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1.  Development of a Preclinical Orthotopic Xenograft Model of Ewing Sarcoma and Other Human Malignant Bone Disease Using Advanced In Vivo Imaging 
PLoS ONE  2014;9(1):e85128.
Ewing sarcoma and osteosarcoma represent the two most common primary bone tumours in childhood and adolescence, with bone metastases being the most adverse prognostic factor. In prostate cancer, osseous metastasis poses a major clinical challenge. We developed a preclinical orthotopic model of Ewing sarcoma, reflecting the biology of the tumour-bone interactions in human disease and allowing in vivo monitoring of disease progression, and compared this with models of osteosarcoma and prostate carcinoma. Human tumour cell lines were transplanted into non-obese diabetic/severe combined immunodeficient (NSG) and Rag2−/−/γc−/− mice by intrafemoral injection. For Ewing sarcoma, minimal cell numbers (1000–5000) injected in small volumes were able to induce orthotopic tumour growth. Tumour progression was studied using positron emission tomography, computed tomography, magnetic resonance imaging and bioluminescent imaging. Tumours and their interactions with bones were examined by histology. Each tumour induced bone destruction and outgrowth of extramedullary tumour masses, together with characteristic changes in bone that were well visualised by computed tomography, which correlated with post-mortem histology. Ewing sarcoma and, to a lesser extent, osteosarcoma cells induced prominent reactive new bone formation. Osteosarcoma cells produced osteoid and mineralised “malignant” bone within the tumour mass itself. Injection of prostate carcinoma cells led to osteoclast-driven osteolytic lesions. Bioluminescent imaging of Ewing sarcoma xenografts allowed easy and rapid monitoring of tumour growth and detection of tumour dissemination to lungs, liver and bone. Magnetic resonance imaging proved useful for monitoring soft tissue tumour growth and volume. Positron emission tomography proved to be of limited use in this model. Overall, we have developed an orthotopic in vivo model for Ewing sarcoma and other primary and secondary human bone malignancies, which resemble the human disease. We have shown the utility of small animal bioimaging for tracking disease progression, making this model a useful assay for preclinical drug testing.
PMCID: PMC3883696  PMID: 24409320
2.  Determination of key structure–activity relationships in siRNA delivery with a mixed micelle system☆☆☆ 
Journal of Controlled Release  2013;172(3):939-945.
Short interfering ribonucleic acids (siRNAs) offer a highly specific and selective form of therapy for diseases with a genetic component; however the poor pharmacokinetic properties of the molecule have impeded its development into a therapeutic for use in vivo. Several different approaches have been taken to develop a successful siRNA delivery system but these systems lack the flexibility for easy optimisation. Here, we propose a polymeric nanoparticle (PNP) system consisting of two amphiphilic diblock copolymers which allow for the rapid determination of structure–activity relationships involving gene knockdown and toxicity. The diblock copolymers self-assemble into monodisperse micelles of defined hydrodynamic diameters ranging from 30 to 100 nm dependent on the copolymer ratio. A luciferase-based high throughput assay varying PNP composition, concentration and siRNA concentration allowed the rapid identification of efficient PNP formulations for adherent and suspension cell lines. Optimised PNPs efficiently knocked down a fusion oncogene in hard to transfect human leukaemic cells raising the possibility of targeting malignant cells in a cancer-specific fashion. This approach allows the optimum PNP formulation to be identified for different cell types and conditions.
Graphical abstract
PMCID: PMC3898608  PMID: 24140749
AML, acute myeloid leukaemia; PNP, polymeric nanoparticle; P1, PCL-b-PDMAEMA; P2, PCL-b-PEG; siRNA, short interfering ribonucleic acid; NR, Nile Red; FACS, fluorescent activated cell sorting; LSCM, laser scanning confocal microscopy; siRNA; Polymer micelles; Leukaemia; Structure–activity relationships; Gene knockdown
3.  Acute B lymphoblastic leukaemia-propagating cells are present at high frequency in diverse lymphoblast populations 
EMBO Molecular Medicine  2012;5(1):38-51.
Leukaemia-propagating cells are more frequent in high-risk acute B lymphoblastic leukaemia than in many malignancies that follow a hierarchical cancer stem cell model. It is unclear whether this characteristic can be more universally applied to patients from non-‘high-risk’ sub-groups and across a broad range of cellular immunophenotypes. Here, we demonstrate in a wide range of primary patient samples and patient samples previously passaged through mice that leukaemia-propagating cells are found in all populations defined by high or low expression of the lymphoid differentiation markers CD10, CD20 or CD34. The frequency of leukaemia-propagating cells and their engraftment kinetics do not differ between these populations. Transcriptomic analysis of CD34high and CD34low blasts establishes their difference and their similarity to comparable normal progenitors at different stages of B-cell development. However, consistent with the functional similarity of these populations, expression signatures characteristic of leukaemia propagating cells in acute myeloid leukaemia fail to distinguish between the different populations. Together, these findings suggest that there is no stem cell hierarchy in acute B lymphoblastic leukaemia.
PMCID: PMC3569652  PMID: 23229821
acute lymphoblastic leukaemia; cancer stem cells; leukaemia maintenance; leukaemia propagating cells; leukaemia stem cells
5.  AML1/ETO proteins control POU4F1/BRN3A expression and function in t(8;21) acute myeloid leukaemia 
Cancer research  2010;70(10):3985-3995.
A variety of genetic lesions, including chromosomal translocations, internal tandem duplications and mutations have been described in acute myeloid leukaemia (AML). Expression profiling has shown that chromosomal translocations, in particular, are associated with distinctive patterns of gene expression. AML exhibiting the translocation t(8;21), which fuses the AML1 and ETO genes, has such a characteristic expression profile. One gene whose expression is highly correlated with the presence of the AML1/ETO fusion is POU4F1, which encodes the POU homeodomain transcription factor BRN3A. Here we demonstrate using specific siRNA in t(8;21) cells and overexpression studies in progenitor cells that AML1/ETO promotes expression of POU4F1/BRN3A. This effect requires DNA-binding function of AML1/ETO, and accordingly AML1/ETO is bound to the POU4F1 locus in t(8;21) cells. Functionally, while over-expression of Brn3a in murine haematopoietic progenitor cells induces terminal myeloid differentiation, co-expression of AML1/ETO or AML/ETO9a blocks this effect. Furthermore, Brn3a reduction by shRNA impairs AML1/ETO-induced immortalisation of murine progenitors. In summary, we identify POU4F1/BRN3A as a novel potential up-regulated AML1/ETO target gene whose dramatically high expression may co-operate with AML1/ETO in t(8;21) cells.
PMCID: PMC2883733  PMID: 20460523
AML1/ETO; BRN3A; AML; transcription; myeloid
6.  The oncogenic fusion protein RUNX1-CBFA2T1 supports proliferation and inhibits senescence in t(8;21)-positive leukaemic cells 
BMC Cancer  2004;4:44.
The fusion protein RUNX1-CBFA2T1 associated with t(8;21)-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t(8;21)-positive cell lines.
The t(8;21)-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence.
Electroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity.
RUNX1-CBFA2T1 supports the proliferation and expansion of t(8;21)-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches.
PMCID: PMC512292  PMID: 15298716
7.  Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells 
The Journal of Cell Biology  2002;156(4):737-750.
The activity of serum response factor (SRF), an essential transcription factor in mouse gastrulation, is regulated by changes in actin dynamics. Using Srf(−/−) embryonic stem (ES) cells, we demonstrate that SRF deficiency causes impairments in ES cell spreading, adhesion, and migration. These defects correlate with defective formation of cytoskeletal structures, namely actin stress fibers and focal adhesion (FA) plaques. The FA proteins FA kinase (FAK), β1-integrin, talin, zyxin, and vinculin were downregulated and/or mislocalized in ES cells lacking SRF, leading to inefficient activation of the FA signaling kinase FAK. Reduced overall actin expression levels in Srf(−/−) ES cells were accompanied by an offset treadmilling equilibrium, resulting in lowered F-actin levels. Expression of active RhoA-V14 rescued F-actin synthesis but not stress fiber formation. Introduction of constitutively active SRF-VP16 into Srf(−/−) ES cells, on the other hand, strongly induced expression of FA components and F-actin synthesis, leading to a dramatic reorganization of actin filaments into stress fibers and lamellipodia. Thus, using ES cell genetics, we demonstrate for the first time the importance of SRF for the formation of actin-directed cytoskeletal structures that determine cell spreading, adhesion, and migration. Our findings suggest an involvement of SRF in cell migratory processes in multicellular organisms.
PMCID: PMC2174087  PMID: 11839767
SRF; ES cells; actin cytoskeleton; focal adhesion; cell migration

Results 1-7 (7)