Small-Molecule Natural Product Physachenolide C Potentiates Immunotherapy Efficacy by Targeting BET Proteins.

Screening for sensitizers of cancer cells to TRAIL-mediated apoptosis identified a natural product of the 17ß-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), as a promising hit. In this study, we show that PCC was also able to sensitize melanoma and renal carcinoma cells to apoptosis in response not only to TRAIL, but also to the synthetic polynucleotide poly I:C, a viral mimetic and immune activator, by reducing levels of antiapoptotic proteins cFLIP and Livin. Both death receptor and TLR3 signaling elicited subsequent increased assembly of a proapoptotic ripoptosome signaling complex. Administration of a combination of PCC and poly I:C in human M14 melanoma xenograft and a syngeneic B16 melanoma model provided significant therapeutic benefit as compared with individual agents. In addition, PCC enhanced melanoma cell death in response to activated human T cells in vitro and in vivo in a death ligand-dependent manner. Biochemical mechanism-of-action studies established bromo and extraterminal domain (BET) proteins as major cellular targets of PCC. Thus, by targeting of BET proteins to reduce antiapoptotic proteins and enhance caspase-8-dependent apoptosis of cancer cells, PCC represents a unique agent that can potentially be used in combination with various immunotherapeutic approaches to promote tumor regression and improve outcome. SIGNIFICANCE: These findings demonstrate that PCC selectively sensitizes cancer cells to immune-mediated cell death, potentially improving the efficacy of cancer immunotherapies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3374/F1.large.jpg.

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

Inhibiting Janus Kinase 1 and BCL-2 to treat T cell acute lymphoblastic leukemia with IL7-Rα mutations.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Current chemotherapy is quite toxic in growing children and more directed therapeutics are being sought. The IL-7R pathway is a major driver of ALL and here we evaluate two drugs directed to that pathway using a model of T cell ALL. Mutant gain-of-function IL-7Rα was transduced into an IL-7-dependent murine thymocyte line conferring ligand-independent survival and growth. JAK1 is associated with IL-7Rα and mediates signaling from the mutant receptor. In vitro, treating the transformed cell line with the JAK1/2 inhibitor ruxolitinib inhibited ligand-independent signaling and induced cell death. Transfer of the transformed cell line into mice resulted in aggressive leukemia and untreated mice succumbed in about three weeks. Treatment with ruxolitinib incorporated into chow showed a potent therapeutic benefit with reduction in leukemic burden and extension of survival. BCL-2 is an anti-apoptotic downstream mediator of the IL-7R survival mechanism. Venetoclax, an inhibitor of BCL-2, showed activity against the transformed cell line in vitro and could be combined with ruxolitinib in vivo. These findings support the therapeutic potential of treating T-ALL by targeting the IL-7R pathway.

LTßR signalling preferentially accelerates oncogenic AKT-initiated liver tumours.

OBJECTIVES: The relative contributions of inflammatory signalling and sequential oncogenic dysregulation driving liver cancer pathogenesis remain incompletely understood. Lymphotoxin-ß receptor (LTßR) signalling is critically involved in hepatitis and liver tumorigenesis. Therefore, we explored the interdependence of inflammatory lymphotoxin signalling and specific oncogenic pathways in the progression of hepatic cancer. DESIGN: Pathologically distinct liver tumours were initiated by hydrodynamic transfection of oncogenic V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT)/ß-catenin or AKT/Notch expressing plasmids. To investigate the relationship of LTßR signalling and specific oncogenic pathways, LTßR antagonist (LTßR-Fc) or agonist (anti-LTßR) were administered post oncogene transfection. Initiated livers/tumours were investigated for changes in oncogene expression, tumour proliferation, progression, latency and pathology. Moreover, specific LTßR-mediated molecular events were investigated in human liver cancer cell lines and through transcriptional analyses of samples from patients with intrahepatic cholangiocarcinoma (ICC). RESULTS: AKT/ß-catenin-transfected livers displayed increased expression of LTß and LTßR, with antagonism of LTßR signalling reducing tumour progression and enhancing survival. Conversely, enforced LTßR-activation of AKT/ß-catenin-initiated tumours induced robust increases in proliferation and progression of hepatic tumour phenotypes in an AKT-dependent manner. LTßR-activation also rapidly accelerated ICC progression initiated by AKT/Notch, but not Notch alone. Moreover, LTßR-accelerated development coincides with increases of Notch, Hes1, c-MYC, pAKT and ß-catenin. We further demonstrate LTßR signalling in human liver cancer cell lines to be a regulator of Notch, pAKTser473 and ß-catenin. Transcriptome analysis of samples from patients with ICC links increased LTßR network expression with poor patient survival, increased Notch1 expression and Notch and AKT/PI3K signalling. CONCLUSIONS: Our findings link LTßR and oncogenic AKT signalling in the development of ICC.

Serum-based tracking of de novo initiated liver cancer progression reveals early immunoregulation and response to therapy.

BACKGROUND & AIMS: Liver inflammatory diseases associated with cancer promoting somatic oncogene mutations are increasing in frequency. Preclinical cancer models that allow for the study of early tumor progression are often protracted, which limits the experimental study parameters due to time and expense. Here we report a robust inexpensive approach using Sleeping Beauty transposition (SBT) delivery of oncogenes along with Gaussia Luciferase expression vector GLuc, to assess de novo liver tumor progression, as well as the detection of innate immune responses or responses induced by therapeutic intervention. METHODS: Tracking de novo liver tumor progression with GLuc was demonstrated in models of hepatocellular carcinoma (HCC) or adenoma (HCA) initiated by hydrodynamic delivery of SBT oncogenes. RESULTS: Rising serum luciferase levels correlated directly with increasing liver tumor burden and eventual morbidity. Early detection of hepatocyte apoptosis from mice with MET+CAT transfected hepatocytes was associated with a transient delay in HCC growth mediated by a CD8(+) T-cell response against transformed hepatocytes. Furthermore, mice that lack B cells or macrophages had an increase in TUNEL(+) hepatocytes following liver MET transfection demonstrating that these cells provide protection from MET-induced hepatocyte apoptosis. Treatment with IL-18+IL-12 of mice displaying established HCC decreased tumor burden which was associated with decreased levels of serum luciferase. CONCLUSIONS: Hydrodynamic delivery of the SBT vector GLuc to hepatocytes serves as a simple blood-based approach for real-time tracking of pathologically distinct types of liver cancer. This revealed tumor-induced immunologic responses and was beneficial in monitoring the efficacy of therapeutic interventions.

Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment.

The gut microbiota influences both local and systemic inflammation. Inflammation contributes to development, progression, and treatment of cancer, but it remains unclear whether commensal bacteria affect inflammation in the sterile tumor microenvironment. Here, we show that disruption of the microbiota impairs the response of subcutaneous tumors to CpG-oligonucleotide immunotherapy and platinum chemotherapy. In antibiotics-treated or germ-free mice, tumor-infiltrating myeloid-derived cells responded poorly to therapy, resulting in lower cytokine production and tumor necrosis after CpG-oligonucleotide treatment and deficient production of reactive oxygen species and cytotoxicity after chemotherapy. Thus, optimal responses to cancer therapy require an intact commensal microbiota that mediates its effects by modulating myeloid-derived cell functions in the tumor microenvironment. These findings underscore the importance of the microbiota in the outcome of disease treatment.

The pivotal role of IKKα in the development of spontaneous lung squamous cell carcinomas.

Here, we report that kinase-dead IKKα knockin mice develop spontaneous lung squamous cell carcinomas (SCCs) associated with IKKα downregulation and marked pulmonary inflammation. IKKα reduction upregulated the expression of p63, Trim29, and keratin 5 (K5), which serve as diagnostic markers for human lung SCCs. IKKα(low)K5(+)p63(hi) cell expansion and SCC formation were accompanied by inflammation-associated deregulation of oncogenes, tumor suppressors, and stem cell regulators. Reintroducing transgenic K5.IKKα, depleting macrophages, and reconstituting irradiated mutant animals with wild-type bone marrow (BM) prevented SCC development, suggesting that BM-derived IKKα mutant macrophages promote the transition of IKKα(low)K5(+)p63(hi) cells to tumor cells. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs.

Macrophage migration inhibitory factor induces epithelial to mesenchymal transition, enhances tumor aggressiveness and predicts clinical outcome in resected pancreatic ductal adenocarcinoma.

MIF is a proinflammatory cytokine and is implicated in cancer. A higher MIF level is found in many human cancer and cancer-prone inflammatory diseases, including chronic pancreatitis and pancreatic cancer. We tested the hypothesis that MIF contributes to pancreatic cancer aggressiveness and predicts disease outcome in resected cases. Consistent with our hypothesis we found that an elevated MIF mRNA expression in tumors was significantly associated with poor outcome in resected cases. Multivariate Cox-regression analysis further showed that MIF is independently associated with patients' survival (HR = 2.26, 95% CI = 1.17-4.37, p = 0.015). Mechanistic analyses revealed that MIF overexpression decreased E-cadherin and increased vimentin mRNA and protein levels in pancreatic cancer cell lines, consistent with the features of epithelial-to-mesenchymal transition (EMT). Furthermore, MIF-overexpression significantly increased ZEB1/2 and decreased miR-200b expression, while shRNA-mediated inhibition of MIF increased E-cadherin and miR-200b expression, and reduced the expression of ZEB1/2 in Panc1 cells. Re-expression of miR-200b in MIF overexpressing cells restored the epithelial characteristics, as indicated by an increase in E-cadherin and decrease in ZEB1/2 and vimentin expression. A reduced sensitivity to the chemotherapeutic drug, gemcitabine, occurred in MIF-overexpressing cells. Indicative of an increased malignant potential, MIF over-expressing cells showed significant increase in their invasion ability in vitro, and tumor growth and metastasis in an orthotopic xenograft mouse model. These results support a role of MIF in disease aggressiveness, indicating its potential usefulness as a candidate target for designing improved treatment in pancreatic cancer.

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.

Systemic IL-12 administration alters hepatic dendritic cell stimulation capabilities.

The liver is an immunologically unique organ containing tolerogenic dendritic cells (DC) that maintain an immunosuppressive microenvironment. Although systemic IL-12 administration can improve responses to tumors, the effects of IL-12-based treatments on DC, in particular hepatic DC, remain incompletely understood. In this study, we demonstrate systemic IL-12 administration induces a 2-3 fold increase in conventional, but not plasmacytoid, DC subsets in the liver. Following IL-12 administration, hepatic DC became more phenotypically and functionally mature, resembling the function of splenic DC, but differed as compared to their splenic counterparts in the production of IL-12 following co-stimulation with toll-like receptor (TLR) agonists. Hepatic DCs from IL-12 treated mice acquired enhanced T cell proliferative capabilities similar to levels observed using splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression in the RENCA mouse model of renal cell carcinoma. Collectively, the data shows systemic IL-12 administration enables hepatic DCs to overcome at least some aspects of the inherently suppressive milieu of the hepatic environment that could have important implications for the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies and infections.

mTOR kinase inhibitor AZD8055 enhances the immunotherapeutic activity of an agonist CD40 antibody in cancer treatment.

mTOR is a central mediator of cancer cell growth, but it also directs immune cell differentiation and function. On this basis, we had explored the hypothesis that mTOR inhibition can enhance cancer immunotherapy. Here, we report that a combination of αCD40 agonistic antibody and the ATP-competitive mTOR kinase inhibitory drug AZD8055 elicited synergistic antitumor responses in a model of metastatic renal cell carcinoma. In contrast to the well-established mTOR inhibitor rapamycin, AZD8055 increased the infiltration, activation, and proliferation of CD8(+) T cells and natural killer cells in liver metastatic foci when combined with the CD40 agonist. AZD8055/αCD40-treated mice also display an increased incidence of matured macrophages and dendritic cells compared with that achieved in mice by αCD40 or AZD8055 treatment alone. We found that the combination treatment also increased macrophage production of TNFα, which played an indispensable role in activation of the observed antitumor immune response. Levels of Th1 cytokines, including interleukin 12, IFN-γ, TNFα, and the Th1-associated chemokines RANTES, MIG, and IP-10 were each elevated significantly in the livers of mice treated with the combinatorial therapy versus individual treatments. Notably, the AZD8055/αCD40-induced antitumor response was abolished in IFN-γ(-/-) and CD40(-/-) mice, establishing the reliance of the combination therapy on host IFN-γ and CD40 expression. Our findings offer a preclinical proof of concept that, unlike rapamycin, the ATP-competitive mTOR kinase inhibitor AZD8055 can contribute with αCD40 treatment to trigger a restructuring of the tumor immune microenvironment to trigger regressions of an established metastatic cancer.

Coactivation of AKT and ß-catenin in mice rapidly induces formation of lipogenic liver tumors.

Obesity is a risk factor for development of certain cancers but the basis for this risk is unclear. In this study, we developed a novel mouse model that demonstrates directly how lipogenic phenotypes commonly associated with diet-induced metabolic syndromes can influence hepatic cancer development. Activated AKT and ß-catenin (AKT/CAT) genes were hydrodynamically codelivered using the Sleeping Beauty transposon to initiate liver tumorigenesis. AKT/CAT and MET/CAT combination induced microscopic tumor foci by 4 weeks, whereas no tumorigenesis resulted from delivery of AKT, MET, or CAT alone. Primary AKT/CAT tumor cells were steatotic (fatty) hepatocellular adenomas which progressed to hepatocellular carcinomas (HCC) upon in vivo passage, whereas primary MET/CAT tumors emerged directly as frank HCC. Conversion of AKT/CAT tumor cells to frank HCC during passage was associated with induction of the human HCC marker α-fetoprotein and the stem cell marker CD133. Using hierarchical clustering and gene set enrichment analysis, we compared the primary murine AKT/CAT and MET/CAT tumors to a panel of 53 human HCCs and determined that these two mouse models could be stratified as distinct subtypes associated in humans with poor clinical prognosis. The chief molecular networks identified in primary and passaged AKT/CAT tumors were steatosis and lipid metabolic pathways, respectively. Our findings show how coactivation of the AKT and CAT pathways in hepatocytes can efficiently model development of a lipogenic tumor phenotype. Furthermore, we believe that our approach could speed the dissection of microenvironmental factors responsible for driving steatotic-neoplastic transformation to frank carcinoma, through genetic modification of existing immunodefined transgenic models.

Successful immunotherapy with IL-2/anti-CD40 induces the chemokine-mediated mitigation of an immunosuppressive tumor microenvironment.

Treatment of mice bearing orthotopic, metastatic tumors with anti-CD40 antibody resulted in only partial, transient anti-tumor effects whereas combined treatment with IL-2/anti-CD40, induced tumor regression. The mechanisms for these divergent anti-tumor responses were examined by profiling tumor-infiltrating leukocyte subsets and chemokine expression within the tumor microenvironment after immunotherapy. IL-2/anti-CD40, but not anti-CD40 alone, induced significant infiltration of established tumors by NK and CD8(+) T cells. To further define the role of chemokines in leukocyte recruitment into tumors, we evaluated anti-tumor responses in mice lacking the chemokine receptor, CCR2. The anti-tumor effects and leukocyte recruitment mediated by anti-CD40 alone, were completely abolished in CCR2(-/-) mice. In contrast, IL-2/anti-CD40-mediated leukocyte recruitment and reductions in primary tumors and metastases were maintained in CCR2(-/-) mice. Treatment of mice with IL-2/anti-CD40, but not anti-CD40 alone, also caused an IFN-gamma-dependent increase in the expression of multiple Th1 chemokines within the tumor microenvironment. Interestingly, although IL-2/anti-CD40 treatment increased Tregs in the spleen, it also caused a coincident IFN-gamma-dependent reduction in CD4(+)/FoxP3(+) Tregs, myeloid-derived suppressor cells and Th2 chemokine expression specifically within the tumor microenvironment that was not observed after treatment with anti-CD40 alone. Similar effects were observed using IL-15 in combination with anti-CD40. Taken together, our data demonstrate that IL-2/anti-CD40, but not anti-CD40 alone, can preferentially reduce the overall immunosuppressive milieu within the tumor microenvironment. These results suggest that the use of anti-CD40 in combination with IL-2 or IL-15 may hold substantially more promise for clinical cancer treatment than anti-CD40 alone.

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.

Nitric oxide is a key component in inflammation-accelerated tumorigenesis.

Nitric oxide (NO(*)), an important signaling molecule and a component of inflammatory response, is involved in tumorigenesis. However, the quantity of NO(*) and the cellular microenvironment influences the role of NO(*) in tumor development. We used a genetic strategy to test the hypothesis that an inflammatory microenvironment with an enhanced level of NO(*) accelerates spontaneous tumor development. C. parvum-induced inflammation and increased NO(*) synthase-2 (NOS2) expression coincided with accelerated spontaneous tumor development, mostly lymphomas, in p53-/-NOS2+/+ C57BL6 mice when compared with the controls (P = 0.001). However, p53-/-NOS2-/- mice did not show any difference in tumor latency between C. parvum-treated and control groups. In C. parvum-treated p53-/-NOS2+/+ mice, tumor development was preceded by a higher expression of NOS2 and phosphorylated Akt-Ser(473) (pAkt-Ser473) in spleen, increased cell proliferation measured by Ki-67 IHC in spleen and thymus, and a lower apoptotic index and CD95-L expression in spleen and thymus. C. parvum-treated p53-/-NOS2+/+ mice showed an increase in the number of Foxp3(+) T-reg cells, dendritic cells (DC), as well as increased CD80(+), CD86(+), CD40(+), and CD83(+) on DC in the spleen. Regulatory T-cells (T-reg) and the maturation of DC may modulate tumorigenesis. An increase in the FoxP3(+)T-reg cells in C. parvum-treated p53-/-NOS2+/+ mice indicates a role of NO(*) in the regulation of T-reg cells that may contribute to a protumor shift of the immune environment favoring an accelerated tumor development. These data provide genetic and mechanistic evidence that an inflammatory microenvironment and an increased level of NO(*) can accelerate tumor development.

IFN-gamma mediates CD4+ T-cell loss and impairs secondary antitumor responses after successful initial immunotherapy.

Protective cell-mediated immune responses in cancer are critically dependent on T-helper type 1 (T(H)1) cytokines such as interferon-gamma (IFN-gamma). We have previously shown that the combination of CD40 stimulation and interleukin-2 (IL-2) leads to synergistic antitumor responses in several models of advanced metastatic disease. We now report that after this treatment and other immunotherapy regimens, the CD4+ T-cell population, in contrast to CD8+ T cells, did not significantly increase but rather exhibited a substantial level of apoptosis that was dependent on IFN-gamma. Mice immunized with tumor cells and treated with an immunotherapy regimen that was initially protective were later unable to mount effective memory responses compared with immunized mice not receiving immunotherapy. Immunotherapy given to tumor-bearing Ifngr-/- mice resulted in restoration of secondary responses. Thus, although immunotherapeutic regimens inducing strong IFN-gamma responses can lead to successful early antitumor efficacy, they may also impair the development of durable antitumor responses.

Enhanced antitumor response by divergent modulation of natural killer and natural killer T cells in the liver.

The use of interleukin-18 (IL-18) together with IL-12 induced high levels of IFN-gamma in tumor-bearing mice and regression of liver tumors that was abolished in IFN-gamma((-/-)) mice. Natural killer (NK) and NKT cells were the major producers of IFN-gamma in the livers of mice treated with IL-18 and/or IL-12. Liver NK cells were significantly increased by treatment with IL-18/IL-12, whereas the degree of liver NKT cell TCR detection was diminished by this treatment. Reduction of NK cells with anti-asGM1 decreased the antitumor activity of IL-18/IL-12 therapy and revealed NK cells to be an important component for tumor regression in the liver. In contrast, the antitumor effects of both IL-18 and IL-12 were further increased in CD1d((-/-)) mice, which lack NKT cells. Our data, therefore, show that the antitumor activity induced in mice by IL-18/IL-12 is NK and IFN-gamma dependent and is able to overcome an endogenous immunosuppressive effect of NKT cells in the liver microenvironment. These results suggest that immunotherapeutic approaches that enhance NK cell function while eliminating or altering NKT cells could be effective in the treatment of cancer in the liver.

Stimulation through CD40 on mouse and human renal cell carcinomas triggers cytokine production, leukocyte recruitment, and antitumor responses that can be independent of host CD40 expression.

CD40, a member of the TNFR superfamily, is expressed on a variety of host immune cells, as well as some tumors. In this study, we show that stimulation of CD40 expressed on both mouse and human renal carcinoma cells (RCCs) triggers biological effects in vitro and in vivo. Treatment of the CD40+ Renca mouse RCC tumor cells in vitro with an agonistic anti-CD40 Ab induced strong expression of the genes and proteins for GM-CSF and MCP-1, and induced potent chemotactic activity. Similarly, administration of alphaCD40 to both wild-type and CD40-/- mice bearing Renca tumors resulted in substantial amounts of TNF-alpha and MCP-1 in the serum, increased the number of total splenocytes and MHC class II+ CD11c+ leukocytes, and when combined with IFN-gamma, inhibited the progression of established Renca tumors in vivo in both wild-type and CD40-/- mice. Similarly, treatment of CD40+ A704 and ACHN human RCC lines with mouse anti-human CD40 Ab induced strong expression of genes and proteins for MCP-1, IL-8, and GM-CSF in vitro and in vivo. Finally, in SCID mice, the numbers of ACHN pulmonary metastases were dramatically reduced by treatment with species-specific human CD40 Ab. These results show that CD40 stimulation of CD40+ tumor cells can enhance immune responses and result in antitumor activity.

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.

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.