Targeting The Heterogeneity Of Cancer-Associated Fibroblasts Inhibits Metastasis In Colorectal Cancer

Background and objective:Colorectal cancer（CRC）,which is one of the most common malignant tumors clinically,is undergoing an increasing incidence in the general population in our country in recent years.What’s worse,the increasing frequency of metastasis leads to poor prognosis in CRC patients.It is estimated that metastasis may exist in 50%-60% of CRC patients,while the 5-year overall survival rate for patients with metastatic CRC is only 13.3%.Cancer-associated fibroblasts（CAFs）are one of the key factors promoting colorectal cancer metastasis,but the heterogeneity ofCAFs determines that they promote metastasis in different ways.MyofibroblasticCAFs（myCAFs）remodel the tumor microenvironment by releasing collagen and collagenase,providing external conditions for the metastasis of tumor cells.However,although attempts to directly target the marker α-smooth muscle actin（α-SMA）of myCAFs can improve survival in mouse models and reduce tumor growth in patients,there exist huge individual differences.Besides,other researches suggest that depletion of extensive depletion ofCAFs resulted in decreased mouse survival.Through the latest researches,we found that there is a subpopulation ofCAFs with high expression of inflammatory factors but low expression of α-SMA,which promotes tumor cell metastasis through inflammatory factors,defined as inflammatory cancer-associated fibroblasts（iCAFs）according to their characteristics.However,iCAFs are precisely ignored in anti-fibrotic therapy,and anti-fibrotic therapy with a large number of α-SMA pan-markers provides a better proliferation environment for iCAFs.Therefore,the relevant treatment forCAF heterogeneity becomes particularly important,but there is still no therapy targetingCAF heterogeneity.The key factor leading to the heterogeneity ofCAFs is the different activation of signaling pathways among differentCAF subtypes,which not only maintains their phenotype,but also ensures the function of promotingCAFs tumor metastasis.In this study,we will discuss:（1）is there functional heterogeneity in colorectal cancerCAFs promoting tumor metastasis;（2）is the heterogeneity ofCAFs in colorectal cancer determined by different signaling pathways;（3）whether inhibition of signaling pathways can block the pro-metastatic effect ofCAFs or not.Methods:Extract primaryCAFs.The functional heterogeneity ofCAFs were observed via Transwell,Western blot,Elisa,RT-q PCR,phalloidin,collagen shrinkage and gelatin zymogram.The differences in activated signaling pathways among different subtypes ofCAFs were identified via transcriptome sequencing,and verified via Western blot,immunofluorescence（IF）and RT-q PCR.Signal pathway inhibitors were selected for in vitro experiments to observe the functional changes ofCAFs.In vivo experiments were carried out in a mouse model of liver metastasis by spleen injection to observe the regulation of the pro-metastatic function ofCAFs in combination with chemotherapeutic drugs and signaling pathway inhibitors.Results:（1）There is functional heterogeneity in the promotion of tumor cell metastasis byCAFs in colorectal cancer.One type ofCAFs promotes tumor cell metastasis by secreting inflammatory factors.Another type ofCAFs remodels TME by secreting collagenase and collagen.（2）iCAFs promote tumor metastasis by activating the JAK/STAT3 signaling pathway.（3）myCAFs promote tumor metastasis by activating the TGF-β/SMAD signaling pathway.（4）JAK/STAT3 signaling pathway inhibitors block the pro-metastatic effect of iCAFs.（5）TGF-β/SMAD signaling pathway blocks the pro-metastatic effect of myCAF.Conclusion:Through the extraction and function-related experiments ofCAFs,we proved thatCAFs can be stored in the invasion and metastasis of colorectal cancer cells.Interestingly,CAFs have functional heterogeneity.Transcriptome RNA sequencing proved thatCAFs promote differentiation and metastasis in colorectal cancer by activating its own signaling pathway.iCAFs activate the JAK-STAT3 signaling pathway,while myCAFs activate the TGF-β/Smad signaling pathway.Finally,we confirmed the conjecture in vitro by animal experiments.After identifying the subtypes ofCAFs in colorectal cancer by their biomarkers,appropriate signaling pathway inhibitors can be used for adjuvant chemotherapy.