The Mechanism Of Extracellular Matrix Remodeling By Cancer-related Fibroblasts And The Induction Of Abnormal Tumor Metabolism During Obstruction Of Colorectal Cancer

Objective: The obstruction caused by colorectal cancer(OCRC)can lead to rapid deterioration of the disease and significantly higher mortality than that of colorectal cancer without obstruction(CRC).The gross pathological phenotype of colorectal cancer is focal narrowing and thickening,but whether the tumor microenvironment(TME)and extracellular matrix(ECM)changes remains unclear.Therefore,we intend to start with the clinicopathological features and ECM of OCRC and CRC to determine whether there are differences between the clinicopathological features and ECM of OCRC and CRC.Methods: According to inclusion and exclusion criteria,clinicopathologic characteristics of colorectal cancer patients in our center were collected and propensity matching analysis was conducted to screen patients into the group.The clinicopathologic features of OCRC and CRC were compared.Fresh specimens were collected for atomic force microscopy(AFM),Raman spectroscopy and Movat-Pentachrome,Masson and Sirius red staining to determine whether there were differences in ECM between the two groups of tumors.Meanwhile,samples of enrolled patients were collected,and immunohistochemistry was used to determine whether there were differences in interstitial cell components between the two groups.Results: In clinicopathological analysis,the overall survival rate(OS)and disease-free survival rate(DFS)of OCRC patients were worse than those of CRC patients.There were statistical differences in TNMstage and Perineural invasion(PNI)between OCRC and CRC.The Average E-modulus in OCRC group was significantly higher than that in CRC group in ECM analysis.The collagen fibers in OCRC group are more regular and directional than those in CRC group.The content of new collagen and mature collagen in OCRC was significantly higher than that in CRC group.Compared with CRC group,the content of collagen fiber,collagen and glycosaminoglycans(GAGs)in tumor tissue of OCRC group was significantly increased.In the analysis of interstitial cells between the two groups,the content of CAFs,the average number of microvessels and microlymphatic vessels in tumor interstitium of the OCRC group were significantly higher than that of the CRC group,and the degree of tumor epithelialization was higher than that of the CRC group.Concluions: Obstruction is an important factor for poor prognosis of colorectal cancer.ECM of OCRC and CRC are different in terms of composition,interstitial cells and biomechanics.Objective: The first part of the study found that the CAFs content of OCRC and CRC groups were different,and the biological force,collagen and GAGs content and collagen fiber arrangement of the two groups were also different.CAFs are heterogeneous cells,and their characteristics and interactions with other cell types may change dynamically as cancer progresses or ECM matrix changes.Therefore,whether the ECM difference between the OCRC and CRC groups is closely related to the heterogeneity of CAFs or the different response of CAFs to tumor internal stimuli remains to be further studiedMethods: Extraction of primary CAFs,transcriptome sequencing,FlexCell tension stimulation and ELISA assay was used to detect the functional differences of CAFs and their reactivity to biological force stimulation.The effects of CAFs on tumor cells were investigated by CCK8,Transwell,3D culture,angiogenesis and Western Blot.Results: CAFs of the OCRC and CRC groups differ in the biological function of ECM protein secretion and tumor cell progression,and the differential gene pathway of CAFs of the OCRC and CRC groups is enriched in ECM-receptor interaction and Focal adhesion.The Flex Cell tension model experiment suggested that biological force stimulation could promote ECM protein secretion and tumor progression of CAFs in OCRC and CRC tissues.Functional analysis suggested that biological forces could promote the synthesis of CAFs GAGs and chondroitin sulfate.In addition,the function of CAFs in CRC group can be transformed into CAFs in OCRC group after force stimulation.Conclusion: CAFs in OCRC group and CRC group are functionally heterogeneous,and the CAFs in CRC group can promote the function of CAFs after biological force stimulation,and the CAFs in CRC group can transform into CAFs in OCRC group after biological force stimulation.Objective: Although the ECM difference between OCRC and CRC and the heterogeneity of CAFs between the two groups were confirmed,the tumor cells with abnormal hyperplasia also played a significant role in the overall tumor microenvironment.Therefore,this part of the study is to explore the role of tumor cells in obstruction.Methods: The potential metabolites were screened by histotranscriptomics and nontargeted metabolomics.The possible mechanism was verified by in vitro cell experiments and in vivo animal models.Results: It was found that there were differences in fatty acid metabolism between CRC and OCRC tissues,and palmitic acid(PA)increased significantly in OCRC tissues.PA can promote the progression of colorectal cancer.At the same time,it can promote the increase of CAFs,collagen fiber content and matrix biological strength in tumor cells.The possible mechanism is that PA promotes the activation of CAFs by stimulating the secretion of CSF1,CXCL8 and TGF-β1 in tumor cells,thus affecting the obstruction phenotype of the remodeling process of ECM by CAFs.Conclusions: PA induces the expression of CSF-1,CXCL8 and TGF-β1 in tumor,promotes the activation of CAFs,accelerates the tumor progression and ECM remodeling,and thus produces an obstructing phenotype.