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. 2011 Jul 15;17(14):4705-18.
doi: 10.1158/1078-0432.CCR-11-0915. Epub 2011 Jun 1.

Engineering the Brain Tumor Microenvironment Enhances the Efficacy of Dendritic Cell Vaccination: Implications for Clinical Trial Design

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Free PMC article

Engineering the Brain Tumor Microenvironment Enhances the Efficacy of Dendritic Cell Vaccination: Implications for Clinical Trial Design

Yohei Mineharu et al. Clin Cancer Res. .
Free PMC article

Abstract

Purpose: Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Clinical trials for GBM using dendritic cell (DC) vaccination resulted in antitumor immune responses. Herein, we tested the hypothesis that combining in situ (intratumoral) Ad-Flt3L/Ad-TK-mediated gene therapy with DC vaccination would increase therapeutic efficacy and antitumor immunity.

Experimental design: We first assessed the immunogenicity of tumor lysates generated by Ad-TK (+GCV), temozolomide (TMZ), or freeze/thawing cycles (FTC) in a syngeneic brain tumor model. We also assessed phenotypic markers, cytokine release, and phagocytosis of bone marrow-derived DCs generated by fms-like tyrosine kinase 3 ligand (Flt3L) + IL-6 or by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) 4. Inhibition of tumor progression and production of anti-GBM antibodies was assessed following vaccination with (i) tumor cell lysates, (ii) DCs generated with either Flt3L/IL-6 or GM-CSF/IL-4 loaded with either Ad-TK/GCV-, TMZ-, or FTC-generated tumor lysates, or (iii) DCs in combination with in situ Ad-Flt3L/Ad-TK gene therapy.

Results: DCs loaded with tumor cell lysates generated with either Ad-TK/GCV or TMZ led to increased levels of phagocytosis, therapeutic efficacy, and humoral immune response. In situ immunogene therapy in combination with DC vaccination led to brain tumor regression and long-term survival in about 90% of animals, a significant increase when compared with either therapy alone.

Conclusions: Our results indicate that modifying the tumor microenvironment using intratumoral Ad-Flt3L/Ad-TK-mediated gene therapy potentiates therapeutic efficacy and antitumor immunity induced by DC vaccination. These data support novel phase I clinical trials to assess the safety and efficacy of this combined approach.

Figures

Figure 1
Figure 1. Characterization of whole cell tumor lysate to be used in vaccination
A, Electron micrographs showing the ultrastructure of CNS1 cells treated with either Ad-TK plus GCV (Ad-TK/GCV) for 72h, three cycles of freeze-thawing (FTC), or 200μM temozolomide (TMZ) for 72h. A black arrow highlights an autophagosome in CNS1 cells treated with TMZ. A white arrow indicates an autophagosome with a characteristic double membrane structure. A white arrowhead identifies an autophagolysosome with characteristic residual digested materials. B, Acridine orange (AO) staining of CNS1 cells treated with TMZ, Ad-TK/GCV or serum-starvation. Red staining reveals acidic vacuoles corresponding to autophagolysosomes, green indicates non-acidic compartments. C, CNS1 cells were treated with TMZ, Ad-TK/GCV or starvation in the presence or absence of bafilomycin A1 (bafA1). Cells were stained with AO and analyzed by flow cytometry. The proportions of cells with acidic vacuoles (AO red positive) are shown in representative dot plots. D, CNS1 cells were treated with TMZ, Ad-TK/GCV or starvation in the presence or absence of bafA1 and analyzed by Western blotting using a primary antibody for LC-3B; α-tubulin was used as a loading control. E, Cell death in CNS1 cells treated with TMZ, Ad-TK/GCV or FTC was assessed by flow cytometry following staining with Annexin V-FITC for apoptosis and propidium iodide (PI) for necrosis. The proportion of cells in each quadrant are shown in representative samples.
Figure 2
Figure 2. Therapeutic efficacy of vaccination using whole tumor cell lysates
A, Lewis rats were implanted with 5,000 CNS1 glioma cells in the striatum. Animals were systemically vaccinated (s.c.) with whole tumor cell lysates three times before tumor cell implantation (day −14, −7 and 0). B, Kaplan Meier survival curves of rats treated with Ad-TK/GCV lysate, TMZ lysate, FTC lysate or saline. Vaccination with tumor lysates prepared from any of the three methods tested, failed to significantly prolong median survival. C, Serum levels of anti-CNS1 cell antibodies at the time of tumor inoculation were measured by flow cytometry. Overlays show the fluorescence intensity of CNS-1 cells labelled with serum from non-tumor bearing rats (naïve control; filled, grey histogram) or with serum from rats from each experimental group (unfilled histograms). * p < 0.05 vs. saline, ^ p < 0.05 vs. FTC lysate; one-way ANOVA followed by Tukey’s test.
Figure 3
Figure 3. Characterization of bone marrow-derived dendritic cells (DCs) induced by Flt3L+IL-6 or GM-CSF+IL-4
Ten million bone marrow cells were cultured in RPMI conditioned media supplemented with 100 μg/ml Flt3L + 50 μg/ml IL-6 (FL-IL6), 100 μg/mL Flt3L (FL) or 10 μg/mL GM-CSF + 10 μg/mL IL-4 (GM-IL4) every 2–3 days. Loosely attached cells were harvested at day 7–8. A, Surface molecules of FL-IL6-induced and GM-IL-4-induced DCs were stained after 7 days culture (pre-stimulation) or after stimulation with either 100 ng/mL CpG2006, 50ng/ml LPS, or mock (48h stimulation) and then measured by flow cytometry. Overlays from one representative sample are depicted. The proportion of cells positive for each surface marker are shown in representative samples. B, Cytokine production from FL-IL6 -induced and GM-IL4 induced DCs. Immature DCs harvested after 7 days in culture were treated with either CpG, LPS or mock for 48h. Supernatant was collected and cytokines were measured by ELISA. C, Experimental design to assess therapeutic efficacy of FL-IL6 or GM-IL4 generated DC vaccines in rats challenged with brain tumors. Lewis rats were systemically vaccinated three times (day −14, −7 and 0) with either FL-IL6 -induced or GM-IL4 induced DC vaccines (s.c.) loaded with Ad-TK/GCV treated whole tumor lysate, or saline as a control, before CNS1 tumor cell implantation (5,000 cells) in the striatum. D, Kaplan Meier survival curves of rats treated with FL-IL6 induced DC vaccine (n = 5), GM-IL4-induced DC vaccine (n = 5) and saline (n = 5) are shown. * p < 0.05 vs. saline; Mantel log rank test.
Figure 4
Figure 4. Comparative efficacy of Flt3L + IL-6 induced DC vaccines loaded with Ad-TK/GCV, TMZ or FTC tumor lysates
A, Phagocytic activity of FL-IL6-induced DCs. DCs labeled with CD161a-PE were co-cultured for 5h with Qtracker-655 labeled CNS1 tumor cells that were treated with either Ad-TK/GCV, TMZ or FTC. Phagocytosis of tumor cells by DCs was assessed by flow cytometry; representative dot plots and corresponding percentage of double labeled cells are shown. Data are represented as the percentage of double-stained versus CD161a-PE positive cells. * p < 0.05 vs mock, ^ p < 0.05 vs F/T one-way ANOVA followed by Tukey’s test. B, Confocal micrograph shows a representative DC (green, CD161a) that engulfed tumor cell remnants (red, Qtracker 655); nuclei were labeled with DAPI (blue). The large square in the top left is a maximum projection of the confocal Z stack. The images to the right and below are reconstructed vertical sections along the fine green lines shown on the maximum projection. C, Lewis rats were systemically vaccinated with DC vaccines (s.c.) loaded with tumor lysates prepared by Ad-TK/GCV, TMZ, or FTC treatment, or saline as a control, three times before tumor cell implantation (day −14, −7 and 0) with 5,000 CNS1 glioma cells in the striatum. D, Kaplan Meier survival curves of rats treated with FL-IL6-induced DC vaccines loaded with either TK/GCV lysate (DC-TK/GCV, n = 5), TMZ lysate (DC-TMZ, n = 5) or F/T lysate (DC-F/T, n = 5), or unloaded DCs or saline as controls, are shown. * p < 0.05 vs. saline; Mantel log rank test. E, Serum levels of anti-CNS1 cell antibodies at the time of tumor implantation were measured by flow cytometry. * p < 0.05 vs. saline, ^ p < 0.05 vs. DC loaded with FTC lysate; one-way ANOVA followed by Tukey’s test.
Figure 5
Figure 5. Treatment efficacy of in situ immunogene therapy and DC vaccination in a small GBM model
Lewis rats were implanted with 5,000 CNS1 GBM cells. After 4 days, rats were treated with either (A) serial s.c. injections of Flt3L+IL6-induced DC vaccine loaded with either Ad-TK/GCV treated tumor lysate (DC-TK/GCV), or TMZ treated tumor lysate (DC-TMZ) every 7 days, or with (B) intratumoral administration of Ad-TK+Ad-Flt3L followed by GCV administration for 7 days. C, Kaplan-Meier survival curve of rats treated with in situ Ad-TK+Ad-Flt3L (n = 5), DC-TK/GCV vaccine (n = 10), DC-TMZ vaccine (n = 5), or saline (n = 6). * p < 0.05 vs. saline; Mantel log rank test.
Figure 6
Figure 6. Therapeutic efficacy of in situ immunogene therapy and DC vaccination in a large intracranial GBM model
A–B, Lewis rats were implanted with 5,000 CNS1 GBM cells. After 10 days, rats were treated either with (A) in situ (intratumoral) Ad-TK, in situ Ad-TK+Ad-Flt3L, (B) subcutaneous Flt3L+IL-6-induced DC vaccine loaded with Ad-TK/GCV tumor lysate, or a combination of in situ Ad-TK+Ad-Flt3L gene therapy plus DC vaccination. Anti-CNS1 antibodies were measured 1 day after the second DC vaccine administration (day 18). C, Kaplan-Meier survival curve of rats treated with in situ Ad-TK+Ad-Flt3L combined with DC vaccination (n = 10), in situ Ad-TK+Ad-Flt3L (n = 10), in situ Ad-TK combined with DC vaccine (n = 10), in situ Ad-TK (n = 10), DC vaccine (n = 10), or saline (n = 7). * p < 0.05 vs. saline, ^ p < 0.05 vs. Ad-TK, # p < 0.05 vs. Ad-TK+Ad-Flt3L; Mantel log rank test. D, Serum levels of anti-CNS1 antibodies measured by flow cytometry. Histograms show the fluorescence intensity of CNS1 cells labeled with serum from non–mtumor bearing rats (isotype control; gray histogram) or with serum from rats from each of the experimental groups (colored lines) * p < 0.05 vs. saline; one-way ANOVA followed by Tukey’s test.

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