Supplementary Materials1. intratumoral release of both RANTES and IL-15 attracted CAR-T cells and promoted their local survival, respectively, increasing the overall survival of tumor bearing mice. These MCLA (hydrochloride) preclinical data support the use of this innovative biological platform of immunotherapy for solid tumors. we used a first generation GD2.CAR that lacks both CD28 and OX40 signaling domains. Co-culture experiments Tumor cells were seeded in 24-well plates (5 104/well for cytotoxicity assay and 1 105/well for T-cell proliferation assay), infected with Ad524 (50 C 100 vp/cell) and then cultured for 3 days. Control and GD2.CAR-T cells (3 104/well for cytotoxicity assay and 5 104/well for T-cell proliferation assay) were then added and cultured for additional 3 days. Residual GFP+ NB cells and T cells were then counted based on GFP and CD3 expression, respectively, using microbeads (CountBright Absolute Counting Beads, Invitrogen). Normalized residual tumor cells were calculated as 100 tumor cell counts with treatment/tumor cell counts MCLA (hydrochloride) without treatment (%). Confocal microscopic video imaging GFP-labeled CHLA-255 cells were seeded into 8-well chamber slide (Lab-TekII, Thermo scientific) (104 cells/well), infected with Ad524 (100 vp/cell) and cultured for 3 days. Control and GD2.CAR-T cells were then added to the well (105 cells/well). GFP+ NB cells stained with Annexin-V (Invitrogen) were imaged using a spinning disk confocal microscope for 16 hrs. Imaging data were acquired and analyzed using Zen software (Zeiss). Migration assay Migration assays were conducted as previously described(21) with minor modifications using 5 m pore 24-well transwell plates (Corning Life Science). The percentage of migrating cells was calculated as follows: 100[cell count of experimental sample C cell count of negative control] / [cell count of positive control C cell count of negative control]. ELISA and Milliplex assay To measure the production of chemokines and cytokines, tumor cells were plated at 5 105 cells/ml in 24-well plates and infected with viruses (50C100 vp/cell). Supernatants were collected 72 hrs later and analyzed for the production of RANTES, MIP-1, MIP-1, MCP-1, IP-10, and IL-15. To measure the production of RANTES and IL-15, tumor and blood samples were collected 14 C 18 days after virus inoculation. Tumor homogenates and serum were separated and finally assayed using specific ELISA kits (R&D Systems). Human IL-17F, GM-CSF, IFN, IL-10, CCL-20, IL-12p70, IL-13, IL-17, IL-22, IL-9, IL-1, IL-33, IL-2, IL-21, IL-4, IL-23, IL-5, IL-6, IL-25, IL-27, IL-31, TNF, TNF and IL-28, and mouse G-CSF, GM-CSF, IFN, IL-1, IL-1, IL-2, MCLA (hydrochloride) IL-4, IL-5, IL-6, IL-7, MCLA (hydrochloride) IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17 and TNF in the serum were measured using Milliplex assay kits (Millipore) following manufactures protocols. NB xenograft animal model To assess antitumor effects and persistence of GD2.CAR-T cells, we used NOD.Cg-imaging system (Xenogen) as previously described(15). Immunohistochemistry Tumor samples were fixed, processed and stained according to standard procedures. We performed Hematoxylin and Eosin staining and labeling of human T cells using polyclonal rabbit anti-human CD3 mAb (A0452, Dako). For detection we used Dako LSAB + System-HRP (K0679, Dako). Statistical analysis Analysis of variance (ANOVA) with Bonferroni correction and the 2-sided unpaired test were used for comparison of 3 or more groups, or 2 groups, respectively as stated in the figure legends. Mixed-model ANOVA was applied to compare tumor growth in different groups of mice. Survival curves were plotted using the Kaplan-Meier methods, and the differences in the survival between groups were assessed by log rank test. Data are presented as mean SD or SEM as stated in the figure legends. Statistical significance was defined at p 0.05. Statistical analysis was performed with Prism 5 (GraphPad Software). Results Coxackie-adenovirus receptor is functionally expressed in neuroblastoma (NB) cell lines but NCR2 not by GD2.CAR-T cells To determine if Ad524 and CAR-T cells could be combined without toxicity to T lymphocytes in a NB tumor model, we first compared the expression of the Coxackie-adenovirus receptor in 7 NB cell lines and CAR-T cells specifically targeting the GD2 antigen expressed by NB cells. As shown in Fig. 1A, all NB cell lines expressed the virus receptor as assessed by flow cytometry, while CAR-T cells did not. Incubation with the oncolytic adenovirus Ad524 induced cellular toxicity in 5/7 NB cell lines in a dose-dependent MCLA (hydrochloride) manner (Fig. 1B), while GD2.CAR-T cells were unaffected, even at the.