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Poor oxygenation of solid tumours has been linked with resistance to chemo- and radio-therapy and poor patient outcomes. Measuring the functional status of the tumour vasculature, including blood flow fluctuations and changes in oxygenation is important in cancer staging and therapy monitoring. A robust method is needed for clinical non-invasive measurement of the oxygen supply and demand in tumours. Current clinically approved imaging modalities suffer high cost, long procedure times and limited spatio-temporal resolution. Optoacoustic tomography (OT) is an emerging clinical imaging modality that can provide static images of endogenous haemoglobin concentration and oxygenation. In this work, an integrated framework for quantitative analysis of functional imaging using OT is developed and applied in vivo with preclinical cancer models. Oxygen Enhanced (OE)-OT is established here to provide insight into tumour vascular function and oxygen availability in the tissue. Tracking oxygenation dynamics using OE-OT reveals significant differences between two prostate cancer models in nude mice with markedly different vascular function (PC3 & LNCaP), which appear identical in static OT. OE-OT metrics are shown to be highly repeatable and correlate directly on a per-tumour basis to tumour vascular maturity, hypoxia and necrosis, assessed ex vivo. Dynamic Contrast Enhanced (DCE) OT demonstrates the relationship between OE-OT response and tumour perfusion in vivo. Finally, the possibility of using OT data acquired at longer wavelengths to report on tumour water and lipid content is investigated, with a view to future providing intrinsically co-registered imaging of tumour oxygenation and cellular necrosis. These findings indicate that OE-OT holds potential for application in prostate cancer patients, to improve delineation of aggressive and indolent disease, while combined with DCE-OT, it may offer significant advantage for localised imaging of tumour response to vascular targeted therapies. Further work is needed to establish whether OT can provide a new method to detect tumour necrosis in vivo.

Delta-like ligand 4 (DLL4) is a ligand for the Notch family of receptors. DLL4 is an important regulator of angiogenesis and DLL4 blockade promotes non-productive angiogenesis and delays tumour growth. The aim of this thesis was to investigate the effects of anti-DLL4 therapy in solid tumours in combination with a clinically relevant dose of ionising radiation (5 Gy; IR) and to analyse alterations in the Notch pathway induced by the treatments. Combining both treatments resulted in a greater than additive tumour growth delay in LS174T tumours, compared to either treatment alone. DLL4 blockade dysregulated vasculature and increased necrosis in LS174T and HCT-15 (DLL4-expressing and negative cell lines respectively) tumours within 3 days after treatment, but no changes were observed with IR alone. Additionally, combined IR and anti-DLL4 treatment of FaDu tumours (another DLL4-negative cell line) by our colleagues, also resulted in a supra-additive growth delay. These results show that combining IR with DLL4 blockade is an effective strategy for prolonging tumour growth delay and suggest that the stroma/vasculature provide the main therapeutic target for the anti-DLL4 therapy. Analysis of Notch pathway shows that IR upregulated Jag1 in tumour cells, and may inhibit Notch and downregulate DLL4 in the stroma. These changes may potentially affect tumour vessels and response to anti-DLL4 therapy. In vitro, anti-DLL4 therapy induced proliferation in quiescent contact-inhibited endothelial cells and also appeared to abrogate IR-induced inhibition of migration. These results suggest that DLL4 may be important in maintaining vessel quiescence and that IR may in part decrease migration through Notch signalling. Combining IR and DLL4 blockade to target tumour growth is an effective and well tolerated strategy and warrants further validation and refinement to be translated into clinical practice.

Radiation therapy is administered to more than 50% of patients diagnosed with cancer. Mechanisms of interaction between radiation and tumour cells are relatively well understood on a molecular level, but much remains uncertain regarding how radiation interacts with the tumour as a whole. Recent studies have suggested that tumour response to radiation may in fact be regulated by endothelial cell response, consequently stressing the role of tumour blood vessels in radiation treatment response. As a result, various treatment regimens have been proposed to strategically combine radiation with vascular targeting agents. A great deal of effort has been aimed towards developing efficient vascular targeting agents. Nonetheless, no optimal method has yet been devised to strategically deliver such agents. Recent evidence suggesting that these drugs may “normalize” tumour blood vessels and enhance radiosensitivity, is supporting experiments where anti-angiogenic drugs are combined with cytotoxic therapies such as radiotherapy. In contrast, ultrasound-stimulated microbubbles have recently been demonstrated to enhance radiation therapy by biophysically interacting with endothelial cells. When combined with single radiation doses, these microbubbles are believed to cause localized vascular destruction followed by tumour cell death. Finally, a new form of ‘pro-angiogenics’ has also been demonstrated to induce a therapeutic tumour response. The overall aim of this thesis is to study the role of tumour blood vessels in treatment responses to single-dose radiation therapy and to investigate radiation-based vascular targeting strategies. Using pharmacological and biophysical agents, blood vessels were altered to determine how they influence tumour cell death, clonogenicity, and tumour growth, and to study how these may be optimally combined with radiation. Three-dimensional high-frequency power Doppler ultrasound was used throughout these studies to investigate vascular response to therapy.

Il a été démontré que l’acidité de l’environnement tumoral jouait un rôle dans la résistance aux chimiothérapies. L’utilisation de nanovecteurs, tels que les nanocapsules lipidiques (LNC), permet non seulement d’améliorer le temps de biodistribution de substances actives, mais aussi de cibler l’environnement tumoral tout en protégeant les actifs de cet environnement acide. L’objectif de cette thèse porte ainsi sur l’optimisation et l’évaluation de LNC furtives et pH-sensibles dans le contexte du mélanome.Dans un premier temps, ces travaux ont consisté à caractériser la vascularisation de mélanomes humain et murin. Ces études ont permis de comparer différentes tumeurs (densité, taille et structure) et de déterminer si l’usage de nanomédecines est approprié dans ce contexte.La seconde partie s’est orientée sur l’élaboration de polymères combinant furtivité et pH-sensibilité. Ces copolymères composés de N-vinylpyrrolidone (NVP)et de vinylimidazole ont été synthétisés par polymérisation RAFT et post-insérés à la surface des LNC. Ces modifications ont donné lieu à des LNC présentant des charges de surface pH-dépendantes,entrainant une augmentation de leur internalisation à pH acide dans des cellules de mélanome. Finalement, des études de biodistribution ont mis en évidence l’intérêt de la NVP dans le développement de nanovecteurs furtifs. En conclusion, les copolymères développés ont permis de prolonger le temps de circulation, mais aussi d’apporter des propriétés pH-sensibles qui pourraient améliorer l’internalisation tumorale des LNC in vivo et donc de potentialiser l’effet d’une thérapie anticancéreuse. / Tumor acidity has been shown to play a major role in resistance to chemotherapy. The use of nanomedicines, as lipid nanocapsules (LNC), allows to protect drugs from this acidic environment. They can also improve the biodistribution of therapeutics and to target the tumor environment. The aim of this thesis concerns the evaluation and characterization of stealth and pH-sensitive LNC in the context of melanoma. Firstly, these works consisted in characterizing the vascularization of human and mice melanoma. These studies allowed to compare different tumors (density, size and structure), and determine if the used of nanocarrier is suitable in the context of melanoma.The second part of this thesis described the development and the characterization of new copolymers, combining stealth and pH-sensitive properties. These copolymers, composed of Nvinylpyrrolidone(NVP) and vinylimidazole, were synthesized by RAFT polymerization and were post in sertedonto LNC surface. These modifications allowed to obtain charge reversal nanocarriers, leading to increase their melanoma cell uptake underacid pH. Finally, biodistribution of these modified nanoparticles was studied in vivo and highlighted the interest of NVP in the development of stealth nanocarriers. To conclude, the developed copolymers able to extend nanocarrier circulation time and to provide pH-responsive properties which should increase the tumor internalization of LNC invivo and potentiate the effect of anticancer drugs.