Made available in DSpace on 2014-10-09T12:33:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:12Z (GMT). No. of bitstreams: 0 / O diagnóstico precoce de tumores e metástase constitui atualmente o elemento de maior impacto dentro das políticas de saúde públicas contra o câncer. No câncer de mama e melanoma, a técnica de biópsia de linfonodo sentinela, para o diagnostico de metástase, tem sido muito utilizada evitando a dissecção total dos nodos da região anatômica afetada, e permitindo definir com precisão o procedimento terapêutico a utilizar. O objetivo principal deste trabalho centrou-se no desenvolvimento de conjugados radiomarcados de dextran-manose para diagnóstico, utilizando o núcleo de tecnécio altamente estável, [99mTc(CO)3]+. A cisteína, ligante tridentado, foi incorporada na estrutura dos conjugados, como agente quelante do Tecnécio-99m. As condições de marcação definidas para os produtos avaliados garantiram altos valores de pureza radioquímica (>90%) e atividade específica (>59,9 MBq/nmol) assim como uma alta estabilidade in vitro. Os conjugados de dextran-cisteína-manose demonstraram uma captação superior (4 vezes maior) nos nodos linfáticos em relação aos homólogos que não possuíam manose na estrutura. O conjugado de dextran-cisteína-manose de 30 kDa radiomarcado (99mTc-DCM2) foi o traçador com melhor desempenho biológico entre os avaliados à diferentes atividades injetadas. Demonstrou-se que concentrações superiores a 1 M favorecem a retenção do produto nos nodos linfáticos. As comparações com radiofármacos já utilizados no Brasil (Dextran-500 e Fitato) para detecção de linfonodo sentinela evidenciaram a superioridade do 99mTc-DCM2. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:45:37Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:08:41Z (GMT). No. of bitstreams: 0 / O 153Sm-EDTMP é um radiofármaco utilizado em medicina nuclear com resultados promissores no alívio da dor metastática. No entanto, pouco se sabe sobre os efeitos do 153Sm-EDTMP em nível celular. O presente trabalho foi conduzido com o intuito de avaliar os efeitos citogenéticos do 153Sm-EDTMP em linfócitos periféricos de pacientes com metástases ósseas (com e sem radio e/ou quimioterapias anteriores) pela da técnica de detecção de aberrações cromossômicas, tanto in vivo como in vitro. Para tanto, as amostras sangüíneas foram coletadas antes e 1 hora após a administração endovenosa do 153Sm- EDTMP (atividade média de 42,53 + 5,31 MBq/kg de peso corpóreo), levando-se em consideração o rápido clearance sangüíneo. Os principais tipos de aberrações cromossômicas estruturais encontrados foram os gaps e quebras, fragmentos acêntricos, anéis cêntricos, double minutes e dicêntricos. A análise estatística mostrou que o único grupo de pacientes que apresentou uma diferença significativa na freqüência de aberrações cromossômicas 1 hora após o tratamento foi o que recebeu prévio tratamento radio e quimioterápico antes da terapia com 153Sm-EDTMP. Quanto a averiguação do número modal de cromossomos e da cinética do ciclo celular, a análise estatística mostrou que não houve diferença significativa entre os grupos analisados, sugerindo que o tratamento com 153Sm-EDTMP não influenciou nesses parâmetros. A molécula carreadora, EDTMP, não teve qualquer influência na indução de aberrações cromossômicas. Em relação aos ensaios in vitro, os dados obtidos de linfócitos periféricos submetidos às diferentes concentrações radioativas de 153Sm-EDTMP (0,046 – 1,110 MBq/mL) de doadores sadios e de pacientes sem prévio tratamento se ajustaram melhor ao modelo de regressão linear (Y=A+BX). O dano cromossômico induzido pelo 153Sm-EDTMP observado in vitro foi cerca de 2 vezes maior do que o encontrado in vivo para o grupo de pacientes sem prévio tratamento. Os dados obtidos mostraram que a terapia com 153Sm-EDTMP induziu uma pequena quantidade de danos citogenéticos em linfócitos periféricos de pacientes 1 hora após sua administração, embora, teoricamente, um efeito estocástico a longo prazo não possa ser descartado. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Metástases para a região oral podem ocorrer nos tecidos moles ou nos ossos maxilares. Tumores metastáticos para a cavidade oral são raros, compreendendo aproximadamente 1% das neoplasias encontradas na região oral. Devido à sua raridade, o diagnóstico de uma lesão metastática na região oral é difícil; tanto para o clínico como para o patologista, ao reconhecer que uma lesão é metastática e na determinação do local de origem. Foram revisados 9 casos sendo 5 mulheres e 4 homens com idades entre 57 e 80 anos e realizada uma crítica revisão da literatura. No presente estudo não foi possível determinar a prevalência das metástases para os ossos maxilares ou para os tecidos moles da cavidade oral; Entretanto, nosso estudo mostra que o exame das estruturas orais é absolutamente fundamental no acompanhamento desses pacientes, pois a presença de possíveis massas metastáticas pode indicar uma neoplasia oculta ou mesmo a falha terapêutica. / Metastasis to the oral region may occur in the soft tissue or jaw bone. Metastatic tumors to the oral cavity are rare, comprising about 1% of neoplasms found in the oral region. Because of its rarity, the diagnosis of a metastatic lesion in the oral region is difficult; both for the clinician and for the pathologist to recognize that an injury is metastatic and determination of the place of origin. 9 cases with 5 women and 4 men aged between 57 and 80 years and performed a critical review of the literature were reviewed. In the present study could not determine the prevalence of metastasis to the jaw bones or the soft tissues of the oral cavity; However, our study shows that the examination of oral structures is absolutely essential to monitor these patients, because the presence of possible metastatic masses may indicate a hidden cancer or treatment failure.

Hintergrund: Das Mammakarzinom stellt weltweit die häufigste maligne Tumorerkrankung der Frau dar und wird immer noch größtenteils als primär systemische Krebsform angesehen. Nach Primärbehandlung werden 5-Jahresüberlebensraten von 80% erreicht. Jedoch überleben Patientinnen, bei denen ein Stadium IV vorliegt, im median nur 20-30 Monate. 5-15% dieser Patientinnen weisen dabei einen isolierten metastatischen Befall der Lunge auf, der als Oligometastasierung im Sinne eines stabilen Zwischenstadiums der Erkrankung angesehen werden kann und somit einer lokalen Therapie zugänglich ist. Etliche Studien weisen darauf hin, dass gerade diese Frauen von einer Resektion ihrer Lungenmetastasen deutlich mehr profitieren können, als von medikamentöser systemischer Therapie allein. Zudem kann das Rezeptorverhalten (Östrogen-, Progesteron-, HER2-Rezeptor) zwischen primärem Mammakarzinom und dessen Metastasen differieren, was in bisher noch nicht geklärtem Umfang Änderungen des Behandlungsschemas zur Folge hat. Frage- und Zielstellung: Ziel der vorliegenden Arbeit ist es, das Outcome von Patientinnen mit pulmonal metastasiertem Mammakarzinom, deren Lungenmetastasen ausschließlich mit einer neuen parenchymsparenden 1318-nm-Lasertechnik reseziert wurden, im Verlauf zu untersuchen und unabhängige prognostische Faktoren zu identifizieren. Weiterhin soll der Nachweis einer Rezeptordiskordanz speziell für pulmonale Fernmetastasen erbracht und aufgezeigt werden, in welchen Größenordnungen mit solchen Rezeptorwechseln zu rechnen ist. Patientinnen und Methoden: Im Rahmen dieser retrospektiven Studie wurde vom 01.01.1996 bis 31.12.2012 bei insgesamt 102 Patientinnen im Alter von 33 bis 78 Jahren und einem Durchschnittsalter von 58 Jahren eine kurative pulmonale Laser-Metastasenresektion mit systematischer Lymphadenektomie vorgenommen. Vorgegebene Einschlusskriterien waren die vollständige Resektion und Kontrolle des Primärtumors sowie das Fehlen von extrapulmonalen/-thorakalen Metastasen bzw. deren präoperative erfolgreiche Therapie. Eine Limitierung bezüglich der Zahl der Lungenmetastasen wurde nicht vorgegeben, allerdings mussten die technische Resektabilität und die funktionelle Operabilität aus der präoperativen Diagnostik ableitbar sein. Mit Hilfe der Kaplan-Meier-Methode wurde das Gesamtüberleben des Patientenkollektivs sowie ausgewählter Subgruppen analysiert. Das Cox-Proportional-Hazard-Modell wurde verwendet, um im uni- und multivariaten Verfahren prognostische Faktoren zu ermitteln. Zum Vergleich des Rezeptorstatus von Primärtumor und Metastasen kam der McNemare-Test zum Einsatz. Eine statistische Signifikanz wurde bei p-Werten von < 0,05 angenommen. Ergebnisse: Insgesamt wurden 936 Lungentumore entfernt, von denen sich nach histopathologischer Sicherung 716 als Metastasen des anamnestisch bekannten Mammakarzinoms erwiesen. Die Anzahl reichte von einer solitären Metastase bis zu 61 zweizeitig entfernten Metastasen (durchschnittlich 7 pro Patientin). Die Lobektomierate betrug 0,98% (n=1). In 7,8% (n=8) der Fälle waren zusätzlich lappensparende Laser-Segmentresektionen möglich. R0-Resektionen konnten bei 73,5% (n=75) der Patientinnen erreicht werden. Das mediane Gesamtüberleben betrug 43 Monate, die 5-Jahresüberlebensrate belief sich auf 46,1%. Als unabhängige prognostische Faktoren konnten der Resektionsstatus (p=0,02), der intrathorakale Lymphknotenbefall (p=0,001) und die Expression des Östrogenrezeptors (p=0,018) nachgewiesen werden. Das Risiko zu versterben war bei tumorbefallenen Lymphknoten und bei fehlender Ausprägung des Östrogenrezeptors 3,2- bzw. 2-fach erhöht. Die Anzahl der resezierten Metastasen, die Art des Lungenbefalls (uni-/bilateral), das krankheitsfreie Überleben nach Primär-Operation (</> 36 Monate) und die Expression des Progesteronrezeptors hatten keinen signifikanten Einfluss auf das Überleben. Angaben zum primären und metastatischen Hormonrezeptor- bzw. HER2-Status waren bei 88,2% (n=90) bzw. 62,7% (n=64) der Patientinnen verfügbar. Es fanden sich Diskordanzraten bzgl. des Östrogen-, Progesteron- und HER2-Rezeptors von 26,7%, 41,1% bzw. 28,1%. Eine Signifikanz der Abweichung zwischen Primärtumor und Metastasen konnte lediglich für den Östrogenrezeptor nachgewiesen werden (p=0,002). In einer Nebenbetrachtung der vorliegenden Arbeit konnten bei 157 Mammakarzinom-Patientinnen mit neu aufgetretenen, radiologisch detektierten Lungenrundherden in 65,6% der Fälle Metastasen des Mammakarzinoms histologisch gesichert werden. Bei den übrigen Befunden handelte es sich um andere therapiebedürftige maligne Tumore und zu etwa 20% um benigne Befunde. Schlussfolgerungen: Die vorliegenden Ergebnisse bekräftigen den positiven Einfluss der Lungenmetastasektomie auf das Überleben ausgewählter Mammakarzinom-Patientinnen mit isolierter pulmonaler Oligometastasierung. Dabei können mit der Anwendung der parenchymsparenden 1318 nm -Lasertechnik, auch bei Vorhandensein von multiplen und beidseitigen Lungenmetastasen, in größerem Umfang als bisher berichtet, vollständige Resektionen ohne wesentlichen Funktionsverlust und somit guter Lebensqualität erreicht werden. Ungeachtet höherer Zahlen resezierter pulmonaler Metastasen werden gegenüber konventionellen Operationstechniken, auch beim pulmonal metastasierten Mammakarzinom, vergleichbare Überlebensraten erreicht. Die Anzahl der präoperativ diagnostizierten Lungenmetastasen sollte daher einen geringen Einfluss auf die Indikationsstellung zur Operation haben, weshalb diesbezüglich eine Erweiterung der Einschlusskriterien sinnvoll erscheint. Eine R0-Resektion konnte erneut als wichtigster prognostischer Parameter bestätigt werden und sollte deshalb stets oberstes Ziel des Operateurs sein. Das wesentlich schlechtere Outcome unvollständig operierter Patientinnen sowie der Vergleich mit der Literatur zur alleinigen systemischen Therapie zeigen, dass das analysierte Patientenkollektiv von einer Resektion der pulmonalen Mammakarzinom-Metastasen deutlich mehr profitieren kann, als von medikamentöser Behandlung allein. Ein intrathorakaler Lymphknotenbefall wurde, nach unserem Wissen, erstmals bei Patientinnen mit isolierten Lungenmetastasen eines Mammakarzinoms, trotz radikaler Ausräumung, als signifikante negative Einflussgröße auf das Überleben nachgewiesen. In Anlehnung an die Therapie des Lungenkarzinoms sollte trotz dessen, zumindest bis zum Vorliegen weiterführender Studien, standardmäßig eine intraoperative systematische Lymphadenektomie durchgeführt werden. Bei positivem Tumornachweis ist eine komplette Lymphknotendissektion zu erwägen, um keine Patientin von einer potenziell kurativen Therapie auszuschließen. Den vorliegenden Ergebnissen zufolge, darf des Weiteren speziell bei Verdacht auf pulmonale Mammakarzinom-Metastasen nicht von einer Konstanz der Expression der Steroidhormon- bzw. HER2-Rezeptoren, insbesondere der des Östrogenrezeptors, ausgegangen werden. Änderungen zum primären Befund treten dabei in relevanten Größenordnungen auf. Die Bestimmung eines aktuellen Rezeptorstatus sollte nach Metastasektomie obligat durchgeführt werden. Bezüglich der Frage des Ursprungs pulmonaler Rundherde bei bekanntem Mammakarzinom kann darüber hinaus durch deren Resektion mit nachfolgender histopathologischer Analyse sicher zwischen Metastasen, Lungenkarzinomen und benignen Tumoren differenziert werden. Insgesamt ermöglicht dies konkrete Therapieentscheidungen zu treffen. Um Patientinnen jedoch in zeitlich limitierter oligometastatischer Tumorausbreitung zu diagnostizieren und einer bestmöglichen Therapie, einschließlich der Resektion, zuzuführen, ist zufolge unserer Daten sowie der neueren Literatur eine konsequente, engmaschige und zudem apparative Nachsorge notwendig. Dieser Problematik wird gegenwärtig in den aktuellen Leitlinien nicht adäquat Rechnung getragen, da sich die Autoren auf ältere, heutzutage kritisch zu hinterfragende Analysen beziehen. Als Limitationen der vorgelegten Arbeit sind das retrospektive Studiendesign und die Form der Kohortenanalyse, die uneinheitliche Bestimmung des primären Rezeptorstatus sowie die Heterogenität der postoperativen Anschlusstherapien anzusehen. Zukünftig sind größere, multizentrische und randomisierte Studien notwendig, um weiterführende Daten zu generieren und die pulmonale Lasermetastasektomie beim Mammakarzinom im Rahmen multimodaler Therapien möglicherweise weiter zu etablieren sowie den Wert einer erweiterten Nachsorge zu evaluieren. / Background: Breast carcinoma is the most common type of cancer in women worldwide and is still regarded as a systemic disease. After primary treatment five-year survival rates around 80% are reported. However, the mean survival time of stadium-IV classified patients is 20-30 months. 5-15% of patients appear with isolated metastases of the lungs which can be considered as an oligometastatic and, therefore, stable intermediate stage in disease process. Several studies point out that especially these women are more likely to benefit from resection of lung metastases than from systemic therapy alone. Furthermore, there is the possibility of a discordant expression of typical receptors (Estrogen-, Progesterone- and HER2-receptor) between primary breast cancer and its paired metastases. As a result a change in treatment regimen might be necessary. Objective: The aim of the present study was to evaluate long-time survival of patients with lung metastases from breast cancer who have been operated exclusively with a new parenchyma-saving and lobe-sparing 1318-nm-lasertechnique. Additionally, the identification of independent prognostic factors was of interest. Furthermore, existence and magnitude of receptor discordance, specifically for distant pulmonary metastases, should be proved. Patients and methods: Within this retrospective study between 1996 and 2012 102 patients (mean age 58; range 33-78 years) underwent curative laser metastasectomy and systematic lymphadenectomy. Inclusion criteria were complete resection of primary breast cancer and absence of extrapulmonary/-thoracal metastases or its previous total treatment. Although there were no limitations regarding the number of metastases, technical resectability and functional operability had to be assumed after the preoperative diagnostics. Kaplan-Meier-analysis was performed to assess overall survival in all patients and selected subgroups. Uni- and multivariate analyses of prognostic factors were performed using the Cox-proportional-hazard model. Comparison of the receptor status of primary breast cancer and paired lung metastases was assessed by the McNemare method. Significant results were assumed if p-values were <0.05. Results: In total 936 intrapulmonary nodules had been resected, including 716 histopathologically confirmed breast cancer metastases. The amount reached from a single metastasis up to 61 two-staged removed pulmonary nodules (mean 7 per patient). The lobectomy rate was 0.98%. In 7.8% of all cases segment-resections, also performed by laser, were possible. Complete resection was achieved in 73.5% (n=75). The median overall survival time was 43 months and the five-year survival rate was 46.1%. As independent prognostic factors resection status (p=0.02), involvement of intrathoracal lymph nodes (p=0.001) and expression of estrogen receptor (p=0.018) were identified. The mortality rate in case of lymph node involvement and negative estrogen receptor status was increased by 3.2- and 2-fold, respectively. The number of resected metastases, type of lung affection (uni-/bilateral), disease free interval after primary breast surgery (</> 36 months), and expression of progesterone receptor had no significant influence on survival. Data concerning the primary and metastatic hormone receptor- and HER2-status were available in 88.2% (n=90) and 62.7% (n=64) of all cases, respectively. Discordant results appeared in 26.7%, 41.1%, and 28.1% regarding the estrogen-, progesterone- and HER2-receptor. Significance of these findings had only been proved for estrogen receptor (p=0.002). A subanalysis of the present study revealed that 65.5% of 157 breast cancer patients who presented with newly occurred radiologically detectable pulmonary nodules had histopathologically confirmed paired metastases. The remaining results showed malignancies other than known breast cancer, and in approximately 20% of all cases there were benign lesions. Conclusions: The results of the present study emphasize the favorable effect of the lung metastasectomy on survival of selected breast cancer patients with isolated pulmonary oligometastatic disease. Via the use of the parenchyma-saving 1318nm-lasertechnique even in case of distinct and bilateral pulmonary metastatic involvement, increased rates of complete resection without substantial loss of lung function can be achieved. Therefore, an adequate quality of life is provided. In comparison with conventional surgery practices, this procedure creates similar survival rates despite higher numbers of resected lung metastases. That is why the number of preoperatively diagnosed metastases should have little influence on decision upon surgery. Thus, an extension of inclusion criteria seems reasonable. Again, complete resection appeared as one of the most important prognostic parameters and should, therefore, be the main objective of the surgeon. The poorer outcome for women with incomplete resections and the results of studies on systemic therapy implicate once more that breast cancer patients are more likely to benefit from the resection of their pulmonary metastases than from medical treatment alone. Furthermore, for the first time according to our knowledge, despite radical excision intrathoracal lymph node involvement has been proved as a significant negative predictive determinant in a collective of patients with isolated pulmonary metastases of breast cancer. Nevertheless, an intraoperative systematic lymph node sampling should be considered, at least until further studies are presented. In reference to the surgical approach of lung carcinoma, as circumstances require, a complete lymph node dissection should be performed to provide potential curative treatment to those affected. Moreover, according to the present findings, in case of the appearance of lung metastases the constancy of metastatic steroid hormone- and HER2 receptor expression, especially of the estrogen receptor, cannot always be assumed. Changes in comparison to the primary carcinoma appear in a relevant number of cases. Thus, the current metastatic receptor status should be evaluated obligatorily after pulmonary metastasectomy. Regarding the origin of pulmonary nodules of patients with history of breast cancer, their surgical resection with subsequent histopathological analysis can reliably differentiate between metastases, lung carcinoma or benign tumors. Altogether this facilitates specific and accurate treatment decisions. However, to identify patients with a limited and stable oligometastatic state of disease and to introduce optimal treatment, including surgical resection, an early, continuous, and also instrument-based follow-up is necessary. This matter is still only slightly taken into account, while the authors of the current guidelines refer to out of date studies, which have to be seen critically. As limitations of the present investigation, the retrospective study design, inconsistent evaluation of the primary receptor status, and also heterogeneity of postoperative medical therapy must be mentioned. In the future larger, multicentric, prospective, randomized trials are necessary to acquire further data, to conceivably continue to establish the pulmonary laser metastasectomy in multimodal therapy settings and also to determine the value of an extended follow-up.

As solid tumor types, breast and prostate cancer are rivalled only by lung cancer in their propensity to metastasize to bone in the later stages of disease. At advanced stages of disease, approximately 80% of breast and 90% of prostate cancer patients will present with bone metastases. Bone metastases are often a painful conclusion to the lives of these patients, resulting in bone pain, hypercalcemia, pathological fractures and spinal cord compression. The culmination of these comorbidities considerably reduces a patient’s quality of life and prolonged survival. Hormone depletion is used as a first line of treatment in the majority of cases, negatively regulating bone health due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts. Not only is bone integrity undermined, but this action of increased bone turnover is beneficial for the colonization of metastasizing cells which co-opt and enhance the same mechanisms to establish and maintain their own growth. This is termed ‘the vicious cycle’ of osteolytic bone metastasis. Current research approaches aim to identify bone-targeted therapies which not only inhibit tumor growth but concurrently protect bone. In this study, Dickkopf-1 (DKK-1), mechanistic target of rapamycin (mTOR) and p38 mitogen-activated kinases (p38 MAPK) are presented as novel targets. Pro-tumor roles have been described for all and clinical trials are currently investigating their efficacy in different cancer types. In normal bone biology DKK-1 is an inhibitor of the canonical Wnt signaling pathway which promotes osteoblastogenesis while mTOR signaling is a promoter of osteoclastogenesis. P38 MAPK inhibitors have been shown to regulate DKK-1 expression and bone destruction in preclinical models of multiple myeloma. The aims of this current study were to 1) investigate the role of DKK-1 in the biology of osteotropic breast cancer, 2) to assess the potential bone protective effects of mTOR inhibition by everolimus in the context of osteotropic cancers and 3) to test the hypothesis that p38 MAPK is a regulator of DKK-1 expression in prostate cancer, potentially supporting an osteolytic phenotype by impairing osteoblastogenesis. In aim 1, analysis of a breast cancer tissue microarray demonstrated that DKK-1 expression was elevated in advanced and invasive tumor stages. Strikingly, positive DKK-1 expression correlated with a significantly reduced survival rate only in estrogen receptor-negative (ER-) breast cancer patients compared to patients with tumors which were negative for DKK-1 expression. In MDA-MB-231 breast cancer xenograft models, neutralization of secreted DKK-1 by treating mice with the monoclonal DKK-1 antibody BHQ880 or knocking out the expression of DKK-1 in MDA-MB-231 cells using CRISPR-Cas9 mediated gene editing, resulted in reduced tumor growth and burden by ≥ 50% (p < 0.05). In aim 2, the mTOR inhibitor everolimus is presented as an anti-tumor and bone-protective agent. The anti-tumor effects of everolimus were confirmed in two subcutaneous tumor models and a model of breast cancer bone metastasis, were tumor burden in the bone was reduced by 45.4% (p < 0.01). Bone loss induced by a hormone-deprived environment in ovariectomized mice was prevented with everolimus treatment as was bone destruction in the metastasis model. In more detail, it could be shown that everolimus maintained osteoblast function while specifically inhibiting osteoclast function. In aim 3, p38 MAPK is presented as a regulator of DKK-1 in prostate cancer. While the activation of p38 MAPK upregulated DKK-1, inhibition of p38 MAPK using small molecule inhibitors and siRNAs inhibited DKK-1 expression. Furthermore, assessment of different p38 MAPK isoforms revealed MAPK11 as the most effective regulator of DKK-1 and inhibition of DKK-1 by interfering with p38 MAPK signaling was sufficient to prevent the inhibitory effects of prostate cancer-derived DKK-1 on osteoblastogenesis in vitro. This study has assessed multiple targets and their concurrent roles in cancer and bone cell biology. Specifically, DKK-1 has been proven to be a tumor promoter in ER- breast cancer and can be targeted therapeutically to inhibit tumor growth. MTOR inhibition by everolimus has been shown to be an effective mono-therapy in ER- breast cancer, inhibiting the growth of subcutaneous tumor and bone metastases and preventing bone loss induced by estrogen ablation. This further supports its use in postmenopausal women with breast cancer who are predisposed to developing osteoporosis and bone metastases. It also supports the use of everolimus in hormone receptor-negative or triple receptor-negative breast cancer, for which it has not yet been approved. A clear link has been made between p38 MAPK signaling and DKK-1 expression in prostate cancer and its consequent regulation of osteoblastogenesis. A future focus on the inhibition of a specific MAPK isoform, MAPK11 in particular, may help in translating these encouraging in vitro results into promising pre-clinical trials in vivo. As a whole, these investigations provide a foundation for further research and could be valuable for the design of future clinical trials, leading to improvements in the treatment and prognosis of osteolytic bone metastases.

Les métastases osseuses sont une complication majeure des cancers du sein. Les bisphosphonates (BPs) bloquent la progression des lyses osseuses. Des effets anti-tumoraux ont été mis en évidence mais à des doses importantes incompatibles avec un usage clinique. La forte affinité des BPs pour le minéral osseux limite leur biodisponibilité et amoindrit donc leur potentiel antitumoral in vivo. Mon travail de thèse s'est inscrit dans le cadre de la potentialisation des effets anti-tumoraux des BPs. Les BPs ont été combinés avec des ultrasons de faible intensité (LIUS) dont le rôle est d'induire une stimulation mécanique et une augmentation modérée de la température du milieu insonifié sans effet de cavitation. Les LIUS favorisent la pénétration des BPs dans des cellules tumorales en augmentant le phénomène d'endocytose. In vivo, un traitement répété aux LIUS associé à une dose clinique de BPs entraine une diminution des lyses osseuses ainsi que de la masse tumorale. L'accumulation d'un marqueur, retrouvé dans les moelles des souris traitées aux LIUS, suggère que la pénétration des BPs a été favorisée sous l'effet des LIUS. L'effet de l'association BPs avec les LIUS a ensuite été évalué sur un modèle de tumeur sous-cutanée mammaire. Un ralentissement de la croissance tumorale lors des premiers jours de traitements a été mis en évidence. Une étude menée avec de la doxorubicine et des BPs a permis d'élargir le champ d'application des LIUS pour le traitement du cancer du sein. En conclusion, ces travaux montrent que les LIUS apparaissent comme une solution d'intérêt pour augmenter la pénétration de drogues dans des tumeurs osseuses et mammaires et augmenter leur potentiel antitumoral / Bone metastases are common complications of advanced breast cancer. They increase morbidity of patients and alter their quality of life. Bisphosphonates (BPs) stop the progression of osteolysis. However, BPs do not affect the tumor burden located inside the bone marrow cavity. Antitumoral effects have been shown but with high doses incompatibles with a clinical use. BPs bind also avidly to bone mineral which limits their bioavailability and reduce their antitumoral potential in vivo. This work is incorporated within the framework of the enhancement of antitumoral effects of BPs. BPs were combined with low intensity ultrasound (LIUS), which are known to induce a mechanical stimulation and a slight increase of temperature without involving cavitationnal effect. Initially, LIUS were found to increase the penetration of BPs inside several mammary tumor cell lines without affecting their viability by increasing endocytosis. In vivo, a daily repeated treatment of LIUS associated with a single and clinical dose of BPs lead to a decrease in osteolysis as well as tumor burden. The accumulation of unprenylated Rap1A form was found in bone marrow of mice suggesting that LIUS promote BPs penetration inside cells of the bone cavity. The effect of BPs and LIUS was evaluated in a subcutaneous mammary tumor xenograft. Tumor growth was slowed during the first days of LIUS treatment. A study was performed using doxorubicin and BPs leading to a better penetration of both compounds when LIUS were used. This last result allows increasing the field of application of LIUS for breast cancer treatment. In conclusion, this work showed that LIUS are an interesting method to enhance the penetration of drugs inside bone and mammary tumors leading to an increase of their antitumoral activity

Chez les patients atteints d’un cancer du sein ou de la prostate, le tissu osseux est souvent le siège de métastases qui présentent un phénotype essentiellement lytique dans le cas des métastases dérivant du cancer du sein, ou mixte (combinaison de lyse et de formation osseuse) dans le cas d’un cancer de la prostate. Le récepteur ERRalpha est impliqué dans la physiologie osseuse et il est considéré comme un facteur de mauvais pronostic dans ces deux types de cancers. Nous avons donc émis l’hypothèse qu'il pourrait être impliqué dans la formation des métastases osseuses associées à ces deux cancers. Nous avons observé que, dans les deux cas, ERRalpha stimule la progression tumorale au site primaire via la stimulation de l'angiogenèse et de l'expression du VEGF. Concernant les métastases osseuses dérivant du cancer du sein, ERRalpha inhibe les lésions ostéolytiques via la modulation de l'ostéoclastogenèse et de l'expression de son inhibiteur, l’OPG. A l'inverse, l'expression de ERRalpha stimule la formation de lésions lytiques induites par les cellules de cancer de la prostate en induisant l'expression du TGF-beta, de MCP-1 et de la cathépsine K, tout en induisant des zones de formation osseuse via la régulation de l'expression de l'OPG, de l’endothéline-1 et de membres de la famille Wnt. Ces résultats confirment la valeur de facteur de mauvais pronostic de ERRalpha dans la tumeur primaire. De plus, ils révèlent, pour la première fois, son implication dans le développement des métastases osseuses et suggèrent une dualité fonctionnelle de ERRalpha, comme inhibiteur et stimulateur du développement des métastases osseuses du cancer du sein et de la prostate, respectivement. Ces résultats suggèrent des mécanismes d'action différents pouvant dépendre des cellules tumorales mais aussi du microenvironnement et du statut hormonal / Breast cancer and prostate cancer patients, often developed bone metastases. As ERRalpha, an orphan nuclear receptor, is involved in bone physiology and is considered as a bad prognosis factor in breast and prostate cancer, we hypothesize that it can be implicated in bone metastasis development that derived from breast and prostate cancers. While we found that, in both cases, ERRalpha stimulates the development of the primary tumor through regulation of angiogenesis and VEGF expression, we show that ERRalpha inhibits osteolytic lesions from breast cancer cells via the regulation of osteoclastogenesis and OPG expression. On the other side ERRalpha stimulates osteolytic lesions from prostate cancer through regulation of TGFbeta, MCP-1 and cathepsin K expression while inducing new bone formation combine with OPG, endothelin-1, and Wnts regulation. All together, our results confirmed ERRalpha as a bad prognosis factor in breast and prostate primary tumors. They also show a dual function of ERRalpha in bone metastasis development as an inhibitor and a stimulator of bone metastasis derived from breast and prostate cancer respectively which suggest different ERRalpha mechanisms that may depend of cancer cells but also of the microenvironment and hormonal status

Les métastases sont une cause majeure de décès dans le cas du cancer. En effet, ces tumeurs secondaires peuvent se développer dans divers organes, distants de la tumeur primaire, et surviennent à des temps différents au cours de la croissance de la tumeur primaire. De nombreuses techniques d’imagerie biomédicale peuvent être utilisées pour les détecter. Parmi celles-ci, l’Imagerie par Résonance Magnétique (IRM) a l’avantage de ne pas utiliser de rayonnements ionisants et permet de forts contrastes entre des tissus mous différents. Toutefois il est encore nécessaire de développer de nouvelles techniques IRM pour mieux caractériser les tumeurs, obtenir des données quantitatives, et réduire drastiquement les durées d’examen. Parmi les caractéristiques biophysiques mesurables par IRM, le temps de relaxation T1 semble être un bio-marqueur de l’efficacité d’une thérapie anti-cancéreuse. Cependant, sa mesure est généralement trop chronophage pour être utilisée en imagerie préclinique sur des cohortes d’animaux, ou pour une utilisation en routine clinique. Cette thèse a porté sur le développement d’une séquence de quantification T1 fiable et rapide. Elle a été appliquée pour détecter et caractériser des métastases chez la souris avec comme pré-requis de générer des images avec une résolution spatiale élevée, d’être insensible aux mouvements respiratoires et de permettre des mesures reproductibles du T1. La séquence Magnetization Prepared 2 Rapid Acquisition Gradient Echoes (MP2RAGE) à encodage cartésien a donc été choisie pour son fort potentiel de quantification T1, sa robustesse aux hétérogénéités de champ magnétique, et pour obtenir rapidement des cartes paramétriques en 3D. L’influence des paramètres de la séquence a d’abord été évaluée par simulations. Puis la séquence a été modifiée pour être compatible avec une méthode d’accélération appelée acquisition comprimée. Cette méthode a alors été utilisée soit pour réduire le temps d’acquisition des cartes T1, soit pour en améliorer la résolution spatiale. Cette nouvelle séquence MP2RAGE a alors été utilisée à 7T pour détecter et caractériser des métastases cérébrales disséminées dans le cerveau de souris. Pour détecter des métastases hépatiques, l’encodage cartésien initial s’est avéré trop sensible aux mouvements respiratoires. Il a donc été remplacé par un encodage radial, nécessitant une adaptation du schéma de reconstruction des cartes T1. Ainsi, des cartes T1 3D de l’abdomen entier de souris ont été obtenues en 9 minutes. Un suivi longitudinal de métastases hépatiques a montré des hétérogénéités de T1 inter- et intra-métastatiques. Pour une accélération supplémentaire, la séquence a été développée avec un encodage multi-coupe 2D, permettant ainsi d’utiliser les nombreux temps-morts présents dans le chronogramme. Des optimisations des paramètres de la séquence ont permis d’obtenir 6 cartes T1 en 9 s in vivo sur le cerveau et l’abdomen de souris. De plus, une étude préliminaire a montré qu’elle permettait de réaliser de la thermométrie. Une première perspective de ces travaux consiste à transférer cette séquence sur un aimant de recherche clinique. Une autre perspective serait de développer une séquence multi-coupe 3D radiale, accélérée par acquisition comprimée, applicable sur le petit-animal comme chez l’humain. Celapermettrait d’allier efficacité de la séquence, forte résolution spatiale et robustesse aux mouvements pour un large éventail d’applications. / Metastases are a leading cause of death in the case of cancer. Indeed, these secondary tumors can develop in various organs, distant from the primary tumor, and occur at different times during the growth of the primary tumor. Many biomedical imaging techniques can be used to detect them. Among these, Magnetic Resonance Imaging (MRI) has the advantage of not using ionizing radiation and allows strong contrasts between different soft tissues. However, it remains necessary to develop new MRI techniques to better characterize tumors, obtain quantitative data and to drastically reduce exam times. Among the biophysical characteristics measurable by MRI, the T1 relaxation time seems to be a biomarker of the efficiency of an anti-cancer therapy. However, its measurement is generally too time consuming to be used in preclinical imaging on cohorts of animals, or for routine clinical use. This thesis therefore had the challenge of developing a reliable and rapid T1 quantification sequence. It has been applied to detect anc characterize metastases in mice with the prerequisites of generating images with high spatial resolution, being insensitive to respiratory movements and allowing reproducible T1 measurements. The Magnetization Prepared 2 Rapid Acquisition Gradient Echoes sequence (MP2RAGE) with Cartesian encoding was therefore chosen for its high T1 quantitation potential, its robustness to magnetic field heterogeneities, and its ability to quickly obtain 3D parametric maps. First, simulations were performed to evaluate the influence of sequence parameters. Then the sequence was modified to be compatible with an acceleration method called Compressed Sensing. This method was then used either to reduce the acquisition time of the T1 maps, or to improve the spatial resolution. This new MP2RAGE sequence was then applied at 7T to detect and characterize disseminated brain metastases in the mouse. The initial Cartesian encoding proved to be too sensitive to respiratory movements to detect liver metastases. It was therefore replaced by a radial encoding, which required an adaptation of the reconstruction scheme of the T1 maps. Thus, 3D T1 maps of the entire abdomen of mice were obtained in 9 minutes. Longitudinal follow-up of hepatic metastases showed inter and intra-metastatic T1 heterogeneities. For an additional acceleration, the sequence was developed with a 2D multi-slice encoding, thus allowing to use the many dead times present in the chronogram. Optimizations of the parameters of the sequence made it possible to obtain 6 T1 maps in 9 s in vivo on the brain and the abdomen of mice. In addition, a preliminary study showed a possible application for thermometry. A first perspective of this work is to transfer this sequence to a clinical research MRI system. Another perspective would be to develop a 3D radial multi-slice sequence, accelerated by Compressed Sensing, applicable to small animals as in humans. This would combine sequence efficiency, high spatial resolution and robustness to movements for a wide range of applications.

As solid tumor types, breast and prostate cancer are rivalled only by lung cancer in their propensity to metastasize to bone in the later stages of disease. At advanced stages of disease, approximately 80% of breast and 90% of prostate cancer patients will present with bone metastases. Bone metastases are often a painful conclusion to the lives of these patients, resulting in bone pain, hypercalcemia, pathological fractures and spinal cord compression. The culmination of these comorbidities considerably reduces a patient’s quality of life and prolonged survival. Hormone depletion is used as a first line of treatment in the majority of cases, negatively regulating bone health due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts. Not only is bone integrity undermined, but this action of increased bone turnover is beneficial for the colonization of metastasizing cells which co-opt and enhance the same mechanisms to establish and maintain their own growth. This is termed ‘the vicious cycle’ of osteolytic bone metastasis. Current research approaches aim to identify bone-targeted therapies which not only inhibit tumor growth but concurrently protect bone. In this study, Dickkopf-1 (DKK-1), mechanistic target of rapamycin (mTOR) and p38 mitogen-activated kinases (p38 MAPK) are presented as novel targets. Pro-tumor roles have been described for all and clinical trials are currently investigating their efficacy in different cancer types. In normal bone biology DKK-1 is an inhibitor of the canonical Wnt signaling pathway which promotes osteoblastogenesis while mTOR signaling is a promoter of osteoclastogenesis. P38 MAPK inhibitors have been shown to regulate DKK-1 expression and bone destruction in preclinical models of multiple myeloma. The aims of this current study were to 1) investigate the role of DKK-1 in the biology of osteotropic breast cancer, 2) to assess the potential bone protective effects of mTOR inhibition by everolimus in the context of osteotropic cancers and 3) to test the hypothesis that p38 MAPK is a regulator of DKK-1 expression in prostate cancer, potentially supporting an osteolytic phenotype by impairing osteoblastogenesis. In aim 1, analysis of a breast cancer tissue microarray demonstrated that DKK-1 expression was elevated in advanced and invasive tumor stages. Strikingly, positive DKK-1 expression correlated with a significantly reduced survival rate only in estrogen receptor-negative (ER-) breast cancer patients compared to patients with tumors which were negative for DKK-1 expression. In MDA-MB-231 breast cancer xenograft models, neutralization of secreted DKK-1 by treating mice with the monoclonal DKK-1 antibody BHQ880 or knocking out the expression of DKK-1 in MDA-MB-231 cells using CRISPR-Cas9 mediated gene editing, resulted in reduced tumor growth and burden by ≥ 50% (p < 0.05). In aim 2, the mTOR inhibitor everolimus is presented as an anti-tumor and bone-protective agent. The anti-tumor effects of everolimus were confirmed in two subcutaneous tumor models and a model of breast cancer bone metastasis, were tumor burden in the bone was reduced by 45.4% (p < 0.01). Bone loss induced by a hormone-deprived environment in ovariectomized mice was prevented with everolimus treatment as was bone destruction in the metastasis model. In more detail, it could be shown that everolimus maintained osteoblast function while specifically inhibiting osteoclast function. In aim 3, p38 MAPK is presented as a regulator of DKK-1 in prostate cancer. While the activation of p38 MAPK upregulated DKK-1, inhibition of p38 MAPK using small molecule inhibitors and siRNAs inhibited DKK-1 expression. Furthermore, assessment of different p38 MAPK isoforms revealed MAPK11 as the most effective regulator of DKK-1 and inhibition of DKK-1 by interfering with p38 MAPK signaling was sufficient to prevent the inhibitory effects of prostate cancer-derived DKK-1 on osteoblastogenesis in vitro. This study has assessed multiple targets and their concurrent roles in cancer and bone cell biology. Specifically, DKK-1 has been proven to be a tumor promoter in ER- breast cancer and can be targeted therapeutically to inhibit tumor growth. MTOR inhibition by everolimus has been shown to be an effective mono-therapy in ER- breast cancer, inhibiting the growth of subcutaneous tumor and bone metastases and preventing bone loss induced by estrogen ablation. This further supports its use in postmenopausal women with breast cancer who are predisposed to developing osteoporosis and bone metastases. It also supports the use of everolimus in hormone receptor-negative or triple receptor-negative breast cancer, for which it has not yet been approved. A clear link has been made between p38 MAPK signaling and DKK-1 expression in prostate cancer and its consequent regulation of osteoblastogenesis. A future focus on the inhibition of a specific MAPK isoform, MAPK11 in particular, may help in translating these encouraging in vitro results into promising pre-clinical trials in vivo. As a whole, these investigations provide a foundation for further research and could be valuable for the design of future clinical trials, leading to improvements in the treatment and prognosis of osteolytic bone metastases.

Metastasizing is responsible for 90% of death in cancer patients. Metastatic tumour cells have several strategies that they use to invade surrounding tissues - they can migrate together or individually. When individual cells migrate, tumour cells adopt two different morphologies. They are either elongated and migrate using the proteolytically active mesenchymal mode, or they are rounded and migrate in the amoeboid mode. Metastatic tumour cells can switch between these modes, which complicates the development of effective migrastatics. In this work, we focused on the effect of inflammatory signalling on metastatic cell migration. We worked with cell lines of malignant human melanoma, which adopt a mixed morphology and show both amoeboid and mesenchymal phenotype during migration. Upon stimulation of melanoma human cells with interferon beta, a mesenchymal to amoeboid transition occurs. Interferon beta appears to induce amoeboid morphology by maintaining high levels of the ISGF3 complex, which is composed of the heterodimer of STAT 1 and STAT 2 proteins and the IRF9 protein. Upon blocking of Jak / Stat signalling pathway by negative regulators, human melanoma cells return to mesenchymal morphology. Key words - invasiveness, mesenchymal-ameboid transition, interferons, inflammation, migration, metastases