TGF-β Stimulates MSCs To Acquire Cancer-Associated Fibroblast Phenotype To Promote The Progression Of B-Cell Acute Lymphoblastic Leukemia

Objective:B cell acute lymphoblastic leukemia(B-ALL)is a clonal hematological malignancy originating from B lymphocyte precursor cells.At present,the complete remission rate of B-ALL patients can reach 80% after receiving standard treatment,but disease progression in the later stage of treatment is still a major challenge for the treatment of B-ALL patients.The tumor microenvironment(TME)is a dynamic network composed of stromal cells,extracellular matrix(ECM),soluble cytokines,growth factors,etc.,which can provide nutrition and support for the occurrence and development of tumor cells.The study found that the dynamic evolution of the bone marrow microenvironment is a key factor mediating the progression of B-ALL.Cancer-associated fibroblasts(CAF)are important stromal cells in the TME,and bone marrow mesenchymal stem cells(BM-MSCs)are important sources of CAF.Studies have shown that CAFs can mediate changes in the bone marrow microenvironment to promote the growth,proliferation and infiltration of tumor cells.But its role in B-ALL is unclear.This article will explore the role of CAF in the progression of B-ALL and the underlying molecular mechanisms of its interaction with leukemia cells.Methods:1.In clinical sample detection,this study collected bone marrow mononuclear cells from healthy controls(n=17),B-ALL-newly diagnosed patients(n=8),B-ALL-complete remission patients(n=23)and B-ALL-relapsed patients(n=7),respectively.And the expression levels of CAF markers α-SMA and FAP in each group were detected by RT-PCR and Western blot.2.For the acquisition of BM-MSCs,the bone marrow blood from B-ALL patients and healthy controls were collected respectively,and the BM-MSCs were separated and cultured by Ficoll gradient centrifugation.The phenotype,adipogenic and osteogenic differentiation functions of BM-MSCs were identified,and P2-P4 generation BM-MSCs were used for experiments.3.In vitro,RT-PCR and Western blot were used to compare the expression levels of CAF markers α-SMA and FAP in healthy control-derived and B-ALL-derived BM-MSCs,respectively.Subsequently,BM-MSCs were co-cultured with B-ALL cell lines(Nalm-6 or RS4;11 cell lines)to detect the expression changes of α-SMA and FAP.And The expression levels of CAF-related secreted factors in the co-culture system were detected by RT-PCR and ELISA.4.Chemotherapeutic drug daunorubicin(DNR)was used to treat leukemia cell lines alone or co-cultured with MSCs that obtained the CAF phenotype,and leukemia cells were collected after 24 hours.Then trypan blue staining and cell apoptosis kits were used to detect the cell viability and apoptosis of B-ALL cells.Further explore the possible mechanism of CAF activation in the B-ALL microenvironment.5.The mouse models of Nalm-6 cell injection group,Nalm-6+MSCs combined injection group and Nalm-6+ MSCs with CAF phenotype combined injection group were established respectively.Then observe the changes of tumor tissue volume and weight in each group of mice,and analyze the effects of MSCs and MSCs with CAF phenotype on the growth and proliferation of leukemia cells.6.Through in vitro transwell experiments,three-dimensional cell culture models,and lentiviral transfection,the potential molecular mechanism of the interaction between MSCs with CAF phenotype and leukemia cells to promote the progression of B-ALL was explored.Results:1.The expression levels of CAF markers α-SMA and FAP in B-ALL-relapsed patients were significantly higher than those in B-ALL-complete remission group and healthy control group(P<0.05).2.In the in vitro cell experiment,there was no significant difference in the m RNA and protein expressions of CAF markers α-SMA and FAP between the bone marrow-derived BM-MSCs of normal controls and B-ALL-derived BM-MSCs(P>0.05).However,after BM-MSCs were co-cultured with Nalm-6 and RS4;11 cell lines for 48 hours(simulating the leukemia microenvironment in vivo),the m RNA and protein expression levels of α-SMA and FAP in BM-MSCs increased(P<0.05).,while the expression levels of CAF-related secretory factors Fn,SDF-1,IL-6 and TGF-β increased in the co-culture system.3.Compared with Nalm-6/RS4;11 cell lines cultured alone,the co-culture system of MSCs with CAF phenotype and Nalm-6/RS4;11 cell lines could significantly reduce the apoptosis rate of leukemia cells exposed to DNR(P<0.05)and increase cell activity(P<0.05);4.Adding TGF-β inhibitor LY2109761 to the co-culture system of leukemia cells and MSCs can reduce the expression of α-SMA and FAP(P<0.05);in the drug treatment experiment,compared with LY2109761 and DNR alone treatment group,the apoptosis rate of leukemia cells in the combined drug(DNR+LY2109761)group was significantly increased(P<0.05).5.The use of recombinant human transforming growth factor β(rh TGF-β)to stimulate BM-MSCs can increase the expression levels of α-SMA and FAP in MSCs(P<0.05).6.The MSCs obtained with CAF phenotype were compared with untreated MSCs,and there was no significant difference in reducing the apoptosis of leukemia cells by flow cytometry(P>0.05).7.Transwell laboratory experiments found that compared with blank control group and untreated MSCs,MSCs with CAF phenotype significantly promoted the migration and invasiveness of leukemia cells(P<0.05).8.In in vivo animal experiments,MSCs with CAF phenotype significantly promoted the growth and proliferation of leukemia cells in mice compared with untreated MSCs(P<0.05).9.The SDF-1/CXCR4 signaling axis acts as a signaling axis that promotes the interaction between stromal cells and tumor cells.Adding the CXCR4 inhibitor AMD3100 to the transwell chamber can attenuate the promotion effect of ALL cell migration and invasion by MSCs with CAF phenotype(P<0.05).By scanning electron microscope observation of cells in three-dimensional cell culture system,AMD3100 can reduce the adhesion of Nalm-6/RS4;11 cells to MSCs with CAF phenotype compared with untreated group.10.Using AMD3100 in the co-culture system of MSCs with CAF phenotype and Nalm-6/RS4;11 cells can reduce the expression of integrinα 5 and integrin β1 in leukemia cells(P<0.05),and further down-regulation of integrin β1 in leukemia cells could reduce the promotion of ALL cell migration and invasion by MSCs with CAF phenotype(P<0.05).Conclusions:1.The expression levels of CAF markers α-SMA and FAP are related to the progression of B-ALL.2.Co-culture of BM-MSCs and B-ALL cell lines can promote BM-MSCs to acquire CAF phenotype,which significantly promotes the migration and invasion of leukemia cells by interacting with leukemia cells;and promote the growth and proliferation of leukemia cells in vivo.3.TGF-β might be the key factor for MSCs to acquire CAF phenotype in B-ALL microenvironment.4.The SDF-1/CXCR4 axis plays an important role in mediating the interaction between MSCs with CAF phenotype and B-ALL cells.5.Blocking the SDF-1/CXCR4 axis or targeting integrin β1 may be an effective intervention to prevent the interaction between leukemia cells and MSCs with CAF phenotype.