Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Durante las periodontitis, la respuesta inmune del hospedero involucra la producción de citoquinas, metaloproteinasas de la matriz, quimioquinas y receptores de quimioquinas que determinan la constitución de un ambiente inflamatorio local y la estimulación de la destrucción de tejidos periodontales, pudiendo derivar en la pérdida de los dientes. Las quimioquinas (CCLs) son citoquinas quimiotácticas que cumplen un rol fundamental en la regulación de la migración de células del sistema inmune. La interacción de las quimioquinas CCL19 y CCL21 con su receptor CCR7 en las células inmunes, incrementa la secreción de citoquinas proinflamatorias y activa la diferenciación de los linfocitos Th1 y Th17. Por lo tanto, el objetivo de este estudio es determinar los niveles de expresión de las quimioquinas CCL19 y CCL21 en biopsias gingivales de pacientes afectados de periodontitis crónica. Los resultados demuestran los elevados niveles de expresión de las quimioquinas CCL19 y CCL21 en biopsias de pacientes afectados con periodontitis en comparación a los sujetos sanos y sugieren un rol pro-inflamatorio en la regulación del sistema inmune y el desarrollo de la enfermedad.

Notre laboratoire a démontré que le récepteur de chimiokine CCR7 joue un rôle majeur dans la migration des monocytes en réponse à ses ligands naturels, les chimiokines CCL19 et CCL21. Plusieurs études ont permis de mettre en évidence que les monocytes en réponse à des infections, des traumatismes ou des maladies neurologiques, migrent vers les tissus en inflammation du cerveau par un mécanisme jusqu’à présent inconnu. D’un autre côté, l’activation de certains récepteurs TLR (de l’anglais « Toll-like receptors ») par des composants bactériens est caractéristique aux infections bactériennes et tient un rôle primordial dans la réponse immunitaire. L’objectif principal de ce projet de maîtrise est de déterminer si l’activation des TLR bactériens module l’expression de CCR7 chez les monocytes, les rendant ainsi aptes à transmigrer à travers la barrière hémato-encéphalique (BHE). Pour ce faire, nous avons d’abord développé un modèle in vitro de la BHE à partir de co- culture d’astrocytes humains et de cellules endothéliales microvasculaires humaines. Les résultats de résistance électrique transendothéliale (de l’anglais « Transendothelial Electrical Resistance » (TEER)) démontrent que ce modèle est fonctionnel et possède des jonctions serrées rendant la BHE imperméable et plus près de la réalité. Des essais de transmigration de monocytes démontrent que le modèle de la BHE construite limite le passage des monocytes à travers la barrière. Ces résultats laissent supposer que la BHE limite donc le passage des monocytes à travers le SNC. Cette imperméabilité aux cellules, propre à la BHE in vivo, rend le modèle idéal à des essais chimiotaxiques de transmigration. Par la suite, nos travaux ont permis de déterminer si l’activation de certains TLR par des composants provenant de bactéries Gram + et Gram – sont capables de moduler l’expression iv et la fonctionnalité de CCR7 chez les monocytes. Des PCR quantitatives effectuées avec la lignée MONO-MAC-1 ont démontré que l’activation des TLR1/2 et TLR4 promeut l’expression de l’ARNm codant pour CCR7. Des essais de cytométrie en flux ont démontré que l’activation de TLR1/2 et TLR9 chez la lignée MONO-MAC-1 ainsi que l’activation de TLR4 chez les monocytes sanguins augmentait le pourcentage de cellules exprimant CCR7 à leur surface. Des essais de migration ont démontré que le récepteur CCR7 était fonctionnel puisque la lignée MONO-MAC-1 et les monocytes sanguins traités avec l’agoniste de TLR4 migraient en plus grand nombre en réponse aux agonistes de CCR7, les chimiokines CCL19 et CCL21. Nos travaux ont aussi démontré que le récepteur CCR7 exprimé par les MONO- MAC-1 et monocytes sanguins avait une plus grande affinité envers CCL19 que CCL21. L’activation de TLR4 promeut la migration CCR7 dépendante des MONO-MAC-1 et des monocytes sanguins à travers le modèle in vitro d’une barrière hémato-encéphalique en réponse à CCL19. Nos résultats suggèrent une implication importante des récepteurs TLR4 dans la migration CCR7 dépendante des monocytes à travers la BHE et donc qu’une infection bactérienne ou que la présence de LPS dans le sang peut influencer la migration des monocytes au cerveau exacerbant ainsi des conditions neurologiques pathologiques. Davantage d’études portant sur les rôles des TLR dans la migration cellulaire pourraient permettre de développer de nouvelles thérapies.

Chemokines are a family of chemotactic cytokines that play a multifaceted role in human biology. Chemokine receptor CCR7, activated by its ligands CCL19 & CCL21, is known to play a significant role in cancer metastasis. In view of the wider roles that chemokines play in the biology of the cell, we hypothesised that CCR7 could influence cancer progression through other mechanisms in particular increased proliferation and/or increased chemo- and radioresistance, and whether these effects may have a physiological/clinical relevance. Interestingly, CXCR4 involvement in cancer recurrence, metastasis and proliferation is already well established. Thus, it was used as a point of reference to compare whether CCR7 axis effect on cancer proliferation and chemoresistance is similar to that observed in CXCR4 axis. It is proven that hypoxia is a driving factor in increased CCR7 expression. This study shows that CCR7 expression is upregulated as a response to a number of other stress factors, in particular that caused by different chemotherapeutic treatments. In addition, we showed that CCL21, one of the two endogenous ligands for CCR7 is similarly produced under these conditions We used several techniques to establish the expression and functionality of CCR7 and CXCR4 in different cancer cell lines. We then showed that activation of the CCR7 axis by physiologically relevant concentrations of CCL21 induces cancer cell proliferation and chemo- and radio-resistance. Furthermore, these effects are abrogated by small molecule antagonists (ICT13069), neutralizing monoclonal antibody, or CCR7 knockdown. Our findings support the hypothesis that antagonising CCR7 receptor will not only inhibit cancer metastasis, as it is well-illustrated in the literature, but it would also lead to alternative therapeutic approaches as well as potential clinical endpoints. / University of Tobruk, and the Ministry of Higher Education of Libya

Yes / The CCR7 chemokine axis is comprised of chemokine ligand 21 (CCL21) and chemokine ligand 19 (CCL19) acting on chemokine receptor 7 (CCR7). This axis plays two important but apparently opposing roles in cancer. On the one hand, this axis is significantly engaged in the trafficking of a number of effecter cells involved in mounting an immune response to a growing tumour. This suggests therapeutic strategies which involve potentiation of this axis can be used to combat the spread of cancer. On the other hand, the CCR7 axis plays a significant role in controlling the migration of tumour cells towards the lymphatic system and metastasis and can thus contribute to the expansion of cancer. This implies that therapeutic strategies which involve decreasing signaling through the CCR7 axis would have a beneficial effect in preventing dissemination of cancer. This dichotomy has partly been the reason why this axis has not yet been exploited, as other chemokine axes have, as a therapeutic target in cancer. Recent report of a crystal structure for CCR7 provides opportunities to exploit this axis in developing new cancer therapies. However, it remains unclear which of these two strategies, potentiation or antagonism of the CCR7 axis, is more appropriate for cancer therapy. This review brings together the evidence supporting both roles of the CCR7 axis in cancer and examines the future potential of each of the two different therapeutic approaches involving the CCR7 axis in cancer.

Chemokines are responsible for the directed migration of leukocyte chemotactic cytokines, coordinating cell movement during inflammation and the transport of hematopoietic cells. In addition to leukocytes, chemokine receptors are also found in neoplastic cells and tumors associated with stromal cells. Among chemokines, and the CXCR4/CXCL12 CCR7/CCL21 systems have been shown the involvement of lymph node metastases or distant metastases in different cancers. Thus, aim of this study was to evaluate the expression of CXCR4, CXCL12, CCR7, CCL21 and Ki-67 in oral squamous cell carcinoma (SCC) and to correlate these markers with clinicopathological indicators, lymph node metastasis and survival. We conducted a survey of reports and paraffin blocks of excisional biopsies of patients with SCC treated at the Hospital Haroldo JuaÃaba (2001-2009). Data on anatomic location of the lesion, sex, age, patient survival, degree of histological differentiation of the tumor, tumor stage and presence or absence of lymph node metastasis, lymphovascular and perineural invasion, nuclear grade and depth of invasion were collected. For immunohistochemical analysis, followed by the technique of streptavidin-biotin-peroxidase using the anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 and Ki-67 antibody. Histological sections were photomicrographed in 10 fields chosen randomly and measured for the number of labeled tumor cells and determined the percentage of each labeling antibody. The marking of CXCR4 was detected in the cytoplasm and nucleus, CXCL12, CCR7 and CCL21 were only cytoplasmic, their expression was observed in 18 (60%) 8 (22.66%) 16 (53.3%) and 3 (12%) cases, respectively. We found a significant positive association between lymphovascular invasion and immunostaining of CXCR4 (p = 0.007) and CCR7 (P = 0.01) and among these cases metastasis was present in 62.5% and 37.5%, respectively. When in combination with Ki67, we found a significant positive correlation between CXCR4 (p = 0.0086), CXCL12 (p = 0.036) and CCR7 (p = 0:04). Among patients CXCR4 + over 111 months, only 38.4% were alive (p = 0.845), whereas both patients CCR7 + (p = 0.398) as well as CXCR4 +, and CCR7 + (p = 0.441) after 62 months, everyone had already died. We conclude that these chemokines are associated with lymphovascular invasion and cell proliferation, perhaps favoring the development of metastasis and poor prognosis. / As quimiocinas sÃo citocinas quimiotÃticas responsÃveis pela migraÃÃo direcionada de leucÃcitos, coordenando o movimento celular durante a inflamaÃÃo e o transporte de cÃlulas hematopoiÃticas. AlÃm dos leucÃcitos, os receptores de quimiocinas tambÃm sÃo encontrados em cÃlulas neoplÃsicas e em tumores associados com cÃlulas estromais. Dentre as quimiocinas, os sistemas CXCR4/CXCL12 e CCR7/CCL21 tÃm sido demonstrado no envolvimento de metÃstases linfonodais ou à distÃncia em diferentes tipos de cÃncer. Dessa forma, foi objetivo desse trabalho avaliar a expressÃo de CXCR4, CXCL12, CCR7, CCL21 e Ki-67 em carcinoma de cÃlulas escamosas orais (CEC) e correlacionar estes marcadores com indicadores clÃnicopatolÃgicos, metÃstase linfonodal e sobrevida. Realizou-se um levantamento de laudos e blocos parafinados de biopsias excisionais de pacientes portadores de CEC tratados no Hospital Haroldo JuaÃaba (2001 a 2009). Foram coletados dados sobre localizaÃÃo anatÃmica da lesÃo, sexo, idade, sobrevida do paciente, grau de diferenciaÃÃo histopatolÃgica do tumor, estadiamento tumoral e presenÃa ou ausÃncia de metÃstase linfonodal, invasÃo linfovascular e perineural, grau nuclear e profundidade de invasÃo. Para reaÃÃo de imunohistoquÃmica, seguiu-se a tÃcnica da estreptavidina-biotina-peroxidase, utilizando os anticorpos anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 e Ki-67. As secÃÃes histolÃgicas foram fotomicrografadas em 10 campos escolhidos aleatoriamente e quantificadas quanto ao nÃmero de cÃlulas tumorais marcadas e determinado o percentual de marcaÃÃo de cada anticorpo. A marcaÃÃo de CXCR4 foi detectada em citoplasma e nÃcleo, CXCL12, CCR7 e CCL21 tiveram marcaÃÃo apenas citoplasmÃtica, sendo observada suas expressÃes em 18 (60%), 8 (22,66%), 16 (53,3%) e 3 (12%) casos, respectivamente. Encontrou-se uma associaÃÃo significativa positiva entre a invasÃo linfovascular e a imunomarcaÃÃo do CXCR4 (p=0.007) e CCR7 (p=0.01) e dentre esses casos a metÃstase esteve presente em 62,5% e 37,5%, respectivamente. Quando em associaÃÃo com o Ki67, encontrou-se uma correlaÃÃo positiva significante entre o CXCR4 (p=0.0086), CXCL12 (p=0.036) e CCR7 (p=0.04). Dentre os pacientes CXCR4+, ao longo de 111 meses, apenas 38,4% estavam vivos (p=0.845), ao passo que tanto para pacientes CCR7+ (p = 0.398), quanto CXCR4+ e CCR7+ (p = 0.441), apÃs 62 meses, todos haviam ido a Ãbito. Conclui-se que essas quimiocinas estÃo associadas com a invasÃo linfovascular e proliferaÃÃo celular, talvez favorecendo o desenvolvimento de metÃstases e um pior prognÃstico.

Chemokines are chemotactic cytokines, which mediate cell trafficking and play a key role in mobilisation of leukocytes. More recently, chemokines and their cognate receptors have been described as key players in different aspects of cancer biology contributing to proliferation, angiogenesis and metastasis. In particular, chemokines CCL19 and CCL21 acting on their associated receptor CCR7 are postulated to be key drivers of lymph node metastasis in a number of malignancies including breast, colon, gastric, & thyroid cancers. It has been reported that the cleavage of the pre-cysteine bridge N-terminal residues of CCL21 (SDGGAQD) and of CCL19 (GTNDAED) renders both peptides incapable of fully activating CCR7. However, little is known about the nature of the interactions that occur between the N-terminal residues of CCL19 or CCL21 and the CCR7 receptor, or the role they have in activation of CCR7. The aim of this study is to investigate the role of the residues in the N-terminus of CCL19 and in particular CCL21 in the context of CCR7 activation and to use this information in the discovery of novel CCR7 antagonists or agonists. To achieve this, we synthesised a number of short (three to seven amino acids) peptides and peptidomimetics inspired by the seven N-terminal amino acid residues of CCL19 and CCL21 and pharmacologically characterised their ability to activate CCR7 or block the activation of CCR7 using a number of in vitro assays such as calcium flux, trans-well (Boyden chamber), and Western blotting. We also carried out computational studies to better understand and predict the activity of these peptides. Our results demonstrate that some of these peptides are indeed capable of acting as agonists or antagonists of CCR7. / Kuwait Health Ministry and Kuwait Civil Services Commission

The lymphatic and blood vascular systems are two important vessel networks that serve different roles in healthy states and in cancer. In breast cancer the most common cancer amongst women, mortality remains high despite increased treatment response due to metastatic spread, preferentially through the lymphatics. One aggressive subtype, triple negative breast cancer (TNBC) contributing to 15 to 30 percent of cases and is characterized by the absence of expression of three therapeutic biomarkers. As targeted therapy is limited, treatment relies on standard of care via surgery, radiotherapy, and chemotherapy with limited efficacy and increase in survival. Chemotherapies negatively alter the lymphatic vasculature benefiting the tumor, through lymphangiogenesis. This dissertation seeks to understand how the mechanisms of commonly used chemotherapeutics, like carboplatin, and a novel 2nd generation ablative therapy called High Frequency Irreversible Electroporation (H-FIRE), which utilizes electric pulses to ablate tumor cells, affect the lymphatic and blood microvasculature in the tumor, surrounding fat pad, tumor draining lymph node (TDLN) using multiple analysis methods. This occurred through three main methods 1) identification of oxidative stress effects of chemotherapeutic application of carboplatin on lymphatic endothelial cells in vitro, 2) characterization of lymphatic and blood microvascular dynamics in a 4T1 breast cancer mouse model treated with sub-ablative H-FIRE, 3) through the development of a novel habitat imaging method to identify treatment specific changes in the tumor draining lymph node, and the development of a hybrid agent-based model (ABM) to test cancer cell flow mediated invasion in brain cancer. Herin the work showed that carboplatin induced lymphatic phenotypic changes occurred through generation of reactive oxygen species dependent on VEGFR3 and was reversed through treatment with the antioxidant N-acetylcysteine. In the 4T1 model, sub ablation with H-FIRE induced temporal remodeling of the lymphatic and blood vasculature within the viable tumor, in the surrounding fat pad, and in the tumor draining lymph node over seven days, suggesting an optimal time of application of adjuvant therapy. The development of a habitat imaging analysis method to identify TDLN vascular habitats and the perturbation to treatment in a retrospective analysis of prior work. Lastly, the development of a hybrid ABM through the incorporation of experimentally measured fluid flow fields from dynamic contrast enhanced MRI imaging building upon existing work, and showing the usefulness in comparing mechanisms of cancer cell invasion mediated fluid flow. Altogether, this work presents novel insight into the lymphatic system in cancer within various treatments contexts and new methods of quantifying changes due to treatment. Hopefully, these findings can be used to further inform the field towards a more comprehensive understanding of treatment effects in breast cancer. / Doctor of Philosophy / The lymphatic and blood vascular systems are two important vessel networks that serve different purposes in healthy states and in the disease called cancer. In breast cancer , a common form of cancer in women , spread of this cancer tends towards the lymphatic vasculature and eventually to other parts of the body. Triple negative breast cancer (TNBC) a less common, but more aggressive form, relies on clinical standard treatments with anti-tumor drugs called chemotherapies. These chemotherapies negatively alter the lymphatic vasculature to the tumors benefit, leaving a lack new methods of treatment. This dissertation seeks to understand how the mechanisms of commonly used chemotherapeutics and a new promising pulsed electric field therapy , High frequency Irreversible Electroporation (H-FIRE), change the lymphatic and blood vessels over time and in different locations using different tools. This occurred through three main methods 1) the effects on lymphatic vascular cells treated with chemotherapy, 2) in a breast cancer mouse model treated with H-FIRE, 3) in math models of the draining lymphatic organ, called the lymph node and an agent-based math model (ABM) of cancer cell movement due to fluid flow. The work showed that in the lymphatic cells, carboplatin a type of chemotherapeutic used to treat breast cancer, changed lymphatic vasculature through generating stress through oxidation and was reversed through treatment with an anti-oxidant. In the breast cancer mouse model, incomplete ablation with H-FIRE caused time dependent changes to the lymphatic and blood vasculature in the tumor, in the surrounding tissue, and in the lymph node over seven days. This work shows the novel findings of pulsed electric field therapy causing changes to the lymphatic vasculature. The creation of a new method of identifying habitats of the lymph node was used to compare changes to the lymphatic and blood vasculature to treatment. Lastly, the creation of an ABM added measured fluid flow maps from medical imaging methods to build upon existing work, and showed the usefulness in comparing mechanisms of cancer cell invasion due to fluid flow. Altogether, this work presents novel insight into the lymphatic system in cancer within after various treatments are applied and new methods of measuring these changes because of treatment using multiple methods. It is our hope that these findings can be used to further inform the field towards a more comprehensive understanding of treatment effects in breast cancer.

Το λεμφαγγειακό σύστημα διαδραματίζει σημαντικό ρόλο στη διατήρηση της ομοιόστασης των ιστών, στην ανοσολογική απόκριση του οργανισμού, στην απορρόφηση των λιπών από τον πεπτικό σωλήνα, και στη διασπορά των καρκινικών κυττάρων. Η πρόσφατη ανακάλυψη ειδικών για τα λεμφαγγειακά ενδοθηλιακά κύτταρα δεικτών και αναπτυξιακών παραγόντων, όπως επίσης και η δημιουργία γενετικών μοντέλων ποντικιών με διαταραγμένη τη λειτουργία του λεμφαγγειακού συστήματος, παρείχαν σημαντικότατες πληροφορίες για τη μοριακή ρύθμιση της εμβρυικής ανάπτυξης του λεμφαγγειακού συστήματος και της φυσιολογίας του. Τα σχετικά πρόσφατα αναγνωρισμένα μοριακά σηματοδοτικά μονοπάτια από τα οποία ρυθμίζεται η λεμφαγγειογένεση επέτρεψαν τη μελέτη της σχετιζόμενης με όγκους λεμφαγγειογένεσης. Οι μελέτες αυτές κατέδειξαν ότι η σχετιζόμενη με τους όγκους λεμφαγγειογένεση αποτελεί σημαντικό στοιχείο της μεταστατικής διαδικασίας, ενώ παράλληλα αναδεικνύουν συνεχώς καινούρια μόρια/σηματοδοτικά μονοπάτια-ρυθμιστές της εν λόγω διαδικασίας. / The lymphatic vascular system plays an important role in the maintenance of fluid homeostasis, in the afferent immune response, in the intestinal lipid uptake and in the metastatic spread of malignant cells. The recent discovery of specific markers and growth factors for lymphatic endothelium and the establishment of genetic mouse models with impairment of lymphatic function have provided novel insights into the molecular control of the lymphatic system in physiology and in embryonic development. Recently, molecular pathways that signal for lymphangiogenesis have been described allowing analyses of tumor lymphangiogenesis to be performed. These studies demonstrate that tumor lymphangiogenesis is a major component of the metastatic process, while at the same time new molecules and transduction pathways are discovered to regulate tumor lymphatics growth.