Jin Li1, Emma L. Reeves1, Jessica K.R. Boult1, Craig Cummings1, Jeffrey C. Bamber1, Ralph Sinkus2, Yann Jamin1, and Simon P. Robinson1
1Division of Radiotherapy & Imaging, The Institute of Cancer Research, London, United Kingdom, 2Division of Imaging Sciences and Biomedical Engineering, King's College London, King’s Health Partners, St. Thomas’ Hospital, London, United Kingdom
Synopsis
MRE-derived shear stiffness |G*| of orthotopic pancreatic tumours was
sensitive to a heterogeneous treatment response induced by the vascular
disrupting agent ZD6126.
Introduction / Purpose
Stromal-dense
pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive deposition
of extracellular matrix (ECM) components, loss of tensional homeostasis and
hypovascularity, impairing efficient drug delivery.1-3 Collagen, the principal component of the
fibrillary protein network within the ECM, is the major contributor to ECM
stiffening, is strongly implicated in tumour evolution and progression, and is
associated with poor patient prognosis. Patients
with pancreatic cancer continue to have poor 5-year survival rates of ~3%. A major contributory factor to this dismal
prognosis is the lack of early diagnosis using current imaging
techniques/biomarkers, as well as limited treatment options.
Magnetic resonance
elastography (MRE) is an emerging imaging technique being used to directly
visualise and quantify the viscoelastic properties of tumours in vivo. We recently demonstrated the use of MRE, coupled with computational
pathology, to interrogate the contribution of collagen to the tumour
biomechanical phenotype in a range of pre-clinical tumour models in vivo4. The study highlighted that the markedly high stiffness quantified by MRE in orthotopically-propagated
PDAC tumours was associated with high collagen deposition, providing a strong rationale for using MRE to assess the response
of PDAC to ECM-targeted drugs. As a
prelude to this, MRE was used to quantify the elasticity and viscosity of orthotopic
PDAC xenografts prior to and following treatment with the vascular disrupting
agent ZD6126, previously shown to induce extensive tumour necrosis in a range
of pre-clinical tumour models.5,6 Methods
All
experiments were performed in accordance with the UK Animals (Scientific
Procedures) Act 1986. Anaesthetised CD1 nu/nu mice bearing orthotopic PDAC tumours
derived from PANC-1 cells were imaged prior to and 24 hours after treatment
with 200 mg/kg ZD6126, using a 3 cm birdcage coil on a 7T Bruker MicroImaging
horizontal MRI system (Bruker Instruments, Ettlingen, Germany). MRE data were acquired in the axial plane and a
purpose built platform as previously described.6 Maps of the
absolute value of complex shear modulus |G*| (G* = Gd + iGl, Gd:
elasticity, Gl: viscosity, all in kPa) and the loss tangent Gl/Gd
were reconstructed with an isotropic pixel size of 300 µm, and mean |G*| were determined
from a region of interest covering the tumour. Paraffin-embedded tissue sections were cut and
stained with H&E (necrosis), picrosirius red (collagen I&III) and endomucin
(microvasculature). Images of picrosirius red stained sections were also acquired
with polarized light microscopy to investigate collagen fibre architecture.Results
Orthotopic PDAC xenografts revealed a heterogeneous
regional distribution of |G*|, characterised by foci of elevated values.
Treatment with ZD6126 over 24 hours resulted in regional reduction in |G*|
in all three tumours (Fig. 1 & 2A).
ZD6126-induced regional reductions in |G*| were associated with
expansive areas of low endomucin staining and necrosis, surrounded by a thin
rim of viable cells (Fig. 3). In the
largest tumour from mouse #1, the
change in viscoelastic properties was spatially more heterogeneous, with regions
showing the smallest reduction in |G*| corresponding to areas of viable tumour
with markedly higher collagen deposition (Fig. 3). Detection of endomucin-stained
microvasculature post-treatment was restricted to the tumour periphery. Elevated signal in the loss tangent Gl/Gd
maps was spatially associated with thick collagen fibre deposition revealed by
polarized light microscopy, while there were regions of high |G*| value associated
with relatively low collagen fibre deposition and dense viable cellularity (Fig.
4).Discussion
Treatment with the vascular disrupting agent ZD6126
induced a clear reduction in the shear modulus |G*| of orthotopic PANC-1 xenografts
associated with pathologically-confirmed tumour necrosis. This response is
consistent with response to vascular disrupting agents in other pre-clinical tumour
models following treatments6,7, and
highlights that MRE can provide sensitive biomarkers of treatment response in
this model of PDAC.
Tumour viscoelastic properties are determined by
cellularity, vascularity, and ECM components.6,8 Generally, responsive regions of PANC-1
tumours exhibited relatively high |G*|
values pre-treatment. It is thus reasonable to assume that these regions represented
viable tumour tissue with relatively patent vasculature and collagen network
before treatment, as previously observed.4 In
comparison, regions that showed no MRE response to ZD6126 were mostly
avascular, with viable tissue supported by a dense collagen network. The absence of any detectable microvasculature
within these non-responding regions reiterates the negative impact of the
stromal dense microenvironment of PDAC on efficient drug delivery.
Interestingly, the case study from mouse #1 illustrated that the less studied
MRE-derived parameter loss tangent Gl/Gd, related
to energy dissipation, appeared to reflect the distribution of collagen fibres,
while |G*| value was affected by both cell viability and collagen deposition.
PDAC is
considered hypovascular, so the marked MRE-response and induction of necrosis
following treatment was somewhat surprising, as ZD6126 targets established
tumour blood vessels.5
A similar pathological response to ZD6126 was reported in orthotopic L3.6pl
pancreatic xenografts.9 Collectively these data suggest that
therapeutic targeting of the vascular compartment in PDAC may be an effective
treatment strategy.Conclusions
Quantitation of the shear modulus |G*|
with MRE provides a sensitive biomarker of the extent and heterogeneous response
of stromal rich orthotopic PDAC xenografts to the vascular disrupting agent
ZD6126 in vivo. The study provides a rationale
for performing MRE-embedded pre-clinical trials of novel therapeutics that
target the ECM being developed for the treatment of PDAC, a cancer of unmet
need.Acknowledgements
We
acknowledge funding from the Cancer Research UK Imaging Centre at the ICR, and
funding from the European Union’s Horizon 2020 research and innovation
programme under grant agreement No 668039, and Cancer Research UK Centre at the
ICR, the Cancer Research UK grant C16412/A27725, in association with a Children with
Cancer UK Research Fellowship. References
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