Journal: Molecular Therapy Oncolytics

Article Title: T Cell-Activating Mesenchymal Stem Cells as a Biotherapeutic for HCC

doi: 10.1016/j.omto.2017.07.002

Figure Lengend Snippet: GPC3-ENG MSCs Expressing CD80 and 41BBL Induce T Cells to Produce IL-2 (A and B) GPC3-ENG, GPC3-ENG.CD80, GPC3-ENG.41BBL, or GPC3-ENG.CD80+41BBL MSCs were cocultured with GPC3 + (G401; HUH7) or GPC3 − (A549) cell lines in 1:10 MSC to tumor cell ratios in the presence of human T cells (10:1 T cell to tumor cell ratio). After 24 hr, IFNγ (A) or IL-2 (B) was determined by ELISA (n = 6, assay performed in duplicates; GPC3-ENG.CD80+41BBL versus GPC3-ENG MSCs: *p < 0.05, **p < 0.01, ***p < 0.001). (C) Luciferase-based cytotoxicity assays using GPC3-ENG, GPC3-ENG.CD80, GPC3-ENG.41BBL, or GPC3-ENG.CD80+41BBL MSCs and GPC3 + (G401.ffLuc; HUH7.ffLuc) or GPC3 − (A549.ffLuc) cell lines as targets at a MSC:tumor ratio of 1:10 in the presence of human T cells (10:1 T cell to tumor cell ratio; n = 6; performed in triplicates; box graph; whiskers: min, max; ***p < 0.001).

Article Snippet: For phenotyping MSCs, the following MAbs were used: CD105-PE (Miltenyi Biotec), CD45-PerCP and CD90-APC (BD Biosciences), 41BBL-PE (BD Biosciences), and CD80-PerCP (eBioscience).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Luciferase

Journal: Molecular Therapy Oncolytics

Article Title: T Cell-Activating Mesenchymal Stem Cells as a Biotherapeutic for HCC

doi: 10.1016/j.omto.2017.07.002

Figure Lengend Snippet: GPC3-ENG MSCs Induce Antigen-Dependent T Cell Proliferation (A–D) GPC3-ENG, GPC3-ENG.CD80, GPC3-ENG.41BBL, or GPC3-ENG.CD80+41BBL MSCs were cocultured with GPC3 + (G401; HUH7) or GPC3 − (A549) cell lines at a 1:10 MSC to tumor cell ratio in the presence of human T cells (10:1 T cell to tumor cell ratio). After 7 days, absolute number of CD3 + cells (A), CD8/CD4 T cell ratio (B), and presence of naive (CD45A + ,CCR7 + ), central memory (CM; CD45A − ,CCR7 + ), effector memory (EM; CD45A − ,CCR7 − ), or effector memory RA (EMRA; CD45A + ,CCR7 − ) T cells (C and D) was determined by FACS analysis (n = 4; performed in duplicates; GPC3-ENG.CD80+41BBL versus GPC3-ENG MSCs: *p < 0.05, **p < 0.01).

Article Snippet: For phenotyping MSCs, the following MAbs were used: CD105-PE (Miltenyi Biotec), CD45-PerCP and CD90-APC (BD Biosciences), 41BBL-PE (BD Biosciences), and CD80-PerCP (eBioscience).

Techniques:

Journal: Molecular Therapy Oncolytics

Article Title: T Cell-Activating Mesenchymal Stem Cells as a Biotherapeutic for HCC

doi: 10.1016/j.omto.2017.07.002

Figure Lengend Snippet: Time-Lapse Confocal Microscopic Imaging of GPC3-ENG MSCs Induced Killing of GPC3 + Tumor Cells (A) Representative images (at 0 hr and 12 hr) for real-time detection of cytolytic activity of T cells in the presence of GPC3 + (HUH7) or GPC3 − (A549) cell lines and GPC3-ENG.CD80+41BBL MSCs (green; 1:10 MSC to tumor cell ratio; 10:1 T cell to tumor cell ratio). Cell death was visualized by incorporation of PI (red). (B) Tumor cell killing at various time points was determined as an area of corrected mean pixel intensity (PI channel; intensity × area × 10 6 pixel). The solid lines in the graph represent the “best-fit” linear regression analysis (HUH7 coculture with GPC3-ENG [blue] or GPC-ENG.CD80+41BBL MSCs [red]; A549 cells coculture with GPC3-ENG [green] or GPC-ENG.CD80+41BBL MSCs [yellow]. The dots show individual experimental values (n = 5; experiments = 2; ***p < 0.001).

Article Snippet: For phenotyping MSCs, the following MAbs were used: CD105-PE (Miltenyi Biotec), CD45-PerCP and CD90-APC (BD Biosciences), 41BBL-PE (BD Biosciences), and CD80-PerCP (eBioscience).

Techniques: Imaging, Activity Assay

Journal: Molecular Therapy Oncolytics

Article Title: T Cell-Activating Mesenchymal Stem Cells as a Biotherapeutic for HCC

doi: 10.1016/j.omto.2017.07.002

Figure Lengend Snippet: GPC3-ENG MSCs Redirect T Cells In Vivo to Kill HCC Antitumor activity of GPC3-ENG MSCs in an HCC xenograft model. Mice were s.c. coinjected with 5 × 10 6 HUH7.eGFP.ffLuc cells and 5 × 10 5 GPC3-ENG (n = 5), GPC3-ENG.CD80 (n = 4), GPC3-ENG.41BBL (n = 5), GPC3-ENG.CD80+41BBL (n = 5), EGFRvIII-ENG (n = 5), or EGFRvIII-ENG.CD80+41BBL (n = 5) MSCs on their left lower flank. On day 7, mice received an i.v. dose of 1 × 10 7 T cells. Tumor growth was followed by bioluminescence imaging. (A) Representative images of animals (red arrow: T cell injection). (B) Quantitative bioluminescence imaging results (solid lines: individual mice; radiance = photons/s/cm 2 /sr). (C) Kaplan-Meier survival curve (control versus treatment groups; *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: For phenotyping MSCs, the following MAbs were used: CD105-PE (Miltenyi Biotec), CD45-PerCP and CD90-APC (BD Biosciences), 41BBL-PE (BD Biosciences), and CD80-PerCP (eBioscience).

Techniques: In Vivo, Activity Assay, Imaging, Injection

Journal: Molecular Therapy Oncolytics

Article Title: T Cell-Activating Mesenchymal Stem Cells as a Biotherapeutic for HCC

doi: 10.1016/j.omto.2017.07.002

Figure Lengend Snippet: GPC3-ENG MSCs Have Anti-tumor Activity in Established Tumors Mice were s.c. injected with 1 × 10 6 HUH7.eGFP.ffLuc cells on their left lower flank (n = 10). On day 7, mice received a peritumoral injection of 1 × 10 6 GPC3-ENG.MSC.CD80+41BBL. On day 10, five mice received an i.v. dose of 1 × 10 7 T cells. Tumor-bearing mice injected with EGFRvIII-ENG MSC.CD80+41BBL (n = 5) or T cells (n = 5) served as controls. Tumor growth was followed by bioluminescence imaging. (A) Representative images of animals (green arrow: MSC injection; red arrow: T cell injection). (B) Quantitative bioluminescence imaging results (solid lines: individual mice; radiance = photons/s/cm 2 /sr). (C) Kaplan-Meier survival curve (control versus treatment groups; *p < 0.05).

Article Snippet: For phenotyping MSCs, the following MAbs were used: CD105-PE (Miltenyi Biotec), CD45-PerCP and CD90-APC (BD Biosciences), 41BBL-PE (BD Biosciences), and CD80-PerCP (eBioscience).

Techniques: Activity Assay, Injection, Imaging