Volume 6, Issue 5, Pages 609-614 (November 2002) The Organotypic Multicellular Spheroid Is a Relevant Three-Dimensional Model to Study Adenovirus Replication and Penetration in Human Tumors in Vitro  Jacques Grill, Martine L.M. Lamfers, Victor W. van Beusechem, Clemens M. Dirven, D.Shareen Pherai, Mathijs Kater, Paul Van der Valk, Ronald Vogels, W.Peter Vandertop, Herbert M. Pinedo, David T. Curiel, Winald R. Gerritsen  Molecular Therapy  Volume 6, Issue 5, Pages 609-614 (November 2002) DOI: 10.1006/mthe.2002.0713 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Immunohistochemistry for coxsackievirus adenovirus receptor (CAR) on primary tumors and derived spheroids. Frozen sections were stained using an anti-CAR mouse monoclonal antibody raised against the CAR ectodomain and counterstained with H&E. Spheroids were processed after 2 weeks of culture, that is, at the time they are routinely used for experiments. (A) Glioma tumor VU-61. CAR expression is detected in > 50% of the tumor cells with a perinuclear and cytoplasmic distribution. Analysis of the whole-tumor sample showed clear heterogeneity in the intensity of the staining. (B) Glioma spheroid VU-61. A similar pattern of CAR staining is detected as in the primary tumor. (C) Meningioma tumor VU-59. CAR expression is detected in ≈ 50% of the cells. Staining was mostly cytoplasmic. (D) Meningioma spheroid VU-59. Similar CAR expression is found in the spheroids. Note that spheroids conserved the architecture of the tumor they were derived from in both cases. Original magnifications: ×40. Molecular Therapy 2002 6, 609-614DOI: (10.1006/mthe.2002.0713) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Transgene expression in spheroids after infection with replication-defective adenovirus. (A) Spheroids prepared from primary glioblastoma multiforme tissue (VU-109) were infected with increasing doses of AdCMVLacZ (106, 107, 108, and 5 × 108 pfu/spheroid) for 24 hours, and X-Gal staining was done on day 1 after infection according to the manufacturer's instructions (β-Gal Staining Set; Boehringer Mannheim, Almere, The Netherlands). En face photographs of the spheroids were taken under light microscopy. The scale bars represent 100 μm. (B) X-gal staining (Beta-Gal Staining Set; Boehringer Mannheim) on a cryosection of a spheroid prepared from primary glioblastoma multiforme tissue (VU-19) was done 24 hours after infection of the spheroid in a virus concentration of 5 × 108 pfu/spheroid AdCMVLacZ. The section was counterstained with H&E. Only the first one or two peripheral layers of the spheroid are infected by the adenovirus as visualized by the expression of the transgene. Scale bars, 100 μm. Molecular Therapy 2002 6, 609-614DOI: (10.1006/mthe.2002.0713) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Transgene expression in organotypic multicellular spheroids during the course of propagation of a replication-competent adenovirus. (A) Cryosections of primary glioblastoma spheroids of patient VU-26 were made at 4, 8, and 12 days post infection with AdE1+Luc (5 × 107 pfu/ml) and stained with a new anti-luciferase monoclonal antibody, LUC-Y. (B) Hematoxylin staining of parallel sections demonstrated the presence of necrotic cells due to viral replication in the periphery of the spheroid and viable cells in the core of the spheroid 12 days post infection. (C) For comparison, a section of VU-26 spheroids infected with replication-defective AdCMVLuc was stained for luciferase expression 12 days post infection. Scale bars, 100 μm. Molecular Therapy 2002 6, 609-614DOI: (10.1006/mthe.2002.0713) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Dose-dependent oncolysis of organotypic glioma spheroids with a replication-competent adenovirus. Spheroids (n = 7 per group) derived from the glioblastoma tumor of patient VU-65 were infected with increasing concentrations of AdE1+Luc, and viability was measured 10 days later with the WST-1 assay according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Data are presented as mean ± SEM of the OD450 after subtraction of the background. A significant dose-dependent oncolysis was observed 10 days after infection (P = 0.0001, ANOVA). Dose-dependent oncolysis could also be observed as early as day 6 (data not shown). Molecular Therapy 2002 6, 609-614DOI: (10.1006/mthe.2002.0713) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Oncolysis of organotypic multicellular spheroids from five glioblastoma multiforme patients after infection with a replication-competent adenovirus. Spheroids from the tumor of five different patients were infected with 108 pfu/spheroid of AdE1+Luc, and viability was assessed 2 weeks after infection. Results are given as the OD450 value of the WST-1 assay, 24 hours after initiation of the reaction. The graph represents the distribution in percentiles of the values observed for each spheroid (n = 5 to 8 per group), according to Cleveland and Tukey using StatView software. The control groups are shown in white and the treated groups are shown in gray. The boxes represent 50% of the distribution. The horizontal lines represent the medians. The bars encompass 90% of the distribution and the circles represent the extreme values. The following differences in metabolic activity of untreated controls and treated spheroids compared by Student's t-test were observed: P > 0.05 for VU-19 and VU-21, P = 0.02 for VU-33, P = 0.005 for VU-35, and P = 0.002 for VU-65. *P lt; 0.05. Molecular Therapy 2002 6, 609-614DOI: (10.1006/mthe.2002.0713) Copyright © 2002 American Society for Gene Therapy Terms and Conditions