Ana Gomes1, Diana Passaro1, Dominique Bonnet1, and Bernard Siow1
1Francis Crick Institute, London, United Kingdom
Synopsis
Acute
Myeloid Leukemia (AML) is the most common acute leukemia in adults. While the
clinical presentation is quite uniform, it is a highly heterogeneous disease at
the genetic level.
Using intravital two-photon microscopy, we have previously showed that AML patient-derived
samples belonging to different genetic subgroups induced a common pathologic bone
marrow (BM) vascular phenotype.
To understand the translational potential of our findings we have optimized
DCE-MRI for the assessment of bone marrow vascular permeability upon leukaemia development.
INTRODUCTION
Acute
Myeloid Leukemia (AML) is the most common acute leukemia in adults. While the
clinical presentation is quite uniform, it is a highly heterogeneous disease at
the genetic level. Mouse models of AML are extensively used to better
understand the pathobiology of the disease, to test potential novel therapies,
and for the development of diagnostic and prognostic imaging tools [1-3].
Using intravital two-photon microscopy, we have previously showed that AML patient-derived
samples belonging to different genetic subgroups induced a common pathologic bone
marrow (BM) vascular phenotype [4].
To understand the translational potential of our findings we have optimized
DCE-MRI for the assessment of bone marrow vascular permeability upon leukemia.METHODS
HL60,
U937 and ML1 cell lines were grown in RPMI1640, and were tested for mycoplasma
prior to commencing experiments. Media were supplemented with 10% FBS and 1x
Penicillin-Streptomycin and all reagents were from Gibco-Life Technologies
(Paisley, UK). Human AML patient samples (n=4) were obtained after informed
consent at St Bartholomew’s Hospital (London, UK).
All
animal experiments were performed under the project license (PPL 70/8904)
approved by the Home Office of UK and in accordance to The Francis Crick Institute
animal ethics committee guidelines.
NSG
mice were injected with 2.0x106 HL60 cells in the tail vein. Two to
three weeks after injection, MRI was performed followed by BM aspiration and
flow cytometry to monitor the engraftment.
T-cell
depleted human AML cells (2.0x106 cells per mouse) were injected in
NSG mice. Engraftment was assessed at 10 weeks from the injection. Once hCD45% in
the BM was above 50%, mice underwent MRI followed by AraC (cytosine
arabinoside) treatment (10 mg/kg/day) for 7 days. MRI and BM puncture were
repeated one week after treatment cessation. Mice were classified as responders
or not responders if after treatment the hCD45% in the BM was below or above 30%
of the starting value respectively. Non-injected
NSG mice were used as controls.
MRI
was performed on a 9.4T horizontal bore system (Bruker GMBH) equipped with a
B-GA12SH gradient coil system. RF transmission and reception was performed with
a 40mm ID quadrature birdcage coil (Bruker GMBH).
A
series of Fast Low Angle Shot (FLASH) scans were used for femur localization
and for slice positioning.
DCE
scans were performed using a FLASH with the following parameters: TR = 17.639ms;
TE = 1.859 ms; FA = 10° ; Repetition = 1100; FOV 30x30x0.5mm3;
matrix 128x128, and resolution of 234µm. Dotarem (0.4mL/Kg) was injected 4 mins
after the start of the scan. Total scan duration was 41 mins. All
mice were placed in a head-first prone position for imaging. Anaesthesia was
induced and maintained using isoflurane (1–4%) in room air supplemented with
oxygen (80%/20%). Temperature and respiration rate were monitored using SA
Instruments system.
Non-model based
parameters were quantified by drawing regions of interest over the bone marrow
and muscle, and using in-house developed Matlab scripts (MathWorks; Natick,
MA).RESULTS
We
first measured DCE-derived vascular parameters in control versus leukemic
animals using different leukaemia cell lines. There is a clear difference in
the BM kinetics in the presence of leukaemia, and it follows leukaemia progression
(Fig.1). Also, changes in DCE-related parameters that are linked to poor
vascular function are found to be correlated with disease progression
(Fig.2A-D). This is also seen in patient-derived leukemic mice (Fig.2E-H).
DISCUSSION / CONCLUSION
Our
DCE-MRI results are in agreement with our previous result using intra-vital
microscopy [4].
Our
results further suggest that the detection of a pathologic vascular phenotype
in the BM of AML patients could be of used to response to treatment and clinical
outcome.
Acknowledgements
The
authors would like to thank Veronique Birault and members of the Translation
team, Biological Research Facility, Flow Cytometry and In Vivo Imaging core
facilities at the Francis Crick Institute for their valuable help. The authors
are grateful to Prof. John Gribben (Barts) for providing human AML samples. A.L.G.
was supported by an i2i translational grant scheme from the Francis Crick
Institute. D.P. was supported by a non-clinical junior research fellowship from
EHA. This work was supported by The Francis Crick Institute, which receives its
core funding from Cancer Research UK (FC001045), The UK Medical Research
Council (FC001045), and the Welcome Trust (FC001045).References
1. Cook
GJ, Pardee TS. Animal models of leukemia: any closer to the real thing? Cancer
Metastasis Rev. 2013;32(1-2):63-76. Epub 2012/10/20. doi:
10.1007/s10555-012-9405-5. PubMed PMID: 23081702; PubMed Central PMCID:
PMCPMC3568447.
2. Kohnken R, Porcu P, Mishra A.
Overview of the Use of Murine Models in Leukemia and Lymphoma Research. Front
Oncol. 2017;7:22. Epub 2017/03/08. doi: 10.3389/fonc.2017.00022. PubMed PMID:
28265553; PubMed Central PMCID: PMCPMC5317199.
3. Zuber J, Radtke I, Pardee TS, Zhao
Z, Rappaport AR, Luo W, et al. Mouse models of human AML accurately predict
chemotherapy response. Genes Dev. 2009;23(7):877-89. Epub 2009/04/03. doi:
10.1101/gad.1771409. PubMed PMID: 19339691; PubMed Central PMCID:
PMCPMC2666344.
4. Passaro
D, Di Tullio A, Abarrategi A, Rouault-Pierre K, Foster K, Ariza-McNaughton L,
et al. Increased Vascular Permeability in the Bone Marrow Microenvironment
Contributes to Disease Progression and Drug Response in Acute Myeloid Leukemia.
Cancer Cell. 2017;32(3):324-41 e6. Epub 2017/09/06. doi:
10.1016/j.ccell.2017.08.001. PubMed PMID: 28870739; PubMed Central PMCID:
PMCPMC5598545.