Emmet McCormack

Long-term Cultures of Bone Marrow-Derived Human Mesenchymal Stem Cells Frequently Undergo Spontaneous Malignant Transformation

Human mesenchymal stem cells (hMSC) aid in tissue maintenance and repair by differentiating into specialized cell types. Due to this ability, hMSC are currently being evaluated for cell-based therapies of tissue injury and degenerative diseases. However, extensive expansion ex vivo is a prerequisite to obtain the cell numbers required for human cell-based therapy protocols. Recent studies indicate that hMSC may contribute to cancer development and progression either by acting as cancer-initiating cells or through interactions with stromal elements. If spontaneous transformation ex vivo occurs, this may jeopardize the use of hMSC as therapeutic tools. Whereas murine MSC readily undergo spontaneous transformation, there are conflicting reports about spontaneous transformation of hMSC. We have addressed this controversy in a two-center study by growing bone marrow-derived hMSC in long-term cultures (5-106 weeks). We report for the first time spontaneous malignant transformation to occur in 45.8% (11 of 24) of these cultures. In comparison with hMSC, the transformed mesenchymal cells (TMC) showed a significantly increased proliferation rate and altered morphology and phenotype. In contrast to hMSC, TMC grew well in soft agar assays and were unable to undergo complete differentiation. Importantly, TMC were highly tumorigenic, causing multiple fast-growing lung deposits when injected into immunodeficient mice. We conclude that spontaneous malignant transformation may represent a biohazard in long-term ex vivo expansion of hMSC. On the other hand, this spontaneous transformation process may represent a unique model for studying molecular pathways initiating malignant transformation of hMSC.

Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival

Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.

Spontaneous Malignant Transformation of Human Mesenchymal Stem Cells Reflects Cross-Contamination: Putting the Research Field on Track - Letter

Several groups, including ours, have published results showing spontaneous transformation of human mesenchymal stem cells (MSC). Recently, we reported in this journal spontaneous transformation of bone marrow-derived human MSC (hMSC), isolated and expanded independently in two laboratories ([1][1

Implantable microenvironments to attract hematopoietic stem/cancer cells

The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.

SIRT1 activation by a c-MYC oncogenic network promotes the maintenance and drug resistance of human FLT3-ITD acute myeloid leukemia stem cells.

The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes.

MiR‐182 and miR‐203 induce mesenchymal to epithelial transition and self‐sufficiency of growth signals via repressing SNAI2 in prostate cells

MicroRNAs play critical roles in tumorigenesis and metastasis. Here, we report the dual functions of miR‐182 and miR‐203 in our previously described prostate cell model. MiR‐182 and miR‐203 were completely repressed during epithelial to mesenchymal transition (EMT) from prostate epithelial EP156T cells to the progeny mesenchymal nontransformed EPT1 cells. Re‐expression of miR‐182 or miR‐203 in EPT1 cells and prostate cancer PC3 cells induced mesenchymal to epithelial transition (MET) features. Simultaneously, miR‐182 and miR‐203 provided EPT1 cells with the ability to self‐sufficiency of growth signals, a well‐recognized oncogenic feature. Gene expression profiling showed high overlap of the genes affected by miR‐182 and miR‐203. SNAI2 was identified as a common target of miR‐182 and miR‐203. Knock‐down of SNAI2 in EPT1 cells phenocopied re‐expression of either miR‐182 or miR‐203 regarding both MET and self‐sufficiency of growth signals. Strikingly, considerable overlaps of changed genes were found between the re‐expression of miR‐182/203 and knock‐down of SNAI2. Finally, P‐cadherin was identified as a direct target of SNAI2. We conclude that miR‐182 and miR‐203 induce MET features and growth factor independent growth via repressing SNAI2 in prostate cells. Our findings shed new light on the roles of miR‐182/203 in cancer related processes.

Synergistic induction of p53 mediated apoptosis by valproic acid and nutlin-3 in acute myeloid leukemia

Although TP53 mutations are rare in acute myeloid leukemia (AML), wild type p53 function is habitually annulled through overexpression of MDM2 or through various mechanisms including epigenetic silencing by histone deacetylases (HDACs). We hypothesized that co-inhibition of MDM2 and HDACs, with nutlin-3 and valproic acid (VPA) would additively inhibit growth in leukemic cells expressing wild type TP53 and induce p53-mediated apoptosis. In vitro studies with the combination demonstrated synergistic induction of apoptosis in AML cell lines and patient cells. Nutlin-3 and VPA co-treatment resulted in massive induction of p53, acetylated p53 and p53 target genes in comparison with either agent alone, followed by p53 dependent cell death with autophagic features. In primary AML cells, inhibition of proliferation by the combination therapy correlated with the CD34 expression level of AML blasts. To evaluate the combination in vivo, we developed an orthotopic, NOD/SCID IL2rγnull xenograft model of MOLM-13 (AML FAB M5a; wild type TP53) expressing firefly luciferase. Survival analysis and bioluminescent imaging demonstrated the superior in vivo efficacy of the dual inhibition of MDM2 and HDAC in comparison with controls. Our results suggest the concomitant targeting of MDM2-p53 and HDAC inhibition, may be an effective therapeutic strategy for the treatment of AML.

Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157–165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157–165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157–165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.

A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer

BACKGROUND The primary aim of our study was to evaluate the safety and potential toxicity of gemcitabine combined with microbubbles under sonication in inoperable pancreatic cancer patients. The secondary aim was to evaluate a novel image-guided microbubble-based therapy, based on commercially available technology, towards improving chemotherapeutic efficacy, preserving patient performance status, and prolonging survival. METHODS Ten patients were enrolled and treated in this Phase I clinical trial. Gemcitabine was infused intravenously over 30min. Subsequently, patients were treated using a commercial clinical ultrasound scanner for 31.5min. SonoVue® was injected intravenously (0.5ml followed by 5ml saline every 3.5min) during the ultrasound treatment with the aim of inducing sonoporation, thus enhancing therapeutic efficacy. RESULTS The combined therapeutic regimen did not induce any additional toxicity or increased frequency of side effects when compared to gemcitabine chemotherapy alone (historical controls). Combination treated patients (n=10) tolerated an increased number of gemcitabine cycles compared with historical controls (n=63 patients; average of 8.3±6.0cycles, versus 13.8±5.6cycles, p=0.008, unpaired t-test). In five patients, the maximum tumour diameter was decreased from the first to last treatment. The median survival in our patients (n=10) was also increased from 8.9months to 17.6months (p=0.011). CONCLUSIONS It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available equipment with no additional toxicities. This combined treatment may improve the clinical efficacy of gemcitabine, prolong the quality of life, and extend survival in patients with pancreatic ductal adenocarcinoma.

Review: genetic models of acute myeloid leukaemia

The use of genetically engineered mice (GEM) have been critical in understanding disease states such as cancer, and none more so than acute myelogenous leukaemia (AML), a disease characterized by over 100 distinct chromosomal translocations. A substantial proportion of cases exhibiting recurrent reciprocal translocations at diagnosis, such as t(8;21) or t(15;17) have been exhaustively studied and are currently employed in clinical diagnosis. However, a definitive conclusion regarding the leukaemogenic potential of defined transgenes for this disease remains elusive. While it is increasingly apparent that a number of cooperating mutations are necessary to develop a leukaemic phenotype, the number of models reflecting these synergisms remains few. Furthermore, little emphasis has been paid to the effect of chromosomal translocations other than recurrent genetic abnormalities, with no models reflecting the multiple abnormalities observed in high-risk cases of AML accounting for 8–10% of adult AML. Here we review the differing technologies employed in generation of GEM of AML. We discuss the relevance of GEM AML from embryonic stem cell-mediated (for example retinoic acid receptor-α fusions and AML1/ETO) models; through to the valuable retroviral-mediated gene transfer models. The latter have been used to great effect in defining the transforming properties of chromosomal translocation products such as MLL (found in 5–6% of all AML cases) and NUP98 (denoting poor prognosis in therapy-related disease) and particularly when co-transduced with bad prognostic factors such as Flt3 mutations. Finally, we comment on the emergence of newer transduction technologies, which can regulate the level of expression to defined cell lineages in both primary murine and human xenografts, and discuss how combining multiple genetic modalities, more relevant models of this complex disease are being generated.