Single-cell next-generation sequencing of circulating tumor cells in patients with neuroblastoma

Circulating tumor cells (CTCs) derived from any tumor tissue could contribute to metastasis and resistance to cancer treatments. In this study, we performed single-cell next-generation sequencing of CTCs and evaluated their usefulness for characterizing tumor biology and the mechanisms of metastasis in neuroblastomas (NB). We aimed to isolate CTCs from 10 patients with NB at diagnosis before any treatments and four patients at relapse. GD2⁺CD90⁺CD45⁻CD235a⁻DAPI⁻ cells were isolated as neuroblastoma CTCs using fluorescence-activated cell sorting. In five patients with advanced stages (M stage), DNA and RNA sequencing of CTCs at single-cell level were performed. NB CTCs were isolated from eight of the 10 patients at diagnosis and three of the four patients at relapse. More CTCs could be isolated from patients with advanced stages. In one patient, ALK mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with MYCN amplification, this gene was amplified in 12 of 13 CTCs. Using single-cell RNA sequencing, angiogenesis-related and cell cycle-related genes together with CCND1 and TUBA1A genes were found to be upregulated in CTCs. In one patient, CTCs were divided into two subgroups showing different gene expression profiles. In one subgroup, cell cycle-related and proliferation-related genes were differentially upregulated compared with the other group. In conclusion, next-generation sequencing of CTCs at single-cell level might help to characterize the tumor biology and the mechanisms of metastasis in NB.     

循环肿瘤细胞及循环肿瘤DNA检测在乳腺癌中的研究进展

乳腺癌是中国女性最常见的癌症,其早期检测、个体化治疗及实时疾病监测一直是科研及临床工作者关注的重点。癌症患者血液中循环肿瘤细胞(CTC)和循环肿瘤DNA(ctDNA)的检测微创、简便,易实时获取信息。目前,有关CTC富集鉴定方法多种多样,CTC及ctDNA检测的敏感度和特异度较前有所提高,未来研究面临的最大挑战为肿瘤细胞特异性标志物的开发以及ctDNA临床意义的研究。CTC及ctDNA在基础研究、检测方法、CTC培养、早期肿瘤检测、个体化治疗及临床预后等方面的研究均有开展。笔者对乳腺癌CTC及ctDNA的研究进展作一总结。     

Breast cancer circulating tumor cells

Metastasization of breast cancer involves various mechanisms responsible for progression from invasive lesion to dissemination in distant organs. Regional lymph node metastasization was considered an initial step in this process, but it is now recognized that hematogenous dissemination is a deviation from lymphatic circulation. The detection of circulating tumor cells (CTC) is an aim in several oncology areas. For this purpose, several techniques have been used to detect CTC, including the use of antibodies and techniques with nucleic acids. This study reviews the published studies considering the detection of breast cancer CTC. There are focused the difficulties in identifying a CTC in a heterogeneous population, the handling of the sample, criteria of positivity, analytical techniques, and specific markers. There are systematized various specific markers of breast cancer cells also the problems with false positive results. Finally, we hypothesize clinical applications either as a prognostic marker or as a therapeutic response monitor.     

趋化因子与循环肿瘤细胞

趋化因子(chemokine,CK)和循环肿瘤细胞(circulating tumor cells,CTC)在肿瘤定向转移中发挥重要作用.CK通过诱导肿瘤细胞上调基质金属蛋白酶(matrix metalloproteinase,MMP),降解细胞外周基质(extracellular matrix,ECM),发生上皮间质转化(epithelial-mesenchymal transition,EMT),抵抗失巢凋亡(anoikis)和免疫逃避等机制,最终形成CTC,发生“信号”归巢和器官定植,其中每一个步骤都有不同CK的正向或反向参与,其中有共性,也有差异.本文综合阐述CK在CTC的形成及其完成转移过程中的作用及可能机制;分析CK在CTC中的作用差异;探讨靶向CK信号轴个体化治疗肿瘤的研究现状及主要方向;为建立基于靶向CK信号轴治疗肿瘤的个体化治疗模式提供理论基础.     

循环肿瘤细胞检测技术

循环肿瘤细胞(CTC)检测在肿瘤患者的实时个体化医疗中具有独特的优势.外周血中CTC稀少而又异质,这成为CTC研究的主要技术挑战.CTC检测技术往往首先利用细胞的物理性质或生物学特性等分离富集CTC,再进行基因型或表型分析等以计数CTC或鉴定其分子特征.单细胞分子分析的进展使CTC检测能够提供更准确全面的信息.CTC侵袭转移时上皮间质转化和聚集成簇等生物进程是促进CTC检测的临床应用需要考虑的问题.     

结直肠癌循环肿瘤细胞及DNA的研究进展

结直肠癌是最常见的恶性肿瘤之一,尽管联合应用手术、化疗、放疗可提高患者的生存率,但仍有大量患者因术后转移和复发死亡。循环肿瘤细胞及循环肿瘤DNA对结直肠癌的早期诊断、疗效监测、预后评估以及个体化治疗方案制定均有重要意义,已成为近年来研究的热点。本文对近年来结直肠癌循环肿瘤细胞及循环肿瘤DNA的检测技术及其在早期诊断、疗效监测和预后判断中的应用进展作一综述。     

Capturing circulating tumor cells of hepatocellular carcinoma

Early metastases of hepatocellular carcinoma (HCC) may be detected by the isolation of circulating tumor cells (CTCs) in the bloodstream. During the course of therapeutic attempts, monitoring CTC changes in patients with HCC is helpful for the efficacy assessment. Nevertheless, the markers used for the detection, such as α-feto protein, asialoglycoprotein receptor or epithelial cell adhesion molecule, CD133 or CD90, are not specific for HCC CTCs. In spite of these limitations, a timely determination of the existence of CTCs will be beneficial for the monitoring of distant metastases, the evaluation of therapeutic attempts, and the prediction of prognosis.     

转移性循环肿瘤细胞的特征

循环肿瘤细胞（circulating tumor cells,CTCs）是肿瘤转移的潜在种植者。这些引起肿瘤转移的CTCs具有干性、转化、簇群、亲器官性和变形等特征,最终在靶器官形成转移灶。研究CTCs的特征可能揭开肿瘤转移的机制。     

循环肿瘤细胞检测的临床应用

循环肿瘤细胞（CTC）计数在判断肿瘤的临床分期、评估肿瘤预后、进行疗效监测中的应用价值已获得广泛认可,但还需要大量研究来建立标准化评价体系并进行临床试验验证.CTC的分子生物学检测不仅可以对CTC计数进行完善,更因为其在肿瘤的辅助诊断和指导个体化治疗中的巨大潜力以及对于了解肿瘤转移的生物学行为的重大意义将成为未来的研究热点.     

Tumorigenic potential of circulating prostate tumor cells

Circulating tumor cells (CTCs) have received intense scientific scrutiny because they travel in the bloodstream and are therefore well situated to mediate hematogenous metastasis. However, the potential of CTCs to actually form new tumors has not been tested. Popular methods of isolating CTCs are biased towards larger, more differentiated, non-viable cells, creating a barrier to testing their tumor forming potential. Without relying on cell size or the expression of differentiation markers, our objective was to isolate viable prostate CTCs from mice and humans and assay their ability to initiate new tumors. Therefore, blood was collected from transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and from human patients with metastatic castration-resistant prostate cancer (PCa). Gradient density centrifugation or red cell lysis was used to remove erythrocytes, and then leukocytes were depleted by magnetic separation using CD45 immunoaffinity beads. CTCs fractions from TRAMP mice and PCa patients were verified by immunocytochemical staining for cytokeratin 8 and EpCAM, and inoculated into immunodeficient mice. TRAMP tumor growth was monitored by palpation. Human tumor growth formation was monitored up to 8 months by ultrasensitive PSA assays performed on mouse serum. We found viable tumor cells present in the bloodstream that were successfully isolated from mice without relying on cell surface markers. Two out of nine immunodeficient mice inoculated with TRAMP CTCs developed massive liver metastases. CTCs were identified in blood from PCa patients but did not form tumors. In conclusion, viable CTCs can be isolated without relying on epithelial surface markers or size fractionation. TRAMP CTCs were tumorigenic, so CTCs isolated in this way contain viable tumor-initiating cells. Only two of nine hosts grew TRAMP tumors and none of the human CTCs formed tumors, which suggests that most CTCs have relatively low tumor-forming potential. Future studies should identify and target the highly tumorigenic cells.     

Role of circulating tumor cells and disseminated tumor cells in primary breast cancer

Metastasis remains a main cause of death in patients with breast cancer regardless of improvements in treatment. Prospective clinical studies of this minimal residual disease have shown disseminated tumor cells (DTCs) in bone marrow and circulating tumor cells (CTCs) in peripheral blood, neither of which can be detected by conventional imaging, to be prognostic and predictive markers for responsive treatment in patients with metastatic breast cancer. However, the guideline from the American Society of Clinical Oncology does not recommend measuring CTCs for clinical decisions because of a lack of evidence for an established, sound methodology and with proven clinical relevance. The Southwest Oncology Group trial S0500 to validate the clinical relevance of CTCs for treatment decisions in patients with metastatic breast cancer is ongoing. In patients with primary breast cancer, the low detection rate of CTCs has been overcome by recent advances in technology. Although generally DTCs were more detectable than CTCs and the association between presence of DTCs and poor prognosis has been shown, the invasiveness of sample collection of DTCs from bone marrow is generally hard for patients to accept. In this review, we concentrate on the question of whether we need to consider CTCs and DTCs in the management of primary breast cancer on the basis of the evidence of the clinical relevance of CTCs and DTCs. The promising role of the molecular characterization of CTCs, which does have the potential for being a predictor for tumor behavior and development, is suggested as a new targeting strategy.     

循环肿瘤细胞临床应用的新进展

循环肿瘤细胞(Circulating tumor cells,CTCs)是指释放到血液中的罕见肿瘤细胞,被认为是肿瘤血液转移的关键步骤之一.循环肿瘤细胞的检测对于肿瘤的诊断、治疗和预后判定都具有非常重要的意义,是目前肿瘤研究的热点之一.本文将从预测生物标志物、治疗反应、预后、血浆肿瘤DNA四个方面介绍循环肿瘤细胞临床应用的最新研究进展.     

Identifying cancer origin using circulating tumor cells

Circulating tumor cells (CTCs) have become an established clinical evaluation biomarker. CTC count provides a good correlation with the prognosis of cancer patients, but has only been used with known cancer patients, and has been unable to predict the origin of the CTCs. This study demonstrates the analysis of CTCs for the identification of their primary cancer source. Twelve mL blood samples were equally dispensed on 6 CMx chips, microfluidic chips coated with an anti-EpCAM-conjugated supported lipid bilayer, for CTC capture and isolation. Captured CTCs were eluted to an immunofluorescence (IF) staining panel consisting of 6 groups of antibodies: anti-panCK, anti-CK18, anti-CK7, anti-TTF-1, anti-CK20/anti-CDX2, and anti-PSA/anti-PSMA. Cancer cell lines of lung (H1975), colorectal (DLD-1, HCT-116), and prostate (PC3, DU145, LNCaP) were selected to establish the sensitivity and specificity for distinguishing CTCs from lung, colorectal, and prostate cancer. Spiking experiments performed in 2mL of culture medium or whole blood proved the CMx platform can enumerate cancer cells of lung, colorectal, and prostate. The IF panel was tested on blood samples from lung cancer patients (n = 3), colorectal cancer patients (n = 5), prostate cancer patients (n = 5), and healthy individuals (n = 12). Peripheral blood samples found panCK⁺ and CK18⁺ CTCs in lung, colorectal, and prostate cancers. CTCs expressing CK7⁺ or TTF-1⁺, (CK20/ CDX2)⁺, or (PSA/ PSMA)⁺ corresponded to lung, colorectal, or prostate cancer, respectively. In conclusion, we have designed an immunofluorescence staining panel to identify CTCs in peripheral blood to correctly identify cancer cell origin.     

CTC、ctDNA在结直肠癌诊断中的研究进展

结直肠癌（colorectal cancer,CRC）是世界上第三大最常见的癌症类型,也是导致癌症死亡的第四大原因。CRC的常规治疗策略包括手术、新辅助治疗和辅助治疗。不幸的是,大约50％的CRC患者仅在晚期被诊断,因此显著降低了不同治疗方案的可用性。学者们不断寻找更准确的诊断结直肠癌的方法,除了通过蛋白质组学技术研究新的血清肿瘤生物标记物外,还引入循环肿瘤细胞（CTC）和循环肿瘤DNA（ctDNA）等新概念。本文就血液中CTC、ctDNA在结直肠癌诊断中的研究进展作一综述。     

Circulating tumor cells and circulating tumor DNA in colon cancer

It is known that tumor cells have the ability to penetrate into the bloodstream. The identification of such circulating tumor cells (CTC) determines the prognosis in several tumors, including colon cancer. Tumor DNA (ctDNA), which is only a part of the total circulating DNA obtained from the blood of cancer patients, is also further separated from plasma. This separation of the neoplastic derivatives of the primary tumor and metastases (CTC, ctDNA, RNA, proteome) in plasma is called “liquid biopsy.” CTC increasingly represents the pool of tumor cells that can initiate the growth of metastatic lesions, while the ctDNA provides the information about the whole tumor mass. Traditional tissue biopsy gives information based only on one small section of the primary tumor or metastasis, often retrieved before the start of treatment; however, liquid biopsy provides real-time information about the molecular disorders for the whole tumor mass and allows us to estimate the dynamics of the evolutionary tumor changes, the heterogeneity of the disease, and the effect of chemotherapy. With the possibility of obtaining multiple blood samples for analysis during the therapy, in contrast to traditional biopsy, it also allows us to evaluate the mechanisms of resistance to treatment, which in the future will perhaps lead to modification of the treatment in accordance with the detected molecular defects in tumors. Thus, this would facilitate implementing the principles of personalized therapy. In this literature review, we concentrate on liquid biopsy in patients with colon cancer.     

Progress of circulating tumor cells in lung cancer

Circulating tumor cells (CTCs) are present in the peripheral blood, and play an important role in the recurrence and metastasis of lung cancer. As the researches about CTCs and targeted therapy move along, it is found that CTCs can be used as a "liquid" tumor tissue to guide clinical treatment and have a certain significance in the clinical staging and prognosis evaluation of lung cance.