Erin Lincoln

IL-27 Mediates Complete Regression of Orthotopic Primary and Metastatic Murine Neuroblastoma Tumors: Role for CD8+ T Cells

We have shown previously that IFN-γ-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8+ T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-γ. In addition, marked enhancement of local IFN-γ gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8+, but not CD4+ T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.

Immunologic and Therapeutic Synergy of IL-27 and IL-2: Enhancement of T Cell Sensitization, Tumor-Specific CTL Reactivity and Complete Regression of Disseminated Neuroblastoma Metastases in the Liver and Bone Marrow

IL-27 exerts antitumor activity in murine orthotopic neuroblastoma, but only partial antitumor effect in disseminated disease. This study demonstrates that combined treatment with IL-2 and IL-27 induces potent antitumor activity in disseminated neuroblastoma metastasis. Complete durable tumor regression was achieved in 90% of mice bearing metastatic TBJ-IL-27 tumors treated with IL-2 compared with only 40% of mice bearing TBJ-IL-27 tumors alone and 0% of mice bearing TBJ-FLAG tumors with or without IL-2 treatment. Comparable antitumor effects were achieved by IL-27 protein produced upon hydrodynamic IL-27 plasmid DNA delivery when combined with IL-2. Although delivery of IL-27 alone, or in combination with IL-2, mediated pronounced regression of neuroblastoma metastases in the liver, combined delivery of IL-27 and IL-2 was far more effective than IL-27 alone against bone marrow metastases. Combined exposure to IL-27 produced by tumor and IL-2 synergistically enhances the generation of tumor-specific CTL reactivity. Potentiation of CTL reactivity by IL-27 occurs via mechanisms that appear to be engaged during both the initial sensitization and effector phase. Potent immunologic memory responses are generated in mice cured of their disseminated disease by combined delivery of IL-27 and IL-2, and depletion of CD8+ ablates the antitumor efficacy of this combination. Moreover, IL-27 delivery can inhibit the expansion of CD4+CD25+Foxp3+ regulatory and IL-17-expressing CD4+ cells that are otherwise observed among tumor-infiltrating lymphocytes from mice treated with IL-2. These studies demonstrate that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing widely metastatic neuroblastoma tumors.

Proteasome Inhibition to Maximize the Apoptotic Potential of Cytokine Therapy for Murine Neuroblastoma Tumors

Human neuroblastomas possess several mechanisms of self-defense that may confer an ability to resist apoptosis and contribute to the observed difficulty in treating these tumors in the clinical setting. These molecular alterations may include defects in proapoptotic genes as well as the overexpression of prosurvival factors, such as Akt among others. As a key regulator of the turnover of proteins that modulate the cell cycle and mechanisms of apoptosis, the proteasome could serve as an important target for the treatment of neuroblastoma. The present studies provide the first evidence that bortezomib, a newly approved inhibitor of proteasome function, inhibits phosphorylation of Akt, induces the translocation of proapoptotic Bid, and potently enhances the apoptosis of murine neuroblastoma tumor cells in vitro. Furthermore, in that inhibitors of the Akt pathway can sensitize otherwise resistant TBJ/Neuro-2a cells to apoptosis induced by IFN-γ plus TNF-α, we hypothesized that bortezomib also could sensitize these cells to IFN-γ plus TNF-α. We demonstrate for the first time that bortezomib not only up-regulates the expression of receptors for IFN-γ and TNF-α on both TBJ neuroblastoma and EOMA endothelial cell lines, but also markedly enhances the sensitivity of these cells to apoptosis induced by IFN-γ plus TNF-α in vitro. Furthermore, bortezomib enhances the in vivo antitumor efficacy of IFN-γ/TNF-α-inducing cytokines, including both IL-2 and IL-12 in mice bearing well-established primary and/or metastatic TBJ neuroblastoma tumors. Collectively, these studies suggest that bortezomib could be used therapeutically to enhance the proapoptotic and overall antitumor activity of systemic cytokine therapy in children with advanced neuroblastoma.

High-Throughput Molecular and Histopathologic Profiling of Tumor Tissue in a Novel Transplantable Model of Murine Neuroblastoma: New Tools for Pediatric Drug Discovery

Using two MYCN transgenic mouse strains, we established 10 transplantable neuroblastoma cell lines via serial orthotopic passage in the adrenal gland. Tissue arrays demonstrate that by histochemistry, vascularity, immunohistochemical staining for neuroblastoma markers, catecholamine analysis, and concurrent cDNA microarray analysis, there is a close correspondence between the transplantable lines and the spontaneous tumors. Several genes closely associated with the pathobiology and immune evasion of neuroblastoma, novel targets that warrant evaluation, and decreased expression of tumor suppressor genes are demonstrated. These studies describe a unique and generalizable approach to expand the utility of transgenic models of spontaneous tumor, providing new tools for preclinical investigation.

Multicolor fluorescence-based approaches for imaging cytokine-induced alterations in the neovascularization, growth, metastasis, and apoptosis of murine neuroblastoma tumors.

Neuroblastoma is one of the most common solid tumors in children. The prognosis of patients with advanced neuroblastoma is poor overall despite standard therapeutic modalities and has stimulated substantial interest in the potential role for biologics such as immunotherapeutic and/or antiangiogenic agents for the treatment of neuroblastoma. To facilitate preclinical investigation of the efficacy and mechanisms of action of new biologic agents for the treatment of neuroblastoma, a comprehensive panel of disease-specific fluorescence-based model systems has been developed by our group to image the growth, neovascularization, metastasis, and apoptosis of neuroblastoma tumors. These model systems use fluorescent proteins to monitor cytokine-induced alterations in the growth and metastasis of neuroblastoma and allow for monitoring and/or quantitation of even minimal residual disease that is localized to visceral organ sites such as the liver, lung, and/or bone marrow. Further, based on the differential spectra of red fluorescent protein, green fluorescent protein (GFP), and agents such as 4′-6-diamidino-2-phenylindole (DAPI) (blue) and fluorescein isothiocyanate–dextran (green), multicolor systems have now been established by our group that allow for combined assessment of parameters, including the macroscopic relation of tumors to their associated vasculature and, within tissue sections, simultaneous quantitation of tumor neovascularization and evaluation of therapy-induced apoptosis within the tumor and vascular endothelial compartments. Further, by engineering cells to express specific mediators of apoptosis that have been linked to GFP (ie, BID-EGFP), these systems can also be used to dissect mechanisms by which neuroblastoma cells are induced to undergo apoptosis in vitro as well as in vivo. Collectively, these model systems provide important tools for investigation of the biology of neuroblastoma tumors and evaluation of mechanisms that mediate the regression of these tumors in response to novel therapeutic agents, including cytokines such as interleukin-12.

Proteasome Inhibition To Maximize the Apoptotic Potential of Cytokine Therapy for Murine Neuroblastoma Tumors

Human neuroblastomas possess several mechanisms of self-defense that may confer an ability to resist apoptosis and contribute to the observed difficulty in treating these tumors in the clinical setting. These molecular alterations may include defects in proapoptotic genes as well as the overexpression of prosurvival factors, such as Akt among others. As a key regulator of the turnover of proteins that modulate the cell cycle and mechanisms of apoptosis, the proteasome could serve as an important target for the treatment of neuroblastoma. The present studies provide the first evidence that bortezomib, a newly approved inhibitor of proteasome function, inhibits phosphorylation of Akt, induces the translocation of proapoptotic Bid, and potently enhances the apoptosis of murine neuroblastoma tumor cells in vitro. Furthermore, in that inhibitors of the Akt pathway can sensitize otherwise resistant TBJ/Neuro-2a cells to apoptosis induced by IFN-gamma plus TNF-alpha, we hypothesized that bortezomib also could sensitize these cells to IFN-gamma plus TNF-alpha. We demonstrate for the first time that bortezomib not only up-regulates the expression of receptors for IFN-gamma and TNF-alpha on both TBJ neuroblastoma and EOMA endothelial cell lines, but also markedly enhances the sensitivity of these cells to apoptosis induced by IFN-gamma plus TNF-alpha in vitro. Furthermore, bortezomib enhances the in vivo antitumor efficacy of IFN-gamma/TNF-alpha-inducing cytokines, including both IL-2 and IL-12 in mice bearing well-established primary and/or metastatic TBJ neuroblastoma tumors. Collectively, these studies suggest that bortezomib could be used therapeutically to enhance the proapoptotic and overall antitumor activity of systemic cytokine therapy in children with advanced neuroblastoma.