Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The "classic" prostate cell lines, DU145, PC-3 and LNCaP, have served as a valuable cell biological model for research into prostate cancer. However, their relevance may be limited because they derive from metastatic, and not from primary normal and tumour epithelium. The cell lines (1532T, 1535T, 1542T, 1542N and BPH-l) have been derived from primary benign and malignant human tumour prostate epithelium and may be more representative. Using these cell lines I have examined the role of basic cell damage responses (repair, checkpoint activation, apoptosis and associated signalling proteins, and the influence of androgen status) in cell inactivation, and its relevance to treatment. Numerous studies have suggested that loss of p53 function leads to resistance to chemotherapeutic agents and irradiation. It is shown here that the p53-inactive cell lines are, in fact, the most sensitive to chemotherapeutic agents such as etoposide, vinblastine and estramustine, whilst the p53 wild-type cell line, LNCaP, is the most radiosensitive. Notwithstanding the effects of p53 degradation by the HPV -16 E6 viral protein, the results on chemosensitivity raises the possibility that different chemotherapeutic agents may have different p53-dependent effects in different tumour cells. Androgen deprivation is demonstrated to sensitise prostate cancer cells to chemotherapeutic agents and it is shown that the hormone independent cell lines are the most chemosensitive. The LNCaP cell line displayed an increased resistance to apoptosis induced by etoposide and gamma irradiation, suggesting that androgens are capable of protection against both these DNA damaging agents. The major factors determining radiosensitivity in human tumour cell lines are known to be DNA double-strand break (dsb) induction and repair. In the prostate cell lines I find that cellular radiosensitivity correlates with the number of DNA double-strand breaks measured within 2 hours of irradiation, and that the more radioresistant cell lines show better repair competence. Conclusions as to the influence of androgen dependence on radiosensitivity and repair are not possible at this stage since only the LNCaP cell line was androgen sensitive. The fact that the 2 hour repair period can separate radiosensitive from radioresistant cells in 2 groups of human tumour cell lines highlights the role of non-homologous end-joining repair. This has implications for therapy, and is consistent with the clinical observation that prostate tumours can be successfully controlled by low dose rate-brachytherapy. To evaluate the role of apoptosis, cells were exposed to TD50 concentrations of chemotherapeutic drugs, and 60Co y-irradiation. Apoptosis was found to be low, overall, and ranged from 0.1% - 12.1%,3.0% - 6.0% and 0.1% - 8.5% for etoposide, estramustine and vinblastine, respectively. The percentage of cells undergoing druginduced apoptosis was, on average, higher in the tumour cell lines than in the normal cell lines. Gamma irradiation-induced apoptosis levels ranged from 1.3% - 7%. The LNCaP cell line yielded the lowest percentage of apoptotic cells after exposure. The l532T cell line yielded the highest percentage of apoptotic cells after exposure. Apoptotic propensity did not rank the cell lines according to their radiosensitivity. Immunoblotting demonstrated that the apoptosis-associated proteins, bax and bcl-2, are expressed at a basal level in all the cell lines tested, but no increase was detected after exposure to TD50 doses of etoposide, vinblastine and estramustine. The ratio of bax and bcl-2 also was not altered by DNA damage. No evidence was found that a correlation may exist between reproductive cell death and the expression of genes which control apoptosis. My results show that apoptosis is not a major mechanism of drug- or radiation-induced cell death in prostate cell lines. In conclusion, loss of p53 function and loss of androgen dependence was not found to be correlated with resistance of tumours to chemotherapeutic drugs. Cellular radiosensitivity was found to be correlated with the number of DNA double-strand breaks remaining after 2 hours of repair. The more radioresistant cell lines showed better repair competence. Apoptosis and genes affecting apoptosis, such as p53 and members of the bcl-2 family, do not seem to contribute significantly to the sensitivity of prostate cancer cells to anticancer drugs and irradiation. / AFRIKAANSE OPSOMMING: Die klassieke prostaat sellyne, DU145, PC-3 en LNCaP, het 'n waardevolle bydrae gemaak in die sel biologiese model in prostaat kanker. Die toepaslikheid daarvan mag egter beperk wees, aangesien hierdie sellyne afkomstig is van metastatiese, en nie van primêr normale en tumor epiteel nie. Die sellyne 1532T, 1535T, 1542T, 1542N en BPH-I is afkomstig van primêre benigne en maligne menslike prostaat tumor epiteel en mag moontlik meer verteenwoordigend wees. Deur van hierdie sellyne gebruik te maak, is die rolondersoek van die reaksie op basiese selskade (d.w.s. herstel, beheerpunt aktivering, apoptose en verwante sein proteïene, en die invloed van androgeen status) tydens die proses van sel inaktivering, asook die toepaslikheid ten opsigte van behandeling. Volgens verskeie studies lei die verlies aan p53 funksie tot weerstandigheid teen chemoterapeutiese middels en bestraling. Die resultate van hierdie studie toon dat die p53-onaktiewe sellyne egter die sensitiefste is vir chemoterapeutiese middels, soos etoposied, vinblastien en estramustien, terwyl die p53 natuurlike-tipe sellyn, LNCaP, die meeste radiosensitief is. Ten spyte van die invloed van p53 afbraak deur die HPV -16 E6 virale proteïen, dui die resultate van chemosensitiwiteit op die moontlikheid dat verskillende chemoterapeutiese middels verskillende p53-afhanklike effekte op verskillende tumorselle mag hê. Dit is bewys dat onttrekking van androgeen prostaat kankerselle sensitiseer teen chemoterapeutiese middels en dat hormoon-onafhanklike sellyne die hoogste chemosensitiwiteit vertoon. Die LNCaP sellyn vertoon 'n verhoogde weerstandigheid teen apoptose wat deur etoposied en y-bestraling geïnduseer is, wat 'n aanduiding is dat androgene beskerming kan bied teen beide hierdie DNA beskadigingsfaktore. Die belangrikste faktore wat die radiosensitiwiteit in menslike tumorselle bepaal, IS bekend dat dit die dubbelbande van DNA verbreek en herstel. Hierdie studie het aangetoon dat in prostaat sellyne die sellulêre radiosensitiwiteit korreleer met die aantal DNA dubbelband verbrekings binne 2 uur na bestraling, en dat die meer radioweerstandige sellyne beter herstelvermoë vertoon. Gevolgtrekkings oor die invloed van androgeen se afhanklikheid van radiosensitiwiteit en herstel kan egter nie op hierdie stadium gemaak word nie, aangesien slegs die LNCaP sellyn androgeenafhanklik was. Die feit dat die 2 uur herstelperiode 'n skeiding kan maak tussen radiosensitiewe en radioweerstandige selle in twee groepe menslike tumor sellyne, onderstreep die rol van herstel van nie-homoloë endverbindings. Dit hou implikasies in vir terapie, en stem ooreen met die kliniese waarnemings dat prostaat tumore suksesvol gekontroleer kan word deur lae intensiteit dosis bragiterapie. Ten einde die rol van apoptose te ondersoek, is selle blootgestel aan TD50 konsentrasies chemoterapeutiese middels, asook 60Co y-bestraling. Apoptose was oor die algemeen laag, en het gestrek van 0.1% tot 12.1%,3.0% tot 6.0% en 0.1% tot 8.5% vir etoposied, estramustien en vinblastien onderskeidelik. Die persentasie selle wat middel geïnduseerde apoptose ondergaan het, was gemiddeld hoër in tumor sellyne as in normale sellyne. Die waardes van apoptose geïnduseer deur y-bestraling het gewissel van 1.3% tot 7.0%. Die LNCaP sellyn het die laagste persentasie apoptotiese selle na bestraling gelewer, terwyl die 1532 r sellyn die hoogste persentasie gelewer het. Die volgorde van die radiosensitiwiteit van die sellyne was nie waarneembaar in hulle geneigdheid tot apoptose nie. Immunoblots het aangetoon dat die apoptose-geassosieerde proteïene, bax en bcl-2, uitgeskei word teen 'n basisvlak in al die sellyne wat getoets is, maar dat geen verhoogde uitskeiding waarneembaar was na blootstelling aan TD50 dosisse etoposied, vinblastien en estramustien nie. Die verhouding van bax en bcl-2 is ook nie beïnvloed deur DNA beskadiging nie. Dit blyk daarom dus onwaarskynlik dat daar 'n korrelasie bestaan tussen reproduktiewe seldood en die uitskeiding van gene wat apoptose beheer. Die resultate dui daarop dat apoptose me 'n belangrike meganisme vir middel- of bestralingsgeïnduseerde seldood in prostaat sellyne is nie.

Background: Prostate cancer is the most common type of cancer in Sweden. The three primary treatment types for prostate cancer are prostatectomy, radiotherapy and different types of endocrine therapy. With all treatments mentioned above comes adverse effects that may have big effects on the person treated. Aim: The aim of this study was to highlight men´s experiences of the adverse effects that comes with the treatment for prostate cancer. Method: The research method employed was a literature study based on eleven qualitative articles. The method of analysis was done according to Friberg five-step analysis of qualitative articles and through the analysis, six themes emerged. Results: The themes were A feeling of emotional imbalance, Not prepared enough, The experience of loosing control, Feeling of diminished masculinity, The experience of a feminized body and An altered identity. Conclusion: The result showed that men experience adverse effects of the prostate cancer treatment as difficult in many ways. The changes to the body and mental well-being tend to be difficult to deal with, and the men need relevant information prior to treatment to give them time to adjust to their new life.

Tse, Ho Man. / Thesis (M.Phil)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 166-173). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 撮要 --- p.v / Table of Content --- p.vi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Basis of Prostate Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology and Risk Factors of Prostate Cancer --- p.1 / Chapter 1.1.2 --- Pathology of Prostate Cancer --- p.2 / Chapter 1.1.3 --- Treatment Approaches for Prostate Cancer --- p.4 / Chapter 1.2 --- Sex Hormones and Prostate Cancer --- p.7 / Chapter 1.2.1 --- Prostate Development --- p.7 / Chapter 1.2.2 --- Involvement of Sex Hormones in Prostate Cancer --- p.8 / Chapter 1.2.3 --- Molecular Mechanisms of Sex Hormones --- p.13 / Chapter 1.2.4 --- Hormone Receptor Antagonists --- p.15 / Chapter 1.3 --- Involvement of microRNAs in Cancer --- p.19 / Chapter 1.3.1 --- Basis of microRNAs --- p.19 / Chapter 1.3.2 --- Aberrant microRNA Expressions in Cancers --- p.23 / Chapter 1.3.3 --- Current Understandings on Regulations of micro RN A Expressions --- p.26 / Chapter 1.3.4 --- Regulation of miRNA Expressions by Hormones --- p.29 / Chapter 1.4 --- "Effects of the Anti-estrogen ICI 182,780 on Prostate Cancer Cells" --- p.30 / Chapter 1.4.1 --- "ICI 182,780 Inhibits Cell Growth ofDU145" --- p.30 / Chapter 1.5 --- Objectives of Project --- p.32 / Chapter Chapter 2: --- Materials --- p.34 / Chapter 2.1 --- Bacteria Strain --- p.34 / Chapter 2.2 --- Tissue Culture Media --- p.34 / Chapter 2.3 --- Plasmids --- p.34 / Chapter 2.4 --- Kits and Accessories --- p.35 / Chapter 2.5 --- Reagents and Solutions --- p.36 / Chapter 2.6 --- DNA Oligos --- p.38 / Chapter 2.7 --- Equipments --- p.40 / Chapter Chapter 3: --- Methods --- p.41 / Chapter 3.1 --- Cell Culture Conditions --- p.41 / Chapter 3.2 --- miRNA Expression Profiling of DU145 --- p.41 / Chapter 3.2.1 --- RNA Isolation --- p.41 / Chapter 3.2.2 --- miRNA Microarray Profiling ofDU145 : --- p.42 / Chapter 3.2.2.1 --- Fluorescent Labeling of RNA and Microarray Hybridization --- p.42 / Chapter 3.2.2.2 --- Scanning and Analysis of Signals --- p.46 / Chapter 3.2.3 --- Confirming miR-765 Up-regulation by ICI with qRT-PCR --- p.46 / Chapter 3.2.3.1 --- Assessing ERp Dependency in miR-765 Induction --- p.48 / Chapter 3.2.4 --- Effects of ICI on ARHGEF11 Expression --- p.49 / Chapter 3.2.4.1 --- Reverse Transcription of mRNA --- p.50 / Chapter 3.2.4.2 --- Quantitative Real-Time PCR for Gene mRNA expression --- p.50 / Chapter 3.3 --- Characterizing the Promoter Region of miR-765 --- p.52 / Chapter 3.3.1 --- Cloning of miR-765 Promoter into pGL3-Basic Vector --- p.52 / Chapter 3.3.1.1 --- PCR Amplification of miR-765 Putative Promoter Region --- p.52 / Chapter 3.3.1.2 --- Ligation of the Amplified Regions in pGL3-Basic Vector --- p.55 / Chapter 3.3.1.3 --- Transformation and Screening of pGL3-765 Plasmid --- p.57 / Chapter 3.3.1.4 --- Preparation of pGL3-765 Plasmid DNA --- p.59 / Chapter 3.3.2 --- Preparation of Truncated miR- 765 Promoter Clones --- p.60 / Chapter 3.3.2.1 --- pGL3-765-Trunc#l --- p.61 / Chapter 3.3.2.2 --- pGL3-765-Trunc#2 --- p.62 / Chapter 3.3.2.3 --- pGL3-765-Trunc#3 --- p.62 / Chapter 3.3.3 --- Assessing the miR- 765 Promoter Activities --- p.63 / Chapter 3.3.3.1 --- Optimizing Transfection Conditions --- p.64 / Chapter 3.3.3.2 --- Co-transfection of pGL3-765 and pRL-CMV into DU145 Cells.. --- p.64 / Chapter 3.3.3.3 --- Measuring Luciferase Activities --- p.65 / Chapter 3.3.4 --- Computational Prediction of Transcription Factor Binding Sites on miR-765 Promoter --- p.66 / Chapter 3.4 --- Characterizing the Promoter Region of ARHGEF11.. --- p.67 / Chapter 3.4.1 --- Cloning of ARHGEF11 Promoter into pGL3-Basic Vector (pGL3-ARH) --- p.67 / Chapter 3.4.1.1 --- PCR Amplification of ARHGEF11 Putative Promoter Region --- p.67 / Chapter 3.4.1.2 --- Ligation of the Amplified Regions in pGL3-Basic Vector --- p.68 / Chapter 3.4.1.3 --- Preparation of Plasmid DNA --- p.69 / Chapter 3.4.2 --- Preparation of Truncated ARHGEF11 Promoter Clones --- p.69 / Chapter 3.4.2.1 --- pGL3-ARH-Trunc#l --- p.69 / Chapter 3.4.2.2 --- pGL3-ARH-Trunc#2 --- p.70 / Chapter 3.4.2.3 --- pGL3-ARH-Trunc#3 --- p.71 / Chapter 3.4.3 --- Assessing ARHGEF11 Promoter Activities --- p.72 / Chapter 3.5 --- Identifying Transcription Factor Binding Sites on ARHGEF11 Promoter with EMS A --- p.73 / Chapter 3.5.1 --- Computational Prediction --- p.73 / Chapter 3.5.2 --- Preparation of Biotinylated Probe for use in EMSA --- p.73 / Chapter 3.5.3 --- Preparation of Specific Competitors --- p.74 / Chapter 3.5.4 --- Preparation of DU145 Nuclear and Cytoplasmic Extracts --- p.75 / Chapter 3.5.4.1 --- Preparation of Extracts --- p.75 / Chapter 3.5.4.2 --- Measuring Protein Concentrations --- p.76 / Chapter 3.5.5 --- EMSA Detection of Interaction between Protein and Probe --- p.76 / Chapter 3.6 --- Assessing Biological Significances of miR-765 --- p.78 / Chapter 3.6.1 --- Effects of ICI on DU145 Cells Growth --- p.79 / Chapter 3.6.2 --- Effects of ICI on DU145 Migration Ability --- p.79 / Chapter 3.6.2.1 --- Monolayer Wound Healing Assay --- p.79 / Chapter 3.6.2.2 --- Transwell Migration Assay --- p.80 / Chapter 3.6.3 --- Validating Functionality of Ectopic miR- 765 --- p.81 / Chapter 3.6.3.1 --- miR-765 Recognition Sequence --- p.81 / Chapter 3.6.3.2 --- Preparation of pMIR-765 vector --- p.82 / Chapter 3.6.3.3 --- Ectopic Introduction of miR-765 into DU145 Cells --- p.84 / Chapter 3.6.3.4 --- "Verifying Functionality, of Ectopic miR-765" --- p.84 / Chapter 3.6.4 --- Effects of miR-765 on DU145 Growth --- p.86 / Chapter 3.6.5 --- Effects of miR-765 on DU145 Migration Ability --- p.86 / Chapter 3.7 --- Statistical Analysis --- p.87 / Chapter Chapter 4: --- Results --- p.88 / Chapter 4.1 --- "Identifying ICI 182,780-Regulated miRNA in DU145 Cells" --- p.88 / Chapter 4.1.1 --- miRNA Expression Profiling of DU145 with Microarray --- p.88 / Chapter 4.1.2 --- "Confirming Induction of miR-765 by ICI 182,780 with qRT-PCR" --- p.91 / Chapter 4.1.3 --- "ARHGEF11, Host Gene of miR-765" --- p.95 / Chapter 4.1.4 --- "Induction of ARHGEF 11 by ICI 182,780" --- p.96 / Chapter 4.2 --- Characterization miR-765 Promoter Region --- p.98 / Chapter 4.2.1 --- Cloning of miR- 765 Promoter Region into pGLS-Basic Vector --- p.98 / Chapter 4.2.2 --- Promoter Activity of miR-765 Promoter --- p.100 / Chapter 4.2.3 --- Deletion Mapping of miR- 765 Promoter Region --- p.102 / Chapter 4.2.4 --- Promoter Activities and Inducibitiy of Truncated miR-765 Promoters --- p.103 / Chapter 4.2.5 --- Computational Prediction of Transcription Factor Binding Sites on miR-765 Promoter --- p.105 / Chapter 4.3 --- Characterization of ARHGEF 11 Promoter Region --- p.107 / Chapter 4.3.1 --- Cloning of ARHGEF 11 Promoter --- p.107 / Chapter 4.3.2 --- Promoter Activitiy of ARHGEFll Promoter --- p.109 / Chapter 4.3.3 --- Deletion Mapping of ARHGEFll Promoter --- p.111 / Chapter 4.3.4 --- Promoter Activities and Inducibitiy of Truncated ARHGEF 11 Promoters --- p.113 / Chapter 4.4 --- Identifying Transcription Factor Binding Sites on ARHGEF 11 Promoter --- p.115 / Chapter 4.4.1 --- Computational Prediction of Transcription Factor Binding Sites onARHGEFll Promoter --- p.115 / Chapter 4.4.2 --- Preparation of Probe and Specific Competitors for EMSA --- p.117 / Chapter 4.4.3 --- Interaction between DU145 Nuclear Extract and ARHGEF 11 Promoter Region --- p.119 / Chapter 4.5 --- Biological Significances of miR-765 --- p.122 / Chapter 4.2.1 --- "Effects of ICI 182,780 on DU145 Cell growth" --- p.122 / Chapter 4.2.2 --- "Effects of ICI 182,780 on DU145 Cell Migration" --- p.124 / Chapter 4.2.3 --- Verifying Functionality of Ectopic miR-765 --- p.131 / Chapter 4.2.4 --- Effects of miR-765 on DU145 Cell Growth --- p.133 / Chapter 4.2.5 --- Effects of miR-765 on DU145 Cell Migration --- p.135 / Chapter Chapter 5: --- Discussion --- p.138 / Chapter 5.1 --- "Identifying miR-765 as an Up-regulated miRNA by ICI 182,780" --- p.139 / Chapter 5.1.1 --- "Information about ICI 182,780" --- p.139 / Chapter 5.1.2 --- miRNA Profiling of DU145 --- p.139 / Chapter 5.1.3 --- "Confirming Induction of miR-765 by ICI 182,780 and ERβ dependency with qRT-PCR" --- p.140 / Chapter 5.1.4 --- "Up-regulation of miR-765 Host Gene, ARHGEF11, by ICI" --- p.141 / Chapter 5.2 --- Regulatory Elements of miR-765 Expression --- p.143 / Chapter 5.2.1 --- Own Upstream promoter of miR- 765 --- p.144 / Chapter 5.2.2 --- Promoter of Host Gene ARHGEF11 --- p.146 / Chapter 5.2.3 --- Interaction between ARHGEF11 Promoter Critical Region and Transcription Factors --- p.147 / Chapter 5.2.4 --- Involvement of independent Promoter and Host Gene Promoter in miR-765 Regulation --- p.757 / Chapter 5.3 --- Biological Significances of miR-765 on DU145 --- p.153 / Chapter 5.4 --- Significance of Findings and Future Studies --- p.158 / Chapter 5.4.1 --- Clinical Significance --- p.158 / Chapter 5.4.2 --- Future Studies --- p.161 / Chapter Chapter 6: --- Conclusion --- p.163 / Chapter Chapter 7: --- References --- p.166

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<p>The image-guided radiation therapy (IGRT) protocol used at Juravinski Cancer Center for post-prostatectomy patients involves acquiring a kV cone beam computed tomography (CBCT) image at each fraction and shifting the treatment couch to align surgical clips. This IGRT strategy is promising but its dosimetric impact is unknown, it requires significant resources, and delivers non-negligible doses to normal tissues. The objective of this work is to evaluate this IGRT protocol and investigate possible alternatives.</p> <p>IGRT delivered dose is reconstructed by deforming the planning CT to the CBCT images acquired at each fraction, computing dose on the deformed images, and inversely transforming the dose back to the original geometry. The treatments of six patients were evaluated under four scenarios: no guidance (Non-IGRT), daily guidance as performed clinically (IGRT), guidance on alternating days (Alt-IGRT), and daily automated guidance (Auto-IGRT). For one patient, the impact of reducing the planning target volume (PTV) margin to five (IGRT-5) and eight (IGRT-8) mm isotropic was also evaluated.</p> <p>With the standard clinical PTV margin of ten/seven mm, the evaluated alternatives produced similar results. The minimum dose to the CTV was decreased by 1.6±1.0, 1.2±0.7, and 0.8±0.8 Gy for Non-IGRT, Alt-IGRT, and IGRT, respectively. IGRT with manual shifting did not appear to significantly improve the delivered treatment dose compared to Auto-IGRT (difference in CTV minimum dose was 1.2±2.1Gy). Doses to the organs at risk varied but in general, an increased volume of the bladder and rectum received low doses while smaller portions received high doses. The IGRT-5 and -8 analyses showed the same CTV dose can be delivered with significant reduction in normal tissue exposure. Overall, the desired doses are delivered during IGRT although much of this may be attributed to the large PTV margins currently employed clinically.</p> / Master of Science (MSc)

The development of checkpoint immunotherapy has been a paradigm shift in the treatment of cancer, leading to dramatic improvement in treatment outcomes across a broad range of tumor types. Nevertheless, our current understanding of the tumor immune microenvironment and mediators of resistance to therapy are limited. The recent development of high-throughput single-cell RNA-Sequencing (scRNA-Seq) technology has opened up an unprecedented window into the transcriptional states of distinct tumor-infiltrating immune and stromal cells. However, even this technology has its biological limitations, with very high levels of data dropout induced by low total mRNA molecules and capture efficiency. This thesis explores the application of a transcriptional regulatory protein activity inference approach to single-cell data in order to resolve gene dropout and more deeply characterize upstream drivers of cell state within the micro-environment of several distinct tumor types. To this end, algorithms for inference of protein activity, drug sensitivity, and cell-cell interaction have been adapted to scRNA-Seq data, along with an approach for querying enrichment of single-cell-derived population marker gene sets patient-by-patient in larger bulk-RNA-Seq cohorts. By applying these tools systematically, we have identified distinct cellular sub-populations associated with clinical outcome in different tumor types, including a novel population of C1Q+/TREM2+/APOE+ macrophages associated with post-surgical tumor recurrence in clear cell renal carcinoma, a sub-population of fibroblasts associated with improved response to immunotherapy in head and neck squamous cell carcinoma, tumor cell subpopulations with distinct inferred drug sensitivities in cholangiocarcinoma and prostate cancer, as well as tumor-specific regulatory T-cells (Tregs), active as a mechanism of immunotherapy resistance across a range of tumor types. In ongoing clinical trials from both primary and metastatic prostate cancer as well as clear cell renal carcinoma, we are able to assess which of these populations are enriched in non-responders to checkpoint immunotherapy. The proteomic master regulators of each of these single-cell types have direct utility as potential biomarkers for treatment response, but they may also be therapeutically modulated as novel targets for combination immunotherapy, potentially improving treatment response rates and treatment outcomes in future clinical trials. Finally, this thesis also presents a discovery-to-validation platform to accelerate micro-environment-directed drug repurposing in the context of immunotherapy resistance and rapid CRISPRko validation of novel therapeutic targets. This platform has been developed specifically to validate newly identified master regulators of tumor-specific immunosuppressive regulatory T-cells (Tregs), resulting in discovery of low-dose gemcitabine as a tumor-specific Treg-modulating drug synergistic with anti-PD1 checkpoint immunotherapy and TRPS1 as a proteomic master regulator with clinically significant effect on tumor Treg-infiltrating and tumor growth rate. However, the platform itself may be readily extended in future work to prioritize agents against immunosuppressive macrophage and fibroblast populations for clinical development and trials. As we have discovered, different cancers have different populations of cells driving therapy response and resistance. Taken together, the analytical and validation tools presented in this thesis represent an opportunity to tailor future immuno-therapies at the single-cell level to particular tumor types and to individual patients.

Cai, Ganhui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 160-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In recent years, three dimensional (3D)-culture technique has emerged as a very popular approach to reconstruct tissue architectures and develop experimental models for studying epithelial cancers. However, 3D culture models of prostate epithelial cells to mimic prostate cancer development are relatively rare, making it highly desirable to develop and characterize novel 3D culture models suitable for studying prostate cancer. Recently, epithelial-mesenchymal transition (EMT) has emerged as an important mechanism for cancer cell invasion. The zinc finger transcriptional factor Snail as a key regulator of EMT has been found to contribute to aggressive progression in many types of neoplasms. Even though several studies corroborated that EMT is implicated in prostate cancer, the expression patterns of Snail in normal prostate and prostate cancer, and the functional role of Snail in prostate cancer as well as its relation with EMT are still unknown. Based on this background, my major efforts were to establish a 3D culture model of human prostatic epithelial cells with structural and functional relevance to prostate gland and to employ this model to study the functional role of Snail in the prostate cancer. / When embedded in Matrigel for 3D culture, BPH-1 cells developed into growth-arrested acinar structures with a hollow lumen. Ultrastructural examination of BPH-1 spheroids by electricon microscopy indicated that BPH-1 spheroids displayed a polarized differentiation phenotype. Immunoflurescence analysis of polarized epithelial markers further confirmed that BPH-1 spheroids were polarized. In contrast, tumorigenic BPH-1CAFTD cells exhibited disorganized and continuously proliferating structures in Matrigel, with polarized epithelial markers randomly diffused or completely lost. In addition, BPH-1 CAFTD cells displayed significantly higher invasive capacity in comparison to BPH-1 cells by transwell invasion assay. Moreover, LY294002 treatment of BPH-1CAFTD1 and BPH-1CAFTD3 cells in 3D cultures resulted in impaired cell proliferation as evidenced by reduced colony size and decreased Ki-67 index, and western blot analysis showed that cyclin D1 protein levels were significantly decreased, while p21 protein levels were slightly up-regulated in LY294002-treated 3D cultures. Additionally, LY94002 significantly decreased the invasive capacity of BPH-1CAFTD1 and BPH-1CAFTD3 cells. Interestingly, LY294002 treatment completely reverted the disorganized non-polar 3D structures of BPH-1CAFTD1 cells to well-organized polarized spheroid structures in Matrigel, but failed to restore the polarized differentiation in 3D cultures of BPH-1CAFTD3 cells, which still formed compact aggregates as shown by confocal immunofluorescence analysis. Snail protein was barely detected in the epithelial cells of human benign prostatic tissue but significantly elevated as nuclear protein in primary prostate cancer and bone metastatic specimens by immunohistochemical analysis. Snail transcript levels were weakly expressed in a majority of nonmalignant prostatic epithelial cell lines, while markedly increased in almost all tested cancer cell lines. Snail expression induced a morphological switch to more scattered and spindle-shaped appearance in BPH-1 and BPH-1CAFTD1 cells in 2D culture, and immunofluorescence analysis of several EMT specific markers indicated that Snail-expressing cells underwent EMT. In 3D contexts, Snail-expressing cells developed into more disorganized structures with many cords or protrusions, with a concurrent EMT change as evidenced by reduced E-cadherin and increased vimentin expression. In addition, Snail expression augmented the invasive capacities in both BPH-1 cells and BPH-lCAFTD1 cells, but did not significantly affect the migratory capacities. Snail expression enhanced the MMP2 activity in BPH-1 cells and promoted both MMP-2 and MMP-9 activities in BPH-1CAFTD1 cells. Moreover, Snail expression enhanced anchorage-independent growth capability in BPH-1 cells, but failed to initiate tumor formation in nude-mice. Lastly, Snail expression induced a dramatic increase in FoxC2 and SPARC transcripts but a marked decrease in claudin-1 and p63 transcripts. / Chu, Jianhong. / Adviser: Franky Chan Leung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3448. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 143-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

[Truncated abstract] In recent years a better understanding of the biology of PPAR , a nuclear transcription factor, has emerged, leading to a resurgence in targeting PPAR for chemotherapy. The family of synthetic PPAR agonists, the thiazolidinediones (TZDs), and non-steroidal anti-inflammatory drugs (NSAIDs) have been implicated in the inhibition of cell proliferation, apoptosis and cell cycle arrest of androgen-sensitive (LNCaP) and androgen-independent (PC-3 and DU145) prostate cancer cells generating much interest in their use for potential curative cancer therapies. In light of the potential use of TZDs and NSAIDs in prostate cancer prevention and their ability to induce inhibitory effects in vitro and in vivo, the first aim of this project was to undertake a comprehensive study of the effects of ciglitazone (TZD) and indomethacin (NSAID) on the androgen-independent prostate cancer cell line DU145, using standardised concentrations and time-points to compare the effects of TZDs and NSAIDs on cell proliferation, cell cycle and apoptosis. Treating the cells with either 10 µM ciglitazone or 10 µM indomethacin resulted in a time-dependent decrease in DU145 cell proliferation. The anti-proliferative effects were found to be in-part attributed to the slowing of cell progression through the G1/S-phase checkpoint of the cell cycle, and in the case of ciglitazone, apoptosis also played a role in its anti-proliferative effects in this cell line. Interestingly, although indomethacin failed to induce apoptosis, its antiproliferative effects were more potent than ciglitazone. The second aim of this project was to further investigate the underlying mechanisms responsible for the anti-proliferative effects of ciglitazone and indomethacin by evaluating their ability to modulate PPAR mRNA and protein expression, and to induce PPAR transcriptional activity. ... In addition, ligandinduced regulation of secreted frizzled related protein 4 (sFRP4) expression, a Wnt/ - catenin antagonists, was investigated. It was demonstrated that both ciglitazone and indomethacin attenuated Wnt/ -catenin signalling via the down-regulation of total - catenin levels within the cells, inhibition or slowing of the translocation of cytoplasmic -catenin into the nucleus and inhibition of cyclin–D1 expression An inverse relationship between PPAR and -catenin protein levels was also detected, suggesting that PPAR may directly bind to -catenin itself. sFRP4 expression was transiently upregulated by ciglitazone and indomethacin-treatment, suggesting that the antiproliferative effects of the ligands may be mediated in part through regulation of sFRP4 mRNA and protein levels. In summary, the anti-proliferative effects of ciglitazone and indomethacin on the androgen-independent prostate cancer cell line, DU145, described in this thesis are progressive steps in characterising the role of PPAR in prostate cancer cell proliferation. The identification of indomethacin as a more potent PPAR agonist than ciglitazone represents a novel target for the development of preventative strategies for advanced disease, and the relationship between PPAR and the Wnt/ -catenin signalling pathway provide an insight into the mechanisms involved in the anti-proliferative effects of ciglitazone and indomethacin. Further studies into this relationship would advance help identify novel preventative and curative therapeutic strategies for advanced prostate cancer.

Evidence-based guidelines are important sources of knowledge in everyday clinical practice. In 2005, the German Society for Urology decided to develop a highquality evidence-based guideline for the early detection, diagnosis and treatment of the different clinical manifestations of prostate cancer. The guideline project started in 2005 and involved 75 experts from 10 different medical societies or medical organizations including a patient organization. The guideline was issued in September 2009 and consists of 8 chapters, 170 recommendations, and 42 statements. Due to the broad spectrum of clinical questions covered by the guideline and the high number of participating organizations and authors, the organizers faced several methodological and organizational challenges. This article describes the methods used in the development of the guideline and highlights critical points and challenges in the development process. Strategies to overcome these problems are suggested which might be beneficial in the development of new evidence-based guidelines in the future. / Evidenzbasierte Leitlinien sind wichtige Quellen komprimierten Wissens für die tägliche klinische Praxis. Die Deutsche Gesellschaft für Urologie beschloss im Jahr 2005, eine qualitativ hochwertige evidenzbasierte Leitlinie zur Früherkennung, Diagnose und Behandlung der verschiedenen klinischen Manifestationen des Prostatakarzinoms zu erstellen. Das Leitlinienprojekt begann im Jahr 2005 unter Mitwirkung von 75 Experten und Patientenvertretern aus 10 verschiedenen Fachgesellschaften und Organisationen. Die Leitlinie wurde im September 2009 veröffentlicht und besteht aus 8 Kapiteln mit insgesamt 170 Empfehlungen und 42 Statements. Das breite thematische Spektrum der Leitlinie und die hohe Zahl teilnehmender Autoren und Organisationen stellten die Organisatoren vor verschiedene methodische und logistische Herausforderungen. Dieser Beitrag stellt die angewendete Methodik bei der Leitlinienerstellung dar und betont kritische Punkte und Probleme der Erstellung. Die beschriebenen Lösungsansätze können bei der Planung und Durchführung künftiger evidenzbasierter Leitlinienprojekte hilfreich sein. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.