第一部分 尿液活性肿瘤细胞定量检测在膀胱癌诊断和疗效监测中的应用

背景：膀胱癌是第二大常见的泌尿系统肿瘤，超过70%的病例在确诊时为非肌层浸润性（Non-Muscle Invasive Bladder Cancer，NMIBC）。经尿道膀胱肿瘤切除术（Transurethral Resection of Bladder Tumors，TURBT）联合术后即刻卡介苗（Bacillus Calmette-Guérin，BCG）或化疗灌注是标准疗法，但TURBT术后高达60%-70%的复发率表明有必要改进治疗策略。持续生理盐水膀胱冲洗（Continuous Saline Bladder Irrigation，CSBI）是一种潜在的改进方案，但目前缺乏膀胱灌注后应用CSBI的临床证据。为观察治疗效果和检测复发，亟需一种可靠的肿瘤负荷检测方法。现有的诊断方法，如膀胱镜检查和尿液细胞学检查，均存在局限性，包括侵入性或灵敏度不足。非细胞生物标志物虽有进展，但不能有效量化肿瘤负荷。尿液中脱落的肿瘤细胞能够反映总体肿瘤负荷和复发情况，是一种有前景的生物标记物。有研究表明，PD-L1阳性的尿液肿瘤细胞能够预测免疫疗法的反应和生存结果。微流控技术与表面标志物联用提高了灵敏度，但仍难以区分尿液环境中的活性细胞。

目的：目前临床缺乏高灵敏度、高特异性的非侵入性膀胱癌定量检测方法，且TURBT联合即刻膀胱灌注治疗方案是否需要进一步的临床策略，尚缺乏临床证据支持。本研究旨在建立一种可靠的尿活性肿瘤细胞（Active Urinary Cancer Cells，AUCC）定量检测方法，并通过分析TURBT围手术期AUCC的变化，研究持续生理盐水膀胱冲洗作为一种可行方案的临床疗效。

方法：本研究建立了一种AUCC定量检测方案，并通过单细胞全基因组测序验证了其可靠性。研究纳入了2021年至2023年期间经膀胱镜检查和病理活检确诊的膀胱癌患者（N=324）以及对照组受试者（N=92）。入组的非肌层浸润性膀胱癌患者均接受了TURBT手术，术后即刻膀胱内灌注表柔比星，之后分组接受或不接受CSBI，并在术后第1天和第5天检测AUCC。对患者进行了为期2年的术后复发随访。

结果：研究证明AUCC方案具有良好的诊断效能，灵敏度为0.821，特异性为0.902。无论患者是否接受了CSBI治疗，TURBT联合即刻膀胱灌注术后第一天，AUCC均出现升高。然而，接受CSBI治疗的患者，其AUCC在第五天下降更为迅速，且存在高危因素的患者从CSBI中获益更多。2年的随访结果表明，接受复杂手术的高危患者能够从CSBI中获得显著益处，2年内复发率明显降低。

结论：本研究开创了一种AUCC定量检测方法，为TURBT导致肿瘤细胞播散提供了实验室证据，并证明CSBI可作为降低潜在复发风险的进一步临床策略。

第二部分 基于循环肿瘤细胞类器官单细胞图谱的非小细胞肺癌转移机制的研究

背景：NSCLC（Non-Small Cell Lung Cancer，NSCLC）治疗面临的最大挑战在于肿瘤间和肿瘤内的高度异质性。这种异质性会驱动肿瘤进展，导致复发/转移和耐药，最终影响患者的生存。因此，临床实践亟需一种精准治疗的临床前模型，以个性化和动态的方式指导临床选择最佳治疗方案。目前，三维类器官培养技术展现出极大的应用前景。它保持原始肿瘤细胞的基因变化和表达，从而进行可重复的体外研究，进而成为肿瘤异质性研究和抗肿瘤药物筛选的平台，并对患者进行精确分层，实现个性化精准治疗。然而，目前的三维类器官研究主要来源于肿瘤组织样本，具有一定的时效性，缺乏动态追踪能力。循环肿瘤细胞（Circulating Tumor Cells，CTC）衍生的类器官为解决这一问题提供了可能。与其他样本来源相比，CTC的采集具有方便、无创、动态等特点，特别适合术后无法实时获取肿瘤组织样本或处于疾病晚期的患者，可实时追踪肿瘤进展，获取驱动基因突变的最新状况。更重要的是，基因突变、拷贝数变异和旁路信号通路的异常激活往往与原发肿瘤或转移瘤中观察到的非常相似。CTC衍生的器官完全符合肿瘤长期治疗的个性化、动态化需求，可实时、体外准确复制体内肿瘤进展状态和分子生物学特征，对揭示肿瘤异质性和复发转移的深层机制、指导临床决策具有重要意义。然而，迄今为止，从患者样本中直接分离和富集CTC进行三维类器官培养的研究还很有限。值得一提的是，本课题组既往的研究已成功培养出NSCLC患者肺静脉血来源的CTC类器官。

目的：本研究旨在建立CTC类器官，作为可满足精准治疗临床实践的临床前模型，以个性化和动态的方式指导临床选择最佳治疗方案。同时，利用测序和生物信息学分析，了解CTC分子生物学特征，揭示肿瘤异质性和复发转移的深层机制。

方法：本研究对NSCLC患者肺静脉血来源的CTC类器官及配对的组织来源类器官进行了外显子测序及单细胞转录组测序分析，对其进行全面的比较分析，寻找CTC执行复发转移行为的关键亚群和特征性基因，并进行分子和功能验证。

结果：CTC衍生的类器官能够准确地再现原发性肿瘤的主要特征，且与组织衍生的类器官相比具有更高的干性和上皮-间质转化（Epithelial-Mesenchymal Transition，EMT）表达水平。此外，在CTC衍生的器官组织的特定亚群中，FAM83A的表达显著增加，提示其在CTC的侵袭和转移中发挥了关键作用。

结论：这项研究提供了对NSCLC中CTC和肿瘤组织器官组织的全面了解，揭示了CTC的生物学本质，并为预防和干预转移提供了潜在靶点。

Part I Quantitative active urinary cancer cell assay in the diagnosis and efficacy monitoring of bladder cancer

Background: Bladder cancer, the second most common urologic tumor, often presents as non-muscle invasive (NMIBC) in over 70% of cases at diagnosis. Standard treatment, transurethral resection of bladder tumors (TURBT), is often followed by BCG or chemotherapy instillation, but 60-70% of recurrence rates after TURBT highlight the need for improved strategies. Continuous saline bladder irrigation (CSBI) is among the approaches, though evidence for CSBI post-immediate bladder instillation is lacking.

The quest for a reliable assay to monitor tumor burden over time is essential to observe treatment effects and detect recurrence. Current diagnostic methods, cystoscopy, and urine cytology have limitations including invasiveness or low sensitivity. Non-cellular biomarkers, while advanced, do not quantify tumor burden effectively. Tumor cells exfoliated in urine, reflecting overall tumor burden and indicating recurrence, offer a promising biomarker. A study showed that PD-L1-positive urinary tumor cells predict response to immunotherapy and survival outcomes. Microfluidics combined with surface markers have improved sensitivity, but don't distinguish active cells amidst urine complexity.

Objective: There is a lack of highly sensitive and specific non-invasive quantitative assays for bladder cancer, and there is a lack of clinical evidence as to whether the TURBT combined with immediate bladder perfusion regimen requires further clinical strategies. The aim of this study was to establish a reliable quantitative assay for Active Urinary Cancer Cells (AUCC) and to investigate the clinical efficacy of continuous saline bladder irrigation as a viable regimen by analyzing the perioperative AUCC changes with TURBT.

Methods: An AUCC assay was developed and its reliability was verified by single-cell whole genome sequencing. Bladder cancer patients (N=324) diagnosed by cystoscopy and pathologic biopsy and control individuals (N=92) were included from 2021 to 2023 in the study. Enrolled patients with non-muscle invasive bladder cancer (NMIBC) underwent TURBT followed by immediate bladder instillation of epirubicin, after subgroups received CSBI or not, and AUCCs were tested on the first and fifth postoperative day. The patients were followed up for two years for postoperative recurrence.

Results: The AUCC assay achieved good detection accuracy, with a sensitivity of 0.821 and specificity of 0.902. AUCC increased on the first day after TURBT in combination with immediate bladder instillation, regardless of whether or not the patient received CSBI. However, AUCCs decreased more rapidly on the fifth day in patients treated with CSBI, and patients with concomitant risk factors benefited more from CSBI. The two-year follow-up results showed that high-risk patients with complex surgeries could benefit significantly from CSBI.

Conclusions: We pioneered a quantitative assay for AUCC and provided laboratory evidence that TURBT causes tumor cell dissemination and CSBI can be further clinical strategy to reduce the risk of potential recurrence.

Part II Single-cell profiling of circulating tumor cell organoids reveals molecular signatures associated with metastasis in non-small cell lung cancer

Background: The greatest difficulty in the treatment of non-small cell lung cancer (NSCLC) is its high degree of inter- and intra-tumor heterogeneity, which drives tumor progression, leads to recurrence/metastasis and drug resistance, and ultimately affects patient survival. Therefore, clinical practice requires a preclinical model of precision therapy that can guide clinical selection of the best treatment in a personalized and dynamic manner. At present, 3D organoid culture technology is the most promising application option. By maintaining the genetic changes and expression of the original tumor cells, it can conduct repeatable in vitro studies, and then become a platform for tumor heterogeneity research and anti-tumor drug screening, and accurately stratify patients to achieve personalized and precise treatment. However, the current 3D organoid research mainly comes from tumor tissue samples, which has certain timeliness and lacks dynamic tracking ability. Circulating tumor cell (CTC) derived organoids offer a possible solution to this problem. Compared to other sample sources, the collection ofCTCis convenient, non-invasive, and dynamic, making it particularly suitable for patients who are unable to obtain tumor tissue samples in real time after surgery or who are in an advanced stage of disease, and can track tumor progression in real time to obtain the latest status of driver gene mutations. Importantly, genetic mutations, copy number variants, and abnormal activation of bypass signaling pathways are often very similar to those observed in primary tumors or metastases.CTCderived organoids perfectly meet the personalized and dynamic needs of long-term tumor treatment, and can accurately replicate in vivo tumor progression status and molecular biological characteristics in real time and in vitro, which is of great significance for revealing the deep mechanism of tumor heterogeneity and recurrence and metastasis, and guiding clinical decision-making. However, to date, there have been limited studies on the direct isolation and enrichment ofCTCfrom patient samples for 3D organoid culture.

Objective: To establishCTCorganoids as a preclinical model that can meet the clinical practice of precision therapy, to guide the clinical selection of optimal treatment protocols in a personalized and dynamic manner, and to understand the molecular biological characteristics ofCTCby using sequencing and biosignature analysis to reveal the deep mechanism of tumor heterogeneity and recurrence and metastasis.

Methods: In this study, exome sequencing and single-cell transcriptome sequencing analyses were performed onCTCorganoids derived from pulmonary venous blood and paired tissue-derived organoids from NSCLC patients, to perform comprehensive comparative analyses and search for key subgroups and characteristic genes ofCTCthat perform recurrent metastatic behaviors, as well as to perform molecular and functional validation.

Results: CTC-derived organoids accurately recapitulated key features of primary tumors with higher levels of stemness and epithelial-mesenchymal transition (EMT) expression compared to tissue-derived organoids. In addition, FAM83A expression was significantly increased in a specific subpopulation of CTC-derived organ tissues, playing a key role inCTCinvasion and metastasis.

Conclusions: This study provides a comprehensive understanding of CTC and tumor-derived organ tissues in NSCLC, reveals the biological nature of CTC, and provides potential targets for prevention and intervention of metastasis.