The expression and function of PD-L1 in CD133(+) human liver cancer stem-like cells / 中华肿瘤杂志

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.

Lung Cancer Stem-like Cells and Drug Resistance / 中国肺癌杂志

Lung cancer remains the leading cause of cancer-related death world-wide. Therapy resistance and relapse are considered major reasons contributing to the poor survival rates of lung cancer. Accumulated evidences have demonstrated that a small subpopulation of stem-like cells existed within lung cancer tissues and cell lines, possessing the abilities of self-renewal, multipotent differentiation and unlimited proliferation. These lung cancer stem-like cells (LCSCs) can generate tumors with high effeciency in vivo, survive cytotoxic therapies, and eventually lead to therapy resistance and recurrence. In this review, we would like to present recent knowledges on LCSCs, including the origins where they come from, the molecular features to identify them, and key mechanisms for them to survive and develop resistance, in order to provide a better view for targeting them in future clinic.
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WM130 preferentially inhibits hepatic cancer stem-like cells by suppressing AKT/GSK3β/β-catenin signaling pathway / 中国药理学与毒理学杂志

OBJECTIVE The eradication of cancer stem cells(CSCs)is signifcant for cancer therapy and prevention.METHODS In this study,we evaluated WM130,a novel derivative of matrine,for its effect on CSCs using human hepatocellular carcinoma(HCC)cell lines,their sphere cells,and sorted EpCAM+cells. RESULTS We revealed that WM130 could not only inhibit proliferation and colony formation of HCC cells, but also suppress the expression of some stemness-related genes and up-regulate some mature hepatocyte marker genes, indicating a promotion of differentiation from CSCs to hepatocytes. WM130 also suppressed the proliferation of doxorubicin-resistant hepatoma cells, and markedly reduced the cells with CSC biomarker EpCAM.Moreover,WM130 suppressed HCC spheres,not only primary spheres but also subsequent spheres,indicating an inhibitory effect on self-renewal capability of CSCs.Interestingly,WM130 exhibiteda remarkable inhibitory preference on HCC spheres and EpCAM+cells rather than their parental HCC cells and EpCAM- cells respectively. In vivo, WM130 inhibited HCC xenograft growth, decreased the number of sphere-forming cells, and remarkably decreased the levels of EpCAM mRNA and protein in tumor xenografts. Better inhibitory effect was achieved by WM130 in combination with doxorubicin.Further mechanism study revealed that WM130 inhibited AKT/GSK3β/β-catenin signaling pathway. CONCLUSION Collectively, our results suggest that WM130 remark-ably inhibits hepatic CSCs, and this effect may via the down-regulation of the AKT/GSK3β/β-catenin pathway.These findings provide a strong rationale for the use of WM130 as a novel drug candidate in HCC therapy.

The influence of swainsonine bring in the expression of N-glycan of osteosarcoma tumor stem cells / 实用医学杂志

Objective Detecting the influence of swainsonine bring in the expression of N?glycan of osteosarcoma tumor stem cells. Methods LM8 cells were cultured in the culture medium (free of serum), then selected the LM8 cells of both CD44 and CD133 positive by magnetic activated cell sorting technique. Detecting the levels ofβ1, 6 branch N?glycan expression in the selected LM8 cells (treating by different doses of N?glycosylation inhibitor swainsonine) surface through the lectin binding assay (L?PHA). Results (96.5 ± 1.2)%of LM8 cells were both CD133 and CD44 positive which selected from all the LM8 cells in culture medium (free of serum) by magnetic activated cell sorting technique. Lectin binding assays shown that the bound rare of osteosarcoma tumor stem cells and L?PHA was (90.3 ± 2.1)%, which was higher than normal LM8 cells and L?PHA by (54.3 ± 3.1)%(P<0.05). The positive rare ofβ1, 6 branch N?glycan on the selected LM8 cells(treat with 1mg/mL swainsonine) surface was 90%. The positive rare of β1, 6 branch N?glycan on the selected LM8 cells (treat with 5mg/mL swainsonine) surface was 21%. Conclusion N?glycosylation inhibitor swainsonine plays a role in inhibiting the expression of N?glycan on cell surface of osteosarcoma tumor stem cell.

Influence of stem-like cells on EMT occurrence in mice with triple-negative breast can-cer and on their biological behavior / 中国肿瘤临床

Objective:To discuss the influence of ALDH1+and CD133+phenotypic breast cancer stem-like cells in TA2 triple negative breast cancer on promoting epithelial-mesenchymal transition (EMT) occurrence in TA2 mice with triple-negative breast cancer and on their biological behavior. Methods:Flow cytometry was performed to analyze the markers ALDH1 and CD133 in TA2 mice triple nega-tive breast cancer and breast cancer stem-like cells with ALDH1+, ALDH1?, CD133+, and CD133?phenotypes, which were sorted out. Then, the TA2 mice were inoculated with sorted tumor cells according to cell type. The mice were divided into ALDH1+, ALDH1?, CD133+, and CD133-groups. The tumor-growing conditions were observed. A tumor tissue was sliced for the immunohistochemical testing of ALDH1?, CD133?, and EMT-related Twist1, E-cadherin, and VE-cadherin proteins. The expression difference of breast cancer stem cell surface markers ALDH1 and CD133 in triple-negative breast cancer and EMT-related proteins Twist1, E-cadherin, and VE-cad-herin was analyzed. Results:The expression rates of breast cancer stem cell markers ALDH1 and CD133 in TA2 mice triple negative breast cancer were 31.2%and 6.5%, respectively. The tumor growth ability of TA2 mice from ALDH1+group was obviously stronger than that from ALDH1?group. The CD133+group was evidently stronger than CD133?group. The immunohistochemical results showed that ALDH1, Twist1, and VE-cadherin expression levels in the ALDH1+group were evidently higher than that in the ALDH1?group (all P<0.05). E-cadherin expression decreased (P<0.05). CD133?, Twist1, and VE-cadherin expression levels in CD133+group were higher than that in CD133?group (all P<0.05). Conclusion:In TA2 mice triple negative breast cancer, ALDH1+and CD133+phenotypic breast cancer stem-like cells may influence the expression of EMT-related proteins, and promote the formation of triple-negative breast cancer.

Isolation of stem-like cells from human MG-63 osteosarcoma cells using limiting dilution in combination with vincristine selection.

To isolate stem-like cells from the human MG-63 osteosarcoma cell line, different subpopulations of MG-63 cells were cloned by limiting dilution and passaged to obtain different sublines. The subline with highest clonogenicity was identified using a proliferation assay, cell-cycle analysis, and soft-agar colony-forming assay. The sublines were further selected in serum-free medium containing 20 ng/ml vincristine to identify cells that could form suspended sarcospheres. Identified cells were then characterized based on morphology, cell surface markers, adipogenic and osteogenic differentiation, and tumorigenicity in nude mice. A total of 19 holoclones that could be stably passaged were obtained. Sublines A1, A3, and D1 were markedly different from other sublines and the parental cell line. Subline D1 not only had a higher colony-forming efficiency and formed larger colonies, but also possessed a shorter latency of tumorigenesis in vivo. After subline D1 was cultured in suspension in medium containing vincristine, a highly enriched subpopulation of cells that could form sarcospheres and be stably passaged were obtained. These cells, designated as MG-63-M expressed multiple markers of multipotent or embryonic stem cells and possessed the capacity for self-renewal, multilineage differentiation, and significant multi-drug resistance. Thus, our results suggest that a subpopulation of stem-like cells can be isolated from human MG-63 osteosarcoma cell line.

Cerebral functional improved in rats with severe acute monoxide poisoning by transplantation of neural stem-like cells derived from bone marrow mesenchymal stem cells / 中华急诊医学杂志

Objective To investigate cerebral functional remodeling of the rat with severe acute carbon monoxide poisoning transplanted with neural stem-like cells derived from bone marrow mesenchymal stem cells (MS-NSCs) . Method Forty Sprague-Dawley rats weighing 200 ～ 250 g were divided randomly into 4 groups: the normal control group, the poisoning control group, the bone marrow mesenchymal stem cells(BMSCs) transplantation group and the MS-NSCs transplantation group. BMSCs were harvested from whole bone marrow in vitro, then were differentiated into MS-NSCs under certain growth factors cocktail,and were followed by BrdU labelling.Twenty-four hours after poisoning, the seed cells were infused into brain via left internal carotid and the functional remodeling of cerebrum was assessed by neurological severity score(NSS) and Morris water maze(MWM) tests. Results There was no significant differences in NSS test between groups after transplantation. However, the differences in MWM test were very significant between 5 weeks after transplantation ( P < 0.01). Conclusions Transplantation of MS-NSCs may improved cerebral function of rats after severe acute CO poisoning. Moreover, the cultured and idfferentiated MS-NSCs induced in vitro preliminarily is potentially more efficient than directly transplanted BMSCs without culture and differentiation.

Cerebral white matter remodelling in rats with severe acute carbon monoxide poisoning after transplantation of neural stem-like cells derived from bone marrow mesenchymal stem cells / 中华急诊医学杂志

Objective To investigate changes in cerebral white matter after transplantation of neural stem like cells (MS-NSCs) derived from the bone marrow mesenchymal stem cells, in rats with severe acute carbon monoxide poisoning. Method Forty Sprague-Dawley rats weighing 200 ～ 250 g were divided randomly into 4 groups: the normal control group, the poisoned control group, the BMSCs transplanted group and the MS-NSCs transplanted group (each group 6 rats). BMSCs were harvested from whole bone marrow in vitro, and then differentiated into MS-NSCs under the cock tail of certain growth factors, followed by BrdU labelling. The seed cells were infused into the brain via the left internal carotid artery 24 hours post poisoning. Remodelling of cerebral white matter was assessed using H & E staining, myelin staining and immunohistochemitry assay after 5 weeks later. Results Cellular transplantation improved the compactness and orderliness of cerebral white matter. BrdU-positive cells were found in the focal insulted areas of sparse white matter; and greater numbers of Brdu-Positive ceus were observed in the MS-NSCs group thar in the BMSCs group (P ＜0.05). Conclusions MS-NSCs participates in the remodeling of cerebral white matter in rats with severe acute carbon monoxide poisoning, and shows promising evidence as seed cells transplanted for brain tissue regeneration.

Effects of EGF on Isolation and Cloning of Mouse Embryonic Stem-like Cells from PGCs / 医学研究杂志

Objective To investigate the effects of the EGF on isolation and cloning of mouse embryonic stem-like cells from PGCs. Methods EGF with different density were added to the culture medium of cells .After a culturing period ,the cloning number of mouse embryonic stem-like cells and the topmost passage would be observed and registered. Results There was not obvious effects on the primary culture of mouse ESC and was distinct on the later passage if EGF were used. Conclusions In the primary passage of mouse ESC may survive without EGF,and the impact of the existence of EGF was better than the one of the inexistence to the cloning of mouse ESC.