O câncer colorretal (CCR) é importante causa de morte no Brasil, representando a segunda causa de morbimortalidade por câncer nas regiões Sudeste e Sul. Foi uma das primeiras neoplasias malignas a ter modelo de carcinogênese identificado. Os objetivos deste trabalho foram avaliar a expressão do NF-KB e da survivina na progressão do CCR, sua relação com alvos moleculares envolvidos na sobrevivência celular [proibitina, fator de necrose tumoral (TNFR1), p53, B- catenina]; invasão (metaloproteinases 2 e 9 - MMP), angiogênese (fator de crescimento endotelial vascular VEGF) e apoptose (caspase 3 e método do Túnel). Trata-se de estudo retrospectivo baseado na análise histopatológica de dezoito casos de adenomas com displasia de alto (AG) e baixo grau (BG), respectivamente; dez casos de adenocarcinoma bem, moderado e pouco diferenciado, respectivamente, nove casos de lesões serrilhadas e nove biópsias colorretais (controle) selecionados aleatoriamente no Serviço de Patologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, no período de 2000 a 2009. Foram construídos arranjos de matriz tecidual para representar os casos de adenocarcinomas e cinco casos de adenomas >1 cm de diâmetro. A detecção do NF-KB foi realizada pelo método de Southwestern imunoistoquímica. Avaliou-se a expressão imunoistoquímica da survivina, proibitina, TNFR1, p53, B-catenina, MMPs 2 e 9, VEGF, caspase 3 e Túnel. Não observamos expressão imunoistoquímica do NF-KB em 70% dos casos de adenocarcinomas, em 72% dos adenomas AG e em 89% das lesões serrilhadas; a marcação foi positiva em 66% e 56% dos casos de adenoma do BG e controle, respectivamente. Expressões da survivina, proibitina citoplasmática, TNFR1, p53, MMP 2 e 9 foram crescentes na seqüência adenoma-carcinoma, sem aparente modulação pelo NF-KB. A survivina suprimiu ação da caspase 3, exceto nos adenomas BG e controle, com baixos níveis de apoptose ao túnel. Lesões serrilhadas e controle apresentaram baixa expressão do TNFR1 e da proibitina citoplasmática, ausência de marcação da p53 e MMPs 2 e 9, exceto um caso controle. Identificou-se marcação nuclear da proibitina nos adenocarcinomas pouco diferenciados, adenomas AG e BG. A expressão da p53 relacionou-se ao grau de displasia nos adenomas e à desregulação da survivina. Observou-se expressão citoplasmática e nuclear da B- catenina nos adenocarcinomas, adenomas AG e BG. As lesões serrilhadas exibiram expressão citoplasmática em 44% dos casos. O grupo controle exibiu expressão preservada da B-catenina. Identificou-se expressão do VEGF nos adenocarcinomas, relacionada à perda de diferenciação celular, presença de metástases, não correlacionada ao NF-KB. Com base nos nossos resultados, sugerimos a desregulação da B- catenina como possível responsável pela inibição do NF-KB; além de sua participação na desregulação da survivina juntamente com a p53. As lesões serrilhadas não exibiram indícios sugestivos de inibição do NF- KB pela B-catenina.A survivina, devido propriedades anti-apoptóticas, emerge como potencial alvo terapêutico no tratamento do CCR, confirmando estudos anteriores. / Colorectal cancer (CRC) is an important cause of death in Brazil, representing the second leading cause of cancer mortality in the Southeast and South. It was among the first malignancies that have the carcinogenesis model identified.The aim of this study was to evaluate the expression of NF-KB and survivin in the progression of CRC, its relationship with molecular targets involved in cell survival [prohibitin, tumor necrosis factor (TNFR1), p53,B-catenina], invasion (metalloproteinases 2 and 9 - MMP), angiogenesis (vascular endothelial growth factor VEGF) and apoptosis (caspase 3 and method Tunnel). This is a retrospective study based on the histopathological analyses of eighteen cases of high- (HG) and low- grade dysplastic (LG) adenomas, respectively; ten cases of adenocarcinoma well, moderately and poorly differentiated, respectively, nine cases of serrated lesions e nine biopsies (control); randomly selected in the Pathology Service of the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo from 2000 to 2009. Tissue microarray were constructed to represent the cases of adenocarcinomas and five cases of adenomas > 1cm in diameter. Detection of NF-KB was performed by the Southwestern immunohistochemistry method. We evaluated immunohistochemical expression of survivin, prohibitin, TNFR1, p53, B- catenin, MMPs 2 and 9, VEGF, caspase 3 and method Tunnel. We did not observe immunohistochemical expression of NF-KB in 70% of cases of adenocarcinomas, in 72% of HG adenomas and 89% of lesions serrated; staining was positive in 66% and 56% of cases of LG adenoma and control, respectively. Expressions of survivin, cytoplasmic prohibitin, TNFR1, p53, MMPs 2 and 9 were increasing in adenoma-carcinoma sequence, without apparent modulation by NF-KB. Survivin suppressed action of caspase 3, except in BG adenomas and control, with low levels of apoptosis to the tunnel. Serrated lesions and control showed low expression of TNFR1 and cytoplasmic prohibitin, the absence of staining of p53 and MMPs2 and 9, except for one control. We identified nuclear staining of prohibitin in poorly differentiated adenocarcinomas, HG and LG adenomas. Expression of p53 was related to the grade of dysplasia in adenomas and deregulation of survivin.The expression of the cytoplasmic and nuclear B-catenin was observed in adenocarcinoma, HG and LG adenoma. Serrated lesions exhibited cytoplasmic expression in 44% of cases. Control group exhibited preserved expression of B-catenin. It was identified VEGF expression in adenocarcinomas, related to the loss of cellular differentiation, metastasis, not correlated with NF-KB. According to our results, we suggest that deregulation of catenin is possible responsible for the inhibition of NF-KB besides their participation in the deregulation of survivin with p53.Serrated lesions exhibited no evidence suggestive of inhibition of NF-KB by B-catenin. Survivin emerges as a potential therapeutic target in the treatment of CRC due to their anti-apoptotic properties, confirming previous studies.

Une collaboration entre les chercheurs du laboratoire Pharma-Dev et de l'Institut de Recherche en Santé Digestive a été mise en place depuis plusieurs années avec pour objectif le criblage thérapeutique de nouvelles molécules naturelles issues de la biodiversité végétale. Dans ce contexte, le travail présenté dans ce manuscrit est consacré à l'étude phytochimique du latex de Hura crepitans L. (Euphorbiaceae) et à la valorisation biologique de ses constituants sur des lignées cancéreuses colorectales humaines. La richesse en structures diterpéniques originales de la famille des Euphorbiaceae et le rôle de ces noyaux structuraux dans l'activation de Protéines Kinases C (PKC) impliquées dans la carcinogenèse colorectale, font de H. crepitans une espèce au potentiel thérapeutique important. Au cours de ce travail, une approche déréplicative par UHPLC-MS/MS a permis de mettre en évidence la présence de cérébrosides et une richesse notable en diterpènes de type daphnane chez H. crepitans. Au total, 33 daphnanes dont 25 nouvelles structures ont été détectées. Le fractionnement du latex a permis l'isolement de 7 daphnanes mono-estérifiés (D1) et de 6 dérivés di-estérifiés. L'évaluation biologique a révélé une activité cytostatique des D1 spécifique sur la lignée cancéreuse Caco-2 comparée à d'autres lignées intestinales normales ou cancéreuses. Cette activité est inhibée par les cérébrosides. Le D1 majoritaire, la huratoxine, a montré une activité cytostatique dès 1 µg/ml et comparativement plus spécifique que le 5-Fluoro-Uracile, cytotoxique utilisé comme référence. De plus, nous avons montré que les effets cytostatiques de la huratoxine et de l'ester de phorbol TPA sont corrélés à la régulation de signaux clés de la carcinogenèse colorectale, GSK3ß et AKT notamment, et à des changements morphologiques. Finalement, nous avons pu identifier une isoenzyme PKC comme cible potentielle de ces composés naturels. / A collaboration between the researchers of the laboratory Pharma-Dev and the Research institute in Digestive Health has been established since several years with as main objective the therapeutic screening of new natural molecules from the plant biodiversity. In this context, the work presented in this manuscript deals with the phytochemical study of the latex of Hura crepitans L. (Euphorbiaceae) and the biological valuation of its constituents on human colorectal cancerous cell lines. The wealth in original diterpene like structures within the family of Euphorbiaceae and the role of these structural cores in the activation of Proteins Kinases C (PKC) involved in the colorectal carcinogenesis, make of H. crepitans a species with a promising therapeutic potential. In the course of this work, a dereplicative approach by UHPLC-MS/MS allowed to highlight the presence of cerebrosides and a notable wealth in daphnane type diterpene in H. crepitans. This led to the annotation of 33 daphnanes including 25 new structures. Therefore, 7 mono-esterified daphnanes (D1) and 6 di-esterified derivatives could be isolated. The biological evaluation revealed, for D1, a specific cytostatic activity on the cancerous cell line Caco-2 when compared to other non-cancerous or cancerous intestinal cell lines. This activity is inhibited by cerebrosides. The main D1, huratoxine, is cytostatic from 1 µg/ml and was showed to be more specific than 5-Fluoro-Uracile, a cytotoxic drug used as reference. Furthermore, we demonstrated that the cytostatic effects of huratoxine and phorbol ester TPA are correlated to the regulation of key signals of the colorectal carcinogenesis, particularly GSK3ß and AKT, and in cells morphological changes. Finally, we were able to identify a PKC isoenzyme as a potential target of these natural products.

Despite indications that colorectal cancer (CRC) screening strategies can decrease mortality and morbidity, screening rates among veterans remains to be low. In the Veterans Affairs (VA), the performance measure for CRC screening is lower than the national standard. This quality improvement (QI) project evaluated the effect of a team-based approach, effective electronic information structures, and the provision of education to nurses and patients in increasing CRC screening rate in primary care from 77% to 85%. CRC screening data were retrospectively collected prior to the start of the project and then compared to screening data 3 months after project implementation. The t test showed a statistically significant increase (p = .009) in CRC screening post intervention. Descriptive analysis was performed to evaluate the knowledge and proficiency of nurses with regard to CRC screening by using pre- and posttest questionnaires. The findings showed that emphasizing the importance of CRC screening among team members as well as appropriately dividing the work was effective in contributing to an increase in CRC screening in primary care. This project contributes to positive social change by increasing the nurses' confidence and proficiency in promoting health and disease prevention among the veterans; decreasing patient suffering; and improving collaboration between providers, nurses, and other departments in the VA primary care.

MUCDHL est une cadhérine atypique encore peu étudiée. Les données obtenues à ce jour suggèrent que ce gène joue un rôle suppresseur de tumeurs dans l’intestin, notamment par son interaction et son effet inhibiteur sur la β-caténine, et que son expression est fréquemment diminuée dans les cancers colorectaux (CCR). Parallèlement à cette fonction anti-oncogénique, d’autres travaux ont suggéré que MUCDHL est impliquée dans la structuration de la bordure en brosse (BB) intestinale, en contribuant à la formation d’un complexe d’interaction inter-microvillositaire. Notre objectif était de déterminer la fonction et le mode d’action de MUCDHL dans le système digestif. Par la caractérisation détaillée de l’interaction avec la β-caténine, nous avons montré que le mode d’action anti-oncogénique de MUCDHL est plus complexe qu’une simple séquestration membranaire de la β-caténine. De plus, nous avons confirmé le rôle suppresseur de tumeurs de MUCDHL sur une cohorte importante de CCR humains et montré pour la première fois que sa perte amplifie la tumorigenèse intestinale dans un model murin. Par ailleurs, l’étude phénotypique des souris Mucdhl-/- a démontré son importance dans l’homéostasie du système digestif. En effet, l’absence de MUCDHL cause des altérations morphologiques de la BB intestinale, mais également de nombreuses perturbations métaboliques. Ces travaux apportent donc des informations inédites sur la fonction et le mode d’action de MUCDHL dans le système digestif. / MUCDHL is an atypical cadherin that has been poorly studied. The data obtained so far suggest that this gene has tumor suppressive activity in the intestine, namely by its interaction and inhibitory effect on β-catenin, and that its expression is frequently decreased in colorectal cancers (CCR). In parallel to this anti-oncogenic function, other studies have suggested that MUCDHL is involved in the assembly of the intestinal brush border (BB), by contributing to the formation of an inter-microvilli interaction complex. Our objective was to determine the function and mode of action of MUCDHL in the digestive system, Through a detailed characterization of the interaction with β-catenin, we showed that the anti-oncogenic mode of action of MUCDHL is more complex than a simple membrane sequestration of β-catenin. In addition, we confirmed the tumor suppressive function of MUCDHL on a very large cohort of human CCR and showed for the first time that its loss amplifies intestinal tumorigenesis in a murine model. Moreover, the study of the phenotype of Mucdhl-/- mice allowed us to demonstrate the importance of MUCDHL in the homeostasis of the digestive system. Indeed, the absence of MUCDHL causes morphological alterations of the intestinal BB, but also numerous metabolic disturbances. Thus, this work provides new information on the function and mode of action of MUCDHL in the digestive system.

Par sa fréquence et sa gravité, le cancer colorectal (CCR) demeure un problème de santé publique. Notre objectif global est de mieux comprendre les mécanismes impliqués dans l'homéostasie intestinale au travers de Mucdhl, une cadhérine atypique méconnue mais qui semble jouer un rôle très particulier dans l'épithélium intestinal et être impliquée dans les CCR. De manière intéressante, son expression semble fréquemment perdue dans les CCR, tandis que son maintien dans les cellules cancéreuses coliques diminue leur potentiel tumoral.Pour mieux appréhender le mode d'action de Mucdhl, une caractérisation fonctionnelle de son interaction avec β-caténine oncogénique a été réalisée et de nouveaux partenaires ont été identifiés dans les cellules intestinales. Afin de comprendre le rôle de Mucdhl dans la physiologie intestinale, encore inconnu à ce jour, un modèle murin déficient pour Mucdhl a été étudié. L'analyse des conséquences de la perte d'expression de Mucdhl indique qu'il est impliqué dans la structure et le fonctionnement de l'intestin chez la souris, mais également au niveau de processus métaboliques. De plus, cette perte d'expression de Mucdhl augmente la sensibilité des souris au développement de certaines tumeurs intestinales. Ces travaux ont donc permis de générer des informations inédites sur les fonctions physiopathologiques de Mucdhl, une cadhérine atypique encore mal connue, mais potentiellement impliquée dans les CCR. / Because of its frequency and severity, Colorectal Cancer (CRC) remains an important public health issue. Our objective is to understand mechanisms contributing to intestinal homeostasis through Mucdhl, a poorly characterized atypical cadherin that may play a unusual role in the intestinal , epithelium and be implicated in CRC. lnterestingly, its expression seems to be frequently reduced in CRC, while its retention in colon cancer cells decreases their tumorigenic potential.To better apprehend the mode of action of Mucdhl, a functional characterization of its interaction with oncogen,iç β-catenin was performed and new partners have been identified in intestinal cells.To understand the role of Mucdhl in intestinal physiology, mice genetically-invalidated at the Mucdhl locus were studied. Analysis of the consequences of Mucdhl loss of expression indicates that it is involved in the morphology and function of the mouse intestine, but also in metabolic processes. Moreover, Mucdhl loss of expression increases the sensibility of mice to the development of certain intestinal tumors. Thus, we generated new information on the physiopathological functions of Mucdhl, an intriguing atypical cadherin potentially involved in CRC.

La chimiorésistance est un enjeu majeur dans la prise en charge des patients atteints de cancers digestifs et peut se manifester par l’efflux de molécules cytotoxiques via la surexpression des transporteurs ABC. Le facteur de transcription CDX2 est un régulateur important de l’identité intestinale et agit comme gène suppresseur de tumeur dans le côlon. Il pourrait être impliqué dans des phénomènes de chimiorésistance des cancers colorectaux (CCR) car le transporteur MDR1/ABCB1 correspond à un de ses gènes cibles. Nous avons confirmé le rôle de CDX2 dans la chimiorésistance des CCR. Nous avons montré par une approche translationnelle, que la surexpression de CDX2 était impliquée dans la résistance au 5-fluorouracile (5-FU) dans les CCR. Le transporteur du 5-FU ABCC11 a été identifié comme gène cible de CDX2 dont l’activité contribue à la résistance au 5-FU des cellules cancéreuses coliques. L’expression d’ABCC11 corrèle avec celle de CDX2 dans les CCR humains mais également avec celle de la DYPD, enzyme impliquée dans le catabolisme du 5-FU. Cette étude montre pour la première fois que CDX2 contribue à la chimiorésistance au 5-FU en impliquant des mécanismes régulant son métabolisme. / Chemoresistance represents a major drawback in digestive cancers’ management and may be achieved particularly through active efflux of the drug through overexpression of ABC transporters The transcription factor CDX2 is a master regulator of intestinal identity that acts as a tumor suppressor in the colon and may be important for drug resistance in colorectal cancer (CRC) as MDR1/ABCB1 was recently identified as one of its target genes. Here, we confirmed the role of CDX2 in the chemoresistance of CRC. We showed through a translational approach that CDX2 overexpression is implicated in 5-fluorouracil (5-FU) chemoresistance in CRC and described the molecular mechanisms implicated in this finding. We identified the 5-FU transporter ABCC11 as a new transcriptional target of CDX2 whose activity contributes to the 5-FU-chemoresistance of colon cancer cells. Remarkably, CDX2 expression correlates with the expression of ABCC11 in human colon tumors, but also with the one of the DPYD, enzyme involved in the 5-FU break down. Thus, our study links for the first time CDX2 to the 5-FU metabolism and provides a molecular mechanism for its impact on 5-FU-based chemotherapy.

La Chimiothérapie IntraPéritonnéale (CIP) est utilisée couramment pour traiter le cancer colorectal métastatique. Cependant il n'existe pas de protocole standardisé.Le but de ce projet a été de modéliser cette chimiothérapie in vitro et de comprendre le rôle de l'hypotonie dans ce modèle et son impact sur la mort des cellules cancéreuses.Nous avons déterminé les conditions optimales de traitement sur les cellules cancéreuses coliques humaines HCT116 à savoir une exposition des cellules pendant 30 minutes à 400µM d'oxaliplatine en conditions hypotoniques (G2.5%) à 37°C. Ces résultats ont été validés sur différentes lignées cancéreuses coliques humaines et murine. Nous avons également montré que ces conditions de traitements étaient également capables d’augmenter la cytotoxixité d’autres dérivés du platine comme le cisplatine et le carboplatine.La mort cellulaire induite par ce traitement en hypotonie est de type apoptotique, Et peut s’expliquer par une augmentation de l’incorporation intracellulaire d'oxaliplatine, en partie due à l'activation et à la trimérisation du transporteur du cuivre CTR1.Le traitement par l'oxaliplatine et le cisplatine (mais pas par le carboplatine) en hypotonie entraine également les stigmates de la mort immunogène, à savoir l'exposition de la calréticuline à la membrane, la libération d'ATP et le relargage d'HMGB1, suggérant que l'hypotonie permettrait d'entrainer la mort immunogène et une réponse du système immunitaire lors de cette modélisation de CIP.Enfin, in vivo nous avons pu mettre en évidence que le traitement de métastases intrapéritonéales de souris Balb/c par une injection intrapéritonéale d'oxaliplatine en hypotonie permettait un ralentissement de l’apparition de nodules tumoraux et une augmentation de la survie des souris.Ainsi, nous avons pu mettre en évidence dans ce travail que l'hypotonie est un des paramètres fondamentaux de la CIP et suggère que son utilisation pourrait permettre d'augmenter l’efficacité de la CIP et de prolonger la survie des patients. / IntraPeritoneal Chemotherapy (IPEC) is commonly used to treat colorectal cancer metastases. However there is no standardized protocol.The aim of this work was to model this chemotherapy in vitro and to understand the role of hypotonic conditions in this model and its impact on cell death.We determined that the optimal treatment parameters on HCT116 human colon cancer cells, were an exposure of the cells for 30 minutes to 400μM of oxaliplatin under hypotonic conditions (G2.5%) at 37 °C. These results have been validated on various human and murine colic cancer cell lines. We have also shown that these treatment conditions are also able to increase the cytotoxicity of other platinum derivatives such as cisplatin and carboplatin.The cell death induced by this treatment in hypotonia is apoptosis, and can be explained by an increase in the intracellular incorporation of oxaliplatin, partly due to the activation and trimerization of the CTR1 copper transporter.Treatment with oxaliplatin and cisplatin (but not carboplatin) in hypotonia also leads to the stigmata of immunogenic death, e.i. exposure of calreticulin at the membrane, release of ATP and HMGB1 in the supernatant, suggesting that hypotonia would entail immunogenic death and an immune system response during this IPEC modeling.Finally, we have been able to demonstrate in vivo that the treatment of intraperitoneal metastases of Balb/c mice by an intraperitoneal injection of oxaliplatin in hypotonia slowed down tumor nodules appearance and increased survival of the mice.Thus, in this work we highlighted that hypotonia is one of the fundamental parameters of IPEC which suggests that its use could make it possible to increase the efficacy of IPEC and maybe to prolong the survival of patients.

Ulcerative colitis is a chronic inflammatory disease that mainly affects the colon and rectum. Onset of disease is most common between the ages of 15-35 years. There is an observed increased risk of colorectal cancer associated with the disease. The risk is often described to be 2% after 10 years, 8% after 20 years and 18% after 30 years disease. Since 1977, all known patients with ulcerative colitis in the catchment area of Örnsköldsvik Hospital have been invited to attend a colonoscopic surveillance programme. At endpoint of the studies included in this thesis there were 214 patients that had attended the surveillance programme. The aims of these studies have been to evaluate the efficiency of the surveillance programme, analyse the impact of findings of DNA aneuploidy, and determine the outcome for patients that underwent limited resections instead of complete proctocolectomy. Further, we have studied the long-term outcome for patients who had an early onset of disease and analysed the expression of cytokeratin 7 and 20 in respect to findings of dysplasia, DNA aneuploidy and colorectal cancer. At the end of the studies the prevalence for ulcerative colitis was 261/100 000 and the incidence rate was 7.6/100 000/year. During the period 1977-2005, four patients died of ulcerative colitis. Nine colorectal cancers were diagnosed in eight patients, one of whom died of the cancer. Fifty-two patients had findings of dysplasia and five of these patients developed colorectal cancer. In the subgroup of patients studied (N= 147) for DNA aneuploidy, 20 were found to have specimens with DNA aneuploidy on at least one occasion. The sensitivity of aneuploidy for development of dysplasia (LGD or higher) was found to be 0.50 and the specificity 0.94. The investigation of the outcome for the patients that underwent limited resections of the colon or rectum showed that none of the patients under surveillance died of colorectal cancer or metachronous cancer in their remaining colon or rectum. A separate study concerning early onset of ulcerative colitis revealed no particular increased risk of colorectal cancer in this cohort but a fairly high incidence of primary sclerosing cholangitis was seen. In the analyses of cytokeratins it was found that 7 out of 10 patients with low-grade dysplasia and 3 of 6 with high-grade dysplasia were positive for CK7. Our results indicate a possible relationship between the expression of CK7 and CK20 and neoplastic development of colorectal mucosa in patients with ulcerative colitis. The studies on which this thesis is based, were performed on a relatively small number of patients, however the time of observation was long and, most importantly, the patients were from a well defined catchment area. We conclude that the surveillance programme has been efficient in minimising the risk of lethal colorectal cancer. Analysing DNA ploidy helps to target the patients that need more attention but the method cannot stand alone. Our study on cytokeratins points to a relationship between dysplasia and CK7 but the results are preliminary and further studies needs to be done. We have shown that it is safe to do a limited colorectal resection in respect to lethal colorectal cancer. Early onset of ulcerative colitis as a risk factor for colorectal cancer was not found in the group we have studied, which could be due to effective surveillance and/or medication. A fairly high operation rate in this group may also have contributed. The most important variable in the beneficial outcome regarding lethal colorectal cancer in these studies is, in our opinion, the outstanding compliance of the patients to the colonoscopic surveillance programme.

Background: Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality. About 50 % of patients with CRC will develop subsequent liver metastases (CLM). The survival for untreated CLM is only a few months and liver resection provides the only chance for a lasting cure. It is therefore essential to detect CLM early, enabling successful surgical resection and achieving a long-term cure. There are no optimal tumour markers for CRC or CLM. The best marker available is Carcinoembryonic Antigen (CEA), a marker found elevated in about 50-60% of patients with CLM, but also in many other conditions. The main focus of cancer research has been on the malignant cancer cell. However, a tumour consists of more than cancer cells. A major part of all solid tumours is made up by the stroma. The tumour stroma is defined as the non-malignant cells of a tumour such as fibroblasts, the cells of the vascular and immune systems as well as the extracellular matrix (ECM). The basement membrane (BM) is a specialized form of the ECM in which type IV collagen is the major protein component. All epithelial cells need a contact to the BM and the definition of an invasive cancer is the degradation of the BM and the spread of cancer cells beyond this structure. Different metastatic growth patterns of CLM have previously been described, namely the desmoplastic, pushing and replacement type of CLM. These differ in their stromal reaction in the border, which separates the tumour from the normal liver. In this thesis the tumour stroma of CRC and CLM is studied with a special emphasis on stromal collagens. The aim is to investigate whether stromal collagens/ circulating type IV collagen can be used as tumour markers for CRC and CLM, and to compare this to the conventional marker CEA. The circulating type IV collagen level is also measured in liver metastases from other primary tumours than CRC. Furthermore, the differences between the stroma of a primary CRC that metastasizes to the liver when compared to a CRC that never spreads are analysed. Additionally, the metastatic growth pattern of CLM is studied in relation to the primary tumour, stromal components and survival. We also sought out to find whether CRC cell lines possess the trait to produce ECM proteins endogenously, and in response to a normal liver stroma in a novel organotypic model for CLM. Methods: Expression patterns of type I, III and IV collagen were studied by immunofluorescence (IF), chemical staining and immunohistochemistry (IHC) in normal colorectal tissue, normal liver, CRC, CLM, benign liver lesions and in liver metastases of other origin than CRC. Circulating plasma levels of type IV collagen were analysed in healthy controls, patients with CRC (T stage I-III) and in patients with CLM. Samples were analysed at the time of diagnosis, during and after oncological and surgical treatment and at the time of relapsing or progressive disease. Additionally, circulating levels were analysed in patients with benign liver lesions and in liver metastases of other origin than CRC. The metastatic growth pattern of CLM was classified according to earlier descriptions. CRC cell lines were studied regarding their production of type IV collagen. The growth, invasiveness and stromal production in CRC cell lines were also investigated in a new organotypic model for CLM using human liver specimens. Results: Circulating type IV collagen levels are increased in patients with CLM and other epithelial-derived liver metastases, and is found normal in patients with primary CRC (stage I-III), with liver metastases from tumours of non-epithelial origin, benign liver lesions and in healthy controls. The type IV collagen levels in patients with CLM reflect the tumour burden in the liver, decreases in response to therapy and is found increased in progressive or relapsing disease. The combination of circulating type IV collagen and CEA increased the sensitivity and specificity for detecting CLM. Livermetastatic CRC displayed an increased stromal production when compared to non-metastatic CRC, with an increased type IV collagen expression in the direct vicinity of the CRC cells. The earlier described growth patterns of CLM were verified, with the pushing type of CLM associated with a short survival and poor outcome. Furthermore, CRC cell lines possess the trait of endogenously producing type IV collagen. The novel organotypic liver model revealed that CRC cell lines grown in the context of normal liver stroma, devoid of other cells, does not elicit a desmoplastic reaction. Conclusion: Circulating type IV collagen is a promising tumour marker for CLM, where the levels reflect the hepatic tumour burden and can detect disease relapse after liver surgery. The combination of the tumour markers CEA and type IV collagen is superior to CEA alone. The stromal composition of primary CRC predicts the risk of subsequent CLM and the metastatic growth pattern of CLM is related to survival.

Putrescine, spermidine and spermine are the polyamines biosynthesized by human cells via ornithine decarboxylase (ODC) and are also sourced from the diet. Polyamines are required for malignant and normal cell growth and development. Pharmacological suppression of polyamine biosynthesis, by difluoromethylornithine, and inflammation, via sulindac, has demonstrated ~70% efficacy in preventing premalignant colorectal adenomas (CRA) in a clinical trial; however, high polyamine intakes mitigated this preventative action. Further, dietary polyamines increase the dysplasia of CRA in initiated animal models of colorectal cancer (CRC) and are hypothesized to function as tumor promoters. Human research on dietary polyamines was limited until the development of a dietary database in 2007 but, continues to be limited by the lack of a biomarker of exposure. Chapter 1 of this dissertation tests the hypothesis that dietary polyamines increase risk of CRA in polyp-formers (n = 1164) and found evidence to support this hypothesis. However, only women, younger participants and certain genotypes experienced more risk of CRA with high polyamine exposure. Chapter II tests the hypothesis that dietary polyamines increase the risk for CRC in an average risk cohort of post-menopausal women (n = 87,620) and did not find evidence to support this hypothesis in the whole population. Rather, dietary polyamines were non-significantly protective against CRC and significantly protective when paired with aspirin use and against CRC-specific death. There was some evidence to support an increase in risk of CRC in younger participants with high polyamine exposure. Overall, the first two chapters suggest that dietary polyamines protect the colorectum in normal risk individuals but promote carcinogenesis in high risk individuals. Chapter III tests the hypothesis that dietary polyamine intake correlates with urinary polyamine output in a group of overweight/obese, older men (n = 36) and Chapter IV tests the hypothesis that intake of highly ripe sweet cherries will increase urinary polyamine output in a subgroup of 10 men from Chapter III. The findings from these chapters suggest there may be a positive correlation, but that a better measure of dietary polyamine intake is needed to determine if urinary polyamines are biomarkers of exposure to polyamines.