AbstractThis study aims to evaluate the prognostic utility of Activity-dependent neuroprotective protein (ADNP) expression in Circulating Tumor Cells (CTCs) inpatients with Non-muscle-invasive Bladder Cancer (NMIBC) undergoing Transurethral Resection of Bladder Tumor (TURBT). A prospective cohort of 74 bladder cancer patients and 22 non-cancer controls were enrolled.

摘要本研究旨在评估经尿道膀胱肿瘤切除术（TURBT）住院的非肌层浸润性膀胱癌（NMIBC）患者循环肿瘤细胞（CTC）中活性依赖性神经保护蛋白（ADNP）表达的预后效用。纳入了74名膀胱癌患者和22名非癌症对照的前瞻性队列。

The expression of ADNP mRNA was detected by immunomagnetic beads-droplet digital PCR. The ADNP mRNA expression was evaluated in patients with high-risk NMIBC and those with indeterminate invasion depth post 2nd TURBT. Primary cultured bladder cancer cells and PBMCs from healthy donors were immunofluorescence stained.

通过免疫磁珠液滴数字PCR检测ADNP mRNA的表达。在高危NMIBC患者和第二次TURBT后侵袭深度不确定的患者中评估ADNP mRNA表达。对来自健康供体的原代培养的膀胱癌细胞和PBMC进行免疫荧光染色。

Our findings suggest that baseline ADNP mRNA level in CTCs shows potential as a prognostic marker for NMIBC with a sensitivity of 83.33% and a specificity of 73.58%. In comparison to baseline, ADNP mRNA expression increased post 2nd TURBT in 5 patients, where 2 experienced recurrence. Meanwhile, among the 12 patients with decreased levels, only one patient relapsed.

我们的研究结果表明，CTC中的基线ADNP mRNA水平显示出作为NMIBC预后标志物的潜力，敏感性为83.33%，特异性为73.58%。与基线相比，5例患者第二次TURBT后ADNP mRNA表达增加，其中2例复发。同时，在12名水平下降的患者中，只有一名患者复发。

A considerable limitation of this study entails the small sample size. The Immuno-magnetic beads-ddPCR technique provided a viable method for ADNP mRNA detection in CTCs from bladder cancer patients. The preoperative ADNP mRNA level in CTCs was identified as a prognostic indicator for NMIBC. Longitudinal monitoring of ADNP mRNA in CTCs of bladder cancer patients shows promise in evaluating treatment responses and predicting prognosis..

。免疫磁珠ddPCR技术为膀胱癌患者CTC中ADNP mRNA的检测提供了一种可行的方法。CTC术前ADNP mRNA水平被确定为NMIBC的预后指标。膀胱癌患者CTC中ADNP mRNA的纵向监测在评估治疗反应和预测预后方面显示出前景。。

IntroductionBladder cancer (BC) is one of the most prevalent forms of cancer globally, with an estimated 570,000 new cases and 210,000 deaths reported in 2020 alone1. Dominantly, Non-muscle invasive bladder cancer (NMIBC), including carcinoma in situ (CIS), as well as stages Ta and T1, stands for approximately 75% of all new BC cases.

引言膀胱癌（BC）是全球最普遍的癌症之一，仅2020年就报告了570000例新病例和210000例死亡1。主要是非肌肉浸润性膀胱癌（NMIBC），包括原位癌（CIS），以及Ta和T1期，约占所有新BC病例的75%。

Despite a high recurrence rate of 50–70% within five years of diagnosis, only 10–20% of these patients progress to the more severe Muscle-invasive bladder cancer (MIBC)2. Notably, MIBC presents an even more daunting prognosis, with the metastases potentially developing in up to 50% of patients within two years of operative intervention, despite initial first-line treatment with cisplatin-based neoadjuvant chemotherapy followed by radical cystectomy and lymph node dissection3.

尽管诊断后五年内复发率高达50-70%，但这些患者中只有10-20%进展为更严重的肌肉浸润性膀胱癌（MIBC）2。值得注意的是，MIBC的预后甚至更令人望而生畏，尽管最初使用顺铂为基础的新辅助化疗进行一线治疗，然后进行根治性膀胱切除术和淋巴结清扫，但在手术干预的两年内，高达50%的患者可能会发生转移3。

This metastasis development further coincides with a survival rate of 36–48% at the five-year mark post-surgery4.The principal causal factor for mortality in BC patients is metastatic bladder cancer5,6. The challenge with MBC is the difficulty in early detection of tumor cell spread, implying that timely and potentially effective interventions are often unattainable7,8,9.

这种转移的发展与术后五年的生存率36-48%相吻合[4]。BC患者死亡的主要原因是转移性膀胱癌[5,6]。MBC面临的挑战是难以早期发现肿瘤细胞扩散，这意味着及时和潜在有效的干预措施往往无法实现7,8,9。

Current clinical management strategies for BC heavily rely on data from the tumor obtained via Transurethral Resection of the Bladder Tumor (TURBT), which may not provide sufficient information to determine optimal individualized treatment approaches3,10,11.Radical bladder surgery, currently widely used for early bladder cancer management, offers optimal cure chances for invasive NMIBC yet remains an excessive procedure for a subgroup of patients12.

目前BC的临床管理策略严重依赖于通过经尿道膀胱肿瘤切除术（TURBT）获得的肿瘤数据，这可能无法提供足够的信息来确定最佳的个体化治疗方法3,10,11。根治性膀胱手术目前广泛用于早期膀胱癌管理，为侵袭性NMIBC提供了最佳的治愈机会，但对于亚组患者来说仍然是一种过度的手术12。

Circulating Tumor Cells (CTCs), originating either from primary tumors or metastatic lesions, are perceived as metastatic seeds13,14,15,16. Current evide.

源自原发性肿瘤或转移性病变的循环肿瘤细胞（CTC）被认为是转移性种子13,14,15,16。当前evide。

① Age 18–80 years.

① 年龄18-80岁。

② Patients with Eastern Cooperative Oncology Group (ECOG) score ≤ 2 points.

② 东部肿瘤协作组（ECOG）患者评分≤2分。

③ No bladder cancer patient underwent antitumor therapy before diagnosis. All the treatments were performed in accordance with the Guidelines for the Diagnosis and Treatment of Urinary Surgery Diseases in China (2014 edition).

③ 诊断前没有膀胱癌患者接受过抗肿瘤治疗。所有治疗均按照《中国泌尿外科疾病诊治指南》（2014年版）进行。

The stage of bladder cancer was classified according to the International Union Against Cancer (UICC) TNM staging of bladder cancer (2017, 8th edition) and the grade was assessed according to the World Health Organization 2004 bladder cancer grading system.The ADNP mRNA expression was detected by ddPCR.

根据国际抗癌联盟（UICC）膀胱癌TNM分期（2017年，第8版）对膀胱癌的分期进行分类，并根据世界卫生组织2004年膀胱癌分级系统进行评估。。

(a)

（一）

Immunomagnetic beads enriched peripheral blood CTCs: Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep density gradient centrifugation (STEMCELL, Cat 07851) with 6 ml blood sample. Add EasySep Hu CD45 Depletion Buffer (STEMCELL, Cat 17898) antibody to remove white blood cells and get 100 μl liquid containing CTCs..

富含免疫磁珠的外周血CTC：通过Lymphoprep密度梯度离心（STEMCELL，目录号07851）和6 ml血液样品分离外周血单核细胞（PBMC）。加入EasySep Hu CD45耗尽缓冲液（STEMCELL，Cat＃17898）抗体以去除白细胞，并获得100μl含CTC的液体。。

(b)

（b）

RNA extraction from CTCs: CTC samples were subjected to RNA extraction using the RNeasy Plus Micro Kit (Qiagen, Cat 74034). The collected RNA (20 μl) was used for one-step probe RT-dPCR detection or immediately stored in the refrigerator at − 80 °C.

从CTC中提取RNA：使用RNeasy Plus Micro Kit（Qiagen，目录号74034）对CTC样品进行RNA提取。收集的RNA（20μl）用于一步探针RT-dPCR检测或立即储存在-80℃的冰箱中。

(c)

ddPCR was used to detect ADNP mRNA expression in CTCs.

ddPCR用于检测CTC中ADNP mRNA的表达。

1.

1.

The synthesis of primers was entrusted to Sangon Biotech Co. Ltd., (Shanghai, China) and the primer sequences of differential genes were shown in Table 1.Table 1 The sequences of primers and probes of target and reference genes.Full size table

引物的合成委托给Sangon Biotech Co.Ltd.（中国上海），差异基因的引物序列如表1所示。表1靶基因和参考基因的引物和探针序列。全尺寸表

2.

2.

2. Each RNA sample was analysed in triplicate (5 μl × 3 = 15 μl). All reagents are prepared and thoroughly mixed at room temperature according to the following table (Table 2) (110% of the required amount is recommended).Table 2 Single reaction tube test reaction liquid preparation group.Full size table.

2.每个RNA样品一式三份进行分析（5μl×3＝15μl）。根据下表（表2）制备所有试剂并在室温下充分混合（建议使用所需量的110%）。表2单反应管测试反应液制备组。全尺寸表。

3.

3.

ddPCR was performed by the ddPCR platform system (TargetingOne Biotech. Co. Ltd. Beijing, China) which includes the Drop Maker M1 and the Chip Reader R1. A total of 15 μl RNA was added to the 75 μl mixture to make 90 μl of the reaction solution, which was then added in aliquots to three reaction tubes.

ddPCR由ddPCR平台系统（TargetingOne Biotech.Co.Ltd。Beijing，China）进行，该系统包括滴剂制造商M1和芯片读取器R1。将总共15μlRNA加入到75μl混合物中以制备90μl反应溶液，然后将其等分加入三个反应管中。

30 μl of the reaction solution was added to the aqueous phase reaction well of the microdroplet generation chip, 180 μl of microdroplet generated oil was added to the oil phase well (TargetingOne, Cat 10002). Samples were prepared as nanoliter droplets in the Drop Maker M1 sample preparator. Finally, the microdroplets were stored in 8-row tube..

将30μl反应溶液加入到微滴生成芯片的水相反应孔中，将180μl微滴生成的油加入到油相孔中（TargetingOne，目录号10002）。在Drop Maker M1样品制备仪中将样品制备为纳升液滴。最后，将微滴储存在8排管中。。

4.

4.

The 8-row tube containing microdroplets were placed into an ETC811 PCR apparatus (EASTWIN, Suzhou, China). The steps of reverse transcription and amplification were as follows: Stage 1: 55 °C, 15 min, 1 cycle; Stage 2: 95 °C, 10 min, 1 cycle; Stage 3: 40 cycles at 94 °C for 30 s, 55–60 °C for 1 min; Stage 4: 12 °C, 5 min 1 cycle..

将含有微滴的8排管放入ETC811 PCR装置（EASTWIN，Suzhou，China）中。逆转录和扩增的步骤如下：阶段1:55℃，15分钟，1个循环；第2阶段：95℃，10分钟，1个循环；第3阶段：在94°C下进行40个循环30 s，在55–60°C下进行1分钟；第4阶段：12°C，5分钟1个循环。。

5.

5.

The amplified samples were put into the Chip Reader R1 analyzer to read the fluorescence signal values of ADNP-FAM and beta-actin-VIC. After Poisson statistical analysis, the FAM and VIC copy numbers were obtained. The ratio of ADNP copy number (FAM) to beta-actin copy number (VIC) was used as the relative expression level of ADNP mRNA, and the mean value of three parallel experiments was used as the relative expression level of ADNP mRNA in CTCs of individual peripheral blood.

将扩增的样品放入Chip Reader R1分析仪中，以读取ADNP-FAM和β-肌动蛋白VIC的荧光信号值。经过泊松统计分析，获得了FAM和VIC拷贝数。ADNP拷贝数（FAM）与β-肌动蛋白拷贝数（VIC）的比值用作ADNP mRNA的相对表达水平，三个平行实验的平均值用作个体外周血CTC中ADNP mRNA的相对表达水平。

The coefficient of variation should not exceed 20%..

变异系数不应超过20%。。

Immunofluorescence staining (IF)

免疫荧光染色（IF）

1.

1.

Cells were released in 100 μl PBS and coated specimen frame of microscope slide (Cytelligen, San Diego, CA, USA). The slide was placed in a constant temperature drying oven at 37 °C for 20 min, so that the cells precipitate adhered to the slide.

细胞在100μlPBS中释放，并在显微镜载玻片（Cytelligen，圣地亚哥，加利福尼亚，美国）的包被样品框架中释放。将载玻片置于37℃的恒温干燥箱中20分钟，使细胞沉淀粘附在载玻片上。

2.

2.

Then fixed with 400 μl, 4% PFA (Kingmorn; Cat# KA1440) for 12 min. Slide was washed twice in PBS for 5 min each time.

。将载玻片在PBS中洗涤两次，每次5分钟。

3.

3.

Next, the slide was placed on a wet box and permeabilized with 5% Triton X-100 (Solarbio; Cat T8200) for 20 min, washed three times with PBS for 5 min each time.

接下来，将载玻片置于湿盒上，并用5%Triton X-100（Solarbio；目录号T8200）透化20分钟，每次用PBS洗涤3次，每次5分钟。

4.

4.

Incubated in a 3% BSA blocking solution (5% BSA was diluted to 3%BSA in PBS, Solarbio, Cat SW3015) for 30 min, then washed three times with PBS for 5 min each time.

在3%BSA封闭溶液（将5%BSA在PBS，Solarbio，Cat SW3015中稀释至3%BSA）中孵育30分钟，然后用PBS洗涤三次，每次5分钟。

5.

5.

Followed by incubation with 150 μl ADNP antibody (1:50, Bioss, Cat bs-0039R-FITC) for 60 min at room temperature, then washed three times with PBS for 5 min each time.

然后在室温下与150μlADNP抗体（1:50，Bioss，Cat bs-0039R-FITC）孵育60分钟，然后用PBS洗涤三次，每次5分钟。

6.

6.

Stained with 10 μl DAPI diluted for 3 min, and rinsed twice with PBS. Nuclei were stained with 10 μl DAPI and immediately observed under a fluorescence microscope or preserved in a refrigerator at 4 °C and shielded from light.

用稀释3分钟的10μlDAPI染色，并用PBS冲洗两次。细胞核用10μlDAPI染色，并立即在荧光显微镜下观察或保存在4°C的冰箱中并避光。

Statistical analysisGraphPad Prism 8.0.2 statistical software was used. The Receiver operating characteristic curve (ROC) was used to analyze the subgroups and determine the optimal threshold value. Progression-free survival (PFS) was defined as the duration from the time of surgery to cancer recurrence/progression.

统计分析使用GraphPad Prism 8.0.2统计软件。。无进展生存期（PFS）定义为从手术时间到癌症复发/进展的持续时间。

Kaplan–Meier survival plots were generated based on the relative expression of ADNP before and after surgery. Gehan–Breslow–Wilcoxon test was used. *p < 0.05 and **p < 0.01 were considered statistically significant and significant differences, respectively. All p values are two-sided.ResultsCharacteristics of the study populationThe clinicopathological characteristics of patients are listed in Table 3.Table 3 Clinicopathological features of patients.Full size tableSensitivity and specificity of ddPCR in detecting ADNP mRNA expression in CTCsUtilizing immunofluorescence staining, we determined the ADNP expression in peripheral blood mononuclear cells (PBMCs) derived from healthy donors and primary bladder cancer cells.

根据手术前后ADNP的相对表达生成Kaplan-Meier生存图。使用了Gehan–Breslow–Wilcoxon检验*p<0.05和**p<0.01分别被认为具有统计学意义和显着性差异。所有p值都是双面的。结果研究人群的特征患者的临床病理特征列于表3中。表3患者的临床病理特征。。

The analysis unveiled that only a few cells in PBMCs from healthy donors demonstrated ADNP positivity (Fig. 1A). In contrast, all the primary bladder cancer cells exhibited ADNP positivity post-immunofluorescence staining (Fig. 1B).Figure 1Immunofluorescence staining of ADNP (A) The ADNP of some cells in peripheral blood mononuclear cells of healthy people showed intracellular staining (green).

分析显示，来自健康供体的PBMC中只有少数细胞表现出ADNP阳性（图1A）。。图1 ADNP的免疫荧光染色（A）健康人外周血单核细胞中某些细胞的ADNP显示细胞内染色（绿色）。

(B) Immunofluorescence staining of ADNP on primary bladder cancer cells.Full size imageIn the modeled CTC samples generated by adding 0–20 primary bladder cancer cells to 6 ml of healthy donor peripheral blood, as was shown in Table 4, our assay efficiently detected the ADNP mRNA expression in the CTCs, even when the sample contained 10 or fewer primary .

（B） ADNP对原代膀胱癌细胞的免疫荧光染色。全尺寸图像在通过将0-20个原发性膀胱癌细胞添加到6 ml健康供体外周血中产生的建模CTC样品中，如表4所示，我们的测定有效地检测了CTC中的ADNP mRNA表达，即使样品含有10个或更少的原发性。

Data availability

数据可用性

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

本研究期间使用和分析的数据集可根据合理要求从通讯作者处获得。

AbbreviationsADNP:

缩写ADNP：

Activity-dependent neuroprotective protein

活性依赖性神经保护蛋白

BC:

不列颠哥伦比亚省：

Bladder cancer

膀胱癌

CTCs:

CTC：

Circulating tumor cells

循环肿瘤细胞

ddPCR:

ddPCR:

Droplet digital PCR

液滴数字PCR

ECOG:

ECOG：

Eastern Cooperative Oncology Group

东部肿瘤协作组

IF:

如果：

Immunofluorescence staining

免疫荧光染色

IMBs:

IMBs：

Immunomagnetic beads

免疫磁珠

MIBC:

MIBC：

Muscle-invasive bladder cancer

肌肉浸润性膀胱癌

NMIBC:

NMIBC：

Non-muscle invasive bladder cancer

非肌层浸润性膀胱癌

PFS:

PFS：

Progression-free survival

无进展生存期

qRT-PCR:

定量rt-PCR：

Quantitative reverse transcription PCR

定量逆转录PCR

TURBT:

涡轮机：

Transurethral resection of the bladder tumor

经尿道膀胱肿瘤切除术

UICC:

UICC:

Union Against Cancer

抗癌联盟

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Download referencesAcknowledgementsThis study was approved by the Medical Ethical Committee of Hunan Cancer Hospital (IRB number: KYJJ-2023-127). This work was supported by the National Cancer Center Clinical Research in “Climbing” Foundation of China [NCC201818A55], and the Natural Science Foundation Health Union Foundation of Hunan Province, China [2021JJ70030].Author informationAuthor notesThese authors contributed equally: Yuheng Wen and Zhihao Ming.Authors and AffiliationsDepartment of Urology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/ Hunan Cancer Hospital, Changsha, ChinaYuheng Wen, Zhihao Ming, Hong Li, Shuai Zhu, Jian Cao, Mingji Ye, Tian Gan & Yu XieClinical Translational Research Center, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/ Hunan Cancer Hospital, Changsha, ChinaYong ZengThe Second People’s Hospital of Huaihua, Huaihua, Hunan, ChinaXiangqun SheAuthorsYuheng WenView author publicationsYou can also search for this author in.

下载参考文献致谢本研究经湖南省肿瘤医院医学伦理委员会批准（IRB编号：KYJJ-2023-127）。这项工作得到了中国“攀登”基金会国家癌症中心临床研究[NCC201818A55]和中国湖南省自然科学基金健康联盟基金[2021JJ70030]的支持。作者信息作者注意到这些作者做出了同样的贡献：温玉恒和明志浩。作者和附属机构中南大学湘雅医学院附属肿瘤医院泌尿外科/湖南省肿瘤医院，长沙，中国余恒文，明志浩，洪丽，朱帅，曹健，叶明基，田干和余燮临床转化研究中心，中南大学湘雅医学院附属肿瘤医院/湖南省肿瘤医院，长沙，中国湖南怀化市第二人民医院，湖南怀化，中国香群社作者余恒文观点作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsXY conceived and designed the project. XY and YZ supervised the project. YHW performed microscopic analysis and conducted all dd-PCR analyses. ZHM analyzed data, including all statistical analyses, and prepared all figures and tables. HL, SZ, MJY and JC provided serum samples from patients with BC, as well as clinicopathological data.

PubMed Google ScholarContributionsXY构思并设计了该项目。XY和YZ监督了该项目。YHW进行了显微镜分析并进行了所有dd PCR分析。。HL，SZ，MJY和JC提供了BC患者的血清样本以及临床病理数据。

TG and XQS provide guidance for students. YHW and ZHM wrote the manuscript. All authors critically revised and approved the manuscript.Corresponding authorsCorrespondence to.

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Reprints and permissionsAbout this articleCite this articleWen, Y., Ming, Z., Li, H. et al. Potential utility of ADNP in circulating tumor cells as biomarker for prognostics in non-muscle-invasive bladder cancer.

转载和许可本文引用本文Wen，Y.，Ming，Z.，Li，H。等人。ADNP在循环肿瘤细胞中作为非肌肉浸润性膀胱癌预后生物标志物的潜在用途。

Sci Rep 14, 19352 (2024). https://doi.org/10.1038/s41598-024-70379-6Download citationReceived: 03 March 2024Accepted: 16 August 2024Published: 21 August 2024DOI: https://doi.org/10.1038/s41598-024-70379-6Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

科学报告1419352（2024）。https://doi.org/10.1038/s41598-024-70379-6Download引文接收日期：2024年3月3日接受日期：2024年8月16日发布日期：2024年8月21日OI：https://doi.org/10.1038/s41598-024-70379-6Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。。

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KeywordsNon-muscle invasive bladder cancer (NMIBC)ADNPddPCRLongitudinal monitoringPrognosis

关键词非肌层浸润性膀胱癌（NMIBC）ADNPDDPCR长期监测预后

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Molecular medicineUrology

分子医学泌尿学

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