Sunil Singhal

Hospital of the University of Pennsylvania, Filadelfia, Pennsylvania, United States

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Publications (88)394.76 Total impact

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    ABSTRACT: Myeloid derived suppressor cells (MDSCs) are an immunosuppressive population of immature myeloid cells found in advanced stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allow MDSCs to suppress T cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctnf/f), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38high MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38high MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38low MDSCs, likely as a result of increased iNOS production. Additionally, we have identified novel tumor-derived factors, specifically IL-6, IGFBP-3 and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38-positive MDSCs in peripheral blood of advanced stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy. Copyright © 2015, American Association for Cancer Research.
    Cancer Research 08/2015; DOI:10.1158/0008-5472.CAN-14-3639 · 9.28 Impact Factor
  • Cancer Research 08/2015; 75(15 Supplement):1291-1291. DOI:10.1158/1538-7445.AM2015-1291 · 9.28 Impact Factor
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    ABSTRACT: During lung surgery, identification of surgical margins is challenging. We hypothesized that molecular imaging with a fluorescent probe to pulmonary adenocarcinomas could enhance residual tumor during resection. Mice with flank tumors received a contrast agent targeting folate receptor alpha. Optimal dose and time of injection was established. Margin detection was compared using traditional methods versus molecular imaging. A pilot study was then performed in three humans with lung adenocarcinoma. The peak tumor-to-background ratio (TBR) of murine tumors was 3.9. Fluorescence peaked at 2 h and was not improved beyond 0.1 mg/kg. Traditional inspection identified 30 % of mice with positive margins. Molecular imaging identified an additional 50 % of residual tumor deposits (p < 0.05). The fluorescent probe visually enhanced all human tumors with a mean TBR of 3.5. Molecular imaging is an important adjunct to traditional inspection to identify surgical margins after tumor resection.
    Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging 07/2015; DOI:10.1007/s11307-015-0878-9 · 2.87 Impact Factor
  • Journal of Biomedical Optics 07/2015; 20(7):76002. DOI:10.1117/1.JBO.20.7.076002 · 2.75 Impact Factor
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    ABSTRACT: Recent studies suggest that immunotherapy may offer a promising treatment strategy for early-stage malignant pleural mesothelioma (MPM), but advanced tumor burden may limit the efficacy of immunotherapy. Therefore, we hypothesized that surgical cytoreduction could restore the efficacy of vaccine-based immunotherapy for MPM. We developed a murine model of MPM through transduction of a mesothelioma cell line with mesothelin. We used this model to evaluate the efficacy of a Listeria monocytogenes vaccine expressing mesothelin. Tumor growth was significantly inhibited at four weeks in animals vaccinated two weeks prior to tumor cell inoculation as compared to those given an empty vector control (1371±420mm(3) versus 405±139mm(3); p<0.01). Mice vaccinated one week prior to tumor challenge also displayed significant reduction in tumor volume (1227±406mm(3) versus 309±173mm(3); p<0.01). The vaccine had no effect when administered concurrently with tumor challenge, or after tumors were established. Flow cytometry showed reduced mesothelin expression in large tumors, as well as tumor-associated immunosuppression due to increased myeloid derived suppressor cells (MDSCs). These factors may have limited vaccine efficacy for advanced disease. Surgical cytoreduction of established tumors restored the antitumor potency of the therapeutic vaccine, with significantly reduced tumor burden at post-operative day 18 (397±103mm(3) versus 1047±258mm(3); p<0.01). We found that surgery reduced MDSCs to levels comparable to those in tumor-naïve mice. This study demonstrates that cytoreduction surgery restores the efficacy of cancer vaccines for MPM by reducing tumor-related immunosuppression that impairs immunotherapy. Copyright © 2015. Published by Elsevier B.V.
    Immunology letters 05/2015; 166(1). DOI:10.1016/j.imlet.2015.05.009 · 2.37 Impact Factor
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    ABSTRACT: More than 80,000 people undergo resection of a pulmonary tumor each year, and the only method to determine if the tumor is malignant is histologic analysis. We propose that a targeted molecular contrast agent could bind lung adenocarcinomas, which could be identified using real-time optical imaging at the time of surgery. Fifty patients with a biopsy-proven lung adenocarcinoma were enrolled. Before surgery, patients were systemically administered 0.1 mg/kg of a fluorescent folate receptor alpha (FRα)-targeted molecular contrast agent by intravenous infusion. During surgery, tumors were imaged in situ and ex vivo, after the lung parenchyma was dissected to directly expose the tumor to the imaging system. Tumors ranged from 0.3 to 7.5 cm (mean: 2.6 cm), and 46 of 50 (92%) lung adenocarcinomas were fluorescent. No false uptake occurred, and in 2 cases, intraoperative imaging revealed tumor metastases (3 mm and 6 mm) that were not recognized preoperatively. Four adenocarcinomas were not fluorescent, and immunohistochemistry showed that these adenocarcinomas did not express FRα. Tumor fluorescence was independent of nodule size, uptake of 2-deoxy-2-((18)F)fluoro-D-glucose, histology, and tumor differentiation. Molecular imaging could identify only 7 of the 50 adenocarcinomas in situ in the patient without bisection. The most important predictor of the success of molecular imaging in locating the tumor in situ was the distance of the nodule from the pleural surface. Intraoperative molecular imaging with a targeted contrast agent can identify lung adenocarcinomas, and this technology is currently useful in patients with subpleural tumors, irrespective of size. With further refinements, this tool may prove useful in locating adenocarcinomas that are deeper in the lung parenchyma, in lymph nodes, and at pleural and resection margins. Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.
    The Journal of thoracic and cardiovascular surgery 05/2015; 150(1). DOI:10.1016/j.jtcvs.2015.05.014 · 3.99 Impact Factor
  • The Journal of Urology 04/2015; 193(4):e695. DOI:10.1016/j.juro.2015.02.2002 · 3.75 Impact Factor
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    ABSTRACT: Over-expression of folate receptor alpha on cancer cells has been frequently exploited for delivery of folate-targeted imaging and therapeutic agents to tumors. Because limited information exists on expression of the beta isoform of the folate receptor in human cancers (FR-β), we have evaluated the immunohistochemical staining pattern of FR-β in 992 tumor sections from 20 different human cancer types using a new anti-human FR-β monoclonal antibody. FR-β expression was shown to be more pronounced in cells within the stroma, primarily macrophages and macrophage-like cells than cancer cells in every cancer type studied. Moreover, FR-β expression in both cancer and stromal cells was found to be statistically more prominent in females than males. A significant positive correlation was also observed between FR-β expression on stromal cells and both the stage of the cancer and the presence of lymph node metastases. Based on these data we conclude FR-β may constitute a good target for specific delivery of therapeutic agents to activated macrophages and that accumulation of FR-β positive macrophages in the stroma could serve as a useful indicator of a tumor's metastatic potential.
    Oncotarget 03/2015; · 6.63 Impact Factor
  • Jane J Keating · Gregory T Kennedy · Sunil Singhal
    The Journal of thoracic and cardiovascular surgery 03/2015; 149(3):e51-3. DOI:10.1016/j.jtcvs.2014.10.081 · 3.99 Impact Factor
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    ABSTRACT: A 58-year-old man with a remote history of choking on a chicken bone 5 years earlier presented with chronic cough but had no remarkable clinical examination findings. He was being followed for recurrent pneumonias complicated by a resistant empyema, for which he had undergone thoracotomy and decortication. Imaging studies initially missed a foreign body (the chicken bone), which was found on follow-up studies and was removed with a flexible bronchoscope despite the fact that 5 years had passed since the aspiration.
    Ear, nose, & throat journal 01/2015; 94(1):E27-9. · 0.88 Impact Factor
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    ABSTRACT: Surgery is the most effective method to cure patients with solid tumors. New techniques in near-infrared (NIR) cancer imaging are being used to identify surgical margins and residual tumor cells in the wound. Our goal was to determine the optimal time and dose for imaging solid tumors using Indocyanine Green. Syngeneic murine flank tumor models were used to test NIR imaging of ICG at various doses ranging from 0 to 10 mg/kg. Imaging was performed immediately after injection and up to 72 hours later. Biodistribution in the blood and murine organs were quantified by spectroscopy and fluorescence microscopy. Based on these results, a six patient dose titration study was performed. In murine flank tumors, the tumor-to-background ratio (TBR) for ICG at doses less than 5 mg/kg were less than 2 fold at all time points, and the surgeons could not subjectively identify tissue contrast. However, for doses ranging from 5 mg/kg to 10 mg/kg, the TBR ranged from 2.1 to 8.0. The tumor signal was best appreciated at 24 hours and the background was least pronounced after 24 hours. Biodistribution studies in the blood and murine organs revealed excretion through the biliary tree and gastrointestinal tract, with minimal blood fluorescence at the higher doses. A follow up pilot study confirmed that these findings were applicable to lung cancer patients, and tumor was clearly delineated from surrounding normal tissue by NIR imaging. For non-hepatic solid tumors, we found ICG was optimal when dosed at 5 mg/kg and 24 hours before surgery.
    American Journal of Nuclear Medicine and Molecular Imaging 01/2015; 5(4):390-400. · 3.25 Impact Factor
  • Molecular-Guided Surgery: Molecules, Devices, and Applications. Proceedings of SPIE:, San Francisco; 01/2015
  • International journal of radiation oncology, biology, physics 11/2014; 90(5). DOI:10.1016/j.ijrobp.2014.08.157 · 4.18 Impact Factor
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    ABSTRACT: Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62LloCD54hi) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.
    Journal of Clinical Investigation 11/2014; 124(12). DOI:10.1172/JCI77053 · 13.77 Impact Factor
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    ABSTRACT: Surgical biopsy of potential tumor recurrence is a common challenge facing oncologists, surgeons, and cancer patients. Imaging modalities have limited ability to accurately detect recurrent cancer in fields affected by previous surgery, chemotherapy, or radiation. However, definitive tissue diagnosis is often needed to initiate treatment and to direct therapy. We sought to determine if a targeted fluorescent intraoperative molecular imaging technique could be applied in a clinical setting to assist a surgical biopsy in a "hostile" field. We describe the use of a folate-fluorescein conjugate to direct the biopsy of a suspected recurrent lung adenocarcinoma invading the mediastinum that had been previously treated with chemoradiation. We found that intraoperative imaging allowed the identification of small viable tumor deposits that were otherwise indistinguishable from scar and necrosis. Our operative observations were confirmed by histology, fluorescence microscopy, and immunohistochemistry. Our results demonstrate one possible application and clinical value of intraoperative molecular imaging.
    Molecular Imaging 11/2014; 13:1-6. DOI:10.2310/7290.2014.00051 · 2.19 Impact Factor
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    ABSTRACT: Careful preparation of human tissues is the cornerstone of obtaining accurate data in immunologic studies. Despite the essential importance of tissue processing in tumor immunology and clinical medicine, current methods of tissue disaggregation have not been rigorously tested for data fidelity. Thus, we critically evaluated the current techniques available in the literature that are used to prepare human lung tumors for immunologic studies. We discovered that these approaches are successful at digesting cellular attachments and ECMs; however, these methods frequently alter the immune cell composition and/or expression of surface molecules. We thus developed a novel approach to prepare human lung tumors for immunologic studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This enzymatic digestion cocktail optimized cell yield and cell viability, retrieved all major tumor-associated cell populations, and maintained the expression of cell-surface markers for lineage definition and in vivo effector functions. To our knowledge, we present the first rigorously tested disaggregation method designed for human lung tumors.
    Journal of Leukocyte Biology 10/2014; 97(1). DOI:10.1189/jlb.5TA0814-373 · 4.30 Impact Factor
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    ABSTRACT: Background: Gene therapy is currently under investigation as a means of managing a variety of pulmonary diseases. Unfortunately, gene transfer to bronchial epithelium has been hampered by the lack of stable and efficient transduction. Recent studies have shown that gene vectors could be tethered to the metallic surfaces of intra-arterial stents. This approach enables efficacious and site-specific adenoviral gene delivery to the vascular endothelium. Objectives: We hypothesized that airway mesh stents impregnated with viral gene vectors could be used for local gene delivery to benign and malignant bronchial epithelium. Methods: Serotype 5 adenoviral vectors (Ad5, E1-/E3-) containing the reporter genes green fluorescent protein (Ad.GFP) or β-galactoside/LacZ (Ad.LacZ), or a therapeutic gene, Ad.INF-β, were coupled to either metallic mesh disks or stents via anti-Ad knob antibodies. These platforms were assessed for their ability to transfect bronchial epithelial cells from both rats and humans, as well as murine (L1C2) and human (A549) lung cancer cell lines. Gene transfer was quantified by fluorescent microscopy, scanning fluorimetry for Ad.GFP, and light microscopy studies assessing β-galactosidase staining for Ad.LacZ. Metallic mesh and stent-mediated gene transfer was also performed in a murine flank tumor model and in a rat endotracheal tumor model in order to evaluate the therapeutic potential. Results: In these studies, murine and human non-small cell lung cancer (NSCLC) cells were successfully transfected with reporter genes in vitro. Ad.LacZ-complexed mesh successfully transfected reporter genes into established murine flank NSCLC tumors. In addition, Ad.LacZ-tethered stents could effectively transfect both tracheobronchial epithelium and submucosal glands in rats. Similar epithelial transfection was achieved in ex vivo human bronchial epithelium. Pilot in vivo experimentation provided data supporting the concept that therapeutic genes could also be delivered with this technology. In additional pilot in vivo experiments, the growth of murine flank tumors was inhibited by placement of mesh disks coupled with Ad.muINF-β, and rats bearing endotracheal tumors demonstrated a trend towards prolonged survival with insertion of Ad.ratINF-β-tethered stents. Conclusions: Stent-mediated gene delivery successfully enabled site-specific vector administration to target rat and human airway cells in cell culture, organ culture and in vivo. Local tracheobronchial gene delivery via stents could provide a viable clinical solution for overcoming the difficulties encountered with vector delivery within the lungs, in particular by lowering requisite vector titers and by directing desired vectors to areas of interest. This strategy may prove valuable for treating tumors involving the tracheobronchial tree, as well as other nonmalignant tracheobronchial disorders. © 2014 S. Karger AG, Basel.
    Respiration 10/2014; 88(5). DOI:10.1159/000368164 · 2.92 Impact Factor
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    ABSTRACT: Background Over 80,000 people undergo pulmonary resection for a lung nodule in the United States each year. Small nodules are frequently missed or difficult to find despite preoperative imaging. We hypothesized that near-infrared (NIR) imaging technology could be used to identify and locate lung nodules during surgery. Methods We enrolled 18 patients who were diagnosed with a pulmonary nodule that required resection. All patients had a fine-cut 1-mm computed tomography scan preoperatively. The patients were given systemic 5 mg/kg indocyanine green and then underwent an open thoracotomy 24 hours later. The NIR imaging was used to identify the primary nodule and search for additional nodules that were not found by visual inspection or manual palpation of the ipsilateral lung. Results Manual palpation and visual inspection identified all 18 primary pulmonary nodules and no additional lesions. Intraoperative NIR imaging detected 16 out of the 18 primary nodules. The NIR imaging also identified 5 additional subcentimeter nodules; 3 metastatic adenocarcinomas and 2 metastatic sarcomas. This technology could identify nodules as small as 0.2 cm and as deep as 1.3 cm from the pleural surface. This approach discovered 3 nodules that were in different lobes than the primary tumor. Nodule fluorescence was independent of size, metabolic activity, histology, tumor grade and vascularity. Conclusions This is the first-in-human demonstration of identifying pulmonary nodules during thoracic surgery with NIR imaging without a priori knowledge of their location or existence. The NIR imaging can detect pulmonary nodules during lung resections that are poorly visualized on computed tomography and difficult to discriminate on finger palpation.
    The Annals of Thoracic Surgery 10/2014; 98(4). DOI:10.1016/j.athoracsur.2014.05.026 · 3.63 Impact Factor
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    ABSTRACT: The mechanisms by which tumor-associated neutrophils (TAN) affect tumor growth are to a large extent unknown. Regulatory T cells (T-regs) are functionally immune-suppressive subsets of T-cells. Depletion or inhibition of T-regs can enhance anti-tumor immunity. We demonstrated both by RT-PCR and ELISA, that murine TAN secrete significant amounts of the T-regs chemoattractant, CCL17, much more than circulating or splenic neutrophils, and at a level progressively increasing during tumor development. Migration assays, both in vitro and in vivo, showed recruitment of T-regs by TAN which was inhibited with anti-CCL17 monoclonal antibodies. Systemic neutrophil depletion in tumor-bearing mice using anti-Ly6G monoclonal antibodies reduced the migration of T-regs into the tumors. We further showed, using flow cytometry, that CCL17 secretion by TAN is not limited to mouse models of cancer but is also relevant to human TAN. Our results suggest a new indirect mechanism by which TAN may inhibit anti-tumor immune activity, thus promoting tumor growth. We further describe, for the first time, a clear link between TAN and regulatory T-cells (T-regs) acting together to impair anti-tumor immunity. © 2014 Wiley Periodicals, Inc.
    International Journal of Cancer 09/2014; 135(5). DOI:10.1002/ijc.28770 · 5.01 Impact Factor
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    ABSTRACT: Introduction Defining tumor from non-tumor tissue is one of the major challenges of cancer surgery. Surgeons depend on visual and tactile clues to select which tissues should be removed from a patient. Recently, we and others have hypothesized near-infrared (NIR) imaging can be used during surgery to differentiate tumors from normal tissue. Methods We enrolled 8 canines and 5 humans undergoing cancer surgery for NIR imaging. The patients were injected with indocyanine green (ICG), an FDA approved non-receptor specific NIR dye that accumulates in hyperpermeable tissues, 16–24 hours prior to surgery. During surgery, NIR imaging was used to discriminate the tumor from non-tumor tissue. Results NIR imaging identified all tumors with a mean signal-to-background ratio of 6.7. Optical images were useful during surgery in discriminating normal tissue from cancer. In 3 canine cases and 1 human case, the tissue surrounding the tumor was inflamed due to obstruction of the vascular supply due to mass effect. In these instances, NIR imaging could not distinguish tumor tissue from tissue that was congested, edematous and did not contain cancer. Conclusions This study shows that NIR imaging can identify tumors from normal tissues, provides excellent tissue contrast, and it facilitates the resection of tumors. However, in situations where there is significant peritumoral inflammation, NIR imaging with ICG is not helpful. This suggests that non-targeted NIR dyes that accumulate in hyperpermeable tissues will have significant limitations in the future, and receptor-specific NIR dyes may be necessary to overcome this problem.
    PLoS ONE 07/2014; 9(7):e103342. DOI:10.1371/journal.pone.0103342 · 3.23 Impact Factor

Publication Stats

2k Citations
394.76 Total Impact Points

Institutions

  • 2002–2015
    • Hospital of the University of Pennsylvania
      • • Department of Surgery
      • • Department of Medicine
      Filadelfia, Pennsylvania, United States
  • 2014
    • Emory University
      • Department of Chemistry
      Atlanta, Georgia, United States
  • 2011–2014
    • Treatment Research Institute, Philadelphia PA
      Filadelfia, Pennsylvania, United States
  • 2010–2014
    • William Penn University
      Filadelfia, Pennsylvania, United States
  • 2005–2014
    • University of Pennsylvania
      • Department of Surgery
      Filadelfia, Pennsylvania, United States
  • 2012
    • University of Texas Southwestern Medical Center
      • Medical School
      Dallas, Texas, United States
  • 2003–2006
    • Johns Hopkins University
      • • Department of Surgery
      • • Department of Medicine
      Baltimore, Maryland, United States