[Show abstract][Hide abstract] ABSTRACT: Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule-based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter-tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.
The Journal of clinical investigation 09/2015; 125(10). DOI:10.1172/JCI81086 · 13.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumoural infiltration of T lymphocytes is determined by local patterns of specific chemokine expression. In this report, we examined the roles of CCL4 and CCL20 in the accumulation of CD8(+) cytotoxic T lymphocytes (CTLs) and regulatory T (Treg) lymphocytes in oesophageal squamous cell carcinoma (ESCC), and determined the correlations between chemokine expressions and ESCC patients' survival.
Reverse transcriptase-PCR and immunohistochemistry (IHC) staining were performed to examine the expressions of interested genes. Flow cytometry was adopted to check the expressions of CCL4- and CCL6-specific receptors, CCR5 and CCR6, on CTLs and Treg cells. In addition, transwell assay was carried on.
The CCL4 expression was significantly correlated with the expression of CTL markers (CD8 and Granzyme B), whereas CCL20 was positively correlated with Treg markers (FoxP3 and IL-10). Consistently, CCR5 was found to be mainly expressed on CD8(+) T lymphocytes, while CCR6 showed prevalence on Treg lymphocytes and the frequencies of CCR5(+)CD8(+) CTLs and CCR6(+) Treg cells were higher in TIL compared with PBMC. Respectively, CCL4 and CCL20 recruited CD8(+) and regulatory T cells in vitro. Importantly, high levels of CCL4 in the lesions of ESCC patients predicted prolonged survival. Furthermore, CCL4(high)/CCL20(low) group demonstrated better overall survival, whereas CCL4(low)/CCL20(low) and CCL4(low)/CCL20(high) groups showed the worst overall survival.
Our data showed that CCL4 and CCL20 recruit functionally different T lymphocyte subsets into oesophageal carcinoma, indicating CCL4 and CCL20 are potential predictors of ESCC patients' survival.British Journal of Cancer advance online publication, 18 August 2015; doi:10.1038/bjc.2015.290 www.bjcancer.com.
British Journal of Cancer 08/2015; 113(5). DOI:10.1038/bjc.2015.290 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic inflammation is considered as a critical cause of a host of disorders, such as cancer, rheumatoid arthritis, atherosclerosis, and neurodegenerative diseases, although the exact mechanism is yet to be explored. Imaging tools that can specifically target inflammation are therefore important to help reveal the role of inflammation in disease progression, and allows for developing new therapeutic strategies to ultimately improve patient care. The purpose of this study was to develop a new in vivo inflammation imaging approach by targeting the cannabinoid receptor type 2 (CB2R), an emerging inflammation biomarker, using a unique near infrared (NIR) fluorescent probe. Herein, we report the first in vivo CB2R-targeted NIR inflammation imaging study using a synthetic fluorescent probe developed in our laboratory, NIR760-mbc94. In vitro binding assay and fluorescence microscopy study indicate NIR760-mbc94 specifically binds towards CB2R in mouse RAW264.7 macrophage cells. Furthermore, in vivo imaging was performed using a Complete Freund's Adjuvant (CFA)-induced inflammation mouse model. NIR760-mbc94 successfully identified inflamed tissues and the probe uptake was blocked by a CB2R ligand, SR144528. Additionally, immunofluorescence staining in cryosectioned tissues validated the NIR760-mbc94 uptake in inflamed tissues. In conclusion, this study reports the first in vivo CB2R-targeted inflammation imaging using an NIR fluorescent probe. Specific targeting of NIR760-mbc94 has been demonstrated in macrophage cells, as well as a CFA-induced inflammation mouse model. The combined evidence indicates that NIR760-mbc94 is a promising inflammation imaging probe. Moreover, in vivo CB2R-targeted fluorescence imaging may have potential in the study of inflammation-related diseases.
American Journal of Nuclear Medicine and Molecular Imaging 06/2015; 5(3):246-258. · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumors are complex ecosystems composed of networks of interacting 'normal' and malignant cells. It is well recognized that cytokine-mediated cross-talk between normal stromal cells, including cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and cancer cells, influences all aspects of tumor biology. Here we demonstrate that the cross-talk between CAFs and cancer cells leads to enhanced growth of oncolytic virus (OV)-based therapeutics. Transforming growth factor-β (TGF-β) produced by tumor cells reprogrammed CAFs, dampened their steady-state level of antiviral transcripts and rendered them sensitive to virus infection. In turn, CAFs produced high levels of fibroblast growth factor 2 (FGF2), initiating a signaling cascade in cancer cells that reduced retinoic acid-inducible gene I (RIG-I) expression and impeded the ability of malignant cells to detect and respond to virus. In xenografts derived from individuals with pancreatic cancer, the expression of FGF2 correlated with the susceptibility of the cancer cells to OV infection, and local application of FGF2 to resistant tumor samples sensitized them to virotherapy both in vitro and in vivo. An OV engineered to express FGF2 was safe in tumor-bearing mice, showed improved therapeutic efficacy compared to parental virus and merits consideration for clinical testing.
Nature medicine 04/2015; 21(5). DOI:10.1038/nm.3848 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since cancer is a genetic disease, the possibility of correcting defective genes would appear to be a highly promising approach to its treatment. However, the use of gene therapy has been limited by the inability to deliver a therapeutic gene to every cell in the tumor and by the heterogeneity in the enabling mutations found both between and within tumors. However, recent advances in the design of both viral (including non-replicating and oncolytic, or selectively replicating viruses) and non-viral gene delivery vehicles have allowed for more efficient and selective delivery of therapeutic genes to tumor cells leading to greater and longer term gene expression. In addition, a more realistic assessment of how genetic material most likely produce the greatest anti-tumor effect when expressed from within only a portion of cells in the tumor has resulted in the evolution of vectors that have demonstrated anti-tumor effects in the clinical setting and will likely result in approved therapies that can truly benefit the patient population. In particular, the capacity to induce a bystander effect, in a way that killing cells within the tumor which might not have been transfected directly with the genetic material be affected is an important property of most successful cancer gene therapies. As such, genes whose products induce an anti-tumor immune response within and against the tumor have been especially successful, as well as genes that can sensitize tumors to chemotherapies (such as through pro-drug conversion) or that target angiogenesis within the tumor. These can be used alone, or in combination with other therapies and many have now demonstrated potential in the clinical setting. Here, we describe the advantages and limitations of the current leading viral and non-viral gene delivery systems; assess the potential and proven capabilities of different therapeutic genes expressed from within the tumor, with special focus on immune-modulating genes and vaccines; and provide an overview of the use of oncolytic viruses as both therapies and gene expression vehicles.
Cancer Immunology, Bench to Bedside Immunotherapy of Cancers edited by N Resaei, 01/2015: chapter 7: pages 131-150; Springer Berlin Heidelberg., ISBN: 978-3-662-44945-5
[Show abstract][Hide abstract] ABSTRACT: Background:
Cytokine-induced killer (CIK) cells are ex vivo-expanded immune cells that express NK-cell and T-cell markers and that are routinely used in the treatment of many cancers. One key advantage of CIK cells is their ability to efficiently traffic to many solid tumours. Although likely to be mediated by chemokine receptor (CKR) expression, a thorough examination of the mechanism of tumour targeting has not been previously explored.
Here, human CIK cell expansions were examined for the level, profile and kinetics of CKR expression.
It was found that CIK cells express a panel of CKRs, with considerable variation between donors. Importantly, CKR levels dropped considerably beyond 14 days in culture, being significantly reduced by day 28 (the time at which cytolytic activity peaked). As such, CIK preparations that are used clinically may not have optimal CKR expression. Several approaches were found to re-stimulate CKR cell-surface levels at these later time points. These approaches also enhanced cytolytic activity in vitro and were demonstrated to increase both in vivo tumour trafficking and anti-tumour activity in mouse models.
Simple modifications of the CIK expansion protocol could therefore be used to significantly enhance the anti-tumour effects of this therapy.
British Journal of Cancer 03/2014; 110(8). DOI:10.1038/bjc.2014.140 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: mt is a cross-disciplinary biomedical journal devoted to publishing the most exciting advances in pharmacology and therapeutics, as they pertain to advances in translational and clinical medicine. It is recognized as one of the most prestigious journals in the field. With an impact factor of 6.825*, mt ranks in the top 4.2% of scientific journals in the latest Science Citation Index. Published monthly online and in print.
[Show abstract][Hide abstract] ABSTRACT: Evaluation of: Kanerva A, Nokisalmi P, Diaconu I et al. Antiviral and anti-tumor T-cell immunity in patients treated with GM-CSF coding oncolytic adenovirus. Clin. Cancer Res. 19(10), 2734-2744 (2013). The field of oncolytic viral therapy has been reinvigorated recently with publication of clinical data from two leading vectors, one based on HSV and one on Vaccinia, both of which express GM-CSF. Part of the reason for the improved clinical results with these vectors appears to be the enhanced immunotherapeutic mechanism of tumor destruction mediated by GM-CSF expression itself. The article by Kanerva et al. extends this work to describe early clinical use of an oncolytic adenovirus expressing GM-CSF, although the data are too preliminary to describe significant therapeutic benefits of GM-CSF expression in this backbone. However, the description of enhanced antitumor immunity in those patients that developed greater antiviral immunity after treatment provides a potent demonstration of the immunotherapeutic potential of epitope spreading after treatment with oncolytic viral therapies.
[Show abstract][Hide abstract] ABSTRACT: Efforts to selectively target and disrupt established tumor vasculature have largely failed to date. We hypothesized that a vaccinia virus engineered to target cells with activation of the ras/MAPK signaling pathway (JX-594) could specifically infect and express transgenes (hGM-CSF, β-galactosidase) in tumor-associated vascular endothelial cells in humans. Efficient replication and transgene expression in normal human endothelial cells in vitro required either VEGF or FGF-2 stimulation. Intravenous infusion in mice resulted in virus replication in tumor-associated endothelial cells, disruption of tumor blood flow, and hypoxia within 48 hours; massive tumor necrosis ensued within 5 days. Normal vessels were not affected. In patients treated with intravenous JX-594 in a phase I clinical trial, we showed dose-dependent endothelial cell infection and transgene expression in tumor biopsies of diverse histologies. Finally, patients with advanced hepatocellular carcinoma, a hypervascular and VEGF-rich tumor type, were treated with JX-594 on phase II clinical trials. JX-594 treatment caused disruption of tumor perfusion as early as 5 days in both VEGF receptor inhibitor-naïve and -refractory patients. Toxicities to normal blood vessels or to wound healing were not evident clinically or on MRI scans. This platform technology opens up the possibility of multifunctional engineered vaccinia products that selectively target and infect tumor-associated endothelial cells, as well as cancer cells, resulting in transgene expression, vasculature disruption, and tumor destruction in humans systemically.
Cancer Research 02/2013; 73(4):1-75. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The combination of an oncolytic virus, that directly destroys tumor cells and mediates an acute immune response, with an immune cell therapy, capable of further enlisting and enhancing the host immune response, has the potential to create a potent therapeutic effect. We have previously developed several strategies for optimizing the delivery of oncolytic vaccinia virus vectors to their tumor targets, including the use of immune cell-based carrier vehicles and the incorporation of mutations that increase production of the enveloped form of vaccinia (extracellular enveloped viral (EEV)) that is better adapted to spread within a host. Here, we initially combine these approaches to create a novel therapeutic, consisting of an immune cell (cytokine-induced killer, CIK) preloaded with an oncolytic virus that is EEV enhanced. This resulted in direct interaction between the viral and immune cell components with each assisting the other in directing the therapy to the tumor and so enhancing the antitumor effects. This effect could be further improved through CCL5 expression from the virus. The resulting multicomponent therapy displays the ability for synergistic crosstalk between components, so significantly enhancing tumor trafficking and antitumor effects.Molecular Therapy (2012); doi:10.1038/mt.2012.257.
[Show abstract][Hide abstract] ABSTRACT: Promising phase II clinical results have been reported recently for several oncolytic viral therapeutics, including strains based on vaccinia virus. One reason for this has been an increased appreciation of the critical therapeutic importance of the immune response raised by these viruses. However, the most commonly used approaches to enhance these immunotherapeutic effects in oncolytic viruses, typically though expression of cytokine transgenes, often also result in a reduction in oncolytic activity and premature clearance of the virotherapy from the tumor. Approaches that enhance the immunotherapeutic effects while maintaining oncolytic activity would therefore be beneficial. Here, it is demonstrated that the expression of the chemokine CCL19 (ELC) from an oncolytic vaccinia virus (vvCCL19) results in increased antitumor effects in syngeneic mouse tumor models. This corresponded with increased t cell and dendritic cell infiltration into the tumor. However, vvCCL19 persisted in the tumor at equivalent levels to a control virus without CCL19, demonstrating that oncolytic activity was not curtailed. Instead, vvCCL19 was cleared rapidly and selectively from normal tissues and organs, indicating a potentially increased safety profile. The therapeutic activity of vvCCL19 could be further significantly increased through combination with adoptive transfer of therapeutic immune cells expressing CCR7, the receptor for CCL19. This approach therefore represents a means to increase the safety and therapeutic benefit of oncolytic viruses, used alone or in combination with immune cell therapies.
Neoplasia (New York, N.Y.) 12/2012; 14(12):1115-21. DOI:10.1593/neo.121272 · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biological cancer therapies, such as oncolytic, or replication-selective viruses have advantages over traditional therapeutics as they can employ multiple different mechanisms to target and destroy cancers (including direct cell lysis, immune activation and vascular collapse). This has led to their rapid recent clinical development. However this also makes their pre-clinical and clinical study complex, as many parameters may affect their therapeutic potential and so defining reason for treatment failure or approaches that might enhance their therapeutic activity can be complicated. The ability to non-invasively image viral gene expression in vivo both in pre-clinical models and during clinical testing will considerably enhance the speed of oncolytic virus development as well as increasing the level and type of useful data produced from these studies. Further, subsequent to future clinical approval, imaging of reporter gene expression might be used to evaluate the likelihood of response to oncolytic viral therapy prior to changes in tumor burden. Here different reporter genes used in conjunction with oncolytic viral therapy are described, along with the imaging modalities used to measure their expression, while their applications both in pre-clinical and clinical testing are discussed. Possible future applications for reporter gene expression from oncolytic viruses in the phenotyping of tumors and the personalizing of treatment regimens are also discussed.
[Show abstract][Hide abstract] ABSTRACT: Oncolytic vaccinia viruses have made some impressive advances over the last 5 years, with a range of -different backbones displaying significant antitumor responses in preclinical models, and some exciting clinical results being reported against liver cancers. Because the virus is capable of rapid spread within the tumor, has evolved to spread relatively undetected within the blood stream, does not integrate into the host cell chromosome, and can infect almost any cell type, it is well-suited to the requirements for a successful oncolytic. In addition, the extensive clinical use of this virus means that contraindications to its use are known, and approved and experimental antivirals are available. Furthermore, because the virus has a large array of virulence genes whose deletion may target different properties of the cancer cell, and a large cloning capacity allowing for insertion of multiple transgenes, the possibilities for further development of novel and next-generation oncolytic vectors are multitude.
[Show abstract][Hide abstract] ABSTRACT: Preclinical studies have demonstrated that, unlike oncolytic adenoviruses, oncolytic vaccinia viruses can reach implanted tumors upon systemic injection. However, the biodistribution of this oncolytic agent in in situ autochthonous tumor models remains poorly characterized. In the present study, we assessed this biodistribution in a model of mouse hepatocellular carcinoma (HCC) obtained after injection of the carcinogen diethylnitrosamine (DEN).
Twelve months after DEN administration, histology, quantitative reverse transcription-polymerase chain reaction, in situ hybridization and viral titration were used to characterize tumors, as well as to assess the viral load of the livers upon either intravenous or intraperitoineal injection.
The results obtained showed that the architecture of the liver was lost, with a noticeable absence of sinusoids, as well as the presence of steatosis and α-fetoprotein-positive HCC tumor nodules. Bioluminescence imaging and measures of the infective virus load demonstrated that intravenous injection of 10(8) plaque-forming units of the recombinant vaccinia virus led to a predominant transduction of the liver, whereas intraperitoneal injection resulted in a lower level of liver transduction accompanied by an increased infection of the lungs, spleen, kidneys and bowels. Immunohistochemical analysis of liver sections of animals injected intravenously with the virus revealed a preferential localization of vaccinia-specific immunoreactivity in the tumors.
The findings of the present study emphasize the importance of the route of administration of the vector and highlight the relevance of systemic injection of oncolytic vaccinia virus in the context of hepatocellular carcinoma.
The Journal of Gene Medicine 12/2011; 13(12):692-701. DOI:10.1002/jgm.1624 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oncolytic viruses (OVs) are designed to replicate in, and subsequently lyse cancer cells. Numerous oncolytic virus platforms are currently in development. Here we review preclinical and clinical experience with JX-594, the lead candidate from the targeted and armed oncolytic poxvirus class. JX-594 is derived from a vaccinia vaccine strain that has been engineered for 1) enhanced cancer targeting and 2) has been "armed" with the therapeutic transgene granulocytemacrophage colony stimulating factor (GM-CSF) to stimulate anti-tumoral immunity. Poxviruses have many ideal features for use as oncolytic agents. The development of oncolytic vaccinia viruses is supported by a large safety database accumulated in the smallpox eradication program. In addition, poxviruses have evolved unique capabilities for systemic spread through the blood that can be harnessed for the treatment of metastatic disease. JX-594 demonstrates a high degree of cancer selectivity and systemic efficacy by multiple mechanisms-of-action (MOAs) in preclinical testing. Data from Phase 1 and 2 clinical trials has confirmed that these features result in potent and systemic efficacy in patients with treatment refractory metastatic cancers.
Current pharmaceutical biotechnology 07/2011; 13(9):1768-72. DOI:10.2174/138920112800958922 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There has recently been resurgence in interest for the use of replication-selective (oncolytic) viruses for the treatment of cancers. This has been fueled by positive clinical data and the promise provided by next-generation vectors that are better targeted and display enhanced therapeutic potential. One factor that has led to more effective oncolytic vectors has been a greater appreciation of their immunotherapeutic potential. This is especially true for strains of vaccinia virus, where the capability for rapid and destructive spread through a target tissue makes this virus the ideal backbone for an oncolytic agent, while its known ability to produce a potent immune response makes it a powerful immunotherapeutic. Approaches to developing next-generation vectors that are capable of effectively harnessing both of these mechanisms of action are discussed here.
Immunologic Research 06/2011; 50(2-3):286-93. DOI:10.1007/s12026-011-8211-4 · 3.10 Impact Factor