[Show abstract][Hide abstract] ABSTRACT: Increasing evidence suggests that inflammatory cytokines play a critical role in tumor initiation and progression. A cancer stem cell (CSC)-like subpopulation in neuroblastoma is known to be marked by expression of the G-CSF receptor (G-CSFR). Here, we report on the mechanistic contributions of the G-CSFR in neu-roblastoma CSCs. Specifically, we demonstrate that the receptor ligand G-CSF selectively activates STAT3 within neuroblastoma CSC subpopulations, promoting their expansion in vitro and in vivo. Exogenous G-CSF enhances tumor growth and metastasis in human xenograft and murine neuroblastoma tumor models. In response to G-CSF, STAT3 acts in a feed-forward loop to tran-scriptionally activate the G-CSFR and sustain neuroblastoma CSCs. Blockade of this G-CSF–STAT3 signaling loop with either anti-G-CSF antibody or STAT3 inhibitor depleted the CSC sub-population within tumors, driving correlated tumor growth inhibition , decreased metastasis, and increased chemosensitivity. Taken together, our results define G-CSF as a CSC-activating factor in neuroblastoma, suggest a comprehensive reevaluation of the clinical use of G-CSF in these patients to support white blood cell counts, and suggest that direct targeting of the G-CSF–STAT3 signaling represents a novel therapeutic approach for neuroblas-toma. Cancer Res; 75(12); 1–14. Ó2015 AACR.
[Show abstract][Hide abstract] ABSTRACT: One of the major limitations of modern cancer vaccine vectors is that, unlike infectious pathogens, to which the immune system has evolved to respond, they are not sufficiently effective in delivering tumor-associated antigens (TAAs) in an immunogenic form to intact professional antigen-presenting cells (APCs) at their anatomic location. To overcome this challenge, we exploited Salmonella Pathogenicity Island 2 (SPI2) and its type III secretion system (T3SS) to deliver a TAA of choice into the cytosol of APCs in situ. We have systematically compared candidate genes from the SPI2 locus of Salmonella typhimurium in the vaccine design, using model antigens and a codon-optimized human TAA, survivin (coSVN). In a screen of 20 SPI2 promoter/effector combinations, the PsifB::sseJ pair demonstrated the maximal potency for antigen translocation in the APC cytosol, presentation to CD8 T cells, and immunogenicity in mice. Therapeutic vaccination with the PsifB::sseJ-coSVN construct (p8032) resulted in CXCR3-dependent tumor infiltration with CD8 T cells, reversal of the CD8:Treg ratio at the tumor site, and potent anti-tumor activity in a CT26 colon carcinoma model. The vaccine's immunogenicity and anti-tumor potency were further enhanced by co-administration of an NKT-cell ligand, 7WD8-5, which strongly enhanced production of IL-12 and IFNγ in vaccinated mice. Furthermore, therapeutic vaccination with p8032/7WD8-5 resulted in complete tumor regression in an A20 lymphoma model, with the generation of protective memory. Thus, oral antigen delivery via SPI2-encoded T3SS of Salmonella may be the foundation for an effective cancer vaccine platform.
Cancer Research 09/2014; DOI:10.1158/0008-5472.CAN-14-1169 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advances in the design of chimeric antigen receptors (CARs) have improved the antitumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. We proposed that CAR expression in Vα24-invariant NKT cells (NKTs) can build upon the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKTs were engineered to express a CAR against the GD2 ganglioside (CAR.GD2), which is highly expressed by neuroblastoma. We compared CAR.GD2 constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB co-stimulatory endodomains. CAR.GD2 expression rendered NKTs highly cytotoxic against neuroblastoma cells without affecting their CD1d-dependent reactivity. We observed a striking Th1-like polarization of NKTs by 4-1BB-containing CARs. Importantly, expression of both CD28 and 4-1BB endodomains in the CAR.GD2 enhanced in vivo persistence of NKTs. These CAR.GD2 NKTs effectively localized to the tumor site, had potent antitumor activity and repeat injections significantly improved the long-term survival of mice with metastatic neuroblastoma. Unlike T cells, CAR.GD2 NKTs did not induce graft-versus-host disease. These results establish the potential of NKTs to serve as a safe and effective platform for CAR-directed cancer immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Neuroblastoma is a neural crest-derived embryonal malignancy, which accounts for 13% of all pediatric cancer mortality, primarily due to tumor recurrence. Therapy-resistant cancer stem cells are implicated in tumor relapse, but definitive phenotypic evidence of the existence of these cells has been lacking. In this study, we define a highly tumorigenic subpopulation in neuroblastoma with stem cell characteristics, based on the expression of CSF3R, which encodes the receptor for granulocyte colony-stimulating factor (G-CSF). G-CSF receptor positive (aka G-CSFr(+) or CD114(+)) cells isolated from a primary tumor and the NGP cell line by flow cytometry were highly tumorigenic and capable of both self-renewal and differentiation to progeny cells. CD114(+) cells closely resembled embryonic and induced pluripotent stem cells with respect to their profiles of cell cycle, miRNA, and gene expression. In addition, they reflect a primitive undifferentiated neuroectodermal/neural crest phenotype revealing a developmental hierarchy within neuroblastoma tumors. We detected this dedifferentiated neural crest subpopulation in all established neuroblastoma cell lines, xenograft tumors, and primary tumor specimens analyzed. Ligand activation of CD114 by the addition of exogenous G-CSF to CD114(+) cells confirmed intact STAT3 upregulation, characteristic of G-CSF receptor signaling. Together, our data describe a novel distinct subpopulation within neuroblastoma with enhanced tumorigenicity and a stem cell-like phenotype, further elucidating the complex heterogeneity of solid tumors such as neuroblastoma. We propose that this subpopulation may represent an additional target for novel therapeutic approaches to this aggressive pediatric malignancy.
Cancer Research 07/2013; 73(13):4134. DOI:10.1158/0008-5472.CAN-12-4056 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Current therapies are toxic and not always curative that necessitates development of targeted immunotherapy. However, little is known about immunobiology of this tumor. In this study, we show that MB cells in 9 of 20 primary tumors express CD1d, an antigen-presenting molecule for Natural Killer T cells (NKTs). Quantitative RT-PCR analysis of 61 primary tumors revealed an elevated level of CD1d mRNA expression in a molecular subgroup characterized by an overactivation of Sonic Hedgehog (SHH) oncogene compared with Group 4. CD1d-positive MB cells cross-presented glycolipid antigens to activate NKT-cell cytotoxicity. Intracranial injection of NKTs resulted in regression of orthotopic MB xenografts in NOD/SCID mice. Importantly, the numbers and function of peripheral blood type-I NKTs were preserved in MB patients. Therefore, CD1d is expressed on tumor cells in a subset of MB patients and represents a novel target for immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Neuroblastoma is a neural crest derived embryonal malignancy which accounts for 13% of all pediatric cancer mortality, primarily due to tumor recurrence. Therapy-resistant cancer stem cells are implicated in tumor relapse, but definitive phenotypic evidence of the existence of these cells has been lacking. In this study, we define a highly tumorigenic subpopulation in neuroblastoma with stem cell characteristics, based on the expression of CD114, which encodes the receptor for granulocyte colony-stimulating factor (G-CSF). CD114+ cells isolated from a primary tumor and the NGP cell line by flow cytometry were highly tumorigenic and capable of both self-renewal and differentiation to progeny cells. CD114+ cells closely resembled embryonic and induced pluripotent stem cells with respect to their profiles of cell cycle, microRNA and gene expression. In addition, they reflect a primitive undifferentiated neuroectodermal/neural crest phenotype revealing a developmental hierarchy within neuroblastoma tumors. We detected this de-differentiated neural crest subpopulation in all established neuroblastoma cell lines, xenograft tumors, and primary tumor specimens analyzed. Ligand activation of CD114 by the addition of exogenous G-CSF to CD114+ cells confirmed intact STAT3 upregulation, characteristic of G-CSF receptor signaling. Together our data describe a novel distinct subpopulation within neuroblastoma with enhanced tumorigenicity and a stem-cell like phenotype, further elucidating the complex heterogeneity of solid tumors such as neuroblastoma. We propose this subpopulation may represent an additional target for novel therapeutic approaches to this aggressive pediatric malignancy.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE Children diagnosed at age ≥ 18 months with metastatic MYCN-nonamplified neuroblastoma (NBL-NA) are at high risk for disease relapse, whereas those diagnosed at age < 18 months are nearly always cured. In this study, we investigated the hypothesis that expression of genes related to tumor-associated inflammatory cells correlates with the observed differences in survival by age at diagnosis and contributes to a prognostic signature. METHODS Tumor-associated macrophages (TAMs) in localized and metastatic neuroblastomas (n = 71) were assessed by immunohistochemistry. Expression of 44 genes representing tumor and inflammatory cells was quantified in 133 metastatic NBL-NAs to assess age-dependent expression and to develop a logistic regression model to provide low- and high-risk scores for predicting progression-free survival (PFS). Tumors from high-risk patients enrolled onto two additional studies (n = 91) served as independent validation cohorts. Results Metastatic neuroblastomas had higher infiltration of TAMs than locoregional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation-related genes than those in patients diagnosed at age < 18 months. Expression of genes representing TAMs (CD33/CD16/IL6R/IL10/FCGR3) contributed to 25% of the accuracy of a novel 14-gene tumor classification score. PFS at 5 years for children diagnosed at age ≥ 18 months with NBL-NA with a low- versus high-risk score was 47% versus 12%, 57% versus 8%, and 50% versus 20% in three independent clinical trials, respectively. CONCLUSION These data suggest that interactions between tumor and inflammatory cells may contribute to the clinical metastatic neuroblastoma phenotype, improve prognostication, and reveal novel therapeutic targets.
[Show abstract][Hide abstract] ABSTRACT: Introduction of an exo-methylene group into the C-glycoside analogue of KRN7000 was found to afford a new glycosphingolipid ligand with potent agonistic activity for both human and mouse invariant natural killer T lymphocytes. The key synthetic strategy utilized the Nozaki-Hiyama-Kishi reaction to achieve a high-yield coupling between an α-galactosyl aldehyde and a vinyl iodide.
[Show abstract][Hide abstract] ABSTRACT: Vα24-invariant NKT cells inhibit tumor growth by targeting tumor-associated macrophages (TAMs). Tumor progression therefore requires that TAMs evade NKT cell activity through yet-unknown mechanisms. Here we report that a subset of cells in neuroblastoma (NB) cell lines and primary tumors expresses membrane-bound TNF-α (mbTNF-α). These proinflammatory tumor cells induced production of the chemokine CCL20 from TAMs via activation of the NF-κB signaling pathway, an effect that was amplified in hypoxia. Flow cytometry analyses of human primary NB tumors revealed selective accumulation of CCL20 in TAMs. Neutralization of the chemokine inhibited in vitro migration of NKT cells toward tumor-conditioned hypoxic monocytes and localization of NKT cells to NB grafts in mice. We also found that hypoxia impaired NKT cell viability and function. Thus, CCL20-producing TAMs served as a hypoxic trap for tumor-infiltrating NKT cells. IL-15 protected antigen-activated NKT cells from hypoxia, and transgenic expression of IL-15 in adoptively transferred NKT cells dramatically enhanced their antimetastatic activity in mice. Thus, tumor-induced chemokine production in hypoxic TAMs and consequent chemoattraction and inhibition of NKT cells represents a mechanism of immune escape that can be reversed by adoptive immunotherapy with IL-15-transduced NKT cells.
The Journal of clinical investigation 05/2012; 122(6):2221-33. DOI:10.1172/JCI59535 · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Live attenuated strains of Salmonella enterica have a high potential as carriers of recombinant vaccines. The type III secretion system (T3SS)-dependent translocation of S. enterica can be deployed for delivery of heterologous antigens to antigen-presenting cells. Here we investigated the efficacy of various effector proteins of the Salmonella pathogenicity island (SPI2)-encoded T3SS for the translocation of model antigens and elicitation of immune responses. The SPI2 T3SS effector proteins SifA, SteC, SseL, SseJ, and SseF share an endosomal membrane-associated subcellular localization after translocation. We observed that all effector proteins could be used to translocate fusion proteins with the model antigens ovalbumin and listeriolysin into the cytosol of host cells. Under in vitro conditions, fusion proteins with SseJ and SteC stimulated T-cell responses that were superior to those triggered by fusion proteins with SseF. However, in mice vaccinated with Salmonella carrier strains, only fusion proteins based on SseJ or SifA elicited potent T-cell responses. These data demonstrate that the selection of an optimal SPI2 effector protein for T3SS-mediated translocation is a critical parameter for the rational design of effective Salmonella-based recombinant vaccines.
Infection and immunity 03/2012; 80(3):1193-202. DOI:10.1128/IAI.06056-11 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer vaccine therapies have only achieved limited success when focusing on effector immunity with the goal of eliciting robust tumor-specific T-cell responses. More recently, there is an emerging understanding that effective immunity can only be achieved by coordinate disruption of tumor-derived immunosuppression. Toward that goal, we have developed a potent Salmonella-based vaccine expressing codon-optimized survivin (CO-SVN), referred to as 3342Max. When used alone as a therapeutic vaccine, 3342Max can attenuate growth of aggressive murine melanomas overexpressing SVN. However, under more immunosuppressive conditions, such as those associated with larger tumor volumes, we found that the vaccine was ineffective. Vaccine efficacy could be rescued if tumor-bearing mice were treated initially with Salmonella encoding a short hairpin RNA (shRNA) targeting the tolerogenic molecule STAT3 (YS1646-shSTAT3). In vaccinated mice, silencing STAT3 increased the proliferation and granzyme B levels of intratumoral CD4(+) and CD8(+) T cells. The combined strategy also increased apoptosis in tumors of treated mice, enhancing tumor-specific killing of tumor targets. Interestingly, mice treated with YS1646-shSTAT3 or 3342Max alone were similarly unsuccessful in rejecting established tumors, whereas the combined regimen was highly potent. Our findings establish that a combined strategy of silencing immunosuppressive molecules followed by vaccination can act synergistically to attenuate tumor growth, and they offer a novel translational direction to improve tumor immunotherapy.
Cancer Research 06/2011; 71(12):4183-91. DOI:10.1158/0008-5472.CAN-10-4676 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF)" is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant "Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES)." This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications.
PLoS ONE 05/2011; 6(5):e20267. DOI:10.1371/journal.pone.0020267 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gene delivery and transfection of eukaryotic cells are widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+ CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy.
[Show abstract][Hide abstract] ABSTRACT: Vα24-invariant natural killer T cells (NKTs) are strictly CD1d-restricted, and CD1d expression has been found in several types of leukemia and lymphoma as well as in brain tumors suggesting that these malignancies could be targeted for direct NKT-cell cytotoxicity. Several studies have revealed strong positive associations between the numbers of tumor-infiltrating or circulating NKTs with improved disease outcome in patients with diverse types of CD1d-negative solid tumors. The mechanism by which NKTs mediate anti-tumor activity against CD1d-negative tumors has long remained enigmatic. Recent evidence indicates that NKTs can suppress tumor growth indirectly by targeting CD1d-positive elements of tumor-supportive stroma such as tumor-associated macrophages. This review summarizes the current knowledge about the mechanisms that regulate NKT-cell localization to the tumor site and their interaction with the tumor microenvironment. The discussed strategies for pharmacologic modulation and genetic engineering of NKTs may lead to development of effective and broadly applicable immunotherapies of cancer.
[Show abstract][Hide abstract] ABSTRACT: Tumor infiltration with Valpha24-invariant NKT cells (NKTs) associates with favorable outcome in neuroblastoma and other cancers. Although NKTs can be directly cytotoxic against CD1d+ cells, the majority of human tumors are CD1d-. Therefore, the role of NKTs in cancer remains largely unknown. Here, we demonstrate that CD68+ tumor-associated monocytes/macrophages (TAMs) represented the majority of CD1d-expressing cells in primary human neuroblastomas. TAMs stimulated neuroblastoma growth in human cell lines and their xenografts in NOD/SCID mice via IL-6 production. Indeed, TAMs produced IL-6 in primary tumors and in the BM of patients with metastatic neuroblastoma. Gene expression analysis using TaqMan low-density arrays of 129 primary human neuroblastomas without MYCN amplification revealed that high-level expression of TAM-specific genes (CD14, CD16, IL6, IL6R, and TGFB1) was associated with poor 5-year event-free survival. While NKTs were not cytotoxic against neuroblastoma cells, they effectively killed monocytes pulsed with tumor cell lysate. The killing of monocytes was CD1d restricted because it was inhibited by a CD1d-specific mAb. Cotransfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumor site and suppressed tumor growth compared with mice transferred with monocytes alone. Thus, killing of TAMs reveals what we believe to be a novel mechanism of NKT antitumor activity that relates to the disease outcome.
The Journal of clinical investigation 06/2009; 119(6):1524-36. DOI:10.1172/JCI37869 · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuroblastoma, the second most common solid tumor in children, frequently metastasizes to the bone marrow and the bone. Neuroblastoma cells present in the bone marrow stimulate the expression of interleukin-6 (IL-6) by bone marrow stromal cells (BMSC) to activate osteoclasts. Here we have examined whether stromal-derived IL-6 also has a paracrine effect on neuroblastoma cells. An analysis of the expression of IL-6 and its receptor, IL-6R, in 11 neuroblastoma cell lines indicated the expression of IL-6 in 4 cell lines and of IL-6R in 9 cell lines. Treatment of IL-6R-positive cells with recombinant human IL-6 resulted in signal transducer and activator of transcription-3 and extracellular signal-regulated kinase-1/2 activation. Culturing IL-6R-positive neuroblastoma cells in the presence of BMSC or recombinant human IL-6 increased proliferation and protected tumor cells from etoposide-induced apoptosis, whereas it had no effect on IL-6R-negative tumor cells. In vivo, neuroblastoma tumors grew faster in the presence of a paracrine source of IL-6. IL-6 induced the expression of cyclooxygenase-2 in neuroblastoma cells with concomitant release of prostaglandin-E2, which increased the expression of IL-6 by BMSC. Supporting a role for stromal-derived IL-6 in patients with neuroblastoma bone metastasis, we observed elevated levels of IL-6 in the serum and bone marrow of 16 patients with neuroblastoma bone metastasis and in BMSC derived from these patients. Altogether, the data indicate that stromal-derived IL-6 contributes to the formation of a bone marrow microenvironment favorable to the progression of metastatic neuroblastoma.
Cancer Research 02/2009; 69(1):329-37. DOI:10.1158/0008-5472.CAN-08-0613 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although tumors express potentially immunogenic tumor-associated antigens (TAAs), cancer vaccines often fail because of inadequate antigen delivery and/or insufficient activation of innate immunity. Engineering nonpathogenic bacterial vectors to deliver TAAs of choice may provide an efficient way of presenting TAAs in an immunogenic form. In this study, we used genes of Salmonella pathogenicity island 2 (SPI2) to construct a novel cancer vaccine in which a TAA, survivin, was fused to SseF effector protein and placed under control of SsrB, the central regulator of SPI2 gene expression. This construct uses the type III secretion system (T3SS) of Salmonella and allows preferential delivery of tumor antigen into the cytosol of antigen-presenting cells for optimal immunogenicity. In a screen of a panel of attenuated strains of Salmonella, we found that a double attenuated strain of Salmonella typhimurium, MvP728 (purD/htrA), was not toxic to mice and effectively expressed and translocated survivin protein inside the cytosol of murine macrophages. We also found that a ligand for CD1d-reactive natural killer T (NKT) cells, alpha-glucuronosylceramide (GSL1), enhanced MvP728-induced interleukin-12 production in human dendritic cells and that in vivo coadministration of a NKT ligand with MvP728-Llo or MvP728-survivin enhanced effector-memory cytotoxic T lymphocyte (CTL) responses. Furthermore, combined use of MvP728-survivin with GSL1 produced antitumor activity in mouse models of CT26 colon carcinoma and orthotopic DBT glioblastoma. Therefore, the use of TAA delivery via SPI-2-regulated T3SS of Salmonella and NKT ligands as adjuvants may provide a foundation for new cancer vaccines.
International Journal of Cancer 01/2009; 126(11):2622-34. DOI:10.1002/ijc.24957 · 5.01 Impact Factor