In recent years, cancer stem cells have become a hotspot for global researchers. Cancer stem cell theory deems that cells with self-renewal and differentiation potential play a key role in tumor resistance and relapse. These cells are named cancer stem cells. At present, the sorting methods include the side population cell sorting technique, screening techniques based on cell surface special markers, tumor sphere cultures, label retaining cell, ALDEFLUOR assays and so on. Side population cells are a small part of cells with the capacity of efflux DNA fluorescent dye Hoechst 33342 and present a low staining intensity in flow cytometry plot. Side population cells are rich in cancer stem cells, and its sorting method has been considered simple and effective in cancer stem cell research.
Flow Cytometry ; Humans ; Neoplasms ; Neoplastic Stem Cells ; Side-Population Cells

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

OBJECTIVETo sort and identify side population (SP) cancer stem cells (CSC) in human prostate cancer (PCa) cell lines.

METHODSStem-like cells were isolated from five PCa cell lines Du145, IA8, LNCaP, TSU-Pr and PC-3 using FACS based on CD133+ CD44+ immunophenotype and SP in Hoechst staining. The in vitro growth pattern and tumorigenicity of SP stem cells were verified by soft agar colony-formation trial. LNCaP/SP cells were selected for further identification of stem cell properties using immunostaining, proliferation and invasion assay. Eventually, tumorigenicity and metastasis ability of LNCaP/SP were confirmed by xenograft experiments.

RESULTSThe percentages of CSCs of the CD133 CD44 + immunophenotype were extremely low in the five PCa cell lines. On the contrary, the percentages of the isolated SP cells were significantly higher in Du145 ([0.15 +/- 0.02]%), IA8 ([0.60 +/- 0.07 ]%), LNCaP ([0.8 +/- 0.1]%) and TSU-PrL ([2.0 +/- 0.4]%), but none was detected in PC-3. Besides, IA8/SP, LNCaP/SP and TSU-PrL/SP cells showed a significantly greater colony-forming efficiency than non-side population (NSP) cells (P < 0.05). Compared with LNCaP/NSP cells, LNCaP/SP cells exhibited high expressions of integrin alpha2, Nanog, CD44, OCT4 and ABCG2, remarkably enhanced invasive and proliferative potentials in vitro, and markedly increased tumorigenicity and metastasis (P < 0.01).

CONCLUSIONSP sorting is more suitable than CD133+ CD44+ selection for enriching CSCs from PCa cell lines, and LNCaP/ SP represents a typical CSC population.

Cell Line, Tumor ; cytology ; Cell Separation ; Humans ; Male ; Neoplastic Stem Cells ; cytology ; Prostatic Neoplasms ; Side-Population Cells ; cytology

This study was aimed to sort the side population (SP) cells from human multiple myeloma cell lines, then detect the biological characteristics of those SP cells. After Hoechst33342 staining, intracellular Hoechst33342 fluorescence staining differences of myeloma cell lines observed by the fluorescence microscopy. The fluorescence-activated cell sorting (FACS) technology was used to isolate SP cells and main population (MP) cells; proliferative capacity in vitro was determined by cell growth curve; the cell colony forming ability was compared by colony forming test. The CD138 expression was detected by flow cytometry. The expression of ABCG2 mRNA was detected by reverse transcription PCR; CCK-8 assay and colony forming test were used to evaluate the effect of bortezomib on the cell proliferation, vitality and colony forming ability of the two populations. The results showed that the myeloma cell lines had a small proportion of SP cells, especially, RPMI 8226 cells accounted for the highest proportion of SP cells (7.10 ± 2.69)%, which have also been confirmed under the fluorescence microscope; the proliferative activity and cell colony forming ability of SP cells were significantly higher than those of MP cells (P < 0.05). The expression levels of CD138 in SP and MP cells were not significantly different (P > 0.05). RT-PCR results showed that SP cells expressed the drug-resistance gene ABCG2, but MP cells hardly express these genes. The inhibition rate of bortezomib on SP cells was significantly lower than that on MP cells (P < 0.05), however, the difference was not significant (P > 0.05) at bortezomib 40 nmol/L. Bortezomib could reduce colony formation in the both two cell populations, but more severe reduction appeared in the MP cells. It is concluded that the myeloma cell line contain a small amount of SP cells with the cancer stem cell characteristics.
Cell Line, Tumor ; Cytological Techniques ; methods ; Humans ; Multiple Myeloma ; Neoplastic Stem Cells ; cytology ; Side-Population Cells ; cytology

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

Severe graft-versus-host disease (GVHD) is an often lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). The safety of clinical-grade mesenchymal stem cells (MSCs) has been validated, but mixed results have been obtained due to heterogeneity of the MSCs. In this phase I study, the safety of bone marrow-derived homogeneous clonal MSCs (cMSCs) isolated by a new subfractionation culturing method was evaluated. cMSCs were produced in a GMP facility and intravenously administered to patients who had refractory GVHD to standard treatment resulting after allogeneic HSCT for hematologic malignancies. After administration of a single dose (1x10(6) cells/kg), 11 patients were evaluated for cMSC treatment safety and efficacy. During the trial, nine patients had 85 total adverse events and the rate of serious adverse events was 27.3% (3/11 patients). The only one adverse drug reaction related to cMSC administration was grade 2 myalgia in one patient. Treatment response was observed in four patients: one with acute GVHD (partial response) and three with chronic GVHD. The other chronic patients maintained stable disease during the observation period. This study demonstrates single cMSC infusion to have an acceptable safety profile and promising efficacy, suggesting that we can proceed with the next stage of the clinical trial.
Bone Marrow ; Drug-Related Side Effects and Adverse Reactions ; Graft vs Host Disease* ; Hematologic Neoplasms ; Hematopoietic Stem Cell Transplantation ; Humans ; Mesenchymal Stromal Cells* ; Myalgia ; Population Characteristics

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