptors.The cross-talk networks regulate tumor microenvironment via affecting biological behavior of various cells in tumor microenvironment.However,clinical samples used in most current research was obtained from tumor entity,tumor-stroma crosstalk networks results may be biased due to the heterogeneity of cellular composition.Meanwhile,interaction between tumor cells and overall stroma was concealed by analyzing interaction between tumor cells and single stromal cells.Thus,it's necessary to construct interaction networks between tumor cells and overall stroma via analyzing tumor cells and stroma samples which can be located more accurately.Purpose:To construct tumor-stroma interaction network of breast cancer,by analyzing microarray data of invasive breast cancer and breast ductal carcinoma in situ obtained by microdissection technique.Based on tumor-stroma interaction network in breast cancer,to screen and validate the biological mechanism of CCR7 involved in mediating breast cancer metastasis.Methods:1.Establishment of tumor-stroma interaction network in breast cancer microenvironment: The cDNA microarray datasets GSE35019,was downloaded from GEO database,and remodeled.Differentially expressed genes(DEGs)between invasive breast cancer and breast ductal carcinoma in situ was compared and analyzed in R using Bioconductor and associated packages.In order to analyze the DEGs at the functional level,GO enrichment and KEGG pathway analysis were performed using DAVID(https://david.ncifcrf.gov/)online tool;tumor-stroma interaction network of breast cancer was established and visualized by Cytoscape software;cytokines and its receptors involved in breast cancer metastasis was screened using high throughput database TCGA and METABRIC;2.Exploration of CCR7 mediated biological mechanisms: correlation analysis of CCL19,CCL21 and CCR7 was conducted using dataset GSE35019 and online tool cBioPortal;CCR7 expression analysis of normal breast vs.breast cancer was performed using oncomine database;datasets GSE35019 was used for GSEA analysis of the CCR7 high and low expression groups;expression of CCR7,E-cadherin and Vimentin was detected using immunohistochemistry in breast cancer tissues.Results:1.Based on the GEO database,9 human breast cancer tumor and stroma tissues microarray datasets obtained by microdissection technique was screened,and remodeled in R using Bioconductor and associated packages;2.A total of 1459 DEGs of tumor parenchyma and 1427 DEGs of tumor stroma were identified.GO analysis results showed that,GO-MF of tumor parenchyma DEGs are enriched in protein binding,transcriptional activator activity and integrin and collagen binding,KEGG pathways are enriched in Glutathione metabolism,Wnt signaling pathway and Cytokine-cytokine receptor interaction,stromal differentially expression genes are enriched in integrin and collagen binding,cytokine and its receptor activity,extracellular matrix structural constituent molecular function,and Cytokine-cytokine receptor interaction,Cell adhesion molecules pathways and ECM-receptor interaction pathways;3.To construct tumor-stroma interaction network in breast cancer of cytokinecytokine receptor interaction,cell adhesion molecules and ECM-receptor interaction pathways;5 groups of gene crosstalk are screened,including CCL19-CCR7,CCL21-CCR7,GH1-GHR,PDGFD-PDGFRB,VEGFC-FLT4/FDR;By using TCGA and METABRIC,these 10 genes expression were shown to be correlated with clinical pathology,including CCL19,CCL21,CCR7,GH1,GHR,PDGFD,PDGFRB,VEGFC,FLT4,FDR;4.A close correlation between the expression of CCR7 and its ligands CCL19 and CCL21 was shown in datasets GSE35019(P<0.0001).Moreover,a positive correlation was also shown when analyzed using online tool cBioPortal(R>0.50),and CCR7 was overexpressed in breast cancer when compared with normal breast tissues(P<0.0001);5.GSEA results showed that CCR7 overexpressed group are enriched in IL6/JAK/STAT3,IL2/STAT5,TNF?,KRAS,mTORC1 signaling pathways,and E2 F,MYC transcription factor pathways,and inflammatory,interferon,unfolded protein response pathways,and DNA repair,mitotic spindle,apoptosis,EMT biological behavior pathways;6.Immunohistochemistry detection results showed that the expression of CCR7,E-Cadherin and Vimentin and clinical information had no significant differences in correlation analysis,no correlation between CCR7 and E-Cadherin was shown.However,a positive correlation between the expression of CCR7 and Vimentin was shown.(P=0.029,R=0.392).Conclusion:1.Based on gene expression profiles microarray data of invasive breast cancer and breast ductal carcinoma in situ obtained by microdissection technique,tumor-stroma interaction network was established and visualized;2.Compared with breast ductal carcinoma in situ,the cytokines crosstalk manner between tumor parenchyma and stroma vary significantly in invasive breast cancer;3.CCR7 may mediate apoptosis,G2 M checkpoint and IL6/ JAK/STAT pathways which are associated with other biological behavior;4.CCR7 may be involved in the regulation of breast cancer metastasis process via promoting EMT program.