[Truncated abstract] The design of effective immunotherapies, using tumour antigens to stimulate a functional effector cytotoxic T cell (CTL) response in a tumour bearing host, requires an understanding of the 'real time' in vivo relationship between the host immune system and antigens expressed by the developing tumour. However, effector function of endogenous anti-tumour CTLs generated during tumour progression has largely been assessed by indirect ex vivo assays and often focused on a single antigen. Therefore, studies in this thesis evaluated the endogenous in vivo CTL response to multiple tumour antigenic epitopes in murine tumour models using Lewis lung carcinoma cells transfected with ovalbumin (an antigen that contains several intra-molecular MHC class I epitopes with a defined hierarchy) or a polyepitope (that contains a string of immunodominant MHC class I epitopes). Potent effector CTLs were generated to multiple dominant tumour antigenic epioptes early in tumour progression. However, in general, these CTL effectors only transiently retarded tumour growth, and at the later time points of tumour growth they were no longer generated in tumour draining lymph nodes. This coincided with diminished tumour antigen presentation in the same nodes which was found to be due to antigen loss. In both models antigen loss was the result of two processes; immuno-editing of the tumour by the host immune response and genetic instability resulting in antigen loss variants that could evade immune surveillance. A third model was generated that maintained low level tumour antigen expression throughout tumour progression. ... The impact of pre-existing endogenous dominant-epitope specific CTLs on tumour expressing the same epitope was also assessed, and resulted in a reduced tumour incidence and a CTL response restricted to a single antigen of the same MHC allele. Finally, the effects of two different immunotherapy regimens were examined. Intratumoural IL-2 treatment enhanced pre-existing CTL responses to the dominant epitopes leading to tumour regression. In addition, use of a multiple peptide vaccination regimen that avoided T cells competing for peptide-MHC complexes on APC was far more likely to be effective than one that did not. These results demonstrate that immunotherapies targeting tumours that express several dominant neo antigenic epitopes can be effective. The caveat for this approach is that it will only be effective in tumours that have generated an endogenous CTL response and must be used before antigen loss variants emerge.

Il existe une littérature abondante sur les mécanismes cellulaires de résistance à la chimiothérapie, décrivant notamment les pompes d’efflux, les modifications des cibles (comme les topoisomérases) ou les altérations de l’apoptose. Peu de publications s’intéressent aux mécanismes de chimiorésistance liée au microenvironnement tumoral. Les agents anticancéreux doivent traverser l’interstitium tumoral pour atteindre toutes les cellules (dont les cellules hypoxiques éloignées des vaisseaux sanguins) à des concentrations suffisantes pour être létales. Les modèles de culture cellulaire en couches multiples ont permis de montrer la faible pénétration des molécules de chimiothérapie. Les techniques d’immunohistochimie permettent une mesure quantitative de la distribution de ces molécules à partir des vaisseaux sanguins. Nous avons évalué la pénétration de plusieurs inhibiteurs de topoisomérases : topotécan, doxorubicine, mitoxantrone et banoxantrone. Nous avons comparé la distribution de ces molécules à travers des tissus sains et des tissus tumoraux, démontrant la pénétration limitée des molécules de chimiothérapie dans les tumeurs. Par contre, nous avons montré que la banoxantrone pénètre rapidement et de façon uniforme. Cette pro-drogue est convertit en AQ4 (un inhibiteur de topoisomérase II ressemblant à la mitoxantrone) en condition d’hypoxie. La mitoxantrone cible les cellules bien oxygénées et AQ4 cible les cellules hypoxiques. Cette combinaison de traitement aboutit à une distribution intratumorale complémentaire et à une amélioration de l’activité antitumorale. Ainsi, optimiser la pénétration des chimiothérapies et/ou cibler spécifiquement les cellules hypoxiques peut contourner la chimiorésistance liée au microenvironnement tumoral. / There is a vast literature about mechanisms that lead to drug resistance of individual cancer cells, including drug export pumps, changes in expression of targets (such as topoisomerases) or alterations in apoptosis. A smaller number of publications has drawn attention to causes of drug resistance that depend on the solid tumour microenvironment. Drugs must penetrate the extra-vascular space to reach all of the cancer cells (including cells far from blood vessels in hypoxic condition) in sufficient concentration to cause lethal toxicity. Model systems such as multilayered cell cultures provide direct evidence of poor drug penetration through tumour tissue. In vivo techniques using quantitative immunohistochemistry allow studying drug distribution as a function of distance from the nearest blood vessel. We have evaluated the penetration of several topoisomerase inhibitors: topotecan, doxorubicine, mitoxantrone and banoxantrone (AQ4N). We have compared the distribution of these drugs through normal and tumour tissue, demonstrating the limited perivascular distribution of conventional chemotherapies in tumour. We have also showed the rapid and uniform penetration of banoxantrone. This pro-drug is reduced to AQ4 (a topoisomérase II inhibitor of similar structure to mitoxantrone) under hypoxic condition. The targeting of mitoxantrone to oxygenated regions and AQ4 to hypoxic tumour regions resulted in effective drug exposure over the entire tumour and increased tumour growth delay compared with either drug alone. Improving drug penetration and/or targeting hypoxic tumour cells may overcome chemoresistance due to the tumour microenvironment.

Evaluation using mouse subcutaneous tumour models is a key process in cancer drug development. Tumour material is implanted subcutaneously and tumour growth measured using callipers. However this methodology can have poor reproducibility and accuracy due to observer variation. Furthermore the physical pressure of using callipers can distress the mouse and lead to tumour damage. Non-invasive digital tumour imaging would reduce handling stresses and allow volume determination without physical contact. This thesis focusses on capturing 2D digital images of subcutaneous tumours, then using image processing and machine learning methods to determine 3D volume. The biggest challenge faced was lack of differentiation between tumour and surrounding skin, rendering tumour boundary identification difficult. Whilst image processing methods such as colour segmentation and edge detection were unsuccessful, machine learning proved more successful. Three convolutional neural networks, VGG-Face, VGG-19 and VGG-16 models were evaluated, with VGG-Face producing the best results. Using the layer FC7 before RELU activation for extraction in the VGC-Face model, a tumour recognition rate of 98.86% was achieved. This was increased to 100% through a semi-automatic step with detection repeated on cropped versions of negatively classified images. Finally, volume was determined through extracting image features using the VGG-Face model and conducting partial least squares regression (error of 0.1). This work has successfully demonstrated that with computational methods the volume of subcutaneous tumours can be evaluated through non-invasive digital imaging without need to have contact with the tumour itself, thus offering refinement benefits to the mice as well as eliminating observer bias.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

Bone morphogenetic protein (BMP) signalling plays key roles in skin development and also possesses a potent anti-tumour activity in postnatal skin. To study mechanisms of the tumour-suppressive role of BMPs in the skin, a transgenic (TG) mouse model was utilized, in which a transgenic expression of the BMP antagonist Noggin was targeted to the epidermis and hair follicles (HFs) via Keratin 14 promoter. K14-Noggin mice developed spontaneous HF-derived tumours, which resembled human trichofolliculoma. Initiation of the tumours was associated with a marked increase in cell proliferation and an expansion of the hair follicle stem/early progenitor cells. In addition, the TG mice showed hyperplastic changes in the sebaceous glands and the interfollicular epidermis. The epidermal hyperplasia was associated with an increase in the susceptibility to chemically-induced carcinogenesis and earlier malignant transformation of chemically-induced papillomas. Global gene expression profiling revealed that development of the trichofolliculomas was associated with an increase in the expression of the components of several pro-oncogenic signalling pathways (Wnt, Shh, PDGF, Ras, etc.). Specifically, expression of the Wnt ligands and (β-catenin/Lef1) markedly increased at the initiation stage of tumour formation. In contrast, expression of components of the Shh pathway was markedly increased in the fully developed tumours, compared to the tumour placodes. Pharmacological treatment of the TG mice with the Wnt and Shh antagonists resulted in the stage-dependent inhibition of the tumour initiation and progression, respectively. Further studies revealed that BMP signalling antagonizes the activity of the Wnt and Shh pathways via distinct mechanisms, which include direct regulation of the expression of the tumour suppressor Wnt inhibitory factor 1 (Wif1) and indirect effects on the Shh expression. Thus, tumour suppressor activity of the BMPs in skin epithelium depends on the local concentrations of Noggin and is mediated, at least in part, via stage-dependent antagonizing of the Wnt and Shh signalling pathways.

<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>

När en individ drabbas av hjärntumör kan det vara aktuellt med intervention i form av neurokirurgisk resektion. I de fall där neurokirurgisk intervention krävs görs om möjligt en preoperativ kartläggning av individens språk i syfte att identifiera känsliga språkområden i kortikala strukturer. I dagsläget används nTMS, navigerad transkraniell magnetstimulering, som genom att temporärt slå ut funktionen i specifika kortikala områden är en användbar metod för nämnda syfte. Patienten får då benämna bilder föreställande substantiv samtidigt som fokala delar i hjärnan stimuleras enskilt med magnetstimulering. Syftet med föreliggande studie var att undersöka huruvida benämningsförmågan av substantiv förändrades vid nTMS hos personer med hjärntumör och på vilket sätt, samt om tumörlokalisation påverkade deltagarnas benämning vid nTMS-undersökning. Samtliga deltagare i studien var patienter med hjärntumör som tidigare undersökts med nTMS på en neurofysiologisk klinik vid ett sjukhus i sydöstra Sverige. Undersökningarna filmades och har sedermera utgjort materialet till datainsamlingen i föreliggande studie. Analys gjordes på grupp- och populationsnivå, där grupperna skapades utifrån deltagarnas tumörlokalisationer. Resultatet visar att benämningsförmågan förändras då ett antal olika språkliga fenomen uppkom vid preoperativ testning med nTMS. De fem vanligaste fenomenen på populationsnivå var tvekljud, latens, inskottsljud, uteblivet svar samt oprecis artikulation. Vid jämförelse på gruppnivå framkom att ingen signifikant skillnad föreligger avseende tumörlokalisation. Resultatet kan tyda på att språkets neurofysiologi består av samarbetande komplexa nätverk. Resultatet kan också styrka teorier om tumörinducerad plasticitet. / When an individual is diagnosed with a brain tumour, intervention by neurosurgery might be needed. In case of neurosurgery, a preoperative assessment of the individual’s language functions is carried out, if possible, in order to identify sensitive language areas in cortical structures. Today, nTMS, navigated transcranial magnetic stimulation, is being used, which temporarily shuts down functions in specific cortical areas and thus is a useful method to fulfill this aim. The patient is asked to name a series of pictures representing nouns while focal parts of the brain is stimulated with magnetic stimulation. The purpose of the present study was to examine whether the naming ability of nouns changed during nTMS in individuals with brain tumour and in what way, as well as whether the location of the tumour affected the participants naming abilities during nTMS. All participants in the present study were patients with brain tumour, previously examined with nTMS in a neurophysiological clinic at a hospital in the South East of Sweden. The examinations were video recorded and have subsequently formed the material for data analysis in the present study. Analysis was made on group and population level. The groups were created based on the participants tumour localisation. The result shows that the naming ability changes as a number of language related phenomenon aroused in the preoperative examination using nTMS. The five most common phenomena on population level were hesitation sound, latency, sound interjection, no response and inaccurate articulation. Comparison on group level revealed no significant difference regarding tumour location. The result might indicate that the neurophysiology of language consists of collaborating complex networks. The result can also support theories regarding tumour induced plasticity.

Capillaries in tumours are often severely buckled (in a plane perpendicular to the axis) and / or chaotic in their direction. We develop a model of these phenomena using nonlinear solid mechanics. Our model focusses on the immediate surrounding of a capillary. The vessel and surrounding tissue are modelled as concentric annulii. The growth is dependent on the concentration of a nutrient (oxygen) diffusing from the vessel into the tumour interstitium. The stress is modelled using a multiplicative decomposition of the deformation gradient F=F_e F_g. The stress is determined by substituting the elastic deformation gradient F_e (which gives the deformation gradient from the hypothetical configuration to the current configuration) into a hyperelastic constitutive model as per classical solid mechanics. We use a Blatz-Ko model, parameterised using uniaxial compression experiments. The entire system is in quasi-static equilibrium, with the divergence of the stress tensor equal to zero. We determine the onset of buckling using a linear stability analysis. We then investigate the postbuckling behaviour by introducing higher order perturbations in the deformation and growth before using the Fredholm Alternative to obtain the magnitude of the buckle. Our results demonstrate that the growth-induced stresses are sufficient for the capillary to buckle in the absence of external loading and / or constraints. Planar buckling usually occurs after 2-5 times the cellular proliferation timescale. Buckles with axial variation almost always go unstable after planar buckles. Buckles of fine wavelength are initially preferred by the system, but over time buckles of large wavelength become energetically more favourable. The tumoural hoop stress T_{ThetaTheta} is the most invariant (Eulerian) variable at the time of buckling: it is typically of the order of the tumoural Young's Modulus when this occurs.

Malignant pleural mesothelioma (MPM) is a form of aggressive tumour that is almost always associated with prior exposure to asbestos. Currently responsible for over 47,000 deaths worldwide each year and rising, it poses a serious threat to global public health. Many clinical studies of MPM, including its diagnosis, prognostic planning, and the evaluation of a treatment, necessitate the accurate quantification of tumours based on medical image scans, primarily computed tomography (CT). Currently, clinical best practice requires application of the MPM-adapted Response Evaluation Criteria in Solid Tumours (MPM-RECIST) scheme, which provides a uni-dimensional measure of the tumour's size. However, the low CT contrast between the tumour and surrounding tissues, the extensive elongated growth pattern characteristic of MPM, and, as a consequence, the pronounced partial volume effect, collectively contribute to the significant intra- and inter-observer variations in MPM-RECIST values seen in clinical practice, which in turn greatly affect clinical judgement and outcome. In this thesis, we present a novel computer-assisted approach to evaluate MPM patient response to treatments, based on the volumetric segmentation of tumours (VTA) on CT. We have developed a 3D segmentation routine based on the Random Walk (RW) segmentation framework by L. Grady, which is notable for its good performance in handling weak tissue boundaries and the ability to segment any arbitrary shapes with appropriately placed initialisation points. Results also show its benefit with regard to computation time, as compared to other candidate methods such as level sets. We have also added a boundary enhancement regulariser to RW, to improve its performance with smooth MPM boundaries. The regulariser is inspired by anisotropic diffusion. To reduce the required level of user supervision, we developed a registration-assisted segmentation option. Finally, we achieved effective and highly manoeuvrable partial volume correction by applying a reverse diffusion-based interpolation. To assess its clinical utility, we applied our method to a set of 48 CT studies from a group of 15 MPM patients and compared the findings to the MPM-RECIST observations made by a clinical specialist. Correlations confirm the utility of our algorithm for assessing MPM treatment response. Furthermore, our 3D algorithm found applications in monitoring the patient quality of life and palliative care planning. For example, segmented aerated lungs demonstrated very good correlation with the VTA-derived patient responses, suggesting their use in assessing the pulmonary function impairment caused by the disease. Likewise, segmented fluids highlight sites of pleural effusion and may potentially assist in intra-pleural fluid drainage planning. Throughout this thesis, to meet the demands of probabilistic analyses of data, we have used the Non-Parametric Windows (NPW) probability density estimator. NPW outperforms the histogram in terms of its smoothness and kernel density estimator in its parameter setting, and preserves signal properties such as the order of occurrence and band-limitedness of the sample, which are important for tissue reconstruction from discrete image data. We have also worked on extending this estimator to analysing vector-valued quantities; which are essential for multi-feature studies involving values such as image colour, texture, heterogeneity and entropy.