Journal: International Journal of Molecular Sciences

Article Title: NK Cells Lose Their Cytotoxicity Function against Cancer Stem Cell-Rich Radiotherapy-Resistant Breast Cancer Cell Populations

doi: 10.3390/ijms22179639

Figure Lengend Snippet: The larger population of CD24 −/low /CD44 + BCSCs in RT-R-breast cancer cells shows increased proliferation, colony formation, migration and invasion abilities. ( A ) CD24 −/low /CD44 + BCSCs were isolated from RT-R-MDA-MB-231 cells as described in the Materials and Methods section, and the CD24 −/low /CD44 + BCSC populations among MDA-MB-231 and RT-R-MDA-MB-231 cells were compared to the isolated CD24 −/low /CD44 + BCSCs. The same number (1 × 10 5 cells) of MDA-MB-231, RT-R-MDA-MB-231, and CD24 −/low /CD44 + cells isolated from RT-R-MDA-MB-231 cells were labeled with both an APC-conjugated anti-CD24 antibody and a PE-conjugated anti-CD44 antibody, and the population of CD24 −/low /CD44 + cells was then quantified by flow cytometry for 5 s at the same flow rate as described in the Materials and Methods section. ( B ) Cell proliferation was measured 0, 24, 48, and 72 h after seeding using a CCK-8 assay kit as described in the Materials and Methods section. ( C ) Cells seeded in 6-well plates were cultured for approximately 2 weeks with the medium replaced with fresh complete medium every 2–3 days. Then, the colony formation ability was determined as described in the Materials and Methods section. ( D , E ) Cells were seeded on an 8 μm-pore size insert with a Matrigel-coated membrane for the migration assay ( D ) or with an endothelial cell-Matrigel-coated membrane for the invasion assay ( E ). After 20 h of incubation, the migrated ( D ) or invaded cells ( E ) were stained with DAPI and counted under a fluorescence microscope. The values are presented as the mean ± SD of five independent experiments. ** p < 0.01 compared to MDA-MB-231 cells; # p < 0.05, ## p < 0.01 compared to RT-R-MDA-MB-231 cells.

Article Snippet: MDA-MB-231 and RT-R-MDA-MB-231 cells (1.4 × 10 6 ) and isolated CD24 −/low /CD44 + cells (1.4 × 10 6 ) were stained with an APC-conjugated human anti-CD24 antibody (#FAB5247A, R&D Systems, Minneapolis, MN, USA) and a PE-conjugated human anti-CD44 antibody (#FAB3660P, R&D systems).

Techniques: Migration, Isolation, Labeling, Flow Cytometry, CCK-8 Assay, Cell Culture, Pore Size, Membrane, Invasion Assay, Incubation, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Highly Efficient Elimination of Colorectal Tumor-Initiating Cells by an EpCAM/CD3-Bispecific Antibody Engaging Human T Cells

doi: 10.1371/journal.pone.0013474

Figure Lengend Snippet: Tumorigenicity of HT-29 colorectal cancer cell populations and primary colorectal TICs.

Article Snippet: Cells were trypsinized, washed with PBS containing 2% FCS and stained with 5–10 μg/ml of the following antibodies for 30 min at 4°C: CD44-PE (Chemicon), CD24-APC (Immunostep), CD44v6-FITC (Abcam), CD133/2-PE (Miltenyi), CD166-ALEXA 647 (Serotec), ABCG2-PE (eBioscience), E-Cadherin (SantaCruz) and EpCAM (MT110, Micromet AG).

Techniques:

Journal: PLoS ONE

Article Title: Highly Efficient Elimination of Colorectal Tumor-Initiating Cells by an EpCAM/CD3-Bispecific Antibody Engaging Human T Cells

doi: 10.1371/journal.pone.0013474

Figure Lengend Snippet: (A) Scheme of cytotoxicity assay with soft agar-based assay readout. (B) Photographs of soft agar plates with crystal violet-stained colonies growing from CD44 high /CD24 high /EpCAM high cells treated in the presence of T cells at the indicated concentrations of MT110, control BiTE antibodies at 100 ng/ml, or T cells alone. (C) Effect of MT110 on the number of CFUs per cm 2 . Bar graphs are mean values after 14 days from triplicate determinations +/− SD. Unstimulated PBMC were used as effector T cell source at an PBMC to HT-29 cell ratio of 10∶1.

Article Snippet: Cells were trypsinized, washed with PBS containing 2% FCS and stained with 5–10 μg/ml of the following antibodies for 30 min at 4°C: CD44-PE (Chemicon), CD24-APC (Immunostep), CD44v6-FITC (Abcam), CD133/2-PE (Miltenyi), CD166-ALEXA 647 (Serotec), ABCG2-PE (eBioscience), E-Cadherin (SantaCruz) and EpCAM (MT110, Micromet AG).

Techniques: Cytotoxicity Assay, Soft Agar Assay, Staining, Control

Journal: PLoS ONE

Article Title: Highly Efficient Elimination of Colorectal Tumor-Initiating Cells by an EpCAM/CD3-Bispecific Antibody Engaging Human T Cells

doi: 10.1371/journal.pone.0013474

Figure Lengend Snippet: (A) Mixtures of 5×10 5 TICs and 1×10 6 human PBMCs were inoculated into NOD/SCID mice (N = 3 for vehicle; N = 5 for vehicle + PBMCs; N = 4 for MT110 5 µg/kg; N = 3 for MT110 50 µg/kg and N = 3 for MT110 500 µg/kg group) and mice daily treated i.v. for 12 days with the indicated doses of MT110, or with vehicle controls in the absence and presence of PBMCs. The mean tumor volume is given. P-value was determined by Student's t-test. (B) Mixtures of 50,000 highly tumorigenic HT-29 xenograft-derived CD44 high /CD24 high /EpCAM high cells and 1×10 5 human PBMCs were inoculated into 5 NOD/SCID mice per group and mice daily treated i.v. for 12 days with the indicated doses of MT110, or with vehicle controls in the presence of PBMCs. The mean tumor volume is given. P-value was determined by Student's t-test. (C) Elimination of established tumors in NOD/SCID mice by treatment with MT110. Mixtures of 5×10 6 TICs and 1×10 7 human PBMCs were inoculated into 5 NOD/SCID mice per group to allow solid tumor formation. After tumor establishment at day 4 mice were treated i.v. for 14 days with 2.5 mg/kg of MT110, or with vehicle control in presence of PBMCs. The mean tumor volume is given. P-value was determined by Student's t-test.

Article Snippet: Cells were trypsinized, washed with PBS containing 2% FCS and stained with 5–10 μg/ml of the following antibodies for 30 min at 4°C: CD44-PE (Chemicon), CD24-APC (Immunostep), CD44v6-FITC (Abcam), CD133/2-PE (Miltenyi), CD166-ALEXA 647 (Serotec), ABCG2-PE (eBioscience), E-Cadherin (SantaCruz) and EpCAM (MT110, Micromet AG).

Techniques: Derivative Assay, Control