Use of fluorescent imaging to monitor drug responses in mouse models of tumourigenesis
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2014-11-28Author
Balderstone, Lucy Anne
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As our understanding of the complexities of cancer biology has increased, the ability
to exploit unique features of tumour cells with molecularly targeted therapies has
become a reality. However, despite unprecedented volumes of new molecules in
clinical trials, the number of highly effective drugs approved by the regulatory
authorities remains disappointingly low. Moreover, oncology drug development is
plagued by high levels of attrition in late phase clinical development. Failure due to
poor efficacy and toxicity issues are not believed to be a result of the development of
molecules with inadequate pharmaceutical properties, but rather due to a lack of
understanding of their full mechanism of action. All of this points to imprecise
analysis of the drugs during the preclinical phase, highlighting the need for better
preclinical drug development tools.
Animal models provide a key preclinical tool, and as a therapeutic area, oncology is
characterised by models which are not predictive of the true human pathology.
Overexpression of the human epidermal growth factor receptor two (HER2)
oncogene, and inactivation of the phosphatase and tensin (PTEN) tumour suppressor,
are two important events in human breast cancer. A novel conditional mouse model
driven by overexpression of HER2 coupled with / without the loss of PTEN has been
characterised to interrogate the importance of these two cellular perturbations.
Multifocal tumours arose in mice from both lines, while luminal tumour
characteristics were shown to be reduced and basal characteristics increased with a
reduction in PTEN expression. Disruption of PTEN rapidly accelerated tumour onset
(from 138 to 82 days) and tumour growth (with the time from tumour onset to
maximum tumour size reduced from 38 to 21 days), significantly reducing overall
survival (from 165 to 102 days). The ability of tumour cells to colonize the lungs was
not significantly affected by the loss of PTEN. Tumours arising in both mice
genotypes were utilized to generate cell lines. These failed to provide an in vitro
representation of the tumours, and found little utility in drug efficacy studies with
HER family targeted agents, a situation which could be improved by the use of
different culture methods. Since suppression of apoptosis is a hallmark of human cancer, and a desired endpoint
of many anticancer therapies is the induction of cell death, the generation of cell lines
inherently capable of sensing caspase-mediated apoptotic cell death would be a
valuable drug development tool. Given that fluorescence imaging is also emerging as
a potentially powerful modality for preclinical drug development, a novel fluorescent
in house apoptosis reporter construct was generated (pCasFSwitch). Initial validation
of pCasFSwitch by transient transfection into murine mammary carcinoma cells
proved difficult due to transfection associated toxicity, yet proof-of-principle was
indicated. Transfer of pCasFSwitch into a retroviral backbone vector enabled the
generation of stably transfected squamous carcinoma cells more suitable for further
analysis. Incubation of lysates from these cells with recombinant enzymes revealed
the construct could be cleaved by caspase-3, but not by other members of the
cysteine protease family. Furthermore, assessment of apoptosis levels in the cells
upon staurosporine treatment proved the utility of the construct to quantify cell death,
and was validated against data generated with a commercial competitor, NucView.
Further comparison of the specificity of the imaging agents using caspase inhibitors
was limited by the functionality of currently available inhibitors, but did reveal that
in common with NucView, construct quantified levels of apoptosis were affected by
inhibition.
This thesis details the development of two preclinical drug development tools. A
novel mouse model enables biological interrogation of two key events in human
breast carcinogenesis. Since PTEN loss is associated with resistance to HER2
targeted therapies, it is ideally suited for efficacy testing to overcome such resistance.
The in house fluorescent apoptosis imaging agent allows a temporal read-out of drug
effects in live single cells. While the use of intravital imaging of stable cell lines
implanted under imaging windows would allow in vivo validation of in vitro data.
Taken together, such facilitation of thorough evaluation of therapies at the preclinical
stage, will reduce the adverse effects felt by the pharmaceutical industry of failure
late in the drug development pipeline.