Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer/testis antigens can be immunological targets in clonogenic CD133 + melanoma cells

doi: 10.1007/s00262-009-0672-0

Figure Lengend Snippet: Ratio of expression of CT antigens in CD133 + clonogenic melanoma cells versus CD133 neg cells by quantitative real time PCR (ratio of ΔCt(CD133 + )/ΔCt(CD133 neg ))

Article Snippet: For flow cytometry, cells were blocked in 10% normal human serum in PBS, stained with anti-CD133 antibody (AC133-PE or 293C3-APC; Miltenyi Biotec, Bergisch Gladbach, Germany) at 1:50 dilution for 15 min at 4°C, washed in PBS, resuspended and collected on a FACSCalibur (Becton Dickson, San Jose, CA) with data analysis by FloJo (Tree Star Inc, OR).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer/testis antigens can be immunological targets in clonogenic CD133 + melanoma cells

doi: 10.1007/s00262-009-0672-0

Figure Lengend Snippet: CD133+ cells are clonogenic and have stem-like properties compared to CD133neg cells. LM-Mel-34 separated by MACS to CD133neg (0.3% CD133) and CD133+ (95%) cells in soft agar (a, b) show differential clonogenicity compared to unsorted cells (c). CD133 is a marker for clonogenicity in other cell lines (Supplementary Fig. 2a–e). (d) When CD133 separated cells are returned to culture, and reanalyzed by flow cytometry several weeks later (right panels), CD133+ cells (top panels) have given rise to CD133neg cells, but CD133neg cells (bottom panels) have been unable to form CD133+ cells, showing a hierarchical relationship

Article Snippet: For flow cytometry, cells were blocked in 10% normal human serum in PBS, stained with anti-CD133 antibody (AC133-PE or 293C3-APC; Miltenyi Biotec, Bergisch Gladbach, Germany) at 1:50 dilution for 15 min at 4°C, washed in PBS, resuspended and collected on a FACSCalibur (Becton Dickson, San Jose, CA) with data analysis by FloJo (Tree Star Inc, OR).

Techniques: Marker, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer/testis antigens can be immunological targets in clonogenic CD133 + melanoma cells

doi: 10.1007/s00262-009-0672-0

Figure Lengend Snippet: CD133+ clonogenic melanoma cells can express cancer/testis antigens. Immunohistochemistry (400X) of CD133 separated cell blocks (CD133+ left, CD133neg right) showing differential expression of (a) NY-ESO-1 in LM-Mel-34 and (b) MAGE-A3/4 in SK-Mel-28. Other CT and differentiation antigens were equally expressed. (c) Cell counts from randomly selected high-power fields of CD133+ and CD133neg melanoma cell blocks (mean ± 2SD)

Article Snippet: For flow cytometry, cells were blocked in 10% normal human serum in PBS, stained with anti-CD133 antibody (AC133-PE or 293C3-APC; Miltenyi Biotec, Bergisch Gladbach, Germany) at 1:50 dilution for 15 min at 4°C, washed in PBS, resuspended and collected on a FACSCalibur (Becton Dickson, San Jose, CA) with data analysis by FloJo (Tree Star Inc, OR).

Techniques: Immunohistochemistry, Quantitative Proteomics

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer/testis antigens can be immunological targets in clonogenic CD133 + melanoma cells

doi: 10.1007/s00262-009-0672-0

Figure Lengend Snippet: Isolated cells in the melanoma metastasis from which the LM-Mel-34 cell line was derived stain double-positive for CD133 (red) and NY-ESO-1 (green) showing in vivo co-expression (merge, with DAPI)

Article Snippet: For flow cytometry, cells were blocked in 10% normal human serum in PBS, stained with anti-CD133 antibody (AC133-PE or 293C3-APC; Miltenyi Biotec, Bergisch Gladbach, Germany) at 1:50 dilution for 15 min at 4°C, washed in PBS, resuspended and collected on a FACSCalibur (Becton Dickson, San Jose, CA) with data analysis by FloJo (Tree Star Inc, OR).

Techniques: Isolation, Derivative Assay, Staining, In Vivo, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer/testis antigens can be immunological targets in clonogenic CD133 + melanoma cells

doi: 10.1007/s00262-009-0672-0

Figure Lengend Snippet: Chemotherapy selects for CD133+ clonogenic melanoma cells which can be eliminated by NY-ESO-1 CTL but not by Melan-A/MART-1 specific CTL. Three treatments of the LM-Mel-34 cell line by DTIC selected progressively chemoresistant cells which showed (a) increased expression of CD133, suggesting selection of cells with a stem-like phenotype, and (b) showed increased number of cells expressing CT antigens (mean ± 2SD of 3 random high-power fields), but decreased intensity of expression of the differentiation antigens Melan-A/MART-1 and tyrosinase (data not shown). IHC of unsorted LM-Mel-34 melanoma cell line showing a minority of cells stain positive for NY-ESO-1 (c) compared to Melan-A (d; 100X). (e) Cytotoxicity as measured by chromium release. Melan-A-specific effectors can lyse LM-Mel-34 target cells, whereas EBV irrelevant control or NY-ESO-1 specific CTL do not cause cell killing unless exogenous cognate peptide (10−6 M) is added. (f) Flow cytometry of LM-Mel-34 melanoma cells after co-incubation with CTLs. NY-ESO-1 specific CTL specifically target and eliminate CD133+ clonogenic melanoma cells which express NY-ESO-1

Article Snippet: For flow cytometry, cells were blocked in 10% normal human serum in PBS, stained with anti-CD133 antibody (AC133-PE or 293C3-APC; Miltenyi Biotec, Bergisch Gladbach, Germany) at 1:50 dilution for 15 min at 4°C, washed in PBS, resuspended and collected on a FACSCalibur (Becton Dickson, San Jose, CA) with data analysis by FloJo (Tree Star Inc, OR).

Techniques: Expressing, Selection, Staining, Control, Flow Cytometry, Incubation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer/testis antigens can be immunological targets in clonogenic CD133 + melanoma cells

doi: 10.1007/s00262-009-0672-0

Figure Lengend Snippet: Melanoma and melanoma cell lines contain CD133+ cells. (a) Lymph node metastasis melanoma paraffin section (b) LM-Mel-34 cell line passage 5 cell block (c) flow cytometry of passage 5 LM-Mel-34 unsorted cells and (d) passage 7 LM-Mel-34 after magnetic bead enrichment

Article Snippet: For flow cytometry, cells were blocked in 10% normal human serum in PBS, stained with anti-CD133 antibody (AC133-PE or 293C3-APC; Miltenyi Biotec, Bergisch Gladbach, Germany) at 1:50 dilution for 15 min at 4°C, washed in PBS, resuspended and collected on a FACSCalibur (Becton Dickson, San Jose, CA) with data analysis by FloJo (Tree Star Inc, OR).

Techniques: Paraffin Section, Blocking Assay, Flow Cytometry

Journal: Genes & Cancer

Article Title: Potential role of CXCL9 induced by endothelial cells/CD133+ liver cancer cells co-culture system in tumor transendothelial migration

doi: 10.18632/genesandcancer.116

Figure Lengend Snippet: (A) and (B): Representative images depicted the proliferation of CD133+ liver cancer cells in co-culture system using fluorescence microscopy. The numbers of CD133+ liver cancer cells (not expressing eGFP) were quantified. (Data are represented as the mean ± SEM, * P < 0.05, ** P < 0.01).

Article Snippet: CD133+ PLC/PRF/5 and Hep-3B cells (hereinafter called CD133+ liver cancer cells) were isolated from PLC/ PRF/5 and Hep-3B liver cancer cell lines by fluorescence- activated cell sorting (FACS) (BD Company, New Jersey, USA) using CD133 antibody (CD133/2(293C3)-APC, 1 test for 107 cells) (Catalog Number: 130-090-854, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Co-Culture Assay, Fluorescence, Microscopy, Expressing

Journal: Genes & Cancer

Article Title: Potential role of CXCL9 induced by endothelial cells/CD133+ liver cancer cells co-culture system in tumor transendothelial migration

doi: 10.18632/genesandcancer.116

Figure Lengend Snippet: (A) ELISA assays were performed to detect the CXCL9 expression. (B) and (C): The western blot results showed that NF-kB signaling pathway was activated in CD133+ liver cancer cells of the direct co-culture group. NF-kB was upregulated in HUVEC (CD133+ Hep-3B), but downregulated in HUVEC (CD133+ PLC/PRF/5). (D) The ELISA results showed that the CXCL9 expression reduced significantly in the supernatants of direct co-culture group treated with NF-kB inhibitor MG132. (Data are represented as the mean ± SEM, * P < 0.05, ** P < 0.01).

Article Snippet: CD133+ PLC/PRF/5 and Hep-3B cells (hereinafter called CD133+ liver cancer cells) were isolated from PLC/ PRF/5 and Hep-3B liver cancer cell lines by fluorescence- activated cell sorting (FACS) (BD Company, New Jersey, USA) using CD133 antibody (CD133/2(293C3)-APC, 1 test for 107 cells) (Catalog Number: 130-090-854, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Co-Culture Assay

Journal: Genes & Cancer

Article Title: Potential role of CXCL9 induced by endothelial cells/CD133+ liver cancer cells co-culture system in tumor transendothelial migration

doi: 10.18632/genesandcancer.116

Figure Lengend Snippet: (A) and (B): Representative images of transwell assays to show the migration and invasion abilities of CD133 liver cancer cells. The numbers of invasive and migratory cells were quantified. (Data are represented as the mean ± SEM, * P < 0.05, ** P < 0.01).

Article Snippet: CD133+ PLC/PRF/5 and Hep-3B cells (hereinafter called CD133+ liver cancer cells) were isolated from PLC/ PRF/5 and Hep-3B liver cancer cell lines by fluorescence- activated cell sorting (FACS) (BD Company, New Jersey, USA) using CD133 antibody (CD133/2(293C3)-APC, 1 test for 107 cells) (Catalog Number: 130-090-854, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Migration

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Simultaneously target of normal and stem cells-like gastric cancer cells via cisplatin and anti-CD133 CAR-T combination therapy

doi: 10.1007/s00262-021-02891-x

Figure Lengend Snippet: Cisplatin treatment induces enrichment of stem cell-like gastric cancer cells. a Immunohistochemical staining for CD133 in three primary gastric cancer (GC) samples before and post-cisplatin treatment. Scale bar = 100 μm. b Detection of CD133 expression in three human GC samples via western blot. c Detection of CD133 expression in cisplatin-exposed human BGC-823 cell line. BGC-823 was treated with 7.5 μM cisplatin (the value of IC50) for 48 h, and then the mRNA and protein levels of CD133 were assessed by western blot. d Percentages of CD133 positive cells post-cisplatin treatment. BGC-823 cells were treated as indicated above and collected for CD133 antibody staining. The percentage of CD133 positive cells were examined by flow cytometry. f CD133 level in apoptotic and living BGC-823 cells post-cisplatin treatment. BGC-823 was treated with 7.5 μM cisplatin (the value of IC50) for 48 h, then stained with Annexin-V-FITC, propidium iodide, and anti-CD133 antibody. Data represent means ± SD. n = 3 samples. ***p < 0.001, versus PBS control group. A two-way ANOVA analysis with Tukey’s post hoc test was performed to evaluate the statistical significance

Article Snippet: The antibodies utilized in this study included anti-human CD133-APC antibody (ab253259, Santa Cruz Biotechnology, clone 293C3), anti-human CCR7-FITC antibody (Biolegend, clone 3D12), anti-human CD62L-PE-Cy7 antibody (Biolegend, clone DREG-56), anti-human CD45RA-BV510 antibody (Biolegend, clone HI100), anti-human CD45RO-PE-Cy7 antibody (Biolegend, clone UCHL1), anti-human CD27-BV650 antibody (Biolegend, clone M-T271), anti-human CD69-APC antibody (Biolegend, clone FN50), and anti-human CD3-BV510 antibody (Biolegend, clone UCHT1).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot, Flow Cytometry, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Simultaneously target of normal and stem cells-like gastric cancer cells via cisplatin and anti-CD133 CAR-T combination therapy

doi: 10.1007/s00262-021-02891-x

Figure Lengend Snippet: Generation of anti-CD133 CAR-T cells. a The discrete CAR units of anti-CD133 CAR-T cells. b Representative plot of flow cytometry detection of transfected T cells. c The rate of anti-CD133 CAR-T cells proliferation within 10 days. d T cell activation markers, such as CCR7, CD62L, CD45RA, and CD45RO, were detected with flow cytometry

Article Snippet: The antibodies utilized in this study included anti-human CD133-APC antibody (ab253259, Santa Cruz Biotechnology, clone 293C3), anti-human CCR7-FITC antibody (Biolegend, clone 3D12), anti-human CD62L-PE-Cy7 antibody (Biolegend, clone DREG-56), anti-human CD45RA-BV510 antibody (Biolegend, clone HI100), anti-human CD45RO-PE-Cy7 antibody (Biolegend, clone UCHL1), anti-human CD27-BV650 antibody (Biolegend, clone M-T271), anti-human CD69-APC antibody (Biolegend, clone FN50), and anti-human CD3-BV510 antibody (Biolegend, clone UCHT1).

Techniques: Flow Cytometry, Transfection, Activation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Simultaneously target of normal and stem cells-like gastric cancer cells via cisplatin and anti-CD133 CAR-T combination therapy

doi: 10.1007/s00262-021-02891-x

Figure Lengend Snippet: Anti-CD133 CAR-T cells exhibit dramatic antitumor efficacy ex vivo. a BGC-823 cells were treated with cisplatin for 48 h, and the CD133+ cells were isolated via flow cytometry and then incubated with anti-CD133 CAR-T cells at the indicated effector-to-target ratios. Cytotoxicity assays evaluated the cytotoxicity of T cells. The concentrations of IL-2 b, IFN-γ c, GM-CSF d, and TNFα e released by anti-CD133 CAR-T cells after coculture with normal and CD133+ BGC-823 cells overnight at an E/T ratio of 1:1 were shown. Data represent means ± SD. n = 3 samples. ***p < 0.001. f, g Canonical T cell markers were detected by flow cytometry at a recommended E: T ratio of 1:1 after coculturing anti-CD133 CAR-T cells with target cells. A two-way ANOVA analysis with Tukey’s post hoc test was performed to evaluate the statistical significance

Article Snippet: The antibodies utilized in this study included anti-human CD133-APC antibody (ab253259, Santa Cruz Biotechnology, clone 293C3), anti-human CCR7-FITC antibody (Biolegend, clone 3D12), anti-human CD62L-PE-Cy7 antibody (Biolegend, clone DREG-56), anti-human CD45RA-BV510 antibody (Biolegend, clone HI100), anti-human CD45RO-PE-Cy7 antibody (Biolegend, clone UCHL1), anti-human CD27-BV650 antibody (Biolegend, clone M-T271), anti-human CD69-APC antibody (Biolegend, clone FN50), and anti-human CD3-BV510 antibody (Biolegend, clone UCHT1).

Techniques: Ex Vivo, Isolation, Flow Cytometry, Incubation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Simultaneously target of normal and stem cells-like gastric cancer cells via cisplatin and anti-CD133 CAR-T combination therapy

doi: 10.1007/s00262-021-02891-x

Figure Lengend Snippet: Anti-CD133 CAR-T cells and cisplatin efficiently reduce tumor progression in BGC-823 xenograft models. a Schematic representation of the treatment strategy. The subcutaneous BGC-823 tumor model was established by injecting 1*106 BGC-823 cells into the right flank, and the antitumor study started when tumor volume reached about 50 mm3, five mice per group. b Tumor growth curve. Data represent means ± SD. n = 5 mice. **p < 0.01, ***p < 0.001. c Tumor weight was assayed. Mice were sacrificed at the 22-day post the first treatment, and tumor tissues were collected and weighted. Data represent means ± SD. n = 5 samples. ***p < 0.001. Representative FACS plots d and the percentage of T cells in the peripheral blood e in different groups at the end of the antitumor study. Data represent means ± SD. n = 5 samples. ***p < 0.001. f Percentage of T cells in the tumor tissues in different groups at the end of the antitumor study. Data represent means ± SD. n = 5 samples. ***p < 0.001. g The protein level of CD133 in the tumor tissues receiving different treatments. Representative FACS plots h and percentages of CD133 positive cells in tumor tissues i in different groups at the end of the antitumor study. Data represent means ± SD. n = 5 samples. ***p < 0.001

Article Snippet: The antibodies utilized in this study included anti-human CD133-APC antibody (ab253259, Santa Cruz Biotechnology, clone 293C3), anti-human CCR7-FITC antibody (Biolegend, clone 3D12), anti-human CD62L-PE-Cy7 antibody (Biolegend, clone DREG-56), anti-human CD45RA-BV510 antibody (Biolegend, clone HI100), anti-human CD45RO-PE-Cy7 antibody (Biolegend, clone UCHL1), anti-human CD27-BV650 antibody (Biolegend, clone M-T271), anti-human CD69-APC antibody (Biolegend, clone FN50), and anti-human CD3-BV510 antibody (Biolegend, clone UCHT1).

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Simultaneously target of normal and stem cells-like gastric cancer cells via cisplatin and anti-CD133 CAR-T combination therapy

doi: 10.1007/s00262-021-02891-x

Figure Lengend Snippet: Anti-CD133 CAR-T cells and cisplatin efficiently reduce tumor progression in KATO III and MKN-28 xenograft models. a Schematic representation of the time course of the experiment. Tumor volume of KATO III b and MKN-28 c subcutaneously injected mice. Data represent means ± SD. n = 5 mice. ***p < 0.001. A two-way ANOVA analysis with Tukey’s post hoc test was performed to evaluate the statistical significance

Article Snippet: The antibodies utilized in this study included anti-human CD133-APC antibody (ab253259, Santa Cruz Biotechnology, clone 293C3), anti-human CCR7-FITC antibody (Biolegend, clone 3D12), anti-human CD62L-PE-Cy7 antibody (Biolegend, clone DREG-56), anti-human CD45RA-BV510 antibody (Biolegend, clone HI100), anti-human CD45RO-PE-Cy7 antibody (Biolegend, clone UCHL1), anti-human CD27-BV650 antibody (Biolegend, clone M-T271), anti-human CD69-APC antibody (Biolegend, clone FN50), and anti-human CD3-BV510 antibody (Biolegend, clone UCHT1).

Techniques: Injection