<em>In Vivo</em> Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Georgetown University Medical Center

To evaluate the outcome of patients with retinoblastoma and postlaminar optic nerve invasion (PLONI).

Outcome of patients with retinoblastoma and postlaminar optic nerve invasion.

Inhibition of tumor growth by thrombospondin (TSP) 1 is generally attributed to its antiangiogenic activity, but effects on tumor immunity should also be considered. We show that overexpression of TSP1 in melanoma cells increases macrophage recruitment into xenograft tumors grown in nude or beige/nude mice. In vitro, TSP1 acutely induces expression of plasminogen activator inhibitor-1 (PAI-1) by monocytic cells, suggesting that TSP1-induced macrophage recruitment is at least partially mediated by PAI-1. Tumor-associated macrophages (TAM) can either promote or limit tumor progression. The percentage of M1-polarized macrophages expressing inducible nitric oxide synthase is increased in TSP1-expressing tumors. Furthermore, soluble TSP1 stimulates killing of breast carcinoma and melanoma cells by IFN-gamma-differentiated U937 cells in vitro via release of reactive oxygen species. TSP1 causes a significant increase in phorbol ester-mediated superoxide generation from differentiated monocytes by interaction with alpha(6)beta(1) integrin through its NH(2)-terminal region. The NH(2)-terminal domain of TSP2 also stimulates monocyte superoxide production. Extracellular calcium is required for the TSP1-induced macrophage respiratory burst. Thus, TSP1 may play an important role in antitumor immunity by enhancing recruitment and activation of M1 TAMs, which provides an additional selective pressure for loss of TSP1 and TSP2 expression during tumor progression.

Thrombospondin 1 promotes tumor macrophage recruitment and enhances tumor cell cytotoxicity of differentiated U937 cells.

Limitations to biomarker discovery are not only technical or bioinformatic but conceptual as well. In our attempt to offer a solution, we are highlighting three issues that we think are limiting progress in biomarkers discovery. First, the confusion stemming from the imposition of a pathology-type immunohistochemical marker (IHCM) concept on omics data without fully understanding the characteristics and limitations of IHCMs as applied in clinical pathology. Second, the lack of serious consideration for the scope of disease heterogeneity. Third, the refusal of the biomedical community to borrow from other biological disciplines their well established methods for dealing with heterogeneity. Therefore, real progress in biomarker discovery will be attained when we recognize that an omics biomarker cannot be assigned and validated without a priori data modeling and subtyping of the disease itself to reveal the extent of its heterogeneity, and its omics' clonal aberrations (drivers) underlying its subtypes and pathways' diversity. To further support our viewpoints, we are contributing a novel a systems biology method such as parsimony phylogenetic approach for disease modeling prior to biomarker circumscription. As an analytical approach that has been successfully used for a half of a century in other biological disciplines, parsimony phylogenetics simultaneously achieves several objectives: it provides disease modeling in a hierarchical phylogenetic classification, identifies biomarkers as the shared derived expressions or mutations--synapomorphies, constructs the omics profiles of specimens based on the most parsimonious arrangement of their heterogeneous data, and permits network profiling of affected signaling pathways as the biosignature of disease classes.

Biomarkers in the age of omics: time for a systems biology approach.

Lexatumumab is an agonistic, fully human monoclonal antibody against tumor necrosis factor-related apoptosis-inducing ligand receptor 2 with preclinical evidence of activity in pediatric solid tumors.

Phase I trial and pharmacokinetic study of lexatumumab in pediatric patients with solid tumors.

Disseminated Candida albicans infection results in high morbidity and mortality despite treatment with existing antifungal drugs. Recent studies suggest that modulating the host immune response can improve survival, but specific host targets for accomplishing this goal remain to be identified. The extracellular matrix protein thrombospondin-1 is released at sites of tissue injury and modulates several immune functions, but its role in C. albicans pathogenesis has not been investigated. Here, we show that mice lacking thrombospondin-1 have an advantage in surviving disseminated candidiasis and more efficiently clear the initial colonization from kidneys despite exhibiting fewer infiltrating leukocytes. By examining local and systemic cytokine responses to C. albicans and other standard inflammatory stimuli, we identify a crucial function of phagocytes in this enhanced resistance. Subcutaneous air pouch and systemic candidiasis models demonstrated that endogenous thrombospondin-1 enhances the early innate immune response against C. albicans and promotes activation of inflammatory macrophages (inducible nitric oxide synthase⁺, IL-6(high), TNF-α(high), IL-10(low)), release of the chemokines MIP-2, JE, MIP-1α, and RANTES, and CXCR2-driven polymorphonuclear leukocytes recruitment. However, thrombospondin-1 inhibited the phagocytic capacity of inflammatory leukocytes in vivo and in vitro, resulting in increased fungal burden in the kidney and increased mortality in wild type mice. Thus, thrombospondin-1 enhances the pathogenesis of disseminated candidiasis by creating an imbalance in the host immune response that ultimately leads to reduced phagocytic function, impaired fungal clearance, and increased mortality. Conversely, inhibitors of thrombospondin-1 may be useful drugs to improve patient recovery from disseminated candidiasis.

Endogenous thrombospondin-1 regulates leukocyte recruitment and activation and accelerates death from systemic candidiasis.

Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.

Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects.

Ewing sarcoma (ES) is driven by fusion of the Ewing sarcoma breakpoint region 1 gene (EWSR1) with an E26 transformation-specific (ETS) transcription factor (EWS-ETS), most often the Friend leukemia integration 1 transcription factor (FLI1). Neuropeptide Y (NPY) is an EWS-FLI1 transcriptional target; it is highly expressed in ES and exerts opposing effects, ranging from ES cell death to angiogenesis and cancer stem cell propagation. The functions of NPY are regulated by dipeptidyl peptidase IV (DPPIV), a hypoxia-inducible enzyme that cleaves the peptide and activates its growth-promoting actions. The objective of this study was to determine the clinically relevant functions of NPY by identifying the associations between patients' ES phenotype and their NPY concentrations and DPP activity.

Systemic levels of neuropeptide Y and dipeptidyl peptidase activity in patients with Ewing sarcoma--associations with tumor phenotype and survival.

Ewing sarcoma (ES) develops in bones or soft tissues of children and adolescents. The presence of bone metastases is one of the most adverse prognostic factors, yet the mechanisms governing their formation remain unclear. As a transcriptional target of EWS-FLI1, the fusion protein driving ES transformation, neuropeptide Y (NPY) is highly expressed and released from ES tumors. Hypoxia up-regulates NPY and activates its pro-metastatic functions. To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model. ES cells were injected into gastrocnemius muscles of SCID/beige mice, the primary tumors excised, and mice monitored for the presence of metastases. SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%). Bone dissemination in SK-ES1 xenografts associated with increased NPY expression in bone metastases and its accumulation in bone invasion areas. The genetic silencing of NPY in SK-ES1 cells reduced bone degradation. Our study supports the role for NPY in ES bone invasion and provides new models for identifying pathways driving ES metastases to specific niches and testing anti-metastatic therapeutics.

High neuropeptide Y release associates with Ewing sarcoma bone dissemination - in vivo model of site-specific metastases.

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2(+) sarcoma and neuroblastoma cell lines in vitro However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2(+) sarcoma, despite effectively controlling GD2(+) neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869-80. ©2016 AACR.

Reduction of MDSCs with All-trans Retinoic Acid Improves CAR Therapy Efficacy for Sarcomas.

Neuroblastoma (NB) is a pediatric malignant neoplasm of sympathoadrenal origin. Challenges in its management include stratification of this heterogeneous disease and a lack of both adequate treatments for high-risk patients and noninvasive biomarkers of disease progression. Our previous studies have identified neuropeptide Y (NPY), a sympathetic neurotransmitter expressed in NB, as a potential therapeutic target for these tumors by virtue of its Y5 receptor (Y5R)-mediated chemoresistance and Y2 receptor (Y2R)-mediated proliferative and angiogenic activities. The goal of this study was to determine the clinical relevance and utility of these findings. Expression of NPY and its receptors was evaluated in corresponding samples of tumor RNA, tissues, and sera from 87 patients with neuroblastic tumors and in tumor tissues from the TH-MYCN NB mouse model. Elevated serum NPY levels correlated with an adverse clinical presentation, poor survival, metastasis, and relapse, whereas strong Y5R immunoreactivity was a marker of angioinvasive tumor cells. In NB tissues from TH-MYCN mice, high immunoreactivity of both NPY and Y5R marked angioinvasive NB cells. Y2R was uniformly expressed in undifferentiated tumor cells, which supports its previously reported role in NB cell proliferation. Our findings validate NPY as a therapeutic target for advanced NB and implicate the NPY/Y5R axis in disease dissemination. The correlation between elevated systemic NPY and NB progression identifies serum NPY as a novel NB biomarker.

Susana Galli

Department of Biochemistry and Molecular & Cellular Biology

Georgetown University Medical Center

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Susana Galli

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular & Cellular Biology

Georgetown University Medical Center

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Department of Biochemistry and Molecular & Cellular Biology