| Objective Bone mesenchymal stem cells(BMSCs),which are characterized by their ability to self-renew and differentiate into tissues of mesodermal and nonmesodermal cell lineages,play crucial roles in supporting hematopoiesis.Studies have increasingly shown that BMSCs play an important role in tumor development and progression.Evidences have proven that BMSCs have a good tumor tendency,and exogenous b FGF can promote the migration of BMSCs by activating AKT signaling pathway.Cancer-associated fibroblasts(CAFs),as the primary stromal cells in the tumor microenvironment,can secrete collagen and multiple growth factors,which are necessary for tumor growth and metastasis to participate in tumor progression.At present,the origin of CAFs is mainly as follows: in situ resting fibroblasts,epithelial mesenchymal transition,endothelial mesenchymal transition and bone marrow-derived mesenchymal stem cells.More and more studies have focused on the transdifferentiation of BMSCs into CAFs.However,the mechanism of specific inducer and its transdifferentiation process are still unclear.Therefore,the aim of this study was to investigate the effect of b FGF on the migration of BMSCs under tumor environment and the effect of BMSCs on the breast cancer cells proliferation and in vivo growth of breast cancer xenograft transplantation model.Furthermore,we also aimed to investigate the role of b FGF in the transdifferentiation of BMSCs into CAFs and the related signaling pathways.Methods BMSCs from BALB/C mice were isolated and identified in vitro.Cell phenotype was identified by flow cytometry analysis.Mouse BMSCs,purchased from Cyagen Biotechnology Co.,Ltd.,were also performed for flow cytometric phenotyping and differentiation functional identification.4T1 breast cancer cell tumor conditioned medium(TCM)and BMSCs conditioned medium(BMSC-CM)were prepared.The effects of 4T1 cells,50%TCM,TCM,specific b FGF neutralizing antibody and exogenous b FGF on the migration ability of BMSCs were detected by Wound-healing and Transwell chamber test.The effect of BMSC-CM on the proliferation of 4T1 breast cancer cells in normal serum(10% FBS)and low serum(1% FBS)medium was detected by trypan blue assay and MTS colorimetric assay.4T1 breast cancer cells in mice transplanted tumor model was used to verify the effects of BMSCs in vivo growth of breast cancer.The expressions of PCNA and CD34 were detected by immunohistochemical staining in each transplanted tumor model group.The expression of α-SMA was detected by immunohistochemical staining in each transplanted tumor model group.Immunofluorescence assay and Western blot assay were used to detect the expressions of α-smooth muscle protein and vimentin in BMSCs cells after 48 h treatment with 50% TCM,10ng/ml b FGF,10ng/ml TGF-β and 50% TCM added with specific b FGF neutralizing antibody(20ng/ml).Picric acid-Sirius red staining was performed to detect collagen expression in BMSCs cells after 48 h treatment with 50% TCM,10ng/ml b FGF,10ng/ml TGF-β and 50% TCM added with specific b FGF neutralizing antibody(20ng/ml).RT-PCR assay was used to detect the m RNA expressions of α-smooth muscle protein,vimentin,collagen I,and collagen III in BMSCs cells after 48 h treatment with 50% TCM,10ng/ml b FGF,10ng/ml TGF-β and 50% TCM added with specific b FGF neutralizing antibody(20ng/ml).The protein expression of α-smooth muscle protein,vimentin,ERK,p-ERK,Smad3 and p-Smad3 in BMSCs was detected by Western blotting after treatment with 10ng/ml b FGF and/or PD98059 for 48 hours.Results 1.BMSCs were isolated by the whole bone marrow adherent cell separation method.The third generation of cultured BMSCs showed fibroblast-like,arranged in a certain direction,strong refraction,and adherent growth.The passage 8 BMSCs had strong proliferative activity,the cell purity was higher and the cell phenotype was CD29+(82.1%)CD44+(93.8%)CD29+ CD44+(82.3%).However,because of low cell yield and poor cell viability after cryopreservation,only part of the pre-experiment was carried out using self-priming cells.2.BALB/C mouse BMSCs purchased from Cyagen Biotechnology Co.,Ltd.had typical BMSCs morphology.The cell phenotype was Sca-1+CD29+CD44+CD105+CD11b-CD34-CD45-CD117-.These BMSCs cells can differentiate into adipocytes and osteoblasts under the induction of adipogenic and osteogenic medium.In order to guard the scientific nature of this study,the experimental results were all from purchased BMSCs in this paper.3.Wound-healing test results showed that there was no difference between 20% TCM and control group in the migration distance of BMSCs(P>0.05).The migration distance of BMSCs in the 50% TCM group was significantly increased than that in the control group(P <0.001).4.The results of Transwell chamber showed that the number of migrating BMSCs after 4T1 breast cancer cells,50% TCM and TCM treated for 12 hours were 38±3,50±2,53±8,respectively.The migration of BMSCs was promoted by 4T1 breast cancer cells(P<0.05),50% TCM(P<0.01)and TCM(P<0.01)compared with the control group(22±6).5.Wound-healing test results showed that the migration distance of BMSCs in the 50% TCM group was increased than that in the control group(P<0.05).And,when 20 ng/ml specific b FGF neutralizing antibody was added in 50% TCM,the migration of TCM was weakened(P<0.05).6.The results of Transwell chamber showed that the migrating cell numbers in 50%TCM,TCM,50%TCM+FGF-Ab,TCM+FGF-Ab and control groups were 90±13,111±5,57±6,72±18,61±20,respectively.The migration of BMSCs was promoted by 50% TCM(P<0.05)and TCM(P<0.05)compared with the control group.However,when 20 ng/ml specific b FGF neutralizing antibody was added in 50% TCM and TCM group,the migration of BMSCs was weakened(P<0.05).7.Wound-healing test results showed that the migration distances of BMSCs in the 10ng/ml and 50ng/ml b FGF group were increased compared with that in the control group(P<0.01).With the increase of b FGF concentration,the migration distance of BMSCs was gradually increased in a dose-dependent manner(P<0.01).8.The results of Transwell chamber assay showed that the number of migrating BMSCs were 55±6 and 78±8,respectively,after treated with 10ng/ml and 50ng/ml exogenous b FGF for 12 hours.The migrating number of BMSCs after 10ng/ml and 50ng/ml b FGF exposure was significantly higher than that in the control group(P<0.05 and P<0.001,respectively).And the number of migrated BMSCs was increased in a dose-dependent manner(P<0.001).9.Trypan blue staining results showed that there was no difference in the cell number of BMSCs after treatment with 50% TCM,50% TCM+FGF Ab,TCM,TCM+FGF Ab,10ng/ml b FGF,50ng/ml b FGF for 12 h when compared with control group(all P<0.05).10.Western Blot assay results showed that mouse BMSCs expressed FGFR1,and b FGF did not affect the expression of FGFR1.11.Trypan blue staining results showed that the number of 4T1 breast cancer cells after treatment with 20% BMSC-CM and 50% BMSC-CM for 72 hours under normal serum culture conditions were 6.20±0.46(×104)and 10.58±0.94(×104),respectively,and both of which was significantly higher than that in the control group(4.43±0.5(×104),P<0.05).The number of 4T1 breast cancer cells after treatment with 20% BMSC-CM and 50% BMSC-CM for 72 hours under low serum culture conditions were 5.75±0.25(×103)and 10.83±2.92(×103),respectively,and both of which was significantly higher than that in the control group(1.83±0.38(×103),P<0.05).With the increase of BMSC-CM concentration,the cell number of 4T1 breast cancer cells was gradually increased in a concentration-dependent manner(P<0.01).12.MTS colorimetric assay showed that the absorbance value(A value)of 4T1 breast cancer cells in 20% BMSC-CM and 50% BMSC-CM group under normal serum culture conditions were 1.467±0.162 and 1.645±0.084,respectively,which were significantly higher than those in control group(0.900±0.008)(P<0.05).13.To determine if BMSCs support tumor growth in vivo,a set of xenograft experiments were performed using mouse 4T1 breast cancer cells.Tumor volume and weight in 4T1 alone group,BMSCs and 4T1 group,and TCM-pretreated BMSCs and 4T1 group were 688.86±105.85,973.90±95.68 and 1223.17±167.39(mm3);475.59±73.50,697.73±39.93 and 834.77±119.82(mg),respectively.4T1 cells mixed with BMSCs or TCM-pretreated BMSCs generated tumors of greater volume and weight when compared to 4T1 cells-alone group(P<0.05).And,although there was no statistical significance,TCM-pretreated BMSC group had bigger tumor volume and weight compared to BMSCs group(P>0.05).14.To quantify PCNA expression,the tumor sections were photographed with a digital camera,and the integral optical density of PCNA in each section was analyzed using Image-J image analysis software.We found that the expression of PCNA in tumor tissues significantly increased in the BMSCs(P<0.001)and TCM-treated BMSC groups(P< 0.001)compared to the 4T1 group.15.Measuring and calculating the necrosis rate of tumor found the necrosis rate in 4T1 alone group,BMSCs and 4T1 group,and TCM-pretreated BMSCs and 4T1 group were 26.86±8.23(%),5.96±3.46(%)and 5.05±3.10(%),respectively.4T1 cells mixed with BMSCs or TCM-pretreated BMSCs generated a smaller necrosis rate when compared to 4T1 cells-alone group(P<0.001).16.To evaluated microvessel density in the tumor sections the tumor sections,we performed immunohistochemical staining for CD34.We found that microvessel density in tumor tissues significantly increased in the BMSCs(P<0.001)and TCM-treated BMSC groups(P<0.001)compared to the 4T1 group.17.Immunohistochemical staining showed that strong positive staining for α-SMA was seen in tumors derived from the BMSCs(P<0.001)and TCM-pretreated BMSC groups(P<0.001).18.Immunofluorescence assay showed that native BMSCs expressed a SMA and vimentin at a low level,while the expression levels of α-SMA and vimentin increased after exposure to the 50%TCM,indicating that the TCM induced BMSCs to acquire CAFs like phenotype.10ng/ml b FGF also significantly increased the expression of α-SMA and vimentin.TGF-β mildly increased the expression of α-SMA and vimentin.The addition of specific b FGF neutralizing antibody inhibited the expression of α-SMA and vimentin protein in 50% TCM-stimulated BMSCs.19.Western blotting analysis showed that BMSCs exposed to the 50%TCM resulted in the increased expression of α-SMA and vimentin.10ng/ml b FGF and TGF-β also increased the expression of α-SMA and vimentin.The addition of specific b FGF neutralizing antibody inhibited the expression of α-SMA and vimentin protein in 50% TCM-stimulated BMSCs.20.Prominent collagen deposition was found after picric acid–sirius red staining and the 50%TCM,b FGF and TGF-β secreted more collagen than the control group.The addition of specific b FGF neutralizing antibody inhibited the expression of collagen in 50% TCM-stimulated BMSCs.21.RT-PCR assay indicated that there were 14-fold,19-fold,20-fold,and 20-fold,upregulations in m RNA expression levels of α-SMA,vimentin,collagen I,and collagen III after treatment of 50%TCM,respectively(all P<0.001).The addition of specific b FGF neutralizing antibody(20ng/ml)inhibited the expression of α-SMA,vimentin,and collagen III in 50% TCM-stimulated BMSCs(P<0.05),while on effects on collagen I m RNA expression.In addition,10ng/ml b FGF also upregulated the m RNA expression levels of α-SMA,vimentin,collagen I,and collagen III(all P<0.001).Under the same consideration as b FGF,TGF-β only upregulated the m RNA expression levels of α-SMA and collagen I,but not vimentin and collagen III(P<0.05).22.b FGF upregulated the phosphorylated level of Erk1/2,indicating that Erk1/2 signaling accompanied the upregulation of α-SMA and vimentin in transdifferentiated BMSCs.Additionally,compared to control group,phosphorylation of mothers against decapentaplegic homolog 3(p Smad3)was also elevated in b FGF induced cells.Then,we blocked the activation of Erk1/2 by treating BMSCs with a specific Erk inhibitor PD98059.In the presence of PD98059,b FGF induced Erk and Smad3 phosphorylation was significantly decreased and both α-SMA and vimentin protein levels were dramatically downregulated,which indicated that Erk/Smad3 signaling pathway was involved in b FGF induced BMSC differentiation.Conclusion 1.BMSCs could be successfully isolated by whole-bone marrow culture.The cell phenotype was CD29+CD44+ by flow cytometry analysis,but the cell yield was low and cell viability was poor after cryopreservation.Therefore,cell cryopreservation methods still need improvement.2.Mouse BMSCs was Sca-1+CD29+CD44+CD105+CD11b-CD34-CD45-CD117-,and had the ability of multiple differentiation potential.3.b FGF under breast cancer microenvironment could promote the migration of BMSCs,suggesting that b FGF is involved in the recruitment of BMSCs by breast cancer cells.4.BMSCs promoted breast cancer cells proliferation and xenografted tumor growth.5.BMSCs promoted the angiogenesis of breast cancer xenografted tumor.6.BMSCs promoted the expression of α-SMA in 4T1 breast cancer model,suggesting that they could differentiate into CAFs with high α-SMA expression in the breast tumor microenvironment in vivo.7.b FGF in the breast tumor microenvironment can induce the transdifferentiation of BMSCs into CAFs.8.ERK/Smad3 signaling pathway is involved in b FGF-induced transdifferentiation of BMSCs into CAFs. |
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