Lacey R. McNally

Anti-EMMPRIN Monoclonal Antibody as a Novel Agent for Therapy of Head and Neck Cancer

Purpose: Extracellular matrix metalloprotease inducer (EMMPRIN) is a tumor surface protein that promotes growth and is overexpressed in head and neck cancer. These features make it a potential therapeutic target for monoclonal antibody (mAb)–based therapy. Because molecular therapy is considered more effective when delivered with conventional cytotoxic agents, anti-EMMPRIN therapy was assessed alone and in combination with external beam radiation. Experimental Design: Using a murine flank model, loss of EMMPRIN function was achieved by transfection with a small interfering RNA against EMMPRIN or treatment with a chimeric anti-EMMPRIN blocking mAb. Cytokine expression was assessed for xenografts, tumor cells, fibroblasts, and endothelial cells. Results: Animals treated with anti-EMMPRIN mAb had delayed tumor growth compared with untreated controls, whereas treatment with combination radiation and anti-EMMPRIN mAb showed the greatest reduction in tumor growth (P = 0.001). Radiation-treated EMMPRIN knockdown xenografts showed a reduction in tumor growth compared with untreated knockdown controls (P = 0.01), whereas radiation-treated EMMPRIN–expressing xenografts did not show a delay in tumor growth. Immunohistochemical evaluation for Ki67 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) resulted in a reduction in proliferation (P = 0.007) and increased apoptosis in anti-EMMPRIN mAb–treated xenografts compared with untreated controls (P = 0.087). In addition, we provide evidence that EMMPRIN suppression results in decreased interleukin 1β (IL-1β), IL-6, and IL-8 cytokine production, in vitro and in vivo. Conclusions: These data suggest that anti-EMMPRIN antibody inhibits tumor cell proliferation in vivo and may represent a novel targeted treatment option in head and neck squamous cell carcinoma.

Early Therapy Evaluation of Combined Anti–Death Receptor 5 Antibody and Gemcitabine in Orthotopic Pancreatic Tumor Xenografts by Diffusion-Weighted Magnetic Resonance Imaging

Early therapeutic efficacy of anti-death receptor 5 antibody (TRA-8) combined with gemcitabine was measured using diffusion-weighted magnetic resonance imaging (DWI) in an orthotopic pancreatic tumor model. Groups 1 to 4 of severe combined immunodeficient mice (n = 5-7 per group) bearing orthotopically implanted, luciferase-positive human pancreatic tumors (MIA PaCa-2) were subsequently (4-5 weeks thereafter) injected with saline (control), gemcitabine (120 mg/kg), TRA-8 (200 mug), or TRA-8 combined with gemcitabine, respectively, on day 0. DWI, anatomic magnetic resonance imaging, and bioluminescence imaging were done on days 0, 1, 2, and 3 after treatment. Three tumors from each group were collected randomly on day 3 after imaging, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining was done to quantify apoptotic cellularity. At just 1 day after starting therapy, the changes of apparent diffusion coefficient (ADC) in tumor regions for group 3 (TRA-8) and group 4 (TRA-8/Gem) were 21 +/- 9% (mean +/- SE) and 27 +/- 3%, respectively, significantly higher (P < 0.05) than those of group 1 (-1 +/- 5%) and group 2 (-2 +/- 4%). There was no statistical difference in tumor volumes for the groups at this time. The mean ADC values of groups 2 to 4 gradually increased over 3 days, which were concurrent with tumor volume regressions and bioluminescence signal decreases. Apoptotic cell densities of tumors in groups 1 to 4 were 0.7 +/- 0.4%, 0.6 +/- 0.2%, 3.1 +/- 0.9%, and 4.7 +/- 1.0%, respectively, linearly proportional to the ADC changes on day 1. Further, the ADC changes were highly correlated with the previously reported mean survival times of animals treated with the same agents and doses. This study supports the clinical use of DWI for pancreatic tumor patients for early assessment of drug efficacy.

KISS1 over-expression suppresses metastasis of pancreatic adenocarcinoma in a xenograft mouse model

Identifying molecular targets for treatment of pancreatic cancer metastasis is critical due to the high frequency of dissemination prior to diagnosis of this lethal disease. Because the KISS1 metastasis suppressor is expressed at reduced levels in advanced pancreatic cancer, we hypothesized that re-expression of KISS1 would reduce metastases. Highly metastatic S2VP10 cells expressing luciferase (S2VP10L) were transfected with a FLAG-tagged version of KISS1 (KFM), KFMΔSS (with deleted secretion signal sequence), or pcDNA3 control plasmid (CP) and expression was confirmed by RTQ-PCR. SCID mice were implanted orthotopically with S2VP10L cells or transfectants and tumor growth and metastases were monitored using bioluminescence imaging. Mice with S2VP10L-KISS1 tumors developed fewer liver (98%) and lung (99%) metastases than S2VP10L. Unexpectedly, mice with S2VP10L-KFMΔSS tumors also had reduced liver and lung metastases, but had more metastases than mice with S2VP10L-KISS. KISS1 protein was found in the cytoplasm of both KFMΔSS and KISS1-expressing orthotopic tumors by immunohistochemistry. Metastases were not found in lungs of mice with S2VP10L-KISS1 tumors; whereas, KFMΔSS lung sections had regions of concentrated KISS1 staining, suggesting that secretion of KISS1 is needed to reduce metastasis significantly. These data suggest induction of KISS1 expression has potential as an adjuvant treatment for pancreatic cancer.

Targeted Noninvasive Imaging of EGFR-Expressing Orthotopic Pancreatic Cancer Using Multispectral Optoacoustic Tomography

Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe before MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a nonspecific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at 6 hours postinjection, demonstrating specific intratumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of the EGF receptor.

Current and Emerging Clinical Applications of Multispectral Optoacoustic Tomography (MSOT) in Oncology

Accurate detection and characterization of cancers are key for providing timely intervention and effective treatments. Current imaging technologies are particularly limited when it comes to detecting very small tumors in vivo, i.e., very early cancers or metastases, differentiating viable tumor from surrounding dead tumor tissue, and evaluating tumor metabolism within tissue. Optoacoustic imaging offers potential solutions to these imaging problems because of its ability to image optical absorption properties of both intrinsic tissue chromophores and exogenous contrast agents without the involvement of ionizing radiation. Optoacoustic imaging uses pulsed laser to induce localized thermoelastic expansion that generates acoustic waves detectable by an ultrasound transducer. To date, multispectral optoacoustic tomography (MSOT) has primarily been used in preclinical research; however, its use in translational and clinical research is expanding. This review focuses on current and emerging applications of optoacoustic imaging for molecular imaging of cancer using both exogenous and endogenous contrast agents and sheds light on potential future clinical applications. Clin Cancer Res; 22(14); 3432–9. ©2016 AACR.

Dectin-1 Activation by a Natural Product β-Glucan Converts Immunosuppressive Macrophages into an M1-like Phenotype.

Tumor-associated macrophages (TAM) with an alternatively activated phenotype have been linked to tumor-elicited inflammation, immunosuppression, and resistance to chemotherapies in cancer, thus representing an attractive target for an effective cancer immunotherapy. In this study, we demonstrate that particulate yeast-derived β-glucan, a natural polysaccharide compound, converts polarized alternatively activated macrophages or immunosuppressive TAM into a classically activated phenotype with potent immunostimulating activity. This process is associated with macrophage metabolic reprograming with enhanced glycolysis, Krebs cycle, and glutamine utilization. In addition, particulate β-glucan converts immunosuppressive TAM via the C-type lectin receptor dectin-1–induced spleen tyrosine kinase–Card9–Erk pathway. Further in vivo studies show that oral particulate β-glucan treatment significantly delays tumor growth, which is associated with in vivo TAM phenotype conversion and enhanced effector T cell activation. Mice injected with particulate β-glucan–treated TAM mixed with tumor cells have significantly reduced tumor burden with less blood vascular vessels compared with those with TAM plus tumor cell injection. In addition, macrophage depletion significantly reduced the therapeutic efficacy of particulate β-glucan in tumor-bearing mice. These findings have established a new paradigm for macrophage polarization and immunosuppressive TAM conversion and shed light on the action mode of β-glucan treatment in cancer.

Enhanced penetration into 3D cell culture using two and three layered gold nanoparticles

Nano-scale particles sized 10–400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. Nanoparticle surface modifications provide for increasing tumor targeting and uptake while decreasing immunogenicity and toxicity. Herein, we created novel two layer gold-nanoshell particles coated with alkanethiol and phosphatidylcholine, and three layer nanoshells additionally coated with high-density-lipoprotein. We hypothesize that these particles have enhanced penetration into 3-dimensional cell cultures modeling avascular tissue when compared to standard poly(ethylene glycol) (PEG)-coated nanoshells. Particle uptake and distribution in liver, lung, and pancreatic tumor cell cultures were evaluated using silver-enhancement staining and hyperspectral imaging with dark field microscopy. Two layer nanoshells exhibited significantly higher uptake compared to PEGylated nanoshells. This multilayer formulation may help overcome transport barriers presented by tumor vasculature, and could be further investigated in vivo as a platform for targeted cancer therapies.

Identification of pancreatic tumors in vivo with ligand-targeted, pH responsive mesoporous silica nanoparticles by multispectral optoacoustic tomography.

Despite significant efforts to translate nanotechnology for cancer application, lack of identification of biodistribution/accumulation of these nanovehicles in vivo remains a substantial barrier for successful implementation of theranostic nanoparticles in the clinic. The purpose of the study was to develop a tumor-targeted theranostic nanovehicle for pancreatic cancer detectable by multispectral optoacoustic tomography (MSOT). To improve the tumor specificity of our mesoporous silica nanoparticle (MSN), we utilized a dual targeting strategy: 1) an elevated tumor receptor, urokinase plasminogen activator receptor (UPAR), and 2) the acidic tumor microenvironment. The tumor specificity of the MSN particle was improved with the addition of both chitosan, targeting acidic pH, and urokinase plasminogen activator (UPA), targeting UPAR. Drug release assays confirmed pH responsive release of gemcitabine in vitro. The UPAR specific binding of MSN-UPA nanoparticles was confirmed by reduction in fluorescence signal following MSN-UPA nanoparticle treatment in UPAR positive cells blocked with a UPAR-blocking antibody. Based upon Indocyanine Green encapsulation within the nanoparticles, UPA ligand targeted MSNs demonstrated increased intensity compared to untargeted MSNs at both pH7.4 (7×) and 6.5 (20×); however the signal was much more pronounced at a pH of 6.5 using tissue phantoms (p<0.05). In vivo, MSN-UPA particles demonstrated orthotopic pancreatic tumor specific accumulation compared to liver or kidney as identified using multispectral optoacoustic tomography (p<0.05) and confirmed by ex vivo analysis. By tracking in vivo nanoparticle biodistribution with MSOT, it was shown that pH responsive, ligand targeted MSNs preferentially bind to pancreatic tumors for payload delivery.

Targeting Acidity in Pancreatic Adenocarcinoma: Multispectral Optoacoustic Tomography Detects pH-Low Insertion Peptide Probes In Vivo

Background: pH-low insertion peptides (pHLIP) can serve as a targeting moiety that enables pH-sensitive probes to detect solid tumors. Using these probes in conjunction with multispectral optoacoustic tomography (MSOT) is a promising approach to improve imaging for pancreatic cancer. Methods: A pH-sensitive pHLIP (V7) was conjugated to 750 NIR fluorescent dye and evaluated as a targeted probe for pancreatic adenocarcinoma. The pH-insensitive K7 pHLIP served as an untargeted control. Probe binding was assessed in vitro at pH 7.4, 6.8, and 6.6 using human pancreatic cell lines S2VP10 and S2013. Using MSOT, semiquantitative probe accumulation was then assessed in vivo with a murine orthotopic pancreatic adenocarcinoma model. Results: In vitro, the V7-750 probe demonstrated significantly higher fluorescence at pH 6.6 compared with pH 7.4 (S2VP10, P = 0.0119; S2013, P = 0.0160), whereas no difference was observed with the K7-750 control (S2VP10, P = 0.8783; S2013, P = 0.921). In the in vivo S2VP10 model, V7-750 probe resulted in 782.5 MSOT a.u. signal compared with 5.3 MSOT a.u. in K7-750 control in tumor (P = 0.0001). Similarly, V7-750 probe signal was 578.3 MSOT a.u. in the S2013 model compared with K7-750 signal at 5.1 MSOT a.u. (P = 0.0005). There was minimal off-target accumulation of the V7-750 probe within the liver or kidney, and probe distribution was confirmed with ex vivo imaging. Conclusions: Compared with pH-insensitive controls, V7-750 pH-sensitive probe specifically targets pancreatic adenocarcinoma and has minimal off-target accumulation. The noninvasive detection of pH-targeted probes by means of MSOT represents a promising modality to improve the detection and monitoring of pancreatic cancer. Clin Cancer Res; 21(20); 4576–85. ©2015 AACR. See related commentary by Reshetnyak, p. 4502

Noninvasive monitoring of mRFP1- and mCherry-labeled oncolytic adenoviruses in an orthotopic breast cancer model by spectral imaging.

Genetic capsid labeling of conditionally replicative adenoviruses (CRAds) with fluorescent tags offers a potentially more accurate monitoring of those virotherapy agents in vivo. The capsid of an infectivity-enhanced CRAd, Ad5/3, delta 24, was genetically labeled with monomeric red fluorescent protein 1 (mRFP1) or its advanced derivative, “mCherry,” to evaluate the utility of each red fluorescent reporter and the benefit of CRAd capsid labeling for noninvasive virus tracking in animal tumor models by a new spectral imaging approach. Either reporter was incorporated into the CRAd particles by genetic fusion to the viral capsid protein IX. Following intratumoral injection, localization and replication of each virus in orthotopic breast cancer xenografts were analyzed by spectral imaging and verified by quantitative polymerase chain reaction. Fluorescence in tumors increased up to 2,000-fold by day 4 and persisted for 5 to 7 weeks, showing oscillatory dynamics reflective of CRAd replication cycles. Capsid labeling in conjunction with spectral imaging thus enables direct visualization and quantification of CRAd particles in tumors prior to the reporter transgene expression. This allows for noninvasive control of CRAd delivery and distribution in tumors and facilitates quantitative assessment of viral replication. Although mCherry appeared to be superior to mRFP1 as an imaging tag, both reporters showed utility for CRAd imaging applications.