The Effects And Mechanisms Of Mesenchymal Stem Cells On Tumor Cells With Different Malignancy Phenotype

BackgroundMensenchymal Stem Cell(MSCs) are a class of stem cells that mainly exists in the connective tissue or organ stroma with a highly self-renewal and multi-differentiation ability.Because of its low immunogenicity and immune regulation function, homing ability to damage and inflammation and roles of tissue repair and regeneration, so there is a wide range of potential applications in the treatment of various disease researches. The potential applications of MSCs in cancer biotherapy have gradually been recognized, mainly taking advantage of their immune suppression and homing ability to tumor site,MSCs act as adjuvant or vector cells in bone marrow transplantation in patients with leukemia or tumor-targeted therapy. However, the impact of MSCs on the biological behavior of tumor cells of different malignant phenotype is not clear, therefore the safety of MSCs application remains controversial. In the previous study, We found tumor cells with different malignant phenotype have different genotypes after co-cultured with MSCs, in which poly-pyrimidine region binding protein(PTB) changed significantly. PTB is a protein involved in the mRNA metabolic processes inside the cell,its expression is higher in the majority of tumor tissue, and is considered to be a potential gene target for cancer therapy. Currently there is a dispute of PTB expression level and its impact on tumor malignant degree, this may be associated with the type and biological characteristics of the tumor cells. Study the effects and mechanisms of MSCs on biological characteristics of different malignant phenotype tumor cells and roles of PTB in the process, can provide a theoretical basis for the application of MSCs in the treatment of different types of cancer research. It also provides a experimental reference for the efficacy and safety of PTB as an gene therapy target.Objectives1. To determine the effects of bone marrow mesenchymal stem cells (BMSCs)on human colorectal cancer cells of different the malignant phenotype in vitro and in vivo experiments, and figure out the regulatory roles and mechanisms of MSCs to tumor cells with different malignant phenotype, further to verify it by studying the effects of adipose derived stem cells(ADSCs) on human breast cancer cell lines with different malignant phenotype.2. To clarify the expression levels PTB and its associated signaling pathway molecules in colorectal cancer cells of different malignant phenotype after co-cultured with BMSCs, and investigate the mechanisms that MSCs regulate PTB expression levels in different malignant phenotype tumor cells, further to study PTB functions in tumor cells with different malignant phenotype by lentiviral-mediated shRNA knockdown technology.Methods1. Effects of bone marrow mesenchymal stem cells (BMSCs)on human colorectal cancer cells with different the malignant phenotypeCulture different malignant phenotype human colon cancer cell line HT29 and HCT116 in vitro, and observe cell morphology under an inverted microscope, proliferative capacity of the cells detected by MTS assay, utilize transwell method to detect cell invasion capacity, detect cell colony formation ability using soft agar colony formation assay; And using real-time PCR to detect the expression of epithelial marker (E-cadherin) and mesenchymal marker (Vimentin) in HT29 and HCT116, to determine the biological characterization of HT29 and HCT116 cells. Then using whole blood marrow adherent culture method to isolate BMSCs from iliac bone marrow of healthy volunteers. After two generations in vitro amplification,we co-cultured BMSCs with HT29 and HCT116 separately, differences in cell morphology, proliferation, invasion, soft agar colony formation and tumor malignancy associated signaling pathway proteins of HT-29 and HCT116 cells were compared between co-culture and the control group at day3 and day5, to investigate the effects of BMSCs on biological characteristics of HT-29 and HCT116. Finally, after co-culture with BMSCs for one week in vitro, HT29 and HCT116 of each group were injected into nude mice subcutaneously, and monitor the differences in tumor fomation time,tumor volume and the differentiation degree of the tumor tissue.2. Function of PTB in the process that BMSCs influence HT29 and HCT116 differentlyBy the method of real-time PCR and Western Blot, detect the changes of PTB as well as its upsteam and downstream signaling gene and protein expression,make sure the changes of PTB signaling pathway after co-culture with BMSCs. Using real-time PCR to detect the expression levels of PTB related miRNAs,to study the regulation mechanisms of PTB in the process of MSCs influence biological characteristics of different malignant phenotype tumor cells. Then, after knockdown PTB expression by lentiviral-mediated ShRNA knockdown technology, detect the biological characteristic changes in HT29 and HCT116, use real-time PCR and western blot method to detect changes of EMT genes, cancer sternness genes and signaling pathway protein molecules associated with tumor malignancy. This part aims to investigate the function of PTB in HT29 and HCT116.3. In vitro studies of adipose-derived mesenchymal stem cells’effects on breast cancer cells of different malignant phenotypeCuluture different malignant phenotype breast cancer cell lines MCF7 (estrogen receptor-positive cells) and MDA-MB-231 (triple-negative breast cancer cells) in vitro, clarify their biological characteristics by comparing cell morphology, proliferation, invasion and colony formation in soft agar. Isolate and culture adipose-derived mesenchymal stem cells from discarded adipose tissue from liposuction plastic surgery. After three generations’amplification in vitro, coculture ADSCs with MCF7 and MDA-MB-231 separately, differences in cell morphology, proliferation, invasion, soft agar colony formation ability and EMT marker genes (E-cadherin and N-caherin) expression of MCF7 and MDA-MB-231 cells were compared between co-culture and the control group at day3 and day5.Results1. Effects of BMSCs on human colorectal cancer cells with different the malignant phenotypeMorphological observation showed HT29 has morphological characteristics of epithelial cells and HCT116 exhibit strong characteristics of mesenchymal cells. Cell Biology characterization showed the proliferation, invasion and soft agar colony formation ability of HT29 were significantly lower than HCT116. BMSCs co-cultured with HT29 and HCT116 separately in vitro,can significantly promote the proliferation, invasion and colony formation in soft agar, but not significant effect on HCT116. The mechanism may be mediated by upregulation of GSK3β, AKT, STAT3 and ERK1/2 protein phosphorylation levels in HT29. In vivo experimental results show that BMSCs can shorten the time to form a tumor, increase tumor volume, and reduce the differentiation degree of HT29 tumor tissue. In accordance with in vitro expenriment results, HCT116 had no significant changes in vivo tumorigenicity after co-culture with BMSCs.2. Function of PTB in the process that BMSCs influence HT29 and HCT116 differentlyBMSCs co-cultured with HT29 and HCT116 in vitro can significantly downregulate PTB gene and protein levels,and its upstream transcription factor c-MYC as well as downstream cell cycle regulatory proteins p27 and apoptosis regulating proteins CASP3 gene expression in HT29, but the PTB and PTB related signaling pathway molecules in HCT116 were not significantly affected. In addition, miRNAs (miR-133, miR-124, miR-339, miR-149) that can mediate PTB downregulation were significantly increased in HT29, but their expression in HCT116 changed inconsistently. PTB knockdown significantly inhibited cell proliferation and colony formation capability in HT29,while raised its cell invasion capacity and reduce tumor volume and tumor tissue differentiation level of HT29. The mechanisms may be mediated by changing the FGFR2 and CD44 gene alternative splicing, upregulate Twistl and ALDH1 gene expression and improve the phosphorylation level of GSK3β and AKT proteins, promote synthesis of Notch intracellular domain (NICD) and reduce P27 protein levels. Unlike HT29, knockdown PTB expression levels in HCT116 significantly inhibited cell proliferation, invasion and soft agar colony formation and in vivo tumorigenicity, ALDH1 gene expression as well as NICD expression and STAT3 protein phosphorylation levels also significantly declined.3. In vitro studies on the effects of ADSCs on breast cancer cells with different malignant phenotypeCell morphology results showed MCF7 had a typical epithelial cell morphology and MDA-MB-231 presents a strong characteristic of mesenchymal cell types. Biological characterization showed that the cell proliferation, invasion and soft agar colony formation ability of MCF7 were significantly lower than MDA-MB-231 under startard culture conditions in vitro. After co-culture with ADSCs, the cell proliferation, invasion and soft agar colony formation ability of MCF7 were significantly increased, EMT markers E-cadherin decreased and N-cadherin increased. In addition,the effects of ADSCs on cell proliferation, invasion and soft agar colony formation ability in MDA-MB-231 cell with high malignant phenotype was not obvious.Conclusions1. BMSCs can promote human colon cancer cell line HT29 proliferation, invasion and colony formation in soft agar as well as tumorigenicity in vivo and reduce the tumor tissue differentiation level by activating GSK3β, AKT, STAT3 and ERK1/2 protein activity, but the effect is not obvious in HCT116,indicating that BMSCs influence tumor cells with different malignant phenotype variably, can induce low malignant phenotype HT29 cells to highly malignant phenotype cells, while the effects on highly malignant phenotype cells HCT116 were not obvious.2. In vitro co-culture conditions BMSCs significantly inhibited PTB signaling pathways in HT29, and PTB knockdown can promote HT29 invasive ability in vitro and reduce the differentiation degree of tumor tissue in vivo,the mechanism may be by regulating the alternative splicing of CD44 FGFR2, promoting Twistl and ALDH1 gene expression as well as increasing P-GSK3β,p-AKT, NICD and reducing P27 protein expression levels,indicating that the decline of PTB expression levels may be a "switch" that elevate the malignant phenotype of HT29,and BMSCs can promote HT29 malignant phenotype by inhibiting PTB signaling pathways.3. BMSCs had no significant effect on the PTB and related signaling pathways of highly malignant phenotype HCT116 in vitro transwell co-culture conditions,but knockdown PTB significantly inhibited HCT116 cell proliferation, invasion, soft agar colony formation ability and in vivo tumorigenicity,also reduced expression of cancer stem-related genes ALDH1 as well as malignancy associated signaling pathway proteins NICD and P-STAT3, suggesting that PTB involved in different signaling pathways that relugate tumor biological behavior in different malignant phenotype tumor cells,and correspondingly,BMSCs regulate PTB expresion differently in tumor cells with different malignant phenotype.4. ADSCs can elevate the malignant phenotype of low malignant phenotype breast cancer cells MCF7 by inducing EMT process, but the biological characteristics effects on highly malignant phenotype of MDA-MB-231 cells was not obvious, suggesting that MSCs derived from different tissues may regulate the malignant behavior of different types of cancer cells through different mechanisms. This study can provide a theoretical basis for MSCs application in different types of cancer therapy and tissue reconstruction in breast cancer patients.