Human Adipose Derived Mesenchymal Stem Cells Affect The Invasion And Metastasis Of Breast Cancer Cells By Secreting TGF-?1

Objective: Breast cancer is a type of malignant tumor.Metastasis and invasion are the main causes of death in breast cancer patients and can occur due to inadequate treatment.Epithelial-mesenchymal transition(EMT)is a process by which cells lose epithelial characteristics and acquire a mesenchymal phenotype that is associated with increased invasion and metastasis.Studies show that the tumor microenvironment plays an important role in the regulation of EMT.Mesenchymal stem cells(MSCs)are non-hematopoietic cells characterized by self-renewal and the potential to differentiate into different connective tissue cells,in addition to the ability to home in on the primary tumor site.These cells have immunomodulatory paracrine functions and can secrete nucleotides,growth factors,cytokines,and chemokines to stimulate tumor cells and/or adjacent cells,thereby exerting pro-tumor or anti-tumor effects and affecting the phenotype of tumor cells.Adipose-derived mesenchymal stem cells(ADSCs),which are components of white adipose tissue,are present in local breast fat and may be close to the location of breast tumor occurrence.Therefore,it is important to study the effect of ADSCs on breast cancer cells.The mechanism of action of ADSCs on breast cancer cells remains unclear,and clarifying the interaction between ADSCs and tumor cells in the breast cancer microenvironment is important to improve our understanding of tumor progression and treatment.Methods: MCF7 and MDA-MB-231 breast cancer cells were cultured with Hu-ADSCs for 72 h in the experimental group,and breast cancer cells without co-culture were used as the control group.1.Hu-ADSCs were isolated from human fat using the collagenase digestion method,and cell surface markers and the multidirectional differentiation potential were identified;2.The growth curves of P3,P5,and P7 of ADSCs were plotted using data from cell proliferation assays;3.Transwell migration and invasion experiments were performed at 24 and 48 h in the experimental and control groups to determine whether Hu-ADSCs promote MCF7 and MDA-MB-231 cell invasion and migration;4.Cell proliferation assays were used to examine the effect of Hu-ADSCs on breast cancer cell proliferation to eliminate the effect of proliferation on migration and invasion experiments;5.Real-time PCR was used to quantify the expression of genes encoding the epithelial marker E-cadherin,mesenchymal marker N-cadherin,and the EMT-related transcription factors Snail,Slug,and Twist in the two groups of cells to determine whether Hu-ADSCs promoted EMT in breast cancer cells;6.Hu-ADSC-conditioned medium was compared with the blank control medium at24,48,and 72 h to determine whether Hu-ADSCs secrete TGF-?1;7.Western blot analysis was used to examine the mechanism underlying the effect of Hu-ADSCs on EMT in breast cancer cells.The co-culture system included anti-TGF-?1 neutralizing antibody or the PI3 K inhibitor LY294002.The epithelial marker E-cadherin,the mesenchymal marker N-cadherin,the EMT-related transcription factors Snail,Slug,and Twist,p-Smad,total Smad,p-AKT,and total AKT were detected in each group to determine whether Hu-ADSCs promote EMT in breast cancer cells at the protein level.In addition,we examined whether the paracrine effect of Hu-ADSCs on breast cancer cells is mediated by the TGF?/Smad or TGF?/non-Smad PI3K/AKT signaling pathways.Results:1.Primary cultured Hu-ADSCs showed long spindle-shaped uniform morphology,with pseudopods,monolayers,and vortex-like growth,and the ability to differentiate into adipocytes and osteoblasts;2.Hu-ADSCs examined by flow cytometry were positive for CD73,CD90,and CD105 expression and negative for CD34,CD45,and HLA-DR expression;3.The growth rate of Hu-ADSCs of P3,P5,and P7 was accelerated after 2 d,decreasing after 5 d,followed by a plateau stage.Cell proliferation rates did not differ significantly between P3 and P5 cells(P>0.05).The proliferation rate of P7 cells was significantly lower than that of P3 and P5 cells(P<0.05);4.Both MCF7 cells and MDA-MB-231 cells were adherent to the wall.MCF7 cells were round,whereas MDA-MB-231 cells were spindle-shaped;5.In vitro migration and invasion experiments indicated that co-culture withHu-ADSCs increased MCF7 or MDA-MB-231 cell migration and invasion compared with those in the control group(all P<0.01);6.Hu-ADSCs promoted the proliferation of MDA-MB-231 cells(P<0.05),but had no effect on the proliferation of MCF7 cells(P>0.05).Therefore,we were unable to rule out the effect of cell proliferation on increased migration or invasion of MDA-MB-231 cells,so MCF7 cells were used in subsequent experiments;7.Real-time PCR showed that expression of E-cadherin was significantly down-regulated,and those of N-cadherin and the EMT-related transcription factors Snail,Slug,and Twist were significantly up-regulated in MCF7 cells co-cultured with Hu-ADSCs(all P < 0.01);8.ELISA experiments showed that Hu-ADSCs secreted TGF-?1,and we speculated that this paracrine effect could affect the migration and invasion of breast cancer cells;9.Western blot analysis showed that co-culture with Hu-ADSCs significantly down-regulated the expression of E-cadherin in MCF7 cells(P < 0.01),and this effect was reversed by the addition of neutralizing anti-TGF-?1 antibody or the PI3 K inhibitor LY294002 into the co-culture system(all P <0.01).The mesenchymal marker E-cadherin and the EMT-related transcription factors Snail,Slug,and Twist showed the opposite expression pattern(all P<0.01).Expression of the pathway-associated phosphorylated proteins p-Smad2/3 and p-AKT were significantly up-regulated in co-cultured MCF7cells(all P < 0.01),and this effect was reversed by anti-TGF-?1 or LY294002treatment(all P<0.05).Addition of anti-TGF-?1 into the co-culture system significantly down-regulated expression of p-AKT in MCF7 cells(P<0.01).These results indicated that Hu-ADSCs promoted the EMT process in breast cancer cells not only through the TGF?/Smad signaling pathway,but also through the PI3K/AKT signaling pathway.Conclusions:1.Hu-ADSCs were successfully cultured using the adherent method and the proliferative capacity of P3 and P5 Hu-ADSCs was significantly higher than that of P7 cells.2.Hu-ADSCs promoted the migration and invasion of MCF7 cells and the proliferation of MDA-MB-231 cells;3.TGF-?1 secreted by Hu-ADSCs promoted EMT in MCF7 cells through the Smadand non-Smad PI3K/AKT signaling pathways.