Joseph Molter

Dual MET–EGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer

Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by T790M-EGFR mutation in half of the relapses. We show that MET is highly expressed in lung cancer, often concomitantly with epidermal growth factor receptor (EGFR), including H1975 cell line. The erlotinib-resistant lung cancer cell line H1975, which expresses L858R/T790M-EGFR in-cis, was used to test for the effect of MET inhibition using the small molecule inhibitor SU11274. H1975 cells express wild-type MET, without genomic amplification (CNV=1.1). At 2 μM, SU11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P=0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells. In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay. Using small-animal microPET/MRI, SU11274 treatment was found to induce an early tumour metabolic response in H1975 tumour xenografts. MET and EGFR pathways were found to exhibit collaborative signalling with receptor cross-activation, which had different patterns between wild type (A549) and L858R/T790M-EGFR (H1975). SU11274 plus erlotinib/CL-387,785 potentiated MET inhibition of downstream cell proliferative survival signalling. Knockdown studies in H1975 cells using siRNA against MET alone, EGFR alone, or both, confirmed the enhanced downstream inhibition with dual MET–EGFR signal path inhibition. Finally, in our time-lapse video-microscopy and in vivo multimodal molecular imaging studies, dual SU11274-erlotinib concurrent treatment effectively inhibited H1975 cells with enhanced abrogation of cytoskeletal functions and complete regression of the xenograft growth. Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer. Furthermore, optimised inhibition may be further achieved with MET inhibition in combination with erlotinib or an irreversible EGFR-TKI.

Long-term Transgene Expression in the Central Nervous System Using DNA Nanoparticles

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.

Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging

Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells.

Synergistic Simvastatin and Metformin Combination Chemotherapy for Osseous Metastatic Castration-Resistant Prostate Cancer

Docetaxel chemotherapy remains a standard of care for metastatic castration-resistant prostate cancer (CRPC). Docetaxel modestly increases survival, yet results in frequent occurrence of side effects and resistant disease. An alternate chemotherapy with greater efficacy and minimal side effects is needed. Acquisition of metabolic aberrations promoting increased survival and metastasis in CRPC cells includes constitutive activation of Akt, loss of adenosine monophosphate-activated protein kinase (AMPK) activity due to Ser-485/491 phosphorylation, and overexpression of 3-hydroxy-3-methylglutaryl–Coenzyme A reductase (HMG-CoAR). We report that combination of simvastatin and metformin, within pharmacologic dose range (500 nmol/L to 4 μmol/L simvastatin and 250 μmol/L to 2 mmol/L metformin), significantly and synergistically reduces C4-2B3/B4 CRPC cell viability and metastatic properties, with minimal adverse effects on normal prostate epithelial cells. Combination of simvastatin and metformin decreased Akt Ser-473 and Thr-308 phosphorylation and AMPKα Ser-485/491 phosphorylation; increased Thr-172 phosphorylation and AMPKα activity, as assessed by increased Ser-79 and Ser-872 phosphorylation of acetyl-CoA carboxylase and HMG-CoAR, respectively; decreased HMG-CoAR activity; and reduced total cellular cholesterol and its synthesis in both cell lines. Studies of C4-2B4 orthotopic NCr-nu/nu mice further demonstrated that combination of simvastatin and metformin (3.5–7.0 μg/g body weight simvastatin and 175–350 μg/g body weight metformin) daily by oral gavage over a 9-week period significantly inhibited primary ventral prostate tumor formation, cachexia, bone metastasis, and biochemical failure more effectively than 24 μg/g body weight docetaxel intraperitoneally injected every 3 weeks, 7.0 μg/g/day simvastatin, or 350 μg/g/day metformin treatment alone, with significantly less toxicity and mortality than docetaxel, establishing combination of simvastatin and metformin as a promising chemotherapeutic alternative for metastatic CRPC. Mol Cancer Ther; 13(10); 2288–302. ©2014 AACR.

Ex vivo diffusion tensor MRI reflects microscopic structural remodeling associated with aging and disease progression in normal and cardiomyopathic Syrian hamsters

Dilated cardiomyopathy (DCM) is a major cause of mortality and morbidity in cardiac patients. Aging is often an ignored etiology of pathological conditions. Quantification of DCM and aging associated cardiac structural remodeling is important in guiding and evaluating therapeutic interventions. Diffusion tensor magnetic resonance imaging (DTMRI) has recently been used for nondestructive characterization of three‐dimensional myofiber structure. In this study, we explored the potential of DTMRI in delineating microscopic structural remodeling in aging and DCM hearts. Six month (n = 10) and nine month old (n = 11) DCM (TO‐2) hamsters and their age‐matched controls (F1β) were characterized. Both aging and DCM hearts showed increased diffusivity and decreased diffusion anisotropy. DTMRI images of DCM hearts also revealed a subgroup of imaging pixels characterized by decreased radial diffusivity and increased FA. The location of these pixels showed qualitative agreement with regions of calcium deposition determined by X‐ray CT imaging. Histological analysis confirmed expanded extracellular space in aging and DCM hearts as well as substantial calcium deposition in DCM hearts. These results suggest that DTMRI may provide a noninvasive technique to delineate structural remodeling associated with aging and DCM progression at the tissue and cellular level without the use of an exogenous contrast agent. Copyright

In vivo dual substrate bioluminescent imaging.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

Synthesis, characterization, and X-ray attenuation properties of ultrasmall BiOI nanoparticles: toward renal clearable particulate CT contrast agents.

A unique decelerated hydrolytic procedure is developed and reported here for the preparation of ultrasmall nanoparticles (NPs) of PVP-coated BiOI with a narrow size distribution, i.e., 2.8 ± 0.5 nm. The crystal structure of this compound is determined by X-ray powder diffraction using the bulk materials. The stability, cytotoxicity, and potential use of the PVP-coated ultrasmall BiOI NPs as a CT contrast agent are investigated. Because of the combined X-ray attenuation effect of bismuth and iodine, such NPs exhibit a CT value that is among the best of those of the inorganic nanoparticle-based CT contrast agents reported in the literature.

Lack of dystrophin results in abnormal cerebral diffusion and perfusion in vivo

Dystrophin, the main component of the dystrophin–glycoprotein complex, plays an important role in maintaining the structural integrity of cells. It is also involved in the formation of the blood–brain barrier (BBB). To elucidate the impact of dystrophin disruption in vivo, we characterized changes in cerebral perfusion and diffusion in dystrophin-deficient mice (mdx) by magnetic resonance imaging (MRI). Arterial spin labeling (ASL) and diffusion-weighted MRI (DWI) studies were performed on 2-month-old and 10-month-old mdx mice and their age-matched wild-type controls (WT). The imaging results were correlated with Evans blue extravasation and vascular density studies. The results show that dystrophin disruption significantly decreased the mean cerebral diffusivity in both 2-month-old (7.38± 0.30 × 10−4mm2/s) and 10-month-old (6.93 ± 0.53 × 10−4 mm2/s) mdx mice as compared to WT (8.49±0.24×10−4, 8.24±0.25× 10−4mm2/s, respectively). There was also an 18% decrease in cerebral perfusion in 10-month-old mdx mice as compared to WT, which was associated with enhanced arteriogenesis. The reduction in water diffusivity in mdx mice is likely due to an increase in cerebral edema or the existence of large molecules in the extracellular space from a leaky BBB. The observation of decreased perfusion in the setting of enhanced arteriogenesis may be caused by an increase of intracranial pressure from cerebral edema. This study demonstrates the defects in water handling at the BBB and consequently, abnormal perfusion associated with the absence of dystrophin.

Imaging early stage osteogenic differentiation of mesenchymal stem cells

Stem cells, such as mesenchymal stem cells (MSCs), contribute to bone fracture repair if they are delivered to the injury site. However, it is difficult to assess the retention and differentiation of these cells after implantation. Current options for non‐invasively tracking the transplanted stem cells are limited. Cell‐based therapies using MSCs would benefit greatly through the use of an imaging methodology that allows cells to be tracked in vivo and in a timely fashion. In this study, we implemented an in vivo imaging methodology to specifically track early events such as differentiation of implanted human MSCs (hMSCs). This system uses the collagen type 1 (Col1α1) promoter to drive expression of firefly luciferase (luc) in addition to a constitutively active promoter to drive the expression of green fluorescent protein (GFP). The resulting dual‐promoter reporter gene system provides the opportunity for osteogenic differentiation‐specific luc expression for in vivo imaging and constitutive expression of GFP for cell sorting. The function of this dual‐promoter reporter gene was validated both in vitro and in vivo. In addition, the ability of this dual‐promoter reporter system to image an early event of osteogenic differentiation of hMSCs was demonstrated in a murine segmental bone defect model in which reporter‐labeled hMSCs were seeded into an alginate hydrogel scaffold and implanted directly into the defect. Bioluminescence imaging (BLI) was performed to visualize the turn‐on of Col1α1 upon osteogenic differentiation and followed by X‐ray imaging to assess the healing process for correlation with histological analyses.

Radio-deoxynucleoside Analogs used for Imaging tk Expression in a Transgenic Mouse Model of Induced Hepatocellular Carcinoma.

Purpose: A group of radiolabeled thymidine analogs were developed as radio-tracers for imaging herpes viral thymidine kinase (HSV1-tk) or its variants used as reporter gene. A transgenic mouse model was created to express tk upon liver injury or naturally occurring hepatocellular carcinoma (HCC). The purpose of this study was to use this unique animal model for initial testing with radio-labeled thymidine analogs, mainly a pair of newly emerging nucleoside analogs, D-FMAU and L-FMAU. Methods: A transgeneic mouse model was created by putting a fused reporter gene system, firefly luciferase (luc) and HSV1-tk, under the control of mouse alpha fetoprotein (Afp) promoter. Initial multimodal imaging, which was consisted of bioluminescent imaging (BLI) and planar gamma scintigraphy with [125I]-FIAU, was used for examining the model creation in the new born and liver injury in the adult mice. Carcinogen diethylnitrosamine (DEN) was then administrated to induce HCC in these knock-in mice such that microPET imaging could be used to track the activity of Afp promoter during tumor development and progression by imaging tk expression first with [18F]-FHBG. Dynamic PET scans with D-[18F]-FMAU and L-[18F]-FMAU were then performed to evaluate this pair of relatively new tracers. Cells were derived from these liver tumors for uptake assays using H-3 labeled version of PET tracers. Results: The mouse model with dual reporters: HSV1-tk and luc placed under the transcriptional control of an endogenous Afp promoter was used for imaging studies. The expression of the Afp gene was highly specific in proliferative hepatocytes, in regenerative liver, and in developing fetal liver, and thus provided an excellent indicator for liver injury and cancer development in adult mice. Both D-FMAU and L-FMAU showed stable liver tumor uptake where the tk gene was expressed under the Afp promoter. The performance of this pair of tracers was slightly different in terms of signal-to-background ratio as well as tracer clearance. Conclusion: The newly created knock-in mouse model was used to demonstrate the use of the dual-reporter genes driven by well-characterized cancer-specific transcriptional units in conjunction with in vivo imaging as a paradigm in studying naturally occurring cancer in live animals. While BLI is suitable for small animal imaging with luc expression, PET with L-FMAU seemed be the choice for liver injury or liver cancer imaging with this animal model for future investigations.