Zhenghong Lee

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.

Automatic MR volume registration and its evaluation for the pelvis and prostate.

A three-dimensional (3D) mutual information registration method was created and used to register MRI volumes of the pelvis and prostate. It had special features to improve robustness. First, it used a multi-resolution approach and performed registration from low to high resolution. Second, it used two similarity measures, correlation coefficient at lower resolutions and mutual information at full resolution, because of their particular advantages. Third, we created a method to avoid local minima by restarting the registration with randomly perturbed parameters. The criterion for restarting was a correlation coefficient below an empirically determined threshold. Experiments determined the accuracy of registration under conditions found in potential applications in prostate cancer diagnosis, staging, treatment and interventional MRI (iMRI) guided therapies. Images were acquired in the diagnostic (supine) and treatment position (supine with legs raised). Images were also acquired as a function of bladder filling and the time interval between imaging sessions. Overall studies on three patients and three healthy volunteers, when both volumes in a pair were obtained in the diagnostic position under comparable conditions, bony landmarks and prostate 3D centroids were aligned within 1.6 +/- 0.2 mm and 1.4 +/- 0.2 mm, respectively, values only slightly larger than a voxel. Analysis suggests that actual errors are smaller because of the uncertainty in landmark localization and prostate segmentation. Between the diagnostic and treatment positions, bony landmarks continued to register well, but prostate centroids moved towards the posterior 2.8-3.4 mm. Manual cropping to remove voxels in the legs was necessary to register these images. In conclusion, automatic, rigid body registration is probably sufficiently accurate for many applications in prostate cancer. For potential iMRI-guided treatments, the small prostate displacement between the diagnostic and treatment positions can probably be avoided by acquiring volumes in similar positions and by reducing bladder and rectal volumes.

Imaging of Mesenchymal Stem Cell Transplant by Bioluminescence and PET

Dynamic measurements of infused stem cells generally require animal euthanasia for single-time-point determinations of engraftment. In this study, we used a triple-fusion reporter system for multimodal imaging to monitor human mesenchymal stem cell (hMSC) transplants. Methods: hMSCs were transduced with a triple-fusion reporter, fluc-mrfp-ttk (encoding firefly luciferase, monomeric red fluorescent protein, and truncated herpes simplex virus type 1 sr39 thymidine kinase) by use of a lentiviral vector. Transduced cells were assayed in vitro for the expression of each functional component of the triple-fusion reporter. Transduced and control hMSCs were compared for their potential to differentiate into bone, cartilage, and fat. hMSCs expressing the reporter were then loaded into porous, fibronectin-coated ceramic cubes and subcutaneously implanted into NOD-SCID mice along with cubes that were loaded with wild-type hMSCs and empty cubes. Mice were imaged repeatedly over 3 mo by bioluminescence imaging (BLI), and selected animals underwent CT and PET imaging. Results: Osteogenic, adipogenic, and chondrogenic potential assays revealed retained differentiation potentials between transduced and wild-type hMSCs. Signals from the cubes loaded with reporter-transduced hMSCs were visible by BLI over 3 mo. There was no signal from the empty or wild-type hMSC–loaded control cubes. PET data provided confirmation of the quantitative estimation of the number of cells at one spot (cube). Cubes were removed from some animals, and histologic evaluations showed bone formation in cubes loaded with either reporter-transduced or wild-type hMSCs, whereas empty controls were negative for bone formation. Conclusion: The triple-fusion reporter approach resulted in a reliable method of labeling stem cells for investigation in small-animal models by use of both BLI and small-animal PET imaging. It has the potential for translation into future human studies with clinical PET.

Emerging therapeutic approaches for multipotent mesenchymal stromal cells

Purpose of reviewMultipotent mesenchymal stromal cells (MSCs) are rare cells resident in bone marrow and other organs capable of differentiating into mesodermal lineage tissues. MSCs possess immunomodulatory properties and have extensive capacity for ex-vivo expansion. Early clinical studies demonstrated safety and feasibility of infusing autologous MSCs and suggested a role in enhancing engraftment after hematopoietic cell transplant (HCT). Subsequent pilot studies using allogeneic MSCs showed safety but presented contradictory results regarding efficacy in treating graft-versus-host disease (GVHD). Recent findingsLarger, phase II allogeneic MSC infusion studies, including cells obtained from haploidentical and third-party donors, showed efficacy in GVHD treatment; however, recent randomized, placebo-controlled studies failed to corroborate these results. New investigations include MSC infusions in umbilical cord blood transplantation, MSC therapy for tissue regeneration/repair, harvest and use of MSCs from adipose tissue and cell-tracking/imaging studies using radionuclides, gene and fluorescent dye-labeled MSCs. SummaryMSCs remain the subject of intense investigation in HCT because of their differentiation potential and immunomodulatory properties. Whereas infusions of autologous, allogeneic and third-party donor MSCs are well tolerated, further research is needed to clarify the optimal methods for harvesting and expansion, optimal timing of administration and efficacy in the setting of HCT.

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.

Imaging Stem Cell Implant for Cellular-Based Therapies

Stem cell–based cellular therapy represents a promising outlook for regenerative medicine. Imaging techniques provide a means for noninvasive, repeated, and quantitative tracking of stem cell implant or transplant. From initial deposition to the survival, migration and differentiation of the transplant/implanted stem cells, imaging allows monitoring of the infused cells in the same live object over time. The current review briefly summarizes and compares existing imaging methods for cell labeling and imaging in animal models. Several studies performed by our group using different imaging techniques are described, with further discussion on the issues with these current imaging approaches and potential directions for future development in stem cell imaging.

Three-dimensional segmentation and skeletonization to build an airway tree data structure for small animals

Quantitative analysis of intrathoracic airway tree geometry is important for objective evaluation of bronchial tree structure and function. Currently, there is more human data than small animal data on airway morphometry. In this study, we implemented a semi-automatic approach to quantitatively describe airway tree geometry by using high-resolution computed tomography (CT) images to build a tree data structure for small animals such as rats and mice. Silicon lung casts of the excised lungs from a canine and a mouse were used for micro-CT imaging of the airway trees. The programming language IDL was used to implement a 3D region-growing threshold algorithm for segmenting out the airway lung volume from the CT data. Subsequently, a fully-parallel 3D thinning algorithm was implemented in order to complete the skeletonization of the segmented airways. A tree data structure was then created and saved by parsing through the skeletonized volume using the Python programming language. Pertinent information such as the length of all airway segments was stored in the data structure. This approach was shown to be accurate and efficient for up to six generations for the canine lung cast and ten generations for the mouse lung cast.

KETONES SUPPRESS BRAIN GLUCOSE CONSUMPTION

The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as beta-hydroxybutyrate (beta HB) and acetoacetate (AcAc), as occurs with fasting, prolonged starvation or chronic feeding of a high fat/low carbohydrate diet (ketogenic diet). In this study, the local cerebral metabolic rate of glucose consumption (CMRglu; microM/min/100g) was calculated in the cortex and cerebellum of control and ketotic rats using Patlak analysis. Rats were imaged on a rodent PET scanner and MRI was performed on a 7-Tesla Bruker scanner for registration with the PET images. Plasma glucose and beta HB concentrations were measured and 90-minute dynamic PET scans were started simultaneously with bolus injection of 2-Deoxy-2[18F]Fluoro-D-Glucose (FDG). The blood radioactivity concentration was automatically sampled from the tail vein for 3 min following injection and manual periodic blood samples were taken. The calculated local CMRGlu decreased with increasing plasma BHB concentration in the cerebellum (CMRGlu = -4.07*[BHB] + 61.4, r2 = 0.3) and in the frontal cortex (CMRGlu = -3.93*[BHB] + 42.7, r2 = 0.5). These data indicate that, under conditions of ketosis, glucose consumption is decreased in the cortex and cerebellum by about 10% per each mM of plasma ketone bodies.

Ketosis Proportionately Spares Glucose Utilization in Brain

The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as β-hydroxybutyrate and acetoacetate, as occurs with fasting, starvation, or chronic feeding of a ketogenic diet. The relationship between changes in cerebral metabolic rates of glucose (CMRglc) and degree or duration of ketosis remains uncertain. To investigate if CMRglc decreases with chronic ketosis, 2-[18F]fluoro-2-deoxy-D-glucose in combination with positron emission tomography, was applied in anesthetized young adult rats fed 3 weeks of either standard or ketogenic diets. Cerebral metabolic rates of glucose (μmol/min per 100 g) was determined in the cerebral cortex and cerebellum using Gjedde-Patlak analysis. The average CMRglc significantly decreased in the cerebral cortex (23.0 ±4.9 versus 32.9 ±4.7) and cerebellum (29.3 ± 8.6 versus 41.2 ±6.4) with increased plasma ketone bodies in the ketotic rats compared with standard diet group. The reduction of CMRg|c in both brain regions correlates linearly by ∼9% for each 1 mmol/L increase of total plasma ketone bodies (0.3 to 6.3 mmol/L). Together with our meta-analysis, these data revealed that the degree and duration of ketosis has a major role in determining the corresponding change in CMRglc with ketosis.

Human Mesenchymal Stromal Cells Attenuate Graft‐Versus‐Host Disease and Maintain Graft‐Versus‐Leukemia Activity Following Experimental Allogeneic Bone Marrow Transplantation

We sought to define the effects and underlying mechanisms of human, marrow‐derived mesenchymal stromal cells (hMSCs) on graft‐versus‐host disease (GvHD) and graft‐versus‐leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T cells and treated with hMSCs had reduced systemic GvHD, donor T‐cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72 hours, and target tissues harvested from hMSC‐treated allogeneic BMT (alloBMT) mice had less GvHD than untreated controls. Cryoimaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T‐cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T‐cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T‐cell proliferation, reduced TNFα, IFNγ, and IL‐10 but increased PGE2 levels. Indomethacin and E‐prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC‐mediated in vitro T‐cell suppression, confirming the role for PGE2. Furthermore, cyclo‐oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T‐cell proliferation likely through PGE2 induction. Stem Cells 2015;33:601–614