Developing multi-modal imaging agents for stem cell tracking

Clinical trials using stem cells as a regenerative therapy or a delivery vehicle for anti-cancer agents have been increasing but the outcomes are highly variable. In vivo imaging of stem cell delivery to target organs will help improve their therapeutic efficacy. However, a single imaging modality cannot provide the complete answer. The work in this thesis aims to develop a multi-modal imaging approach to overcome the limitations of each modality. To understand the distribution pattern of transplanted stem cells in vivo, luciferase expressing adipocyte derived mesenchymal stem cells (ADSCs) were labelled with novel bimodal (nuclear/magnetic resonance imaging) nanoparticles and the following hypotheses were tested; 1) that the distribution pattern of transplanted ADSCs would be different between venous and arterial routes, 2) that the arterial route would provide a more efficient way of delivering ADSC to tumours. In addition, ultrasound-guided renal artery injection was developed to improve stem cell delivery to kidney and the efficiency of this injection was assessed using photoacoustic and bioluminescence imaging. Moreover, the applicability of gold nanoparticles (GNP) as cell tracking agents was explored using multi-modal imaging. Results demonstrated the advantages of multi-modal imaging in assessing different cell distribution patterns after two systemic injections and confirmed that the arterial route was more efficient in delivering ADSCs to tumours. The assessment of cell localisation and viability in the kidney suggests that the level of cell engraftment improved after ultrasound-guided renal artery injection. Multi-modal imaging results indicated that GNPs are a promising cell tracking agent for computed tomography but further studies are required to define their specific applications. In conclusion, this work has demonstrated the successful application of multi-modal imaging for stem cell tracking in different organs. The findings from this thesis proved that combining the strengths of each modality can provide greater insight into cell migration and distribution

Requirement for Interaction of PI3-Kinase p110α with RAS in Lung Tumor Maintenance

SummaryRAS proteins directly activate PI3-kinases. Mice bearing a germline mutation in the RAS binding domain of the p110α subunit of PI3-kinse are resistant to the development of RAS-driven tumors. However, it is unknown whether interaction of RAS with PI3-kinase is required in established tumors. The need for RAS interaction with p110α in the maintenance of mutant Kras-driven lung tumors was explored using an inducible mouse model. In established tumors, removal of the ability of p110α to interact with RAS causes long-term tumor stasis and partial regression. This is a tumor cell-autonomous effect, which is improved significantly by combination with MEK inhibition. Total removal of p110α expression or activity has comparable effects, albeit with greater toxicities

Development of lipopolyplexes for gene delivery: a comparison of the effects of differing modes of targeting peptide display on the structure and transfection activities of lipopolyplexes

The design, synthesis and formulation of non‐viral gene delivery vectors is an area of renewed research interest. Amongst the most efficient non‐viral gene delivery systems are lipopolyplexes, in which cationic peptides are co‐formulated with plasmid DNA and lipids. One advantage of lipopolyplex vectors is that they have the potential to be targeted to specific cell types by attaching peptide targeting ligands on the surface, thus increasing both the transfection efficiency and selectivity for disease targets such as cancer cells. In this paper, we have investigated two different modes of displaying cell‐specific peptide targeting ligands at the surface of lipopolyplexes. Lipopolyplexes formulated with bimodal peptides, with both receptor binding and DNA condensing sequences, were compared with lipopolyplexes with the peptide targeting ligand directly conjugated to one of the lipids. Three EGFR targeting peptide sequences were studied, together with a range of lipid formulations and maleimide lipid structures. The biophysical properties of the lipopolyplexes and their transfection efficiencies in a basal‐like breast cancer cell line were investigated using plasmid DNA bearing genes for the expression of firefly luciferase and green fluorescent protein. Fluorescence quenching experiments were also used to probe the macromolecular organisation of the peptide and pDNA components of the lipopolyplexes. We demonstrated that both approaches to lipopolyplex targeting give reasonable transfection efficiencies, and the transfection efficiency of each lipopolyplex formulation is highly dependent on the sequence of the targeting peptide. To achieve maximum therapeutic efficiency, different peptide targeting sequences and lipopolyplex architectures should be investigated for each target cell type

Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress

A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment

Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans.

Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.Postdoctoral fellowships were from EMBO (A LTF 165-2013) to S.D.C, EU Marie Curie (MEIF-CT-2005-010264) to E.T. and EU Marie Curie (PIIF-GA-2009-252846) to I.M.B. M.Z.-T. is supported by the EPSRC Early Career Fellowship of T.L.K. (EP/L006472/1). D.J.S. is a BHF Intermediate Basic Science Research Fellow (FS/15/33/31608). A.L.D is supported by the UK NIHR Joint UCL/University College London Hospitals Biomedical Research Centre. V.E.R.P. was supported by the Wellcome Trust (097721/Z/11/Z). R.K.S. is supported by the Wellcome Trust (WT098498), the Medical Research Council (M RC_MC_UU_12012/5). R.G.K. is supported by the NIHR Rare Diseases Translational Research Collaboration. V.W. is supported by the European FPVI Integrated Project ‘Eurostemcell’. M.F.L. and A.B. are supported by the King’s College London and UCL Comprehensive Cancer Imaging Centre CR-UK and EPSRC, in association with the MRC and DoH (England). W.A.P. is supported by funding from the National Health and Medical Research Council (NHMRC) of Australia. Work in the laboratory of M.G. is supported by research grants SAF2013-46542-P and SAF2014-59950-P from MICINN (Spain), 2014-SGR-725 from the Catalan Government, the People Programme (Marie Curie Actions) from the European Union's Seventh Framework Programme FP7/2007-2013/ (REA grant agreement 317250), the Institute of Health Carlos III (ISC III) and the European Regional Development Fund (ERDF) under the integrated Project of Excellence no. PIE13/00022 (ONCOPROFILE). Work in the laboratory of B.V. is supported by Cancer Research UK (C23338/A15965) and the UK NIHR University College London Hospitals Biomedical Research Centre.This is the author accepted manuscript. The final version is available from the American Association for the Advancement of Science via http://dx.doi.org/10.1126/scitranslmed.aad998

Image-guided Raman spectroscopy probe-tracking for tumor margin delineation

Significance: Tumor detection and margin delineation are essential for successful tumor resection. However, postsurgical positive margin rates remain high for many cancers. Raman spectroscopy has shown promise as a highly accurate clinical spectroscopic diagnostic modality, but its margin delineation capabilities are severely limited by the need for pointwise application. Aim: We aim to extend Raman spectroscopic diagnostics and develop a multimodal computer vision-based diagnostic system capable of both the detection and identification of suspicious lesions and the precise delineation of disease margins. Approach: We first apply visual tracking of a Raman spectroscopic probe to achieve real-time tumor margin delineation. We then combine this system with protoporphyrin IX fluorescence imaging to achieve fluorescence-guided Raman spectroscopic margin delineation. Results: Our system enables real-time Raman spectroscopic tumor margin delineation for both ex vivo human tumor biopsies and an in vivo tumor xenograft mouse model. We then further demonstrate that the addition of protoporphyrin IX fluorescence imaging enables fluorescence-guided Raman spectroscopic margin delineation in a tissue phantom model. Conclusions: Our image-guided Raman spectroscopic probe-tracking system enables tumor margin delineation and is compatible with both white light and fluorescence image guidance, demonstrating the potential for our system to be developed toward clinical tumor resection surgeries

LSC - 2017 - Targeting glucose metabolism in experimental lung injury and fibrosis

peer reviewedIntroduction:Akin to many cancers, enhanced18F-FDG-PET signal has been associated with fibrotic lesions in pulmonary fibrosis. In tumors, elevated glucose uptake is indicative of a metabolic switch to aerobic glycolysis (AG), critical to fuel biosynthetic demand. De novo expression of the low-activity isoform of the ‘glycolytic gatekeeper’ pyruvate kinase, PKM2, is a key driver of AG and pharmacological modulation of PKM2 is known to attenuate tumor proliferation and tumorigenesis.Aim:We hypothesised that the fibroproliferative response to bleomycin lung injury is characterised by metabolic reprogramming driving an enhanced glucose requirement of cells in the injured lesions. We investigated glucose uptake and modulation of the glycolytic marker PKM2 in the bleomycin mouse model.Methods&Results:Autoradiography of18F-FDG uptake in cryofrozen lung sections at day 28 post-bleomycin showed a significant ›50% increase in18F-FDG uptake in bleomycin-challenged compared to uninjured lungs. The regions of highest18F-FDG uptake corresponded to dense fibrotic regions. Immunohistochemistry revealed that PKM2 was localised to multiple cell types in fibrotic lung lesions, including a-SMA-positive myofibroblasts. A small molecule activator of PKM2, TEPP-46, achieved a significant ~50% increase in PKM2 activity in the bleomycin-injured lung. However, chronic TEPP-46 administration from day 5 post-injury had no significant effect on lung fibrosis quantified by microCt at day 28.Conclusions:We show that glucose uptake is significantly increased in fibrotic lung lesions. Although fibrotic lesions express PKM2, increasing the activity of this isoform was not sufficient to influence progression of fibrosis in bleomycin lung injur