Download NUP98-HOXA10HD knock in model: a good tool for the biology and

S60
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3036 - DECLINED PRESENTATION
T-CELL PRECURSORS TO GENERATE CHIMERIC ANTIGEN
RECEPTOR (CAR)-MODIFIED T CELLS
Alla Dolnikov1,2, Sylvie Shen1,2, Ning Xu1,2, Susan Yang2, and Tracey O’Brien1,2
1
Sydney Children’s Hospital, Randwick, New South Wales, Australia; 2Children’s
Cancer Institute, Sydney, New South Wales, Australia
The ability to target cancer cells using genetically modified T cells armed with a
chimeric antigen receptor (CAR) recognising tumour antigen is a novel approach
to the treatment of cancer. Proven success in clinical trials has demonstrated a potent
anti-cancer effect of CART-cells however in most cases, only transient in vivo expansion and persistence of gene modified effector cells is demonstrated, resulting in
tumour recurrence. Pre-clinical studies of adoptive T cell transfer in mice have shown
that although effector T cells (Tem) have robust cytolytic function, T cells with young
phenotype – central memory T-cells(Tcm) or na€ıve T cells (Tn) and the recently
defined T-memory stem cells (Tmsc), are critical for in vivo expansion and longterm persistence. Here we propose to use a novel approach to improve mature
CART-cell persistence through the transduction of CAR into T-cell precursors
(pre-Ts). Pre-Ts were generated ex-vivo from cord blood CD34+ haematopoietic
stem cells using conditions mimicking thymic microenvironment using immobilised
Notch-DL4 ligand. Retroviral vector was used to transduce pre-Ts with CAR targeting human CD19 expressed in B-cell malignancies and normal B-cells. Transduction
with CARs achieved 60-70%. CAR-transduced CD34+CD7+ pre-Ts efficiently engrafted immune-deficient NOD/SCID/IL2Rg-null mice and generated mature T-cells
expressing CARs. T-cells generated from pre-Ts developed memory subsets confirming the expectation of development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CART-cells had decreased CD19+B-cell but not
myeloid or T-cell populations confirming functionality of CAR targeting in vivo. In
addition, delayed leukaemia progression was observed in mice reconstituted with
CAR-transduced pre-Ts and challenged with CD19+ leukaemia Raji-Luc cells.
Importantly, leukaemia burden inversely correlated with the proportion of T-cells
confirming T-cell mediated anti-leukaemic effects. In summary, our data support
the concept of genetic modification of pre-Ts with CARs.
3037 - NUP98-HOXA10HD KNOCK IN MODEL:
A GOOD TOOL FOR THE BIOLOGY AND REGENERATION
OF HEMATOPOIETIC STEM CELLS
Yong Dong, Chengxiang Xia, Shumin Fang, Xiaofei Liu, Dan Yang, and
Jinyong wang
Guangzhou institutes of biomedicine and health, Chinese Academy of Sciences,
Guangzhou, China
Overexpression of Nup98-HOXA10HD (NA) in hematopoietic stem cells (HSCs) by
retroviral transduction has been reported to robustly expand HSCs in vitro. Here, we
construct a NA-Tdtomato conditional knock in model (LSL-NA) in mouse ROSA26
locus. Upon crossing LSL-NA mice to Vav-Cre line, all the white blood cells from
the LSL-NA, Vav-Cre mice (NAfl/+) are Tdtomato positive. NAfl/+ HSCs show stronger competitiveness than WT HSCs (1:1). Interestingly, HSCs from NAfl/fl mice
demonstrate even higher competitiveness than the counterparts from NAfl/+ mice,
which indicates the correlation between doses of NA and the competitiveness that
NA confers. When co-culture NAfl/+ bone marrow cells with an irradiated
AFT024-SCF feeder line over six weeks, the NA cells show better engraftment
than WT bone marrow cells, indicating that the potential of NAfl/+ HSCs is maintained in vitro. Transplantation assay shows that the NAfl/+ HSCs are enriched in
sorted LSKCD48- population. Of note, the NAfl/+ mice show normal hematopoiesis
and are healthy after more than six months. Currently we are analyzing the expression profile of NAfl/+ HSCs based on single cell RNA-seq method, and using the
NAfl/+ ES and NAfl/+ MEF for derivation research of iHSCs. Thus NAfl/+ strain is a
new tool for the biology and regeneration of HSCs.
3038 - A NOVEL 3D PROSTATE CANCER BONE MARROW METASTASIS
IN VITRO MODEL SYSTEM
Eman Mohamed Othman Mosaad, Karen Chambers, Judith Clements, and
Michael R. Doran
Queensland University of Technology, Brisbane, Queensland, Australia
Objective: Prostate cancer (PCa) is the second most common cancer worldwide, and
the most common cancer in men in developed countries. In the primary and metastatic PCa, stromal cell contribution to the tumor progression, invasion and metastasis
has been reported. The lack of an effective in vitro model system of the complex
microenvironment of the PCa, either in the primary or metastatic site, restricts the
effective screening of novel therapeutics. To address this deficiency we are developing 3D in vitro model system that uses a high throughput microwell platform to
manufacture hundreds of microaggregates formed from precise combinations of
PCa and stromal cells. This model system better mimics the composite 3D cellular
microenvironment found in primary and metastatic PCa tumours, and will ultimately
enable more reliable in vitro drug screening.
Methods: Prostate cancer cells were either seeded in mono-culture or co-culture with
a prostate-derived stromal cell line (WPMY-1) or bone marrow stromal cells. Bone
marrow-derived mesenchymal stromal cells (MSCs) were used as a source of
different bone marrow niche cells. Microaggregates were treated with docetaxcel
to determine efficacy of the drug in the presence of stromal cells and this was
compared to the response in mono-cultures.
Results: Prostate cancer cells cultured as 3D micro-aggregates have a higher tolerance to drug compared to monolayer. In a co-culture system, PCa cells responded
differently to drug when maintained in the presence of MSC or other stromal cells
relative to mono-cultures. Changes in stromal cells signalling are hypothesized to
be a critical mediators of the PCa drug sensitivity: this work is on-going.
Conclusions: In summary, our results showed that the use of the microwell 3D coculture system does generate more rational cell organization, and that this different
morphology and stromal cell composition does impact on PCa cell drug response.
Through further work, we hope to determine if this system can be used to understand
which of the factors the complex stromal cells microenvironment contribute to PCa
bone marrow metastasis and the development chemoresistance.
3039 - BLOOD CELLS FORM FROM PHENOTYPICALLY DISTINCT
PRECURSORS DURING HUMAN BLAST COLONY DIFFERENTIATION
Freya Bruveris1,2, Elizabeth Ng1,2, Lisa Azzola1, Edouard Stanley1,2,3, and
Andrew Elefanty1,2,3
1
MCRI, Melbourne, Victoria, Australia; 2Monash University, Melbourne, Victoria,
Australia; 3University of Melbourne, Melbourne, Victoria, Australia
The earliest hematopoietic progenitor, termed the blast colony forming cell [BLCFC], can be isolated from human pluripotent stem cells [hPSCs] differentiated in
response to specific growth factors. This precursor generates blood, endothelium
and smooth muscle through a process believed to involve a transient hemogenic
endothelial intermediate. We investigated the process of human blood cell formation
using differentiation of blast colonies in vitro as a model system. To facilitate these
studies we used a hPSC reporter line [RUNX1CGFP/w SOX17mCherry/w] in which the
genes encoding GFP and mCherry were inserted via homologous recombination
into the RUNX1C and SOX17 loci, respectively in H9 hESCs. In these cells, GFP
marks RUNX1C+ haematopoietic precursors whilst mCherry identifies SOX17 expressing endothelial cells. Previous studies have identified a crucial role for SOX17
in the specification and regulation of blood cell formation from hemogenic endothelium at later developmental time points in both mouse and human. SOX17 expression
was observed during blast colony growth, with some cells in most colonies expressing the mCherry SOX17 reporter within 3 days. Analysis of sorted CD34+ cells early
during blast colony development revealed robust hematopoietic activity in the
SOX17- fraction that generated RUNX1C+ myeloid and GLYPA+ erythroid cells
without a significant residual endothelial component. Although SOX17+ cells lacked
initial clonogenic activity, they formed an endothelial layer from which myeloid and
erythroid progeny were later generated. These data indicate that SOX17 expression
distinguishes two temporally and phenotypically distinct hemogenic precursors during human blast colony development. Transcriptional profiling of these populations
confirms distinct patterns of gene expression, indicating that different starting cell
populations give rise to human blood cells.