Alterations In Mechanical Properties Of Upper Tract Urinary Carcinoma With Different Tumor Architecture And Impact Of Matrix Stiffness On Proliferation In Urothelial Carcinoma Cells

Background and ObjectiveUpper urinary tract urothelial carcinoma(UTUC)is relatively rare,accounting for approximately 5%of all urothelial carcinomas.Despite surgical refinement,oncological outcomes after radical nephroureterectomy(RNU)have not changed substantially over the past three decades.A substantial proportion of patients after RNU with clinically nonmetastatic disease will experience disease recurrence and subsequently die from metastatic UTUC.Although the benefit of adjuvant therapies remains to be proved,identification of patients at high risk of failure with RNU alone is important for accurate prognostic assessment,patient counseling,and design of clinical trials using integrated muLtimodal therapy.The design of this project ideas originated from visual observation of surgical resection of UTUC after RUN and we found that morphology and texture hardness were different from each other.Papillary architecture type always grows towards the tube wall of upper urinary tract with soft texture,however,sessile type grows towards the lumen of upper urinary tract reguLarly with hard texture.There are some differences between PA type UTUC and SA type UTUC in terms of disease progression and survival prognosis,however,few reports have been published in China.Therefore,we firstly explored the relationship between tumor architecture and the impact of disease recurrence and survival prognosis.As our single center study and the limited sample size may influence the clinical significance,we further quantitatively combined the resuLts with those of existing published literature through a meta-analysis compiled from searching several databases to reveal the relationship between tumor architecture and upper urinary tract epithelial carcinoma.After statistics of clinical case,we used shear wave elastic imaging and measurement of tumor matrix ratio to detect external tissue mechanics and internal tumor composition distribution between PA type UTUC and SA type UTUC.The application of immunohistochemical staining the type I collagen,type IV collagen and laminin were stained on the tissue samples of two types of tumors,and the distribution of the internal structure of the matrix in the two types of tumors was explored.The gene expression profiles between PA type UTUC and SA type UTUC were identified by mRNA expression profiling.The system cluster analysis and the bioinformatics tools were used to select the signal pathways related to genes and pathways.After establishing fundamental mechanics research model of urinary epithelial tumors,the morphology of 5637 was observed and Ki67 proliferative index,proliferating protein,such as Cyclin D1,C-myc,Ki67 and its signal pathway were tested with the corresponding matrix hardness of 3 kPa,10 kPa and 19 kPa.The aim of this study was to demonstrate the effect of different hardness cuLture environments on the proliferation of 5637 cell lines in matrix mechanics.Methods1.Analyze the differences of clinicopathological features and prognostic betweenPA type UTUC and SA type UTUCWe retrospectively reviewed the medical records of patients with clinical UTUC who had undergone surgery at first affiliated hospital of Dalian medical university from October 1,2005 to October 1,2015.The clinical data were collected and analyzed by SPSS 21.0 statistical software.As our single center study and the limited sample size may influence the clinical significance,we further quantitatively combined the resuLts with those of existing published literature through a meta-analysis compiled from searching several databases including the PubMed databases?Medline databases,the Dutch Medical Abstracts database?Embase And the Cochrane database.The meta-analysis was done by the Stata 13.0 software provided by the Cochrane Collaboration.2.Determination of the mechanical properties and the proportion of tumor matrixcomponents between PA type UTUC and SA type UTUCBy establishing shear wave elastic imaging model of isolated UTUC,we firstly detected average elasticity value and quantitative analysze between PA type UTUC and SA type UTUC.Then we measured the structural changes of tumor matrix between PA type UTUC and SA type UTUC by HE staining of histopathological sections;Lastly,we used immunohistochemical staining to detect the expression of type I collagen,type IV collagen and laminin.3.Comparison of the expression profiles of the basal mechanics-related gene expression between PA type UTUC and SA type UTUCThe gene expression profiles between PA type UTUC and SA type UTUC were identified by mRNA expression profiling.The differentially distributed genes were identified by volcanic map and scatter gram.The R softwarewas used to cluster the clusters.Using the standard enrichment method,GO analysis and Pathway analysis were done.The mRNA expression of COLIA1,COL3A1,COL4A1,LAMA1,LOX,MMP2,CAV1,VIM,FN1,ITGFB1,EGFR,ITGB3,Ki67,C-myc,CCND1 and CD9 were verified by qRT-PCR.4.Explore the role of matrix mechanics in the reguLation of urothelial carcinoma cell 5637 and its molecuLar mechanism56372 cell was inocuLated on polyacrylamide films at 3 kPa,10 kPa and 19 kPa,respectively.The spreads of the cells were recorded at aspect of circumference,long axis,short axis and so on.The deformation parameters,cell deform factor(CF)and ratio of long diameter to short diameter were measured by Imagetool graphical tool.Proliferation index of Ki67 was calcuLated by immunofluorescence staining.Immunoblotting assay was used to detect protein content of Itegrin ?1,Integrin ?3,Integrin a4,Integrin a5,GSK-3?/?-catenin signaling protein,such as p-GSK-3?,p-?-catenin,Catenin,TCF1,TCF4 and other protein under different matrixes.ResuLt1.Analyze the differences of clinicopathological features and prognostic between PA type UTUC and SA type UTUCIn this retrospective cohort study,101 patients of UTUC were classified as PA type UTUC and SA type UTUC.Chi-square test and spearman's correlation analysis showed that tumor architecture was found to be positively correlated with the tumor location and the histological grade.Kaplan-Meier curves showed that patients with SA type UTUC had significantly poor recurrence free survival(RFS)and cancer specific survival(CSS).Furthermore,Cox muLtivariate analysis suggested that tumor architecture and Ki67 staining index were independent prognostic factors for RFS and pathological stage was independent prognostic factor for CSS in UTUC patients.A meta-analysis of investigating tumor architecture and its effects on UTUC prognosis was conducted.Combined resuLts showed that SA type UTUC was associated with both disease recurrence and cancer-specific mortality.2.Determination of the mechanical properties and the proportion of tumor matrixcomponents between PA type UTUC and SA type UTUCBy shear wave elastic uLtrasound analysis,image of more than 50%of SA type UTUC showed red and yellow with high hardness and image of more than 50%of SA type UTUC showed blue or green with soft texture relatively.The ratio of SA type UTUC was found to be higher than PA type UTUC.We next used IHC staining and scoring to verify COL1A1,LAMA1 and COL4A1 and the resuLt indicated that levels of these markers in SA type UTUC were higher than SA type UTUC.In SA type UTUC,COL1A1 was arranged along the tumor morphology with a vein shape and tumor content was significantly higher than the matrix component,however,in PA type UTUC,COL1A1 was arranged disorderly with a lot of matrix parts.3.Comparison of the expression profiles of the basal mechanics-related geneexpression between PA type UTUC and SA type UTUCThe total hierarchical clustering map was used to visualize the expression of differentiated mRNA between PA type UTUC and SA type UTUC.mRNAa with a total of more than 2 times the difference were 3869,of which a total of 2644 up,accounting for 68.3%of total mRNAs,down a total of 1225,31.7%of total mRNAs.The difference of genes included genes about extracelluLar matrix components,extracelluLar matrix remodeling,matrix mechanics signal pathway,proliferation related factors,etc.We further detected differentially expressed genes by KEGG database and pathway analysis and found that the differential pathway included extracelluLar matrix composition pathway,extracelluLar matrix degradation pathway,cell matrix adhesion pathway,tissue remodeling pathway,tissue remodeling pathway,cell component movement pathway,mechanical stimuLation pathway,cell base adhesion pathway,mechanical stimuLation cell response pathway,mechanical pathway.According to functional importance and difference value,the 16 selected expressed gene included COL1A1,COL3A1,COL4A1,LAMA1,LOX,MMP2,CAV1,VIM,FN1,ITGFB1,EGFR,ITGB3,Ki67,C-myc,CCND1 and CD9,as determined by quantitative real-time PCR,were consistent with the microarray resuLts.4.Explore the role of matrix mechanics in the reguLation of urothelial carcinoma cell 5637 and its molecuLar mechanismIn this study,the urothelial carcinoma cell line 5637 was inocuLated to 3kPa,10 kPa and 19kPa,respectively,we found that the number of 5637 cells was significantly increased with the increase of matrix hardness under the same observation period,accompanied by increased Ki67 proliferation index,higher the fusion area between cells,more slender cell morphology and increased short diameter ratio and spherical index gradually.The contents of Cyclin D1,C-myc,Ki67 and p-GSK-3?,p-?-catenin,TCF1 and TCF4 were increased.Then we tested the expression of Integrin?1,Integrin?3,Integrin a4 and Integrin a5,the resuLts showed that the expression of Integrin ?1 increased with the increase of matrix hardness,but there was no significant difference in the expression of Integrin ?3,Integrin a4 and Integrin a5.The levels of Cyclin D1,C-myc and Ki67 protein in 5637 cells were significantly decreased and the phosphorylation level in GSK-3?/?-catenin pathway was significantly lower after siRNA Integrin ?1 specific antibody.The expression of p-?-catenin and ?-catenin was significantly increased after GSK-3? inhibition,and the expression of Cyclin D1,C-myc and Ki67 protein was increased by binding to transcription factor.Conclusion1.Our retrospective cohort study showed that tumor architecture was found to be positively correlated with the tumor location and the histological grade.Kaplan-Meier curves showed that patients with sessile tumor architecture had significantly poor RFS and CSS.Furthermore,Cox muLtivariate analysis suggested that tumor architecture and Ki67 staining index were independent prognostic factors for RFS.Moreover,a meta-analysis showed that sessile tumor architecture was associated with both disease recurrence and cancer-specific mortality.2.By shear wave elastic uLtrasound analysis,we found that the average elasticity of SA type UTUC was significantly higher than PA type UTUC.Moreover,the ratio of SA type UTUC was found to be higher than PA type UTUC.We next used IHC staining and scoring to verify COL1A1,LAMA1 and COL4A1 and the resuLt indicated that levels of these markers in SA type UTUC were higher than SA type UTUC.3.Comparing SA type UTUC with PA type UTUC,the difference of genes included genes about extracelluLar matrix components,extracelluLar matrix remodeling,matrix mechanics signal pathway,proliferation-related factors,etc.We further detected differentially expressed genes by KEGG database and pathway analysis and found that the differential pathway included extracelluLar matrix composition pathway,extracelluLar matrix degradation pathway,cell matrix adhesion pathway,tissue remodeling pathway,tissue remodeling pathway,cell component movement pathway,mechanical stimuLation pathway,cell base adhesion pathway,mechanical stimuLation cell response pathway,mechanical pathway.4.Matrix stiffness couLd reguLate the morphological changes and proliferative ability of 5637 cells.The expression of Cyclin D1,C-myc,Ki67 and GSK-3?/B-catenin-related signaling pathway were adjusted by mechanical signal factor Integrin ?1.