Le corps humain (adulte) est composé de 206 os (“Anatomy and Physiology | Simple Book Production” n.d.) qui sont des tissus denses et composent la majeure partie du squelette humain. Le squelette, étant hautement vascularisé, est l’endroit le plus communément affecté par le cancer métastatique (Coleman 1997). L’apparition de ces métastases osseuses fragilise l’os et peut provoquer des fractures pathologiques. Toutefois la prédiction de telles fractures est difficile et loin d’être automatique. Une possibilité pour créer un outil de diagnostic plus performant serait les simulations éléments finis (FEA en anglais pour « Finite Elements Analysis »). Des études ont montré que la FEA spécifique au patient était capable de surpasser l’expertise des cliniciens dans le cas d’étude ex vivo avec défauts osseux induits mécaniquement (dont Derikx et al. 2012). Les recherches portant sur le cancer osseux sont toutefois dur à mettre en place, les échantillons étant rare. De manière à contourner la difficulté de trouver des échantillons humains rarement disponibles, la souris a été utilisé comme modèle squelettique dans plusieurs cas, incluant la tenue mécanique d’os atteint de métastases ex vivo (Mann et al. 2008). Ainsi, de manière à pouvoir étudier l’implication du tissu métastatique dans la résistance globale de l’os sur échantillons réels, nous avons utilisé ce modèle animal pour créer des échantillons tumoraux.Notre but était double : premièrement, quantifier l’apport de la prise en compte des propriétés mécaniques de la métastase dans la résistance globale de l’os. Deuxièmement, statuer sur le fait qu’un modèle plus simple que celui proposé dans la littérature (reposant sur des propriétés purement élastiques plutôt qu’élasto-plastiques (Eggermont et al. 2018) pouvait permettre d’améliorer la prédiction de fractures pathologiques.Tout d’abord, les résultats obtenus avec nos modèles hétérogènes (ne prenant pas en compte la tumeur) ont montré une bonne consistance avec la littérature, la corrélation entre tous les modèles hétérogènes (n=43 pattes) quant à la fracture simulée et expérimentale étant du même ordre de grandeur que celles d’une étude analogue menée sur vertèbres de souris (Nyman et al. 2015). Ensuite, le modèle prenant en compte les propriétés des tumeurs n’as pas permis d’améliorer la prédiction de fracture, au contraire, la moyenne des différences de ces modèles étant de 30±21% (n=11 pattes tumorales) contre 12±9% (n=43 pattes). De plus le modèle spécifique (prenant en compte le module des tumeurs) étant plus difficile à obtenir que le modèle hétérogène (ne nécessitant pas de segmentation entre os et tumeur), le premier ne semble pas être judicieux dans la prédiction de fracture d’os long présentant des lyses osseuses. Enfin, un critère de détection reposant sur la différence entre valeurs de forces ultimes globale et locale a permis de détecter la majorité des instabilités mécaniques constatées dans cette étude (sensibilité de 85% et spécificité de 100%). Un autre critère, basé sur le ratio entre poids des individus et la force ultime locale prédite via FEA a permis de correctement diagnostiquer l’ensemble des cas (100% de sensibilité et de spécificité). Ce résultat pourrait s’avérer être d’une grande aide quant à la prise de décision d’intervention chirurgicale dans le cas d’os long atteints de métastases osseuses. Bien sûr, avant cela la route à parcourir reste longue, ce résultat devant d’abord être confirmé cliniquement (possiblement en ayant recours à l’étude d’un cohorte rétrospective, comme cela a déjà pu être fait dans d’autres études (Eggermont et al. 2018). Cette étude vient d’être initiée dans le cas du projet MEKANOS (étude multicentrique en France) porté par le Professeur Cyrille Confavreux (rhumatologue) / The human body (adult) is composed of 206 bones ("Anatomy and Physiology | Simple Book Production" n.d.) that are dense tissues and make up the bulk of the human skeleton. The skeleton, being highly vascularized, is the most commonly affected site for metastatic cancer (Coleman 1997). The development of these bone metastases weakens the bone and can cause pathological fractures. However, the prediction of such fractures is difficult and far from automatic. One possibility for creating a more powerful diagnostic tool would be finite element simulations (FEA). Studies have shown that patient-specific FEA is able to surpass the expertise of clinicians in the case of ex vivo studies with mechanically induced bone defects (including Derikx et al., 2012). Research on bone cancer, however, is hard to put in place as samples are rare. In order to overcome the difficulty of finding human samples that are rarely available, the mouse has been used as a skeletal model in several cases, including the mechanical resistance of bones with ex vivo metastases (Mann et al., 2008). Thus, in order to study the involvement of metastatic tissue in the overall bone resistance of real samples, we used this animal model to create tumor samples. Our goal was twofold: first, to quantify the contribution of taking into account the mechanical properties of metastasis in the overall resistance of the bone. Secondly, to see if a simpler model than that proposed in the literature (based on purely elastic rather than elastoplastic properties (Eggermont et al., 2018) could improve the prediction of pathological fractures. First, the results obtained with our heterogeneous models (not taking tumor into account) showed a good consistency with the literature, the correlation between all the heterogeneous models (n = 43 legs) regarding the agreement of simulated and experimental fracture were of the same order of magnitude as a similar study conducted on mouse vertebrae (Nyman et al., 2015). Then, the model taking into account the properties of the tumors did not make it possible to improve the fracture prediction. The average of the differences of models taking tumor into account being of 30 ± 21% (n = 11 tumor limbs) against 12 ± 9% (n = 43 limbs). In addition, the specific model (taking into account the modulus of the tumors) being more difficult to obtain than the heterogeneous model (not requiring segmentation between bone and tumor), the first does not seem to be a wise choice in the prediction of long bone fracture presenting bone lysis. Finally, a detection criterion based on the difference between global and local ultimate force values made it possible to detect the majority of the mechanical instabilities observed in this study (sensitivity of 85% and specificity of 100%). Another criterion, based on the ratio between individual weights and the local ultimate force predicted via FEA, made it possible to correctly diagnose all cases (100% sensitivity and specificity). This result could prove to be of great help in making surgical decision making in the case of long bone with bone metastases. Of course, before that, the road ahead is long, this result having to be clinically confirmed first (possibly through the study of a retrospective cohort, as has already been done in other studies (Eggermont et al., 2018). This study has just been initiated in the case of the project MEKANOS (multicenter study in France) led by Professor Cyrille Confavreux (rheumatologist)

At present, prostate cancer is a significant public health issue throughout the world and is the second leading cause of cancer deaths in older men. The prostate specific antigen or PSA (which is encoded by the kallikrein 3/KLK3 gene) test is the current most valuable tool for the diagnosis and management of prostate cancer. However, it is insufficiently sensitive and specific for early diagnosis, for staging of prostate cancer or for discriminating between benign prostatic hyperplasia (BPH) and prostate cancer. Recent research has revealed another potential tumour marker, glandular kallikrein 2 (KLK2 gene/hK2 protein), which may be used alone or in conjunction with PSA to overcome some of the limitations of the PSA test. Twelve new kallikrein gene family members have been recently identified and, like hK2 and PSA, many of these genes have been suggested to be involved in carcinogenesis. In this study, the cellular localisation and level of expression of several of these newer kallikreins (KLK4, KLK5, KLK7, KLK8 and KLK11) was examined in prostate tissue, to provide an understanding of the association of their expression with prostatic diseases and their potential as additional biomarkers. Like PSA and hK2, the present observation indicated that each of these proteins, hK4, hK5, hK7, hK8 and hK11, was detected within the cytoplasm of the secretory cells of the prostate glands. For the first time, all of these newly-identified proteins were shown to be expressed in prostatic intraepithelial neoplasia (PIN) lesions, in comparison to normal glands and cancer lesions. In addition to cytoplasmic secretory cell expression, the localisation of hK4 to the basal cells and nuclei in prostatic lesions was intriguing. The intensity of hK4 staining in prostate tissue was strongest in comparison to the other newly-identified kallikrein proteins (hK5, hK7, hK8 and hK11). Therefore, KLK4/hK4 expression was characterised further to define this cellular localisation and examined in non-prostatic tissue and also in a larger number of prostate tissues in an attempt to determine its potential value as a biomarker for prostate disease. Three hK4 antipeptide polyclonal antibodies, derived against N-terminal, mid-region and C-terminal hK4 amino acid sequences, were used. The hK4 N-terminal antipeptide antibody was used to demonstrate the cellular localisation of hK4 in kidney, salivary glands, liver, testis, colon carcinoma, heart, endometrium and ovarian cancer, for the first time. The presence of hK4 in these non-prostate tissues was consistent with the previous reports using RT-PCR. The dual cytoplasmic and nuclear localisation of hK4 observed in the prostate above was also seen in these tissues. Although hK4 was found widely expressed in many human tissue types, indicating that it is not prostate specific in its expression, the highest expression level of hK4 was seen in the prostate. Therefore, detailed expression patterns and levels of KLK4 mRNA and hK4 protein in the normal prostate and prostatic diseases and histopathological lesions were investigated and reported for the first time in this study. Twelve benign prostatic hyperplasia (BPH), 19 adenocarcinoma (Gleason grade 2-5) and 34 bone metastases from prostate cancer were analysed. Using in situ hybridisation, the expression of KLK4 mRNA was detected in the cytoplasm of the secretory cells of both normal and diseased prostate tissue. KLK4 mRNA was also noted in both secretory and basal cells of PIN lesions, but the basal cells of normal glands were negative. Using the hK4 N-terminal and mid-region antipeptide antibodies, hK4 was predominantly localised in the cytoplasm of the secretory cells. The intensity of hK4 staining appeared lowest in normal and BPH, and increased in PIN lesions, high Gleason grade prostate cancer and bone metastases indicating the potential of hK4 as a histopathological marker for prostatic neoplasias. Further studies are required with a larger cohort to determine its utility as a clinical biomarker. Small foci of atypical cells, which were found within normal glands, were also intensely stained. Surprisingly, hK4 protein was found in the nucleus of the secretory cells (but not the basal cells) of high grade PIN and Gleason grade 3 prostate cancer. The detection of KLK4 mRNA and hK4 protein in PIN lesions and small foci of atypical cells suggests that up-regulation of KLK4 expression occurs early in the pathology of prostate carcinogenesis. The finding of basal cell expression is not typical for the kallikreins and it is not clear what role hK4 would play in this cell type. With the use of the hK4 C-terminal antipeptide antibody, the staining was mainly localised in the nuclei of the secretory cells of the prostate glands. Although the nuclear localisation was readily noted in more than 90% of epithelial cells of the prostate gland with the C-terminal antibody, no difference in staining intensity was observed among the histopathological lesions of the prostate. The prominent nuclear localisation with the C-terminal antipeptide antibody was also shown to be distributed throughout the nucleus by using confocal microscopy. Further, by using gold-labelled particles for electron microscopy, the intracellular localisation of these hK4 antipeptide antibodies was reported here for the first time. Similar to the immunohistochemical results, the cytoplasm was the major site of localisation with the N-terminal and mid-region antipeptide antibodies. To further characterise the involvement of KLK4/hK4 in human prostate cancer progression, the transgenic adenocarcinoma mouse prostate (TRAMP) model was used in this study. In this study, mouse KLK4 (also known as enamel matrix serine protease -1, EMSP-1) was shown to be expressed in the TRAMP prostate for the first time. Previous studies had only shown the developing tooth as a site of expression for EMSP-1. The level of EMSP-1 mRNA expression was increased in PIN and prostate cancer lesions of the TRAMP model, while negative or low levels of EMSP-1 mRNA were seen in normal glands or in control mouse prostate tissue. The normal mouse prostate did not stain with any the three hK4 antipeptide antibodies. hK4 N-terminal and mid-region antipeptide antibodies showed positive staining in the cytoplasm of the epithelial cells of PIN and cancer lesions of the mouse prostate. The C-terminal antipeptide antibody showed distinctively nuclear staining and was predominantly localised in the nuclei of the glandular cells of PIN and cancer lesions of the mouse prostate. The expression patterns of both the mRNA and protein level for mouse KLK4 strongly supported the observations of KLK4/hK4 expression in the human prostate and further support the utility of the TRAMP model. Overall, the findings in this thesis indicate a clear association of KLK4/hK4 expression with prostate cancer progression. In addition, several intriguing findings were made in terms of cellular localisation (basal as well as secretory cells; nuclear and cytoplasmic) and high expression in atypical glandular cells and PIN, perhaps indicating an early involvement in prostate disease progression and, additionally, utility as basal cell and PIN histological markers. These findings provide the basis for future studies to confirm the utility of hK4 as a biomarker for prostate cancer progression and identify functional roles in the different cellular compartments.