Whole-body Bioluminescence Imaging Research Articles

Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition.

Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment.

Enhanced anti-tumor efficacy and safety profile of tumor microenvironment-responsive oncolytic adenovirus nanocomplex by systemic administration

Oncolytic adenovirus (Ad) holds great promise as a potential gene therapy for cancer. However, intravenously administered Ad may encounter difficulties due to unfavorable host responses, non-specific interactions, and the heterogeneity of the tumor cell population. As an approach to combine the advantages of oncolytic Ad and synthetic polymers and to address the associated difficulties, Ad was physically complexed with a pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine) (mPEG-b-pHis). The in vitro transduction efficiency at an acidic extracellular pH was remarkably enhanced in cancer cells when treated with the Ad expressing green fluorescent protein (GFP) coated with mPEG-b-pHis (c-dE1/GFP) as compared to that of naked Ad (n-dE1/GFP). Time-lapse total internal reflection fluorescence microscopic imaging revealed a significantly enhanced cellular uptake rate of c-dE1/GFP at acidic tumor pH when compared with that at neutral pH or naked cognate Ad (n-dE1/GFP). In addition, c-dE1/GFP remained relatively stable in human serum-containing media, and considerably reduced both the innate and adaptive immune response against Ad. Moreover, the therapeutic efficacy and survival benefit of mPEG-b-pHis-complexed oncolytic Ad (c-H5mT/Luc) by systemic treatment was significantly enhanced compared to that with naked oncolytic Ad (n-H5mT/Luc) in both coxsackie and adenovirus receptor-positive and -negative tumors. Whole-body bioluminescence imaging showed 7.3-fold higher luciferase expression at the tumor site and 23.0-fold less luciferase expression in liver tissue for c-H5mT/Luc relative to that for naked oncolytic Ad (n-H5mT/Luc). Considering the heterogeneity of tumor tissue, these results are important for guiding the development of more potent and specific treatment of devastating metastatic cancers using this viral system. Statement of significanceAlthough adenoviral systems have shown considerable promise and undergone extensive evaluation attempts to specifically target Ad vectors to cancer cells have met limited success. This shortcoming is due to the strong immune response stimulated by Ad and the hepatotoxicity of the viral particles. To overcome restricted vector issues, we generated Ad/mPEG-b-pHis for tumor microenvironment-targeting hybrid vector systems, an oncolytic Ad coated with a pH-responsive polymer, mPEG-b-pHis. The Ad/mPEG-b-pHis exhibited pH-dependent transduction efficiency and cancer-cell killing effects. Moreover, systemic administration of oncolytic Ad/mPEG-b-pHis led to marked suppression of tumor growth and tumor-specific viral replication. Ad successfully avoided the innate and adaptive immune responses and liver accumulation with the help of mPEG-b-pHis on its surface.

Using a magnetic field to redirect an oncolytic adenovirus complexed with iron oxide augments gene therapy efficacy

Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited by low coxsackievirus and adenovirus receptor (CAR) expression in tumors and non-specifically targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and directing their migration with an external magnetic field (MGF). We electrostatically complexed GFP-expressing, replication-incompetent Ad (dAd) with PEGylated and cross-linked iron oxide nanoparticles (PCION), generating dAd-PCION complexes. The dAd-PCION showed increased transduction efficiency, independent of CAR expression, in the absence or presence of an MGF. Cancer cell killing and intracellular oncolytic Ad (HmT)-PCION replication significantly increased with MGF exposure. Site-directed, magnetically-targeted delivery of the HmT-PCION elicited significantly greater therapeutic efficacy versus treatment with naked HmT or HmT-PCION without MGF in CAR-negative MCF7 tumors. Immunohistochemical tumor analysis showed increased oncolytic Ad replication in tumors following infection by HmT-PCION using an MGF. Whole-body bioluminescence imaging of tumor-bearing mice showed a 450-fold increased tumor-to-liver ratio for HmT-PCION with, versus without, MGF. These results demonstrate the feasibility and potential of external MGF-responsive PCION-coated oncolytic Ads as smart hybrid vectors for cancer gene therapy.

315. Optimization of the Capsid of Recombinant Adeno-Associated Virus 6 (AAV6) Vectors for Liver-, and Muscle-Directed Gene Therapy

Recombinant adeno-associated virus (AAV) vectors are currently in use in a number of Phase I/II clinical trials. However, large vector doses are needed to achieve therapeutic effects. The ubiquitinproteasome pathway plays a critical role in the intracellular trafficking of AAV vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. We have previously reported that substitution of critical amino acid residues dramatically increases the transduction efficiency of vectors based on AAV serotypes 2, 3, 5, and 8 by preventing capsid phosphorylation and subsequent proteasomal degradation.In the present studies, we document that the combination of mutations of specific tyrosine (Y), serine (S), threonine (T) and lysine (K) residues on the capsid of AAV6 vectors also significantly increases the transduction efficiency compared with the wild-type (WT) AAV6 vectors. AAV6 capsid contains 7 tyrosine, 17 serine, 19 threonine and 11 lysine residues, which are surface-exposed. Initially, 17 single-mutants were generated based on the AAV6 crystal structure and overlap of amino acid positions with previously studied serotypes: Y445F, Y705F, Y731F, S455V, S472V, S499V, S551V, S663V, S669V, T251V, T492V, T665V, T722V, K531E, K567E, K666E, K707E. Next, the best performing Y-, S-, T-, and K- mutations were combined onto a single AAV6 capsid to generate 9 double-mutants: Y705+731F, Y731F+T492V, T492V+S663V, T492V+S551V, T665V+S551V, S663V+S551V, T492V+K531E, S663V+K531E, T492V+K666E; 7 triple-mutants: Y445+705+731F, Y705+731F+S663V, Y705+731F+S551V, Y705+731F+T492V, Y705+731F+K531E, Y705+731F+K666E, T492V+S663V+K531E; and two quadruple-mutants: Y705+731F+T492V+S663V, and Y705+731F+T492V+K531E. We identified several AAV6-mutant vectors, such as S663+551V, Y705+731F+T492V and Y705+731F+K531E, which were particularly efficient. These vectors increased the transduction efficiency by approximately 5- to 8-fold in a murine hepatocyte cell line, H2.35, and approximately 3- to 5-fold in a murine myoblast cell line, C2C12, over the WT AAV6 vector in vitro. Similar results were obtained in murine hepatocytes and muscle in vivo after tail-vein or intra-muscular injections of these vectors, followed by whole-body bioluminescence imaging of C57BL/6 mice.These residues are localized towards the base of the protrusions surrounding the icosahedral 3-fold axes of the capsid (T492S, S551V and K531E), in the depression surrounding the 2-fold axes (Y705F and Y731Y), or the HI loop (Y663S). This study thus identifies these capsid surface regions as being critical for the trafficking and transduction properties of the AAV6 vectors.In summary, our studies have led to the generation of novel capsid-optimized AAV6 vectors for their potential use in liver-directed, and muscle-directed gene therapy in humans.

380. Use of Somatotransgenic Bioimaging as a Platform for Studying NFkB Pathway in a Tissue-Specific Manner in Two Mouse Models of Inflammation: Rheumatoid Arthritis and Contact Dermatitis

Somatotransgenic bioimaging is a technology for in vivo imaging using lentiviral mediated delivery of a luciferase construct into neonatal mice. This luciferase marker, under the control of transcription factor binding elements or surrogate promoters, can be activated during physiological and pathological processes and can be quantified as photonic readout by whole-body bioluminescence imaging technology. The technique permits lifelong expression with immune tolerance to the transgenic proteins.In this study, we show the utility of somatotransgenic bioimaging using NFkB signaling as a model of inflammation in mouse models of rheumatoid arthritis and contact dermatitis.An NFkB transcription factor response element was inserted upstream of a mouse minimal promoter and the genes encoding firefly luciferase and green fluorescent protein. The cassette was inserted into a lentivirus vector.VSV-G-pseudotyped lentivirus vector was injected subcutaneously into CD1 outbread neonatal (P0) mice either in the footpad or in the abdominal skin to generate paw or skin specific light emitting transgenic mice.Canonical NFkB signaling (through TLR4) was first validated using ultrapure LPS in both the paw and skin of somatic transgenic mice. Following intraperitoneal LPS injection we observed upregulation of luciferase activity in the paw or the skin which returned to background levels within a few days.As a model of inflammatory mediated rheumatoid arthritis, we injected zymosan, which activates NFkB via TLR2 into the mouse paw fat pad. Induction of contact dermatitis was achieved by applying topically oxazolone, which plays an indirect activator role in the NFkB pathway. Luciferase expression was then monitored by whole body bioluminescence.Stimulation with Zymosan of the paw biosensor mice induced a prolonged upregulation of luciferase expression, specifically into the mouse paw which peaked at day 3 post injection and gradually returned to background level after twenty days.Similarly, after topical application of oxazolone onto the skin biosensor mice, we observed a specific, localised and increased luciferase activity which peaked at day 2 and returned to baseline by day 5.We have therefore shown that targeted gene expression can be achieved in two mouse models of inflammation. This also allowed us to quantify NFkB-mediated response to pro-inflammatory stimuli in a disease- and tissue-specific manner. Applied to collagen-induced arthritis, this technique could improve quantitation of disease severity compared to subjective scoring.

Postchallenge administration of brincidofovir protects healthy and immune-deficient mice reconstituted with limited numbers of T cells from lethal challenge with IHD-J-Luc vaccinia virus.

Protection from lethality by postchallenge administration of brincidofovir (BCV, CMX001) was studied in normal and immune-deficient (nude, nu/nu) BALB/c mice infected with vaccinia virus (VACV). Whole-body bioluminescence imaging was used to record total fluxes in the nasal cavity, lungs, spleen, and liver and to enumerate pox lesions on tails of mice infected via the intranasal route with 10(5) PFU of recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve (AUCs) were calculated for individual mice to assess viral loads. A three-dose regimen of 20 mg/kg BCV administered every 48 h starting either on day 1 or day 2 postchallenge protected 100% of mice. Initiating BCV treatment earlier was more efficient in reducing viral loads and in providing protection from pox lesion development. All BCV-treated mice that survived challenge were also protected from rechallenge with IHD-J-Luc or WRvFire VACV without additional treatment. In immune-deficient mice, BCV protected animals from lethality and reduced viral loads while animals were on the drug. Viral recrudescence occurred within 4 to 9 days, and mice succumbed ∼10 to 20 days after treatment termination. Nude mice reconstituted with 10(5) T cells prior to challenge with 10(4) PFU of IHD-J-Luc and treated with BCV postchallenge survived the infection, cleared the virus from all organs, and survived rechallenge with 10(5) PFU of IHD-J-Luc VACV without additional BCV treatment. Together, these data suggest that BCV protects immunocompetent and partially T cell-reconstituted immune-deficient mice from lethality, reduces viral dissemination in organs, prevents pox lesion development, and permits generation of VACV-specific memory. Mass vaccination is the primary element of the public health response to a smallpox outbreak. In addition to vaccination, however, antiviral drugs are required for individuals with uncertain exposure status to smallpox or for whom vaccination is contraindicated. Whole-body bioluminescence imaging was used to study the effect of brincidofovir (BCV) in normal and immune-deficient (nu/nu) mice infected with vaccinia virus, a model of smallpox. Postchallenge administration of 20 mg/kg BCV rescued normal and immune-deficient mice partially reconstituted with T cells from lethality and significantly reduced viral loads in organs. All BCV-treated mice that survived infection were protected from rechallenge without additional treatment. In immune-deficient mice, BCV extended survival. The data show that BCV controls viral replication at the site of challenge and reduces viral dissemination to internal organs, thus providing a shield for the developing adaptive immunity that clears the host of virus and builds virus-specific immunological memory.

A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.

Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 103 and 5 × 104 cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging. FigOptical imaging using TurboLuc fusion reporter protein

Abstract 2699: Organ-specific signatures of melanoma metastasis.

Abstract We have previously reported on a reproducible and predictive animal model that can be used to elucidate the enigmatic biology of melanoma metastasis, a key prerequisite for therapeutic advances. Here, we have used this model to investigate the molecular drivers responsible for metastasis formation in different organs, with a special emphasis on brain-specific features. A human melanoma brain metastasis cell line (H1) was developed in our laboratory, transduced with the genes for green fluorescent protein and luciferase, and injected into the left cardiac ventricle of seven immunodeficient mice. Whole-body bioluminescence imaging (BLI) and brain magnetic resonance imaging was performed weekly to follow the development of metastases. The animals invariably developed both intracranial and extracranial tumors. At about seven weeks, the animals were euthanized and organ involvement was examined in detail by ex vivo BLI. The brain, femurs, adrenals, and ovaries from five random animals were enzymatically dissociated using organ-tailored protocols. Sample yield was validated by fluorescence microscopy prior to fluorescence-activated cell sorting of tumor cells. Three tumor cell samples from the brain, femurs, adrenals, and ovaries of different mice were then analyzed using next-generation RNA-sequencing (NGS) technology. We also performed baseline NGS on the H1 cell line before injection. Taken together, bioinformatics analysis revealed unique molecular signatures of organ-specific metastasis, validating some previously reported metastasis drivers and introducing other genomic features critical to the detrimental momentum of metastatic melanoma. Citation Format: Terje Sundstrøm, Lars Prestegarden, Clifford Tepper, Elizabeth Ingham, Lisa Even, Sarah Johnson, Kai Ove Skaftnesmo, Morten Lund-Johansen, Rolf Bjerkvig, Katherine Ferrara, Frits Thorsen. Organ-specific signatures of melanoma metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2699. doi:10.1158/1538-7445.AM2013-2699

Optimization of the capsid of recombinant adeno-associated virus 2 (AAV2) vectors: the final threshold?

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2–3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.

Systemic Delivery of Oncolytic Adenoviruses Targeting Transforming Growth Factor-β Inhibits Established Bone Metastasis in a Prostate Cancer Mouse Model

We have examined whether Ad.sTβRFc and TAd.sTβRFc, two oncolytic viruses expressing soluble transforming growth factor-β receptor II fused with human Fc (sTGFβRIIFc), can be developed to treat bone metastasis of prostate cancer. Incubation of PC-3 and DU-145 prostate tumor cells with Ad.sTβRFc and TAd.sTβRFc produced sTGFβRIIFc and viral replication; sTGFβRIIFc caused inhibition of TGF-β-mediated SMAD2 and SMAD3 phosphorylation. Ad(E1-).sTβRFc, an E1(-) adenovirus, produced sTGFβRIIFc but failed to replicate in tumor cells. To examine the antitumor response of adenoviral vectors, PC-3-luc cells were injected into the left heart ventricle of nude mice. On day 9, mice were subjected to whole-body bioluminescence imaging (BLI). Mice bearing hind-limb tumors were administered viral vectors via the tail vein on days 10, 13, and 17 (2.5×10(10) viral particles per injection per mouse, each injection in a 0.1-ml volume), and subjected to BLI and X-ray radiography weekly until day 53. Ad.sTβRFc, TAd.sTβRFc, and Ad(E1-).sTβRFc caused significant inhibition of tumor growth; however, Ad.sTβRFc was the most effective among all the vectors. Only Ad.sTβRFc and TAd.sTβRFc inhibited tumor-induced hypercalcemia. Histomorphometric and synchrotron micro-computed tomographic analysis of isolated bones indicated that Ad.sTβRFc induced significant reduction in tumor burden, osteoclast number, and trabecular and cortical bone destruction. These studies suggest that Ad.sTβRFc and TAd.sTβRFc can be developed as potential new therapies for prostate cancer bone metastasis.

Bioluminescence Imaging Correlates with Tumor Serum Marker, Organ Weights, Histology, and Human DNA Levels during Treatment of Orthotopic Tumor Xenografts with Antibodies

In this study, we correlate results of bioluminescence measurements with established readouts for assessing therapeutic efficacy of antibodies in orthotopic cancer xenografts. An orthotopic tumor model of pancreatic cancer (AsPC-1-luc) and experimental lung metastasis (A549-luc) were established. Whole-body bioluminescence imaging (BLI) was performed to observe tumor progression under therapy with antibodies targeting different receptor kinases (primary readout). For purpose of verification, anti-tumoral efficacy was cross-validated with results obtained by measurement of organ weights, histology, tumor serum marker analysis (CYFRA 21-1), and quantification of human DNA concentration in the organ of interest (secondary readouts). Anti-tumoral efficacy is demonstrated for the antibodies tested. In the pancreas xenograft, a tumor growth inhibition of 95% (p < 0.01) was achieved as compared to control. Therapeutic efficacy could be identified as soon as 1 week after initiation of treatment. In the model of experimental lung metastasis, antibody treatment significantly suppressed tumor growth up to 75% (p < 0.05). All imaging results were confirmed by correlation analysis showing excellent agreement with the secondary readouts. BLI was demonstrated to be a reliable tool for monitoring early drug responses in orthotopic small animal cancer models. BLI allows rapid and non-invasive assessment of tumor load in the animal over time and, thus, provides a suitable method for routine use in preclinical cancer research.

Secreted Luciferase for In Vivo Evaluation of Systemic Protein Delivery in Mice

A naturally secreted Gaussia luciferase (Gluc) has been utilized as a reporter for bioluminescence imaging (BLI) evaluation. However, the potential application of Gluc for in vivo monitoring of systemic protein delivery, as well as its natural biodistribution, has not been studied. To examine Gluc secretion and uptake profile, we injected Gluc-encoding plasmids into mice by hydrodynamic tail-vein injection. Whole-body BLI showed that imaging quantification obtained at pawpad was directly correlated to blood Gluc activities. When gene expression was restricted to the liver by the use of a hepatic promoter, in vivo Gluc biodistribution analysis revealed the kidney/bladder, stomach/intestine, and lung as the major uptake organs. Three-dimensional BLI identified liver/stomach and lung as the main internal luminescent sources, demonstrating the feasibility of detecting major uptake organs in live animals by 3D BLI with high-background signals in circulation. Notably, Gluc levels in capillary-depleted brain samples from Gluc-injected mice were comparable to controls, suggesting that Gluc may not cross the blood-brain barrier. Gluc uptake kinetics and intracellular half-life were assessed in various types of cell lines, implicating the involvement of non-specific pinocytosis. These results suggest that Gluc-based system may provide a useful tool for in vivo evaluation of protein/agent biodistribution following systemic delivery.

A New Method to Determine Antigen-Specific CD8+ T Cell Activity in Vivo by Hydrodynamic Injection.

Hydrodynamic tail vein (HTV) delivery is a simple and rapid tail vein injection method of a high volume of naked plasmid DNA resulting in high levels of foreign gene expression in organs, especially the liver. Compared to other organs, HTV delivery results in more than a 1000-fold higher transgene expression in liver. After being bitten by malaria-infected mosquitoes, malaria parasites transiently infect the host liver and form the liver stages. The liver stages are known to be the key target for CD8+ T cells that mediate protective anti-malaria immunity in an animal model. Therefore, in this study, we utilized the HTV delivery technique as a tool to determine the in vivo cytotoxic effect of malaria antigen-specific CD8+ T cells. Two weeks after mice were immunized with recombinant adenoviruses expressing malarial antigens, the immunized mice as well as naïve mice were challenged by HTV delivery of naked plasmid DNA co-encoding respective antigen together with luciferase using dual promoters. Three days after the HTV challenge, non-invasive whole-body bioluminescent imaging was performed. The images demonstrate in vivo activity of CD8+ T cells against malaria antigen-expressing cells in liver.

Marrow Stromal Cells (MSC) Rescue Hematopoiesis in Lethally Irradiated Mice Despite Rapid Clearance After Infusion,

Abstract 3406Ionizing radiation causes dose dependent damages in many organs with most pronounced effects on those with high proliferative potential such as the hematopoietic system and gastrointestinal tract. Lympho-hematopoietic failure is often the cause of death following moderate to severe exposures to radiation. Effects of low to moderate (sub-lethal) doses of radiation can be mitigated by cytokines such as granuclocyte colony stimulating factor (GCSF) or Flt-3 ligand; higher doses of radiation require a new hematopoietic system by stem cell transplantation (SCT). As SCT for large numbers of radiation victims is not only costly but impractical following mass casualties (given the need for tissue matching prior to transplantation), there is considerable interest in cellular therapies that can be rapidly expanded, have a long shelf life and do not require tissue matching. Mesenchymal stromal cells (MSC, also referred to as mesenchymal stem cells) have been proposed as one such cellular therapy that could improve survival after moderate to high doses of radiation, but direct evidence for such a clinical benefit is scant in pre-clinical models. To determine if infusion of MSCs following ionizing radiation may rescue hematopoiesis after lethal irradiation in the murine model, we first determined the LD50 (lethal dose 50 or the dose at which 50% of animals survive without specific intervention) in C57/BL6 mice to be between 600 and 700 cGy when a X-Ray irradiator (RS2000) was used as the source of ionizing radiation. We then radiated adult female C57/Bl6 mice (age 6 to 8 weeks) with 700 cGy followed by infusion of either pooled immortalized MSC clones (henceforth referred to as cMSC, 1×106 cells each, n=19) or primary MSC (referred to as pMSC, 1×106 cells each, n=20) by tail vein injection 2 hours later. MSC were syngeneic to the recipients. Control mice received equal volume of PBS (n=21). Survival at the end of 7 weeks after radiation was determined using log-rank test which showed that animals that received either cMSC or pMSCs had significantly improved survival rates (p values of 0.017 and 0.041 respectively) when compared to the control animals that received only PBS (Figure 1). [Display omitted] The precise mechanism of action of MSCs after systemic infusion in various tissue injury models is currently undefined. Although trans-differentiation to host tissues has been proposed as one potential mechanism, recent reports have suggested that there is little evidence of persistence of infused MSC after the initial few days in the target tissues making trans-differentiation unlikely. Hence we determined the in vivo distribution kinetics of infused MSC by three techniques: whole-body bioluminescent imaging. (BLI), immune histochemistry (IHC) and quantitative real time polymerase chain reaction (q RT PCR). MSC were labeled with firefly luciferase (ffLuc) using a lentiviral vector and infused to adult female recipients after they were administered 700 cGy radiation (1×106 cells each, n=7). In all mice, strong bioluminescent signals were detected from the chest region at 4 hours after infusion, suggesting an accumulation of infused MSCs in the lungs. The signals rapidly decreased during the first 24 hours, and no bioluminescent signal was detected at 72 hours after infusion. No signals were detectable from other organs (liver, spleen or long bones) at any time point. Ffluc could not be reliably detected in tissues by IHC. Detection of luciferase transcripts in different tissues (lungs, hearts, spleens, livers, guts, muscles, and bone marrow) was performed by quantitative RT PCR at days 1, 4 and 7 (n = 6 each) following infusion. Ffluc transcripts were reliably detected in all animals only in the lungs at 24 hours after MSC infusion, confirming the BLI results. At subsequent time points, ffluc transcripts were detectable at very low levels in a variable proportion of animals from various tissues. In the absence of reliable BLI signals from these extra-pulmonary tissues, we interpret the presence of low levels of ffluc transcripts in these tissues as to have arisen from unviable cells or circulating RNA. Together, these results show that both immortalized MSCs and primary MSCs improve hematopoietic recovery after lethal ionizing radiation, but the infused cells are mostly filtered out by the lungs and their beneficial effect is likely mediated by indirect mechanisms (secondary effector cells in the lungs or secreted cytokines). Disclosures:No relevant conflicts of interest to declare.

Improved Efficacy Upon Combined JAK1/2 and Pan-Deacetylase Inhibition Using Ruxolitinib (INC424) and Panobinostat (LBH589) in Preclinical Mouse Models of JAK2V617F-Driven Disease

Abstract 798▪FN2▪This icon denotes a clinically relevant abstract Background:Ruxolitinib (INC424), a potent and selective oral JAK1 and JAK2 inhibitor, has demonstrated rapid and durable reductions in splenomegaly and improved disease-related symptoms, role functioning, and quality of life in 2 phase 3 studies in patients with myelofibrosis. Panobinostat, a potent oral pan-deacetylase inhibitor (pan-DACi) that increases acetylation of proteins involved in multiple oncogenic pathways, has shown encouraging clinical activity in phase 1/2 trials of myelofibrosis. Here, we investigate the combination of ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. Methods:Scid-beige female mice were randomized to treatment groups on the basis of baseline bioluminescence 4 days after injection of Ba/F3 EpoR JAK2V617F-luciferase cells. Mice were treated with vehicle control, panobinostat (4, 8, or 12 mg/kg intraperitoneal 3 times a week) alone or in combination with ruxolitinib (60 mg/kg orally twice daily), or ruxolitinib alone (n=7 each). Whole-body bioluminescence imaging was conducted on days 7 and 11. Spleens were weighed and extracts were obtained to determine levels of phosphorylated STAT5 (p-STAT5) and lysine acetylation. Results:Imaging on day 11 showed luminescence reductions (P <.05 compared with vehicle control) in all treatment groups (Table)Ruxolitinib, mg/kg060000606060Panobino- stat, mg/kg0048124812n=7n=7n=7n=7n=7n=7n=7n=7Change in biolu- mines- cence (T/C),a %10040*27*20*11*,†22*15*,†3*,†‡Spleen weight, mg183.7 ± 18.176.4 ± 17.0*105.3 ± 19.6107.4 ± 20.594.1 ± 17.6*59.9 ± 10.0*45.7 ± 5.6*,‡40.9 ± 6.1*,‡aCalculated as treated/control (T/C) of fractional change in bioluminescence on day 11/day 4*P <.05 vs vehicle control;†P <.05 vs ruxolitinib;‡P <.05 vs panobinostat at the same dose by Tukey’s test on log10 transformed values for fractional change Biolux and spleen weight.A dose-dependent reduction was observed in mice treated with panobinostat alone. Combination therapy produced the greatest reductions in luminescence, as low as 3% of the untreated control in the ruxolitinib + panobinostat (12 mg/kg) group (P <.05 compared with the same dose of panobinostat alone or ruxolitinib alone). Similarly, reductions in spleen weight (P <.05 compared with control) were observed with ruxolitinib alone and panobinostat (12 mg/kg) alone and in all combination groups. Treatment with ruxolitinib reduced phosphorylation of the JAK2 downstream target STAT5, whereas panobinostat treatment reduced total STAT5 levels. Mice treated with both panobinostat and ruxolitinib had reduced levels of p-STAT5 and total STAT5 in spleen extracts. Lysine acetylation, unchanged in ruxolitinib-treated mice, was markedly increased in panobinostat-treated mice. Together, the pharmacodynamic analyses indicate that ruxolitinib and panobinostat are active and have nonoverlapping and complementary effects on biological pathways. There was no major change in tolerability, as assessed by body weight, between panobinostat alone and in combination with ruxolitinib. The experiment was repeated with the 2 top doses of panobinostat and ruxolitinib and showed similar results. Based on these encouraging data, the combination is currently being tested in a mouse JAK2V617F bone marrow transplant model of myeloproliferative neoplasm (MPN) disease to assess its disease-modifying potential. Conclusions:The combination of ruxolitinib and panobinostat showed significant improvements in anticancer activity compared with either treatment given alone in this JAK2V617F mouse model. Further studies in a murine MPN disease model are underway. Disclosures:Baffert:Novartis Pharma AG: Employment. Evrot:Novartis Pharma AG: Employment. Ebel:Novartis Pharma AG: Employment. Roelli:Novartis Pharma AG: Employment. Andraos:Novartis Pharma AG: Employment. Qian:Novartis Pharma AG: Employment. Romanet:Novartis Pharma AG: Employment. Murakami:Novartis Pharma AG: Employment. Radimerski:Novartis Pharma AG: Employment.

Vascular endothelial growth factor broadens lentivector distribution in the heart after neonatal injection

For some applications, the success of gene therapy depends on the efficiency of gene transfer into target organs, however, delivery to many tissues is limited. Efforts have been made to improve the efficiency of gene transfer into target organs such as the brain by using mannitol or vascular endothelial growth factor (VEGF) prior to gene delivery, since these treatments have been reported to increase vascular permeability in experimental animals. Here, we investigated the effect of VEGF pretreatment of neonatal mice on the ability of injected lentivirus (LV)--engineering expression of firefly luciferase (luc)--to enhance the transduction of various organs, including the brain and heart. LV/luc was delivered to VEGF-treated neonatal mice via the temporal vein. Whole-body bioluminescence imaging (WBLI) of luciferase expression showed that VEGF pretreatment does not diminish transgene expression over time since it remained steady for up to 12 weeks. Ex vivo imaging of the organs and assessments of organ luciferase activity showed that VEGF pretreatment resulted in significantly increased luciferase expression not only in the heart, but also in the brain, lung, and kidney. This study shows that VEGF may have therapeutic importance to enhance the efficiency of viral gene delivery to the heart, as well as to other target organs.

Tracking cardiac engraftment and distribution of implanted bone marrow cells: Comparing intra-aortic, intravenous, and intramyocardial delivery

Cell therapy improved cardiac function after a myocardial infarction in several preclinical studies; however, the functional benefits were limited in the initial clinical trials, perhaps because of inadequate cell engraftment. We used noninvasive molecular imaging to compare the distribution and myocardial retention of cells implanted by using clinical delivery routes. Bone marrow stromal cells isolated from male rats and transfected with a firefly luciferase reporter gene were injected by using 3 increasingly invasive techniques (ie, intravenous, intra-aortic, and intramyocardial) into female rats 3 or 28 days after coronary ligation. Whole-body bioluminescence imaging was performed 2, 24, and 48 hours later; implanted cells were quantified at 48 hours in explanted organs by means of bioluminescence and real-time polymerase chain reaction. Variations in cell distribution among groups were profound, with nearly complete trapping of the injected cells in the lungs after intravenous delivery. Cell delivery into the aortic root (with the distal aorta occluded) produced minimal cell retention in the heart. Direct intramyocardial injection facilitated the best early targeting of the cells (P < .05 vs intravenous and intra-aortic injection). Rapid signal loss over 48 hours indicated very poor cell survival in all 3 groups, although implanted cell retention was greater in mature compared with acute infarcts. This is the first study to correlate live cell imaging with quantitative genetic and histologic techniques. Noninvasive molecular imaging tracked delivered cells and will permit the evaluation of new and improved delivery platforms designed to increase cell homing, retention, and engraftment.

Antibody-based immunotherapy: targeting CSPG4 on human basal breast cancer stem cells.

Abstract Abstract #5046 Introduction: Basal-like breast cancer is associated with high grade, poor prognosis, and younger patient age. Because tumors are estrogen receptor (ER) and HER2 negative, effective targeted treatment for basal breast cancer remains elusive. The lack of efficacy of currently used chemotherapies may reflect their inability to eradicate cancer stem cells (CSC) within a basal breast tumor.&amp;#x2028; Chondroitin sulfate proteoglycan 4 (CSPG4), a molecule initially identified in melanoma cells, is a cell surface antigen involved in migration and invasion of tumor cells. The rat homolog of the human CSPG4, named NG2, is expressed by progenitor cells in several types of tissues. Furthermore, NG2 expression is typically maximal when progenitor cells are mitotic and motile. As one important characteristic of cancer stem cells is their ability to migrate and metastasize, and both CSPG4 and NG2 play a role in tumor cell migration, we tested whether CSPG4 is expressed on cells with the CSC phenotype in ER negative/HER2 negative (basal-like) breast cancer, and can thus serve as a target for immunotherapy of breast CSC.&amp;#x2028; Methods and Results: The mRNA levels of CSPG4 in ER- and Her2- breast cancer were compared to ER-/Her2+, ER+/Her2-, and ER+/Her2+ breast cancer using a publicly available, clinically annotated breast cancer data set. The gene expression level of CSPG4 was elevated in the ER- and Her2- subgroup when compared to ER-/Her2+ (Mann Whitney, p=0.03), ER+/Her2- (Mann Whitney, p=0.01), and ER+/Her2+ (Mann Whitney, p=0.06). These results suggest that CSPG4 expression is best correlated with the basal phenotype of breast cancer. Flow cytometric analysis showed that CSPG4 is expressed on CD44+, CD24-/lo cells in the human basal breast cancer cell lines, i.e., MDA-MD-231, MDA-MB-435, HS578T and SUM149, but is not detectable on the luminal breast cancer cell lines, i.e., MCF-7, SK-BR-3 and T-47D. Furthermore CSPG4 is expressed on the cell surface of putative breast CSC identified as lineage-negative (CD2,3,10,16,18,31,45,64,140b), CD44+, CD24-/lo cells in pleural effusions of 12 patients with breast cancer. Further, inoculation of flow cytometric sorted CD44+CD24-/low CSPG4+ cells from the MDA-MB-231-luciferease expressing cell line, to the mammary fat pads of immunodeficient mice, led to the development of tumors detected by whole-body bioluminescent reporter imaging 3 weeks after the injection. In contrast, inoculation of CD44+CD24-/low CSPG4- cells, sorted from the same cell line, did not generate tumors. More importantly, CSPG4-specific mAb significantly inhibited post-surgery tumor recurrence and lung metastases in SCID mice transplanted with the human basal breast cancer cell line MDA-MB-435, which has the putative breast CSC phenotype (CD44+, CD24-/lo) in more than 99% of cells.&amp;#x2028; Conclusion: These results suggest that the CSPG4 is a potential new immunotherapeutic target for basal-like breast cancer and breast CSC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5046.

Function of Heparanase in Prostate Tumorigenesis: Potential for Therapy

Heparanase is the predominant enzyme that cleaves heparan sulfate, the main polysaccharide in the extracellular matrix. Whereas the role of heparanase in sustaining the pathology of human cancer is well documented, its association with prostate carcinoma remains uncertain. Our research was undertaken to elucidate the significance of heparanase in prostate tumorigenesis and bone metastasis. We applied immunohistochemical analysis of tissue microarray, in vitro adhesion and invasion assays, as well as mouse models of intraosseous growth and spontaneous metastasis of prostate cancer, monitored by whole-body bioluminescent imaging. Electroporation-assisted administration of anti-heparanase small interfering RNA in vivo was applied as a therapeutic approach. We report a highly statistically significant (P < 0.0001) prevalence of heparanase overexpression in prostate carcinomas versus noncancerous tissue, as well as strong correlation between tumor grade and the extent of heparanase expression. We observed >5-fold increase in the metastatic potential of PC-3 prostate carcinoma cells engineered to overexpress heparanase. Notably, overexpression of a secreted form of the enzyme also led to a dramatic increase in intraosseous prostate tumor growth. Local in vivo silencing of heparanase resulted in a 4-fold inhibition of prostate tumor growth, representing the first successful application of anticancer therapy based on heparanase small interfering RNA and validating the potential of heparanase as a target for prostate cancer treatment. Heparanase directly contributes to prostate tumor growth in bone and its ability to metastasize to distant organs. Thus, anti-heparanase strategy may become an important modality in the treatment of prostate cancer patients, particularly those with bone metastases.

Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis

The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.