<p>Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses.</p><p>We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy.</p><p>In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.</p>

<p>This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy.</p><p>The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated <i>ex vivo</i>.</p><p>Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3⁺ to CD3⁺ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications.</p><p>Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. <i>In vitro</i> expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4⁺ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048).</p><p>In conclusion, human solid tumours are recognised in sentinel nodes and <i>in vitro</i> expanded sentinel node-acquired CD4⁺ T lymphocytes seem useful in the treatment of patients with disseminated cancer.</p>

Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses. We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy. In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.

This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy. The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated ex vivo. Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3+ to CD3+ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications. Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. In vitro expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4+ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048). In conclusion, human solid tumours are recognised in sentinel nodes and in vitro expanded sentinel node-acquired CD4+ T lymphocytes seem useful in the treatment of patients with disseminated cancer.

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Background: Prostate cancer is the most common cancer in Sweden with around 10,000 new cases every year. Kidney and bladder cancer are less common with 1,000 and 2,000 new cases annually, respectively. The incidence of these cancer sites is higher in developed, than in developing countries, suggesting an association between lifestyle and cancer risk. The aims of this thesis were to investigate body mass index (BMI), blood pressure, and blood levels of glucose, total cholesterol, and triglycerides as risk factors for prostate, kidney, and bladder cancer. Furthermore, we aimed at assess probabilities of prostate cancer and competing events, all-cause death, for men with normal and high levels of metabolic factors. Material and methods: This thesis was conducted within the Metabolic Syndrome and Cancer project (Me-Can), a pooled cohort study with data from 578,700 participants from Norway, Sweden, and Austria. Data from metabolic factors were prospectively collected at health examinations and linked to the Cancer and Cause of Death registers in each country.  Results: High levels of metabolic factors were not associated with increased risk of prostate cancer, but high levels of BMI and blood pressure were associated with risk of prostate cancer death. The probability of prostate cancer was higher for men with normal levels of metabolic factors compared to men with high levels, but the probability of all-cause death, was higher for men with high levels than for those with normal levels. For both men and women, high levels of metabolic factors were associated with increased risk of kidney cancer (renal cell carcinoma). Furthermore, blood pressure for men and BMI for women were found as independent risk factors of kidney cancer. High blood pressure was associated with an increased risk of bladder cancer for men. Conclusions: High levels of metabolic factors were associated to risk of kidney and bladder cancer and to death from kidney, bladder, and prostate cancer. Compared to men with normal levels, men with high levels of metabolic factors had a decreased probability of prostate cancer but an increased probability of all-cause death. / <p>Ytterligare forskningsfinansiärer: World Cancer Research Fund (2007/09) och Wereld Kanker Onderzoek Fonds (R2010/247)</p> / Me-Can

Electronic medical record databases (e.g. the Clinical Practice Research Datalink, CPRD) are increasingly used in epidemiological research. The CPRD has two formats of data: coded, which is the sole format used in almost all research; and free-text (or ‘hidden’), which may contain much clinical information but is generally unavailable to researchers. This thesis examines the ramifications of omitting free-text records from research. Cases with bladder (n=4,915) or pancreatic (n=3,635) cancer were matched to controls (n=21,718, bladder; n=16,459, pancreas) on age, sex and GP practice. Coded and text-only records of attendance for haematuria, jaundice and abdominal pain in the year before cancer diagnosis were identified. The number of patients whose entire attendance record for a symptom/sign existed solely in the text was quantified. Associations between recording method (coded or text-only) and case/control status were estimated (χ2 test). For each symptom/sign, the positive predictive value (PPV, Bayes' Theorem) and odds ratio (OR, conditional logistic regression) for cancer were estimated before and after supplementation with text-only records. Text-only recording was considerable, with 7,951/20,958 (37%) of symptom records being in that format. For individual patients, text-only recording was more likely in controls (140/336=42%) than cases (556/3,147=18%) for visible haematuria in bladder cancer (χ2 test, p<0.001), and for jaundice (21/31=67% vs 463/1,565=30%, p<0.0001) and abdominal pain (323/1,126=29% vs 397/1,789=22%, p<0.001) in pancreatic cancer. Adding text records reduced PPVs of visible haematuria for bladder cancer from 4.0% (95% CI: 3.5–4.6%) to 2.9% (2.6–3.2%) and of jaundice for pancreatic cancer from 12.8% (7.3–21.6%) to 6.3% (4.5–8.7%). Coded records suggested that non-visible haematuria occurred in 127/4,915 (2.6%) cases, a figure below that generally used for study. Supplementation with text-only records increased this to 312/4,915 (6.4%), permitting the first estimation of its OR (28.0, 95% CI: 20.7–37.9, p<0.0001) and PPV (1.60%, 1.22–2.10%, p<0.0001) for bladder cancer. The results suggest that GPs make strong clinical judgements about the probable significance of symptoms – preferentially coding clinical features they consider significant to a diagnosis, while using text to record those that they think are not.

Submitted by Mirian Yoshiko Matsumoto (mirian_matsumoto@hotmail.com) on 2018-09-13T03:42:01Z No. of bitstreams: 1 Tese de Doutorado BGA Mirian Matsumoto.pdf: 5590776 bytes, checksum: 35a18a86dcfd4a42e7e9c40dbce00a94 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-09-14T16:51:20Z (GMT) No. of bitstreams: 1 matsumoto_my_dr_bot.pdf: 5590776 bytes, checksum: 35a18a86dcfd4a42e7e9c40dbce00a94 (MD5) / Made available in DSpace on 2018-09-14T16:51:20Z (GMT). No. of bitstreams: 1 matsumoto_my_dr_bot.pdf: 5590776 bytes, checksum: 35a18a86dcfd4a42e7e9c40dbce00a94 (MD5) Previous issue date: 2017-05-30 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O câncer de bexiga (CB) é a segunda malignidade geniturinária mais comum. A maioria (75%) dos CBs são primeiramente diagnosticados como não-músculo invasivos (CBNMIs) nos estádios Ta, T1, e carcinoma in situ (CIS). Atualmente, o tratamento mais utilizado contra CBNMIs envolve a imunoterapia com Bacillus Calmette-Guérin (BCG) associada à ressecção transuretral (RTU). No entanto, a utilização de BCG pode causar graves efeitos colaterais e está associada com uma elevada taxa de recorrência após o tratamento. Por conseguinte, várias abordagens têm sido investigadas, incluindo o desenvolvimento de novas moléculas, assim como a melhoria da terapia com medicamentos convencionalmente utilizados no tratamento de tumores e a incorporação de sistemas de carreamento de fármacos. Considerando o uso de novas moléculas, complexos metálicos derivados de bases de Schiff (BSs) são compostos versáteis que apresentam atividade antitumoral, fornecendo assim novas perspectivas para a terapia do CBNMI. Em relação ao carreamento de fármacos, ao longo dos últimos anos, carreadores lipídicos nanoestruturados (CLNs) têm atraído considerável interesse como veículos alternativos para produtos farmacêuticos antitumorais. Assim, com o intuito de adquirir maior conhecimento a respeito da química dos complexos derivados da BS N-Salicilideno anilina (Salan) e também seus efeitos na progressão do CBNMI, esta tese descreve a síntese e caracterização de complexos N-Salicilideno anilina(níquel) [Salan(Ni)] e N-Salicilideno anilina(platina) [Salan(Pt)]. A estrutura proposta desses compostos foi estabelecida por análise elementar (CHN), FTIR, TG, RMN de 1H e 13C. As atividades citotóxicas dos compostos sintetizados [Salan, NiCl2 e Salan(Ni)] foram avaliadas por ensaio MTT e os dados obtidos indicaram que Salan(Ni) demonstrou atividade citotóxica significativa contra linhagens celulares de leucemia e câncer de fígado. Além disso, neste estudo os efeitos histopatológicos e moleculares dos compostos sintetizados [Salan, Salan(Ni) e Salan(Pt)] foram caracterizados e comparados com o tratamento com BCG em um modelo animal de CBNMI. Os resultados obtidos indicaram que o grupo Salan(Ni): apresentou melhor recuperação histopatológica quando comparado ao grupo Câncer; aumentou os níveis da proteína UPIII; aumentou a expressão dos genes supressores de tumor PTEN e p53; causou inibição da angiogênese devido aos níveis proteicos elevados de endostatina e níveis mais baixos da proteína VEGF. No entanto, durante a instilação dos tratamentos, observou-se a precipitação dos compostos. Então, testes utilizando 2% de DMSO em óleo de milho como veículo para complexos do tipo Salan [Salan(Ni) e Salan(Pt)] foram realizados. Adicionalmente, o complexo Salan(Pt) foi incorporado a um carreador lipídico nanoestruturado (CLN) derivado de murumuru. O CLN carregado com Salan(Pt) [Salan(Pt)-CLN] foi preparado através do sistema de agitação mecânica e apresentou diâmetro médio de 165,4 nm e potencial zeta -34,4 mV. Por fim, os compostos obtidos [Salan(Ni), Salan(Pt), CLN livre, Salan(Pt)-CLN] foram administrados in vivo para avaliar seus efeitos contra CBNMI. A análise histológica revelou que o grupo Salan(Ni) apresentou melhor recuperação histopatológica. Análises de Western blotting (WB) indicaram que os tratamentos com Salan(Ni), Salan(Pt) e Salan(Pt)-CLN provavelmente ativam a via p53 diminuindo os níveis proteicos de Akt e PI3K. Em conclusão, os resultados indicaram que o complexo Salan(Ni) apresentou melhores efeitos na redução da agressividade do CBNMI comparado aos demais complexos e BCG. / Bladder cancer (BC) is the second most common genitourinary malignancy. Most (75%) BCs are non–muscle invasive (NMIBC) at first diagnosis [Ta, T1, and carcinoma in situ (CIS)]. Currently, the most used treatment against NMIBC involves the immunotherapy with BCG (Bacillus Calmette-Guérin) associated with the transurethral resection. However, the use of BCG can cause severe side effects and it is associated with high recurrence rate after treatment. Therefore, several approaches have been investigated, including the development of new molecules and also the improvement of the therapy with drugs conventionally used to treat cancers by using drug delivery systems. Considering the use of new molecules, metal complexes derived from Schiff bases (SBs) are versatile molecules with anticancer activity, providing new perspectives for the therapy of NMIBC. Regarding the drug delivery, over the past few years, nanostructured lipid carriers (NLCs) have been attracting considerable interest as alternative carriers for anticancer pharmaceuticals. Thus, in order to acquire more information about the chemistry of the complexes derived from the SB of N-Salicylidene aniline (Salan), as well as its effects on the progression of NIMBC, the present thesis describes the synthesis and characterization of N-Salicylidene aniline(nickel) [Salan(Ni)] and N-Salicylidene aniline(platinum) [Salan(Pt)] complexes. The proposed structure of these compounds was established by elemental analysis (CHN), FTIR, TG, 1H and 13C NMR. Cytotoxic activities of the synthesized compounds [Salan, NiCl2 and Salan(Ni)] were evaluated by the MTT assay and the obtained data indicated that Salan(Ni) showed significant cytotoxic activity against leukemia and liver cancer cells lines. Furthermore, in this study the histopathological and molecular effects of the synthesized compounds [Salan, Salan(Ni) and Salan(Pt)] were characterized and compared with BCG treatment in an animal model of NMIBC. Our results demonstrated that the Salan(Ni) group: improved histopathological recovery when compared with Cancer group; increased UPIII protein levels; increased expression of tumor suppressors genes PTEN and p53; inhibited of angiogenesis assigned to elevated levels of endostatin and lower levels of VEGF. However, during the instillation of treatments, compounds precipitation were observed. Then, tests using 2% DMSO in corn oil as vehicle for Salan-type complexes [Salan(Ni) and Salan(Pt)] were performed. Additionally, Salan(Pt) complex was incorporated into nanostructured lipid carrier (NLC) derived from murumuru. The NLC loaded with Salan(Pt) [Salan(Pt)-NLC] was prepared by using mechanical agitation method and had an average diameter of 165,4 nm as well as zeta potential of -34,4 mV. Ultimately, the obtained compounds [Salan(Ni), Salan(Pt), free NLC, Salan(Pt)-NLC] were administered in vivo to evaluete their effects against NMIBC. The histological analysis revealed that Salan(Ni) group Abstract MATSUMOTO, M. Y. Tese de Doutorado em Biologia Geral e Aplicada – UNESP – Botucatu showed bestter histopathological recovery. Western blotting (WB) analysis indicated that Salan(Ni), Salan(Pt) and Salan(Pt)-NLC treatments probably activate the p53 pathway by decreasing the protein levels of Akt and PI3K. In conclusion, the results showed the Salan(Ni) has better effects in reduction of NMIBC aggressiveness compared to the other complexes and BCG / 2013/04708-8

Orientador: Wagner José Fávaro / Resumo: O câncer de bexiga (CB) é a segunda malignidade geniturinária mais comum. A maioria (75%) dos CBs são primeiramente diagnosticados como não-músculo invasivos (CBNMIs) nos estádios Ta, T1, e carcinoma in situ (CIS). Atualmente, o tratamento mais utilizado contra CBNMIs envolve a imunoterapia com Bacillus Calmette-Guérin (BCG) associada à ressecção transuretral (RTU). No entanto, a utilização de BCG pode causar graves efeitos colaterais e está associada com uma elevada taxa de recorrência após o tratamento. Por conseguinte, várias abordagens têm sido investigadas, incluindo o desenvolvimento de novas moléculas, assim como a melhoria da terapia com medicamentos convencionalmente utilizados no tratamento de tumores e a incorporação de sistemas de carreamento de fármacos. Considerando o uso de novas moléculas, complexos metálicos derivados de bases de Schiff (BSs) são compostos versáteis que apresentam atividade antitumoral, fornecendo assim novas perspectivas para a terapia do CBNMI. Em relação ao carreamento de fármacos, ao longo dos últimos anos, carreadores lipídicos nanoestruturados (CLNs) têm atraído considerável interesse como veículos alternativos para produtos farmacêuticos antitumorais. Assim, com o intuito de adquirir maior conhecimento a respeito da química dos complexos derivados da BS N-Salicilideno anilina (Salan) e também seus efeitos na progressão do CBNMI, esta tese descreve a síntese e caracterização de complexos N-Salicilideno anilina(níquel) [Salan(Ni)] e N-Sa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bladder cancer (BC) is the second most common genitourinary malignancy. Most (75%) BCs are non–muscle invasive (NMIBC) at first diagnosis [Ta, T1, and carcinoma in situ (CIS)]. Currently, the most used treatment against NMIBC involves the immunotherapy with BCG (Bacillus Calmette-Guérin) associated with the transurethral resection. However, the use of BCG can cause severe side effects and it is associated with high recurrence rate after treatment. Therefore, several approaches have been investigated, including the development of new molecules and also the improvement of the therapy with drugs conventionally used to treat cancers by using drug delivery systems. Considering the use of new molecules, metal complexes derived from Schiff bases (SBs) are versatile molecules with anticancer activity, providing new perspectives for the therapy of NMIBC. Regarding the drug delivery, over the past few years, nanostructured lipid carriers (NLCs) have been attracting considerable interest as alternative carriers for anticancer pharmaceuticals. Thus, in order to acquire more information about the chemistry of the complexes derived from the SB of N-Salicylidene aniline (Salan), as well as its effects on the progression of NIMBC, the present thesis describes the synthesis and characterization of N-Salicylidene aniline(nickel) [Salan(Ni)] and N-Salicylidene aniline(platinum) [Salan(Pt)] complexes. The proposed structure of these compounds was established by elemental analysis (CHN), FTIR, TG... (Complete abstract click electronic access below) / Doutor