dc.contributor.advisor |
Hicks, K |
en |
dc.contributor.advisor |
Pruijn, F |
en |
dc.contributor.author |
Abuwarwar, Mohammed |
en |
dc.date.accessioned |
2015-11-18T20:19:37Z |
en |
dc.date.issued |
2015 |
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dc.identifier.citation |
2015 |
en |
dc.identifier.uri |
http://hdl.handle.net/2292/27507 |
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dc.description |
Full text is available to authenticated members of The University of Auckland only. |
en |
dc.description.abstract |
Many tumours contain regions of hypoxia, where cells are resistant to radiation and some chemotherapeutic agents, are associated with poor prognosis and therapeutic resistance. The absence of severe hypoxia in normal tissues has resulted in the development of a number of hypoxia-selective prodrugs. SN30000 is a 2nd generation analogue of the 1,2,4-benzotriazine 1,4-dioxide (BTO) hypoxia-activated prodrug (HAP) tirapazamine (TPZ) with improved tumour transport, hypoxic selectivity and cytotoxic potency, and is in advanced pre-clinical development. SN30000 was developed by a pharmacokinetic model-guided approach based on data from multicellular layers (MCLs), a three-dimensional (3D) model system which mimics many of the features of the tumour extravascular compartment. BTOs are HAPs which are activated at low and intermediate oxygen concentrations but have no bystander effect on the surrounding well-oxygenated cells. Consequently, they must be used in combination with other treatments, such as radiation therapy, to kill hypoxic tumour cells at the time of radiation. To date, no combination studies have been done in 3D cell cultures to investigate the complementarity of TPZ or SN30000 with radiation or to compare the effects of SN30000 with TPZ directly, partly due to the low throughput using MCL cultures. This project sought to develop methods to expose multicellular tumour spheroids to SN30000, TPZ, or radiation and drug/radiation combinations under laboratory conditions similar to those used in monolayer drug screening studies. Their effects were measured by two methods: clonogenic surviving assay and spheroid growth inhibition. Initial optimisations were performed for quantitating spheroid size as a function of time. Optimisation of image analysis using photo microscopy with a macro for open-source image software ImageJ as well as the advanced imaging system ImageXpress and MetaXpress aided automated image analysis by improving the speed and accuracy of spheroid size measurements. SN30000 was less potent than TPZ in spheroids exposed under 20% oxygen by both clonogenic surviving assay and spheroid growth inhibition but was more hypoxia-selective as shown by superior potency in anoxic spheroids and 2D cultures. While there was a good correlation between cell killing and spheroid growth inhibition this occurred only at supra-pharmacological concentrations which would result in killing of the outer well-oxygenated cells. Radiation caused substantial spheroid growth inhibition with 50% inhibition of growth at approximately 6 Gy which was highly correlated with clonogenic cell killing in matched spheroids. The substantial spheroid growth inhibition observed at these radiation doses, taken together with the results of little growth inhibition for SN30000 and TPZ, suggest that growth inhibition was dominated by the rapidly dividing well-oxygenated cells. This conclusion was supported by comparing spheroid survival curves to monolayer survival under oxic and anoxic conditions, and by measuring the surviving fraction of cells 16 days after spheroid irradiation at several of the highest radiation doses given. Combination studies were performed showing that additional growth inhibition was small when SN30000 or TPZ was combined with radiation despite substantial predicted hypoxic cell killing by the drugs in addition to radiation alone. This study has demonstrated that spheroid growth inhibition can be used as an additional endpoint to clonogenic assays in the investigation of effect of HAPs such as SN30000 and radiation. Spheroid growth inhibition has the advantage of allowing monitoring over time, which is further assisted by semi-automated image acquisition & analysis methods. Screening in 3D cultures has advantages over 2D cultures and provides additional clinically relevant endpoints, but requires optimisation and independent mechanistic experiments to interpret the data. |
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dc.publisher |
ResearchSpace@Auckland |
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dc.relation.ispartof |
Masters Thesis - University of Auckland |
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dc.relation.isreferencedby |
UoA99264825612902091 |
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dc.rights |
Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. |
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dc.rights |
Restricted Item. Available to authenticated members of The University of Auckland. |
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dc.rights.uri |
https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm |
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dc.title |
Comparison of Tirapazamine with its improved analogue SN30000 in HCT116 multicellular tumour spheroids by two therapeutic endpoints |
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dc.type |
Thesis |
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thesis.degree.discipline |
Biomedical Science |
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thesis.degree.grantor |
The University of Auckland |
en |
thesis.degree.level |
Masters |
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dc.rights.holder |
Copyright: The Author |
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pubs.elements-id |
505397 |
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pubs.record-created-at-source-date |
2015-11-19 |
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dc.identifier.wikidata |
Q112907896 |
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