Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Optimization of cell culture conditions for generating canine CAR T cells . The impact of cell culture conditions, including T cell activation and T cell expansion, were evaluated to identify the optimal conditions. A Diagram of human B7-H3 CAR construct used to generate canine B7-H3 CAR T cells. In B , flow cytometry was used to determine expression of canine CD5 + T cell CD4 and CD8 subsets by day 10 CAR T cell cultures. In C the relative CAR T cell transduction efficiency was determined by immunostaining with recombinant L-protein. Canine T cells were activated with PHA, CD3/CD28 beads, or aAPCs, and expanded in rhIL-2 (top row) or rcIL-2 (bottom row), and the percentage of L-protein positive cells determined by flow cytometry. In D , CAR T cells were generated from n = 6 dogs (3 healthy, 3 osteosarcoma), and the mean ± SEM cell numbers and percentages of CAR T cells (red) and non-transduced T cells (blue) determined for each of the culture activation and expansion conditions (x-axis) determined, illustrating greatest CAR T generation when rhIL-2 was used for expansion. E Representative CAR T cell cytotoxicity study, using the canine OS cell line Abrams as the target, as described in Methods. Target killing over time determined for B7-H3 CAR T cells cultured in IL-2 + IL-21 (red) or IL-2 + IL-21 + IL-7 and IL15 (green), demonstrating superior killing activity of CAR T cells expanded in the 4-cytokine cocktail. Assays were run in triplicate, and statistical significance was determined using Dunn’s multiple means comparison test; *P < 0.05, *****, P < 0.0001, ns not significant

Article Snippet: Mouse anti-canine CD3 (clone CA17.2A12, Bio-Rad) and mouse anti-canine CD28 (clone 5B8, Invitrogen) antibodies were conjugated to magnetic tosyl-activated Dynabeads (Life Technologies) as previously established at a 1:1 antibody ratio [ , ]

Techniques: Cell Culture, Activation Assay, Construct, Flow Cytometry, Expressing, Transduction, Immunostaining, Recombinant, Generated, Activity Assay, Comparison

Journal: Cancer gene therapy

Article Title: Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor.

doi: 10.1038/sj.cgt.7700416

Figure Lengend Snippet: Figure 7 Immunohistochemical analysis of histone deacetylase 2 (HDAC2). Using a polyclonal anti-HDAC2 antibody, we determined nuclear staining of HDAC2 in LNCaP tumors after treatment with: (a) vehicle control, (b) scrambled control ODNsr750/15, (c) castration, (d) antisense AR ODNas750/15.

Article Snippet: Staining was performed according to a streptavidin–biotin–peroxidase protocol using a mouse monoclonal anti -hAR antibody (clone F39.4; BioGenex Laboratories, San Ramon, CA),17 a mouse monoclonal anti–Ki-67 antibody (DAKO, Glostrup, Denmark), and a rabbit polyclonal anti -HDAC2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Immunohistochemical staining, Histone Deacetylase Assay, Staining, Control