It has been widely verified by various sorting methods that cancer stem cells (CSCs) exist in different types of tumor cells or tissues. However, due to lack of specific stem cell surface markers, CSCs are very difficult to be separated from some cancer cells, which becomes the key barrier of functional studies of CSCs. The sorting method by side population cells (SP) lays a solid foundation for in-depth and comprehensive study of CSCs. To identify the existence of SP in prostate cancer cell lines, we applied flow cytometry sorting by SP to cultures of prostate cancer cell lines (TSU, LnCap, and PC-3), and the cancer stem-like characteristics of SP were verified through experiments in vitro and in vivo. The proportion of SP in TSU cells was calculated to be 1.60%±0.40% [Formula: see text], and that in PC-3 and LnCap cells was calculated to be 0.80%±0.05% and 0.60%±0.20%, respectively. The colony formation assay demonstrated that the colony formation rate of SP to non-SP sorted from TSU via flow cytometry was 0.495±0.038 to 0.177±0.029 in 500 cells, 0.505±0.026 to 0.169±0.024 in 250 cells, and 0.088±0.016 to 0.043±0.012 in 125 cells respectively. In the in vivo experiments, tumors were observed in all the mice on the 10th day after injecting 50 000 cells subcutaneously in SP group, whereas when 5×10(6) cells were injected in non-SP group, tumors were developed in only 4 out of 8 mice until the 3rd week before the end of the experiment. Our results revealed that prostate cancer cells contain a small subset of cells, called SP, possessing much greater capacity of colony formation and tumorigenic potential than non-SP. These suggest that SP in prostate cancer cells may play a key role in the self-renewal and proliferation, and have the characteristics of cancer stem-like cells. Dissecting these features will provide a new understanding of the function of prostate CSCs in tumorigenicity and transformation.
Cell Line, Tumor ; Humans ; Male ; Neoplastic Stem Cells ; pathology ; Prostatic Neoplasms ; pathology ; Side-Population Cells ; pathology

OBJECTIVE: To investigate the optimizing conditions in isolation of the side population in laryngeal carcinoma cell line Hep-2. METHOD: Single-cell suspension cells were detached from the culture flask with trypsin EDTA, at a concentration of 1 x 10(6) cells/ml. (1) The trail Samples were incubated with Hoechst33342 at a concentration of 5 microg/ml, 9 microg/ml, 10 microg/ml, 11 microg/ml for 90 minutes. (2) They were incubated with Hoechst for 50, 70, 90, 110, 130 min in water bath individually. (3) The single-cell suspension were incubated Hoechst in water bath and in thermostat each. (4) The two different density of cells were harvested, which were 100% and 70%, and then di gest into single-cell suspension. Once incubation finished, suspended in phosphate buffered saline (PBS), then test SP% by flow cytometry. Among all groups,Verapamil hydrochloride was added to the control samples, incubated at 37 degrees C for 30 minutes, the other condition were keep the same with their trial groups. RESULT: (1) The percentage of Hoechst-negative cells in trial group was (39.96 +/- 0.24)%, (26.23 +/- 0.39)%. (18.79 +/- 0.02)%, (19.01 +/- 0.14)% at the concentration of 5 microg/ml, 9 microg/ml, 10 microg/ml, 11 microg/ml respectively, when the PI-positive cells were (30.45 +/- 0.63)%, (49.9 +/- 0.42)%, (50.12 +/- 0.68)%, (64.16 +/- 0.39)% separately. (2) Varying the duration of staining incubation showed that there was a typical FACS pattern and SP% was constant when the incubation was at least 90 min. (3) Compare to water bath, SP% was more than in thermostat, the SP% was (18.67 +/- 0.45)%, (22.6 +/- 0.50)% respectively; (4) Cell density is also responsible for SP%. The low density the cell is, the less in SP%. SPSS13.0 was used in statistical analysis, the groups were compared using t-Test. P < 0.05 was considered statistically significant. CONCLUSION The optimum concentration and duration of incubation of Hoechst33342 in isolation of the side population cells in laryngeal carcinoma cell line Hep-2 is 10 microg/ml and 90 min. Incubated in water bath is better than in thermostat. The best staining cell density is around 80%-90%.
Cell Count ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Laryngeal Neoplasms ; pathology ; Neoplastic Stem Cells ; cytology ; Side-Population Cells ; cytology

OBJECTIVETo investigate an approach enriching cancer stem cells (CSCs) more effectively from laryngeal cancer cell line.

METHODSCD133(+)SP and CD133(-)SP subpopulation was detected and isolated from Hep-2 cell line using Hoechst33342 dye and phycoerythrin (PE)-conjugated CD133 monoclonal antibody assisted by fluorescence activated cell sorting technology. Sorted CD133(+)SP and CD133(-)SP cells were compared in CSCs-related assays including proliferation, differentiation, spheroid formation and drug sensitivity.

RESULTSCD133(+)SP cells accounted for a very small fraction of (0.30 ± 0.12)% in Hep-2 cell line, far less than the proportion of CD133(+) subgroup and side population subgroup, which were (3.15 ± 0.83)% and (17.1 ± 2.0)% respectively. Intriguingly, CD133(+)SP cells proliferated much faster than CD133(-)SP cells in RPMI1640 and gave rise to CD133(-)SP cells and other heterogeneous cells that formed the bulk of the tumor. In contrast, CD133(-)SP cells were not able to differentiate into CD133(+)SP cells. In serum-free medium CD133(+)SP cells grew as spherical clusters and remained floating. In addition, CD133(+)SP cells manifested the marked resistance to chemotherapy than CD133(-)SP cells.

CONCLUSIONSCompared with CD133(-)SP cells, CD133(+)SP subpopulation exhibited extraordinary cancer stem-like properties, were enriched for cancer stem cells more effectively and might serve as an ideal putative candidate for CSCs research in laryngeal cancer.

AC133 Antigen ; Antigens, CD ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Cell Separation ; Glycoproteins ; Humans ; Laryngeal Neoplasms ; pathology ; Neoplastic Stem Cells ; cytology ; Peptides ; Side-Population Cells ; cytology

OBJECTIVETo observe the proliferation changes of the side population of gastric cancer cell line SGC-7901 cells (SP), the non-side population (NSP) cells, and unsorted cells (Total) after intervened by Sijunzi Decoction (SD) containing serum.

METHODSSixteen pure bred New Zealand rabbits were equally divided into the normal control group, the low dose SD group (at the daily dose of 7 mL/kg), the middle dose SD group (at the daily dose of 14 mL/kg), and the high dose SD group (at the daily dose of 28 mL/kg) according to the random digit table. Rabbits' serum was extracted after equal volume of corresponding medication was given by gastrogavage twice daily for 2 consecutive weeks. The drug serum was identified using high performance liquid chromatography. SP cells of SGC-7901 were detected using flow cytometry, SP and NSP cells were screened. The proliferation curve of SP, NSP, and Total cells were detected with CCK-8 assay. Changes of their proliferation were also observed.

RESULTSGinsenoside Rg1, an effective ingredient in SD was detected in prepared drug serum. The proliferation of SGC-7901 SP cells was significantly higher than that of NSP cells and Total cells (P < 0.05). Drug serum on gastric cancer cell line SGC-7901 SP, NSP, and Total cells could inhibit their proliferation, but its inhibition on SP cells' proliferation was significantly lower than on NSP and Total cells (P < 0.05).

CONCLUSIONSSD could significantly inhibit the proliferation of gastric cancer cell line SGC-7901 SP, NSP, and Total cells. But there exist obvious difference in the inhibition among the three groups.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Rabbits ; Side-Population Cells ; drug effects ; pathology ; Stomach Neoplasms ; pathology

Objective: To inhibit the stemness maintenance potential of endometrial cancer and increase the sensitivity of endometrial cancer side population cells to chemotherapy drugs by inducing extensive deSUMOylation modification of proteins. Methods: Flow cytometry was used to sort and culture CD133(+) CD44(+) KLE endometrial cancer cell clone spheres. Protein expression level of small ubiquitin-related modifier 1 (SUMO1) and two stemness maintenance genes of tumor side population cells, octamer binding transcription factor-4 (Oct4) and sex determining region Y-box2 (Sox2), were detected by western blotting method. Lentivirus-mediated Sentrin/SUMO-specific proteases 1 (SENP1) gene was stably transfected into KLE side population cells. Western blotting was used to detect the protein expressions of SENP1, SUMO1, Oct4 and Sox2. The clone formation rate was compared between KLE side population cells with or without SENP1 overexpression. Flow cytometry was applied to detect cell cycle changes. 3-(4, 5-Dimethylthiazole-2)-2, 5-diphenyl-tetrazolium bromide (MTT) experiment and flow cytometry apoptosis method were used to detect the chemosensitivity of the side population of endometrial cancer cells to cisplatin. Tumor-bearing mouse models of endometrial cancer were established to detect the effect of SENP1 overexpression on the chemotherapy sensitivity of cisplatin. Results: Compared with CD133(-)CD44(-) KLE cells, CD133(+) CD44(+) KLE side population cells could form clonal spheres and express higher levels of SUMO1, Oct4 and Sox2 proteins (P<0.05). Compared with KLE side population cells that were not transfected with SENP1 gene, the expression level of SENP1 protein in KLE side population cells overexpressing SUMO1、Oct4 and Sox2 were lower. The clonal sphere formation rate was reduced from (25.67±5.44)% to (7.46±1.42)%, and cell cycle shifted from G(0)/G(1) phase to G(2) phase. IC(50) of cisplatin decreased from (55.46±6.14) μg/ml to (11.55±3.12) μg/ml, and cell apoptosis rate increased from (9.76±2.09)% to (16.79±3.44)%. Overexpression of SENP1 could reduce the tumorigenesis rate of KLE side population cells in vivo and increase their chemotherapy sensitivity to cisplatin (P<0.05). Conclusion: Overexpression of SENP1 can induce protein deSUMOylation modification, inhibit the stemness maintenance potential of endometrial cancer side population cells, and enhance their chemotherapy sensitivity, which provides a new reference for gene therapy of endometrial cancer.
Animals ; Female ; Humans ; Mice ; Apoptosis ; Cell Line, Tumor ; Cisplatin/pharmacology* ; Cysteine Endopeptidases/metabolism* ; Endometrial Neoplasms/genetics* ; Side-Population Cells/pathology* ; Sumoylation

OBJECTIVETo isolate the side population (SP) and non-side population (NSP) cells from A431 cells and compare their difference in tumorigenicity in mice and the expression profiles of epithelial-mesenchymal transition (EMT) markers.

METHODSA431 cells stained with Hoechst 33342 were sorted with flow cytometry. The isolated SP cells and NSP cells were inoculated into NOD/SCID mice and the tumorigenicity of the cells was observed. EMT markers E-cadherin, β-catenin, vimentin, AXL, and Erbb3 in the tumor tissues were detected by immunohisto-staining.

RESULTSThe tumors generated by SP cells were larger than those by NSP cells in NOD/SCID mice. Compared with the tumors generated by NSP cells, the cells in the periphery of tumors generated by SP cells showed up-regulated expressions of AXL, vimentin and β-catenin and down-regulated ERBB3 and E-cadherin.

CONCLUSIONThe SP cells in A431 cells have a strong tumorigenicity and show more EMT phenotypes in tissues.

Animals ; Biomarkers, Tumor ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplastic Stem Cells ; metabolism ; Side-Population Cells ; metabolism ; Skin Neoplasms ; metabolism ; pathology

OBJECTIVEIsolate and characterize the side population (SP) cells with potency of stem cells from human gastric carcinoma cell line BGC-823.

METHODSSP and non-SP cells were sorted from BGC-823 cells by fluorescence-activated cell sorting (FACS) using Hoechst33342 staining. The tumorigenic ability of the SP cells was assessed by in vivo transplantation into non-obese diabetic/severe combined immunodeficiency mice.

RESULTSSP cells were isolated from BGC-823 cells in a proportion of 0.9% to 2.1% with respect to the whole cell population. The colony formation assay showed that the colony formation rate of the SP cells was significantly higher than that of the non-SP cells (72.56% vs. 49.00%, P < 0.01). The drug sensitivity test showed that the SP cells showed stronger drug resistance to 5-Fu than the non-SP cells. The in vivo transplantation of SP cells in mice showed that the tumor weight was (0.176 ± 0.034) g, significantly higher than that of non-SP cells (0.045 ± 0.046) g (P < 0.01).

CONCLUSIONSThe results of this study indicate the existence of cancer stem-like SP cells in the human gastric cancer line BGC-823 cells. Further characterization of this SP cell population may provide new insights for diagnosis and treatment of gastric cancer.

Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; Colony-Forming Units Assay ; Drug Resistance, Neoplasm ; Female ; Fluorouracil ; pharmacology ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Side-Population Cells ; drug effects ; pathology ; transplantation ; Stomach Neoplasms ; pathology

Cancer stem cell (CSC) research has increased exponentially to gain further insight into the mechanisms underlying both carcinogenesis and chemotherapy resistance. The present study was performed to explore the potential value of a side population (SP) assay for identifying and characterizing putative CSCs among canine lymphoma cells. Canine lymphoma cells from cell lines and clinical samples were subjected to the SP assay consisting of Hoechst 33342 staining and subsequent flow cytometric analysis. The SP assay revealed various amounts of a SP fraction among the canine lymphoma cells. The percentages of SP were not affected by inhibitors of membrane transporters, verapamil hydrochloride, or fumitremorgin C. Most of the canine lymphoma cells expressed high levels of Bmi-1 and membrane transporter proteins such as ABCG2 and phosphorylated (p)-glycoprotein. This investigation lays the groundwork for further studies of the biological behaviors and molecular characteristics of CSCs in cases of canine lymphoma.
Animals ; Benzimidazoles/*metabolism ; Cell Line, Tumor ; Dog Diseases/*diagnosis/drug therapy/pathology ; Dogs ; Flow Cytometry/*methods/veterinary ; Fluorescent Dyes/*metabolism ; Gene Expression Regulation, Developmental ; Lymphoma/diagnosis/drug therapy/pathology/*veterinary ; Neoplastic Stem Cells/drug effects/*metabolism/pathology ; Side-Population Cells/drug effects/*metabolism/pathology

OBJECTIVETo isolate and characterize the side population cells (SP cells) in the lung adenocarcinomas cell line A549.

METHODSThe protein expression of ABCG2 in human lung adenocarcinoma cell line A549 was detected by immunohistochemistry. SP and NSP cells in the cell line A549 were isolated by FACS, and their differentiation was analysed. ABCG2 expression in the two cell subsets was detected by RT-PCR. The cell growth curves, cell division indexes, cell cycles, plate clone formation tests, migration and invasion assays, chemotherapeutic susceptibility tests, tests of the intracellular drug levels, and the tumor cell implantation experiments on nude mice were applied to study the biological properties of the two cell subsets. The expression of ABCG2 in the transplanted tumor in nude mice was detected by immunohistochemistry and RT-PCR.

RESULTSThe positive rate of ABCG2 expression in the A549 cells by immunohistochemistry was 2.13%. SP and NSP cells were isolated by FACS. The SP cells could produce both SP and NSP cells, while NSP cells only produced NSP cells. SP cells expressed ABCG2, but NSP cells did not. The proliferation and migration abilities of the two cell subsets were similar, but the invasion and tumorigenic ability of SP cells was significantly higher than that of NSP cells. The susceptibilities to DDP and its intracellular levels of the two cell subsets were similar, but the susceptibilities to 5-FU, VP16, NVB and GEM and their intracellular levels of NSP cells were significantly higher than those of the SP cells.

CONCLUSIONSSP cells in the human lung adenocarcinomas cell line A549 is enriched with tumor stem cells. An effective way to get lung adenocarcinomas stem cells is to isolate SP cells by FACS.

ATP-Binding Cassette Transporters ; metabolism ; Adenocarcinoma ; pathology ; Animals ; Cell Cycle ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Transformation, Neoplastic ; metabolism ; Fluorouracil ; metabolism ; Humans ; Lung Neoplasms ; pathology ; Mice, Nude ; Neoplasm Proteins ; metabolism ; Neoplasm Transplantation ; Neoplastic Stem Cells ; drug effects ; Side-Population Cells

Breast cancer stem cells are a group of undifferentiated cells with self-renewal and multidifferentiation potential. Chemotherapeutic and radiotherapeutic resistance, hypoxic resistance, high tumorigenicity, high cell invasion, and metastatic abilities are characteristics of these cells, which are responsible for breast cancer recurrence. Therefore, the correct sorting and identification of breast cancer stem cells is a primary step for research in this field. This article briefly describes the recent progress on sorting and identification technologies for breast cancer stem cells. Sorting technologies include the side population technique, technologies that depend on cell surface markers, ALDEFLUOR assays, and in situ detection. Identification technologies include mammosphere cultures, limited dilution in vitro, and in-vivo animal models. This review provides an important reference for breast cancer stem cell research, which will explore new methods for the treatment of patients with breast cancer.
AC133 Antigen ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; Aldehyde Dehydrogenase ; metabolism ; Animals ; Antigens, CD ; metabolism ; Breast Neoplasms ; pathology ; Female ; Flow Cytometry ; methods ; Glycoproteins ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Integrin alpha6 ; metabolism ; Integrin beta1 ; metabolism ; Integrin beta3 ; metabolism ; Isoenzymes ; metabolism ; Membrane Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Neoplastic Stem Cells ; metabolism ; pathology ; Peptides ; metabolism ; Retinal Dehydrogenase ; metabolism ; Side-Population Cells ; cytology ; metabolism