Nei pi xing yi yang hua dan he cheng mei xin hao tong lu zai qian lie xian ai gan xi bao zeng zhi zhong de gong neng yan jiu

Ph.D.Prostate cancer (PCa) is the most commonly diagnosed cancer in men and one of the major causes of cancer deaths around the world including Hong Kong. Androgen-depletion therapy (ADT) is the mainstay and also effective therapeutic for PCa, but only initially as many patients will eventually relapse and progress to the advanced, castration-resistant and metastatic stage (CRPC), to which current treatments are few and poorly efficient. Cumulative studies indicate that there is a small subpopulation of cancer cells exist in the tumour bulks referred as cancer stem-like cells (CSCs) or tumour-initiating cells as they share many common characteristics with normal stem cells, particularly self-renewal and multipotency. Various methodologies have been developed for the identification and isolation of CSCs, including specific cell markers sorting, reporter system and sphere culture. Non-adherent sphere culture, based on coating of synthetic hydrogels or natural hydrogels, is one of the best methods to produce high quality and quantity of CSCs. However, the disadvantages of each method limit their application as well as the CSCs research progression. Agar-based sphere culture method is also not yet fully developed. CSCs possess the potent multidrug resistance and tumour re-generation ability so that they are considered to contribute to the tumour initiation, treatment resistance and relapse. Prostate CSCs (PCSCs) have been isolated and studied from various in vitro and in vivo sources and are believed to play a critical role in the development of CRPC and also its metastasis. Targeting PCSCs is a promising therapeutic strategy for the treatment of advanced PCa, however, the detailed mechanisms of PCSCs growth as potential targets are still unclear. Nitric oxide (NO) is a small biological gas which acts as a cellular signalling messenger with pleiotropic functions in human health and diseases, including cancer in which NO molecules may promote tumour growth as well as suppress tumour growth depending on the concentration and the microenvironment. In CSCs, NO molecules produced by nitric oxide synthase (NOS) are also found playing an important role in maintaining their stemness and tumorigenic capacity. Our previous work demonstrates that enhanced endothelial NOS (eNOS) activation with increased NO level can contribute to the antiandrogen-resistant growth of PCa cells. Furthermore, the CRPC xenograft animal model (VCaP-CRPC) was found to express much higher PCSCs-associated cell markers. However, the role of NOSs/NO signalling in PCSCs proliferation remains unclear.In this study, a newly improved agar-based non-adherent sphere culture method was developed to enrich CSCs from various cancer cell lines, including prostate cancer (DU145, LNCaP, VCaP and 22Rv1), hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT116), as well as prostate tumour samples. The spheres derived from PCa cells by agar-based non-adherent culture method were shown to grow much better (in term of numbers and sizes) in the 0.6 - 1.2 % agar coated dishes than > 1.2 % agar-coated dishes and have a comparable efficiency to commercial Ultra-low attachment dishes. The tumour spheres grown by the 0.9 % agar-based non-adherent culture method were validated to contain enriched CSCs by their higher expressions of CSC-specific markers, including CD133, NANOG, OCT4, BMI1, AR, PSA, enhanced in vivo tumorigenic capability compared to the adherent 2D cultures as well as positive signals of the CSCs reporter system (SORE6-GFP). Moreover, continual passages of the prostaspheres in the same agar-coated dishes could maintain the stemness, expand the number of CSCs and also eliminate the false-positive spheres derived from non-CSCs.Further study found that the PCa cell line-derived prostaspheres and CD133+ cells sorted from primary cultured prostate tumour tissues exhibited up-regulated eNOS expression and NO production. Interruption of eNOS/NO signalling, cGMP/PKG- dependent, by NOSs inhibitors and eNOS-shRNAs significantly suppressed the sphere formation capability of PCa cells. Moreover, exogenous overexpression of eNOS could induce increased expressions of CSC markers and promote the in vivo tumour growth and metastasis of PCa orthotopic xenografts, while knock-down of eNOS could prevent the tumour growth and metastasis. Combined whole transcriptome sequencing analysis and qPCR validation of PCSCs with eNOS knock-down identified a list of potential targets of eNOS signalling in the growth regulation of PCSCs. Some identified genes (F2RL1, HES6, SCAMP3, DANCR) showed up-regulation in the prostaspheres as compared to adherent 2D cultures, and importantly, their expression levels decreased back to the low levels upon eNOS knock-down. On the contrary, expression of KDM3A was down-regulated in the prostaspheres but was restored when eNOS activity was inhibited.Taken together, this thesis described the development of a novel agar-based non-adherent sphere culture method and its optimization for the enrichment of CSCs with advantages demonstrated including its simplicity, reproducibility, low cost and also the repeated use of the agar-coated plates for serial passages of CSC-enriched spheres. Furthermore, the results also suggest that the involvement of eNOS/NO signalling in the growth regulation of PCSCs via cGMP/PKG dependent pathway. The potential pathways regulated by eNOS were also screened and investigated some of the down-stream targets of eNOS/NO signalling could be the potential therapeutic targets for CRPC.前列腺癌是世界各地，包括香港，男性中最常见的癌症也是癌症死亡的主要誘因之一。雄激素治療是早期前列腺癌的主要和有效療法，但是大多數患者最终都會复发并进展為晚期的，致命的，去勢抵抗性前列腺癌（castration-resistant prostate cancer, CRPC）。目前，針對CRPC的治疗選擇很少且效果差。研究表明，肿瘤细胞中存在着一个小的細胞亞群，它们与正常的干细胞有着许多共同之處，因此被称为癌症類干细胞（cancer stem-like cells, CSCs）或肿瘤起始细胞。对CSCs的鑒定和分离已經建立了多種方法，包括特定的细胞标记篩選、熒光报告系统和微球體培养等。基于合成水凝胶鋪制的微球體懸浮培养技术是培養高质量和数量CSCs的最佳方法之一,但是它們的缺点限制了其应用及CSCs的研究进展。然而基於天然水凝胶（琼脂）的微球體培养方法还没有得到完全的发展及驗證。CSCs具有较强的多药耐药性和肿瘤再生成能力,因此被认为參與了肿瘤的起始、治疗耐药性產生和复发。前列腺CSCs（PCSCs）已經在不同的体外和体内来源的前列腺癌樣本中分離鑒定和研究，并被认为在CRPC的发展和及其转移過程中发挥著关键的作用。因此，靶向PCSCs的療法是治疗晚期前列腺癌的一种很有希望的方法。然而，關於PCSCs生长的详细机制仍不清楚。一氧化氮（nitric oxide, NO）是一种小分子气体，可作为细胞信号信使在人类的健康和疾病中，包括癌症，發揮多樣性的功能。在CSCs的研究中，一氧化氮合酶(nitric oxide synthase, NOS)产生的NO分子在维持其乾性和腫瘤再生能力方面发挥着重要作用。我们之前的研究也表明，增强的内皮型一氧化氮合酶（endothelial NOS, eNOS）活性及NO水平可以促进前列腺癌细胞的耐药性生长。此外，CRPC的动物模型显示出更高的PCSCs特異性標記物表达水平。然而，NOSs/NO信号通路在PCSCs生長中的作用仍然不清楚。本論文發展了一种新改进的基於琼脂板的微球體培养方法，在多種癌细胞系中成功的富集了CSCs，包括前列腺癌（DU145、LNCaP、VCaP和22Rv1）、肝癌（HepG2）、大肠癌（HCT116）以及前列腺肿瘤組織樣本。前列腺癌细胞在0.6-1.2%琼脂板上培養的微球体在数量和直徑大小上都要好于>1.2%的琼脂板培養的微球體。更重要的是，在0.9%琼脂板上悬浮培养的微球體表達了更高的PCSCs特異性細胞標誌物，包括CD133、NANOG、OCT4、BMI1、AR、PSA等，以及加強的腫瘤再生能力。這些結果验证了在瓊脂板上懸浮培養的微球體含有丰富的PCSCs。此外,微球體在相同的琼脂瓊脂板上可以连续傳代培養可以保持其乾性，扩大CSCs的細胞数量, 并消除非CSCs衍生的假阳性微球体。进一步的研究发现，前列腺癌微球體以及CD133+的前列腺肿瘤组织原代培养物具有較高的eNOS表达量和NO水平。使用NOSs抑制剂或eNOS沉默阻断eNOS/NO信号（cGMP/PKG依赖的）显著地抑制了前列腺癌微球體的生長。此外，eNOS高表达可引起CSC特異性标记物的表达增加并促进原位肿瘤的生长和转移,而沉默eNOS可阻止肿瘤的生长和转移。结合對eNOS敲除的PCSCs的转录組测序分析和验证結果，本文确定了一系列eNOS信號通路調控PCSCs生长的潜在通路靶點。其中一些基因（例如 F2RL1、HES6、SCAMP3、DANCR），相較于2D貼壁培養的細胞，在前列腺癌微球體中的有較高的表達量，更重要的是，它们的表达水平在eNOS沉默后卻降低到了低水平。與此相反，KDM3A在前列腺癌微球體中的表达比2D貼壁培養的細胞要低一些，但在抑制eNOS后恢复到高水平。綜上，本研究發展并优化了一种新的瓊脂板懸浮培養微球體的方法來富集CSCs。其优点包括操作简单、結果重现性好、成本低廉以及可以重复使用來培養同一種CSCs。此外，還發現eNOS/NO信号能夠通过cGMP/PKG依赖的途径调控PCSCs的生长，並且eNOS调控的潜在通路也被筛选出来，這些可能是針對CRPC的潜在治疗靶点。Gao, Weijie.Thesis Ph.D. Chinese University of Hong Kong 2018.Includes bibliographical references (leaves 95-115).Abstracts also in Chinese.Title from PDF title page (viewed on …).Gao, Weijie