Study On The Mechanism Of TGF-β1-activated CAFs Promoting Lung Metastasis Of Breast Cancer In Mice

Background:Female breast cancer has become the most prevalent cancer worldwide in 2020.Although good progress has been made in treatment,the prognosis remains poor due to the high recurrence rates and metastasis.Lung metastasis,in particular,is one of the main obstacles in clinical treatment of breast cancer.Metastasis is a fundamental feature of malignant tumors and is regulated by multiple mechanisms.Epithelialmesenchymal transition(EMT)of tumor cells mediated by the tumor stroma is a key step in promoting tumor cell migration and invasion,it plays a crucial role in inducing tumor metastasis.Cancer-associated fibroblasts(CAFs)are the most abundant cellular components in the tumor stroma,it could be activated from normal fibroblasts under the induction of transforming growth factor-β1(TGF-β1),which play a vital role in promoting the invasion and metastasis of tumors.Previous in vitro studies of our group have preliminarily confirmed the effects of autophagy and FAP-α expression of CAFs on the EMT process of tumor cells,further exploration of the effects of autophagy and FAP-α on the EMT process and lung metastasis in mixed xenograft tumors will have more profound clinical significance.Objectives:This study aims to establish a mixed xenograft model of TGF-β1-induced CAFs formation.On the basis of this model,autophagy blocked or FAP-α knockdown was performed to further explore the effect of TGF-β1-activated CAFs on tumor growth and lung metastasis in mice and to explore its mechanism.Methods:1.Female BALB/c mice with mixed inoculation of mouse breast cancer cell 4T1 and mouse embryonic fibroblast NIH3T3 were used to construct the mixed xenograft model.2.The tumor volume and tumor weight of mice were monitored,and the paraffin sections of tumor and lung tissues were pathological analyzed to investigate the tumor growth and lung metastasis in mice.3.The mRNA and protein expression levels of CAFs markers(α-SMA,FAP-α,Cav-1),autophagy markers(Beclin1,LC3β)and EMT markers(E-cadherin,Ncadherin,Vimentin)in tumor tissues of mice were detected by RT-q PCR,Western blotting and immunohistochemistry.4.Immunofluorescence staining was performed on paraffin sections of tumor tissues to investigate the co-localization expression of autophagy marker LC3β and mesenchymal marker Vimentin in tumor tissues of mice.5.The expression of FAP-α in CAFs was silenced with si RNA technology to investigate the effect of FAP-α expression on tumor growth and lung metastasis in mice.Results:1.Compared with the group inoculated with 4T1 alone,the mice inoculated with4T1 and NIH3T3 cells grew faster,the final tumor weight and volume increased,and the necrotic area of tumor tissue decreased.TGF-β1 treatment increased the immunohistochemical expression of CAFS markers in tumor tissues,and the tumor growth was significantly accelerated,and the necrotic area of tumor tissues was significantly reduced.Treatment with autophagy inhibitor 3-MA down-regulated the m RNA and protein expression levels of CAFs and autophagy markers in mixed xenograft tumors.2.Compared with the control group,the growth,tumor volume and tumor weight of mice treated with 3-MA were significantly reduced,and the necrotic area of tumor tissue was also increased.The lung metastasis of 3-MA treated group was relieved,the number of metastatic nodules on the surface of lung tissue was significantly reduced,and the metastasis of tumor cells in lung tissue was also reduced.Treatment with 3-MA down-regulated the m RNA and protein expression levels of EMT markers in tumor tissues,and decreased the co-localization expression of autophagy marker LC3β and stromal marker Vimentin.3.Compared with the group inoculated with normal NIH3T3 cell,the mixed inoculation of NIH3T3/FAP-α+ cells accelerated the growth of tumor,increased the number of lung metastatic nodules and tumor metastases,it also up-regulated the m RNA and protein expression levels of EMT markers.Before FAP-α silencing,high FAP-α expression was associated with faster tumor growth,more lung metastasis,and higher expression levels of EMT marker,and TGF-β1 treatment further enhanced this effect.After FAP-α silencing,the promoting effects of TGF-β1-activated CAFs on promoting tumor growth,lung metastasis,and EMT process were reversed.Conclusions:1.Autophagy mediates TGF-β1-induced CAFs activation in the tumor microenvironment of mixed xenograft tumors.2.TGF-β1-activated CAFs induce the EMT progression in tumor cells through autophagy and further promote the growth and lung metastasis of mixed xenograft tumors.3.TGF-β1-activated CAFs induce the EMT progression in tumor cells by increasing the expression of FAP-α and further promoted the growth and lung metastasis of mixed xenograft tumors.