Search

Acknowledgements: The KiCS program is supported by the Garron Family Cancer Centre at The Hospital for Sick Children through funding from the SickKids Foundation. A.H. received funding from the Canadian Institutes for Health Research (grant no. 162267) and is the Tier 1 Canada Research Chair in Rare Childhood Brain Tumors. D.M. is supported by the CIBC Children’s Foundation Chair in Child Health Research. A.S. is partially supported by an Early Researcher Award from the Ontario Ministry of Research and Innovation; by the Canada Research Chair in Childhood Cancer Genomics; and by funding from the V Foundation and the Robert J. Arceci Innovation Award from the St. Baldrick’s Foundation. We would like to thank the Centre for Applied Genomics, The Hospital for Sick Children, for assistance with RNA sequencing and the Treehouse Childhood Cancer Initiative, University of California, Santa Cruz, for access to their public data repository., The causes of pediatric cancers' distinctiveness compared to adult-onset tumors of the same type are not completely clear and not fully explained by their genomes. In this study, we used an optimized multilevel RNA clustering approach to derive molecular definitions for most childhood cancers. Applying this method to 13,313 transcriptomes, we constructed a pediatric cancer atlas to explore age-associated changes. Tumor entities were sometimes unexpectedly grouped due to common lineages, drivers or stemness profiles. Some established entities were divided into subgroups that predicted outcome better than current diagnostic approaches. These definitions account for inter-tumoral and intra-tumoral heterogeneity and have the potential of enabling reproducible, quantifiable diagnostics. As a whole, childhood tumors had more transcriptional diversity than adult tumors, maintaining greater expression flexibility. To apply these insights, we designed an ensemble convolutional neural network classifier. We show that this tool was able to match or clarify the diagnosis for 85% of childhood tumors in a prospective cohort. If further validated, this framework could be extended to derive molecular definitions for all cancer types.

Mesenchymal stem/stromal cells (MSCs) are known to be the helpers for the healing of tissue damage, often referred to as ambulatory cells. However, MSCs are also recruited by cancer cells to similarly aid in tumor growth and progression. In this review, some of the key steps in cancer progression and metastases are described including the various steps in which MSCs participate and may play important roles. MSCs aid in cancer cells' ability to evade immune attack, while promoting tumor angiogenesis, even being counter-acting against chemotherapeutics and other drugs used to fight various cancers. Furthermore, MSCs participate in many of the crucial steps in invasion and metastasis, including stimulating the epithelial-mesenchymal transition (EMT) and induction of stem-like properties that allow cancer stem cells to increase their survivability through the circulation. These steps are described in detail. Differences between circulating tumor cells (CTCs) and cancer stem cells (CSCs) are discussed, along with descriptions of the formation of a pre-metastatic niche, the role of exosomes from both cancer cells and MSCs in metastasis and tumor reseeding (self-seeding). More and more, MSCs are being proposed as a promising tumor targeting drug-delivery tool. In order to fulfill this promise, further understanding of the precise roles that MSCs play in the process of cancer metastases must be achieved, in attempting to create remedies that will improve the outcome of available therapeutics.

A shared enthusiasm towards the highly promising utility of stem cell research brought together clinicians and scientists from academic institutions, government, and industry at the Second Annual International Stem Cells and Regenerative Medicine meeting presented by GeneExpression Systems, Inc. earlier this year. A common theme was echoed by presenters that a deeper understanding of natural development of the whole organism and individual organ systems is needed. Armed with this greater knowledge, a more efficient and conscientious translation of stem cell research may be achieved. Guest lectures included presentations by Dr. Elizabeth H. Blackburn, Dr. Irving L. Weissman, and Dr. Erin Lavik. Attendees were updated and encouraged by these and several other speakers on such topics as stem cell biology, epigenetics, biotechnology, regenerative medicine, tissue engineering, and bioethics. Biomedical engineers and scientists are making steady strides in designing specific three dimensional scaffolds, tracking and controlling stem cells. The future of stem cell research continues to be promising with applications for treating cardiac and neurodegenerative diseases, hearing loss, diabetes, and so on.

The involvement of G proteins in the mechanism underlying the increased atrial natriuretic factor (ANF) secretion observed after atrial muscle stretch (stretch-secretion coupling) was assessed using a combined pharmacological, immunocytochemical, and tissue fractionation approach. It was found that G(i/o) inhibition by pertussis toxin (PTX) abolished stretch-secretion coupling without affecting baseline secretion through a mechanism that is independent of G(q) signaling agonists. Mastoparan-7, a G(i/o) agonist, significantly increased ANF secretion even in the absence of muscle stretch through a PTX-sensitive mechanism. By confocal and electron immunocytochemistry, ANF and G(o) partially colocalized, whereas ultracentrifugation analysis suggested the presence of two populations of granules, one of which was partially associated with G(o), as demonstrated by Western blotting. PTX did not affect basal or endothelin-1-stimulated ANF secretion, in line with the view that endothelin-1 signals mainly through G(q). It is concluded there are at least two types of regulated secretory processes in atrial cardiocytes: one is acutely responsive to muscle stretch and is PTX sensitive, and the other is G(q)mediated and PTX insensitive and may be responsible for changes in secretion after chronic changes in the neuroendocrine environment.

The cardiac hormone atrial natriuretic factor (ANF or ANP) plays significant, well-established roles in a large number of physiological and pathophysiological processes, including water and electrolyte balance, blood pressure regulation, and cardiovascular growth. Understanding the regulation of its production and secretion by atrial cardiomyocytes is incomplete. We have previously established a significant role of Gi/oprotein signaling in modulating ANF secretion as promoted by stretch of the atrial myocardium. In the present study, we investigated the role of Gqprotein signaling and its relationship to Gi/oprotein signaling using pharmacological manipulation of proximal effectors of Gαqin an ex vivo model of spontaneously beating rat atria. Phospholipase C (PLC) and protein kinase C (PKC) inhibitors dramatically increased basal secretion of ANF. Furthermore, although atrial wall stretch is a potent stimulus for secretion, stretch unexpectedly reduced ANF secretion to basal levels under PLC and PKC inhibitory conditions. Inhibition of the inositol triphosphate receptor did not appear to affect basal secretion but dose-dependently blocked stretch-secretion coupling. The results obtained demonstrate that the PLC and PKC signaling cascades play important albeit unexpected roles in the regulation of basal and stimulated ANF secretion and suggest interplay between the Gqand Gi/oprotein signaling pathways.

The goal of our project is to develop a unique ligand-conjugated nanoparticle (NP) therapy against childhood acute lymphoblastic leukemia (ALL). LLP2A, discovered by Dr. Kit Lam, is a high-affinity and high-specificity peptidomimetic ligand against an activated α4β1 integrin. Our study using 11 fresh primary ALL samples (10 precursor B ALL and 1 T ALL) showed that childhood ALL cells expressed activated α4β1 integrin and bound to LLP2A. Normal hematopoietic cells such as activated lymphocytes and monocytes expressed activated α4β1 integrin; however, normal hematopoietic stem cells showed low expression of α4β1 integrin. Therefore, we believe that LLP2A can be used as a targeted therapy for childhood ALL. The Lam lab has developed novel telodendrimer-based nanoparticles (NPs) which can carry drugs efficiently. We have also developed a human leukemia mouse model using immunodeficient NOD/SCID/IL2Rγ null mice engrafted with primary childhood ALL cells from our patients. LLP2A-conjugated NPs will be evaluated both in vitro and in vivo using primary leukemia cells and this mouse model. NPs will be loaded first with DiD near infra-red dye, and then with the chemotherapeutic agents daunorubicin or vincristine. Both drugs are mainstays of current chemotherapy for childhood ALL. Targeting properties of LLP2A-conjugated NPs will be evaluated by fluorescent microscopy, flow cytometry, MTS assay, and mouse survival after treatment. We expect that LLP2A-conjugated NPs will be preferentially delivered and endocytosed to leukemia cells as an effective targeted therapy.

Abstract 3576 Primary leukemia cells are known to be difficult to culture in vitro. Therefore, mouse xenograft models are commonly used to study human leukemia biology and to develop new therapeutics. Leukemia can be maintained and expanded through serial transplantations. Although many mouse models with different types of leukemia, mouse strains, and inoculation methods, have been used, there are very few studies validating the models. We have established acute lymphoblastic leukemia (ALL) mouse models using primary ALL samples and NOD/SCID/IL2Rg null (NSG) mice. In order to increase engraftment efficiency, we transplanted leukemia cells into healthy adult mice via intra-bone marrow (BM) injection (1 to 100 million cells per mouse to tibia bilaterally). To our knowledge, the model with primary ALL samples in NSG mice via intra-BM injection is novel. A total of 9 samples, 2 T cell ALL and 7 precursor B (preB) ALL, were transplanted into cohorts of 3 to 8 mice each. Of these, 1 T cell ALL and 6 preB ALL samples engrafted and 1 T cell and 1 preB ALL sample failed to engraft. Leukemia engraftment was observed between 10 to 33 weeks after transplantation. In each cohort, each mouse developed leukemia at approximately the same time after leukemia transplantation. Necropsy showed significant hepatosplenomegaly and white BM in all mice, indicating leukemia infiltration. Cells were harvested from BM, spleen and other leukemia-infiltrated organs, and confirmed to be human origin by flow cytometry using anti-HLA-ABC and anti- human CD45 antibodies. Nearly 100% of BM was replaced with human leukemia cells. One T cell and 1 preB ALL sample have been serially transplanted using the same method as the primary transplantation to quaternary and tertiary generations, respectively. We observed that the time to develop leukemia became shorter with each transplantation: 16 weeks for the first transplantation and 10 weeks for the tertiary transplantation in a T cell ALL sample. Some mice transplanted with a preB ALL sample developed chloroma. Interestingly, chloroma development was consistently observed with this sample, which was transferred through serial transplantation although the patient did not develop chloroma. Morphology and immunophenotype were similar to the original leukemia. There were some changes in CD expression patterns; however, immunophenotyping was consistent with the original leukemia. To help understand phenotype progression in transplanted leukemia samples, we are currently comparing the transcriptome profiles of leukemia samples obtained from the patient, primary and quaternary mice for T cell ALL, and primary and tertiary mice for preB ALL samples (preB ALL patient sample not available). Next-generation sequencing (NGS)-based RNA-Sequencing (RNA-Seq) is in progress for this analysis. In addition to obtaining digital expression profiles, and differential gene expression, RNA-Seq analysis will reveal the complete repertoire of splice variants, point mutations, and fusion transcripts in the ALL samples. Complete results of these analyses for our mouse models with both T cell and preB ALL will be presented. Thus, this technique will provide the most detailed transcriptomic analysis and no validation studies have yet been reported on ALL samples sequentially engrafted NSG mice using RNA-Seq. Disclosures: No relevant conflicts of interest to declare.

Abstract 3269 The one-bead-one-compound (OBOC) combinatorial library method was invented by Kit Lam to identify cancer cell surface targeting ligands (Peng L et al. Nat. Chem. Biol. 2006, Aina OH et al., Mol. Pharm. 2007). Using this method, LLP2A, a ligand against activated a4b1 integrin, was discovered that targets malignant lymphoma with high affinity and specificity (Peng L et al., Mol Cancer Ther. 2008). We tested whether childhood acute lymphoblastic leukemia (ALL) cells expressed activated a4b1 integrin and bound to LLP2A using primary leukemia cells and leukemia cells engrafted in NOD/SCID/IL2Rg null (NSG) mice. Expression of activated a4b1 integrin was observed in three primary leukemia samples (1 T-ALL, 1 precursor B (preB) ALL, and 1 relapsed preB ALL) by the rainbow beads (color-coded polystyrene bead mixture displaying unique ligands against known receptors such as a4b1, a3b1, avb3, a5b1 integrins (Luo J et al., J. Comb. Chem. 2008). To quantify activated a4b1 integrin expression, primary leukemia cells were analyzed by flow cytometry using biotinylated LLP2A/streptavidin-PE. Nine samples (7 preB ALL and 2 relapsed preB ALL) were analyzed. All samples, except for one relapsed preB ALL sample, showed expression of activated a4b1 integrin at different levels. In order to determine the specificity of activated a4b1 integrin expression, hematopoietic stem cells from three normal bone marrow (BM) samples were analyzed and showed very low levels of activated a4b1 integrin expression. Furthermore, we have identified 32 analogues of LLP2A using three fresh preB ALL samples to screen two LLP2A-focused libraries. Peptide microarrays using a polystyrene slide coated with neutravidin and biotinylated ligands will be prepared with these analogues. We will determine the binding affinity and specificity of these analogues to a series of primary ALL cells. Binding specificity to leukemia cells will be determined by excluding ligands which bind to normal hematopoietic progenitor (CD34) cells. The property of the selected ligands will be tested in vivo using NSG mice engrafted with primary ALL cells. Updated results with these new ligands will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 3241 Leukemia cells are believed to arise from leukemia stem cells (LSC). It is also known that LSC are responsible for relapse in certain types of leukemia, such as acute myeloid leukemia (AML). However, the existence and role of LSC in acute lymphoblastic leukemia (ALL) is unclear. CD9 was reported to be a marker for LSC in B-ALL using cell lines (Nishida H. et al., 2009). CD9 is a tetraspanin and is believed to be involved in cell adhesion, motility, and signaling events. It is also involved in metastasis; however, the mechanisms are unknown. Since childhood ALL is a heterogeneous group of diseases and cell lines can be different from primary leukemia cells, we tested the role of CD9 as a candidate LSC marker using primary precursor B (preB) ALL cells from pediatric patients. Two methods, Raman spectroscopy and serial transplantation of sorted leukemia cells in NOD/SCID/IL2R g null (NSG) mice, were used to confirm LSC. Raman spectroscopy is a laser-based technique for the single cell analysis of intrinsic molecular vibrations reflecting cellular biochemical information. It can provide a quantitative assessment of the levels of DNA, RNA, proteins, lipids, and carbohydrates in the cell, as well as molecular-level conformational changes. Previous studies by our group showed that unique Raman fingerprints were identified in normal blood cells, ALL cells, and stem cells, including hematopoietic stem cells and embryonic stem cells. Four preB ALL samples were stained for CD9 and sorted by flow cytometry. ALL samples were obtained from patients at diagnosis or freshly harvested from NSG mice engrafted with primary leukemia samples. All samples showed heterogeneous expression of CD9. CD9 high-positive cells and negative cells were flow sorted. Raman spectra of freshly sorted CD9 high-positive and negative cells were obtained. 10 to 20 cells were analyzed in each sample. CD34 positive cells, which were isolated from normal donors, were also analyzed by Raman spectroscopy as a control. No unique Raman fingerprints were identified to separate CD9 high-positive cells from negative cells using Principal Component Analysis (PCA). Furthermore, CD9 high-positive and negative cells from three preB ALL samples were transplanted into NSG mice via intra-bone marrow injection. Equal cell numbers (5×105 to 1.5×106 cells) were used for positive and negative samples in each injection. The majority of the mice from both groups (transplanted with CD9 high-positive or negative cells) developed leukemia 3 to 4 months after injection. Leukemia phenotype was confirmed to be the same as the original leukemia. In conclusion, although CD9 was shown to be a marker for LSC in B-ALL cell lines, it does not appear to be an LSC marker in primary preB ALL. Since childhood preB ALL is a heterogeneous group of diseases, larger cohorts are necessary to confirm our findings. Raman spectroscopy may be a useful screening tool for analysis of cellular intrinsic markers. Disclosures: No relevant conflicts of interest to declare.