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1
Cha, Jiwon, Hyungseok Cho, Jae-Seung Chung, Joon Seong Park, and Ki-Ho Han. "Effective Circulating Tumor Cell Isolation Using Epithelial and Mesenchymal Markers in Prostate and Pancreatic Cancer Patients." Cancers 15, no. 10 (May 18, 2023): 2825. http://dx.doi.org/10.3390/cancers15102825.
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Circulating tumor cells (CTCs) display antigenic heterogeneity between epithelial and mesenchymal phenotypes. However, most current CTC isolation methods rely on EpCAM (epithelial cell adhesion molecule) antibodies. This study introduces a more efficient CTC isolation technique utilizing both EpCAM and vimentin (mesenchymal cell marker) antibodies, alongside a lateral magnetophoretic microseparator. The effectiveness of this approach was assessed by isolating CTCs from prostate (n = 17) and pancreatic (n = 5) cancer patients using EpCAM alone, vimentin alone, and both antibodies together. Prostate cancer patients showed an average of 13.29, 11.13, and 27.95 CTCs/mL isolated using EpCAM alone, vimentin alone, and both antibodies, respectively. For pancreatic cancer patients, the averages were 1.50, 3.44, and 10.82 CTCs/mL with EpCAM alone, vimentin alone, and both antibodies, respectively. Combining antibodies more than doubled CTC isolation compared to single antibodies. Interestingly, EpCAM antibodies were more effective for localized prostate cancer, while vimentin antibodies excelled in metastatic prostate cancer isolation. Moreover, vimentin antibodies outperformed EpCAM antibodies for all pancreatic cancer patients. These results highlight that using both epithelial and mesenchymal antibodies with the lateral magnetophoretic microseparator significantly enhances CTC isolation efficiency, and that antibody choice may vary depending on cancer type and stage.
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Lee, Jae Hyuk, Sung Ho Park, Jihoon Kang, Jihyun Lee, Soee Kim, Jungwon Kim, Young Woong Sohn, and Jong Kil Lee. "Abstract 6692: Identification of circulating tumor cells based on machine learning." Cancer Research 83, no. 7_Supplement (April 4, 2023): 6692. http://dx.doi.org/10.1158/1538-7445.am2023-6692.
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Abstract Circulating tumor cells (CTC) are tumor cells that have shed into the bloodstream from primary tumor and circulate in the blood. CTC could provide better understanding of tumor metastasis and show the possibility as promising biomarker for early detection of cancer, cancer prognosis, noninvasive monitoring of treatment response, and personalized medicine. However, the isolation and characterization has been a major technological challenge due to their rareness among the massive blood cells. CytoGen’s Smart BiopsyTM CTC platform comprised of CTC isolator, IF (Immunofluorescence) Stainer, and Cell Image Analyzer is a unique and competitive technology platform for the isolation and analysis of CTC from blood. Intact live CTC could be isolated gravity-based filtration via high-density micro-porous (HDM) chip with minimized cellular damage and high retrieval rate. For the more accurate and quick analysis of CTC after isolation with Smart BiopsyTM CTC platform, we are developing the analysis method of CTC identification using machine learning technologies. Preliminary results of machine learning showed the accurate separation of CTC and PBMC (peripheral blood mononuclear cells) based on the specific markers of them. CTC identification based on machine learning technologies might be reliable analysis method and give secure information of CTC. Citation Format: Jae Hyuk Lee, Sung Ho Park, Jihoon Kang, Jihyun Lee, Soee Kim, Jungwon Kim, Young Woong Sohn, Jong Kil Lee. Identification of circulating tumor cells based on machine learning. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6692.
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Macaraniag, Celine, Jian Zhou, Ian Papautsky, Jing Li, William Putzbach, and Nissim Hay. "Abstract 5598: Microfluidic isolation and capture of circulating tumor cells and clusters from mouse blood." Cancer Research 83, no. 7_Supplement (April 4, 2023): 5598. http://dx.doi.org/10.1158/1538-7445.am2023-5598.
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Abstract Background. Circulating tumor cells (CTCs) and their clusters have been regarded as potential targets for therapies and subjects for identifying certain mechanisms of cancer metastasis. Mouse models have been especially useful tools to investigate the process of metastasis, since they are easier to control and are less variable compared to clinical patient samples. Therefore, it is of interest to extract and study CTCs and CTC clusters from such mouse models. However, most isolation systems are designed and tested for human blood and cell lines. Sized-based methods of CTC isolation have been optimized for human cell lines, but mouse CTCs are generally smaller in size (i.e., ~14 µm EO771 breast cancer cells). Therefore, mouse cell separation necessitates a varied protocol for CTC isolation. We used mouse blood and EO771, a mouse breast cancer cell line, to demonstrate the isolation of mouse CTCs from mouse blood using a sized-based microfluidic device. We also validated the presence of endogenous CTCs and CTC-neutrophil clusters in a tumor bearing mouse model. Methods. Our microfluidic device employs inertial migration in a straight channel segment (150 µm × 50 µm × 24 mm) to isolate CTCs from blood samples. We spiked 100 and 1000 ZsGreen-expressing EO771 cells in 5 × diluted blood containing tdTomato-expressing neutrophils, easily distinguishing CTCs from the red-colored neutrophils. To enumerate the recovered cells, we used Cytospin to concentrate the cells into a glass slide. Lastly, we used hydrodynamic traps to capture CTC clusters from tumor-bearing mouse blood. The blood was extracted from the posterior vena cava of the mice at the endpoint experiment. Results. We achieved an overall recovery rate of 22-29%, which combines the recovery of the isolation device and the Cytospin. Here, we enumerate cells only after their extraction from the collection tube outside of the isolation device. Other methods with higher capture efficiency enumerate the recovered CTCs before they are harvested from the isolation device which is not always representative of the number of cells that are analyzed. We were also able to discover endogenous CTC-neutrophil clusters from tumor-bearing mice which we distinguished apart with anti-EpCAM staining of CTCs (green) and anti-Ly6G (red) staining of neutrophils. Conclusion. We demonstrated and optimized a procedure for isolating mouse-derived CTCs from naïve mice blood and CTC-neutrophil clusters from tumor-bearing mice with our microfluidic isolation devices, as opposed to the isolation of human CTCs as in other sized-based isolation systems. In future studies, we aim to continue to use our microfluidic devices to capture and study CTC and CTC clusters. Citation Format: Celine Macaraniag, Jian Zhou, Ian Papautsky, Jing Li, William Putzbach, Nissim Hay. Microfluidic isolation and capture of circulating tumor cells and clusters from mouse blood. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5598.
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Cheng, Jie, Yang Liu, Yang Zhao, Lina Zhang, Lingqian Zhang, Haiyang Mao, and Chengjun Huang. "Nanotechnology-Assisted Isolation and Analysis of Circulating Tumor Cells on Microfluidic Devices." Micromachines 11, no. 8 (August 14, 2020): 774. http://dx.doi.org/10.3390/mi11080774.
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Circulating tumor cells (CTCs), a type of cancer cell that spreads from primary tumors into human peripheral blood and are considered as a new biomarker of cancer liquid biopsy. It provides the direction for understanding the biology of cancer metastasis and progression. Isolation and analysis of CTCs offer the possibility for early cancer detection and dynamic prognosis monitoring. The extremely low quantity and high heterogeneity of CTCs are the major challenges for the application of CTCs in liquid biopsy. There have been significant research endeavors to develop efficient and reliable approaches to CTC isolation and analysis in the past few decades. With the advancement of microfabrication and nanomaterials, a variety of approaches have now emerged for CTC isolation and analysis on microfluidic platforms combined with nanotechnology. These new approaches show advantages in terms of cell capture efficiency, purity, detection sensitivity and specificity. This review focuses on recent progress in the field of nanotechnology-assisted microfluidics for CTC isolation and detection. Firstly, CTC isolation approaches using nanomaterial-based microfluidic devices are summarized and discussed. The different strategies for CTC release from the devices are specifically outlined. In addition, existing nanotechnology-assisted methods for CTC downstream analysis are summarized. Some perspectives are discussed on the challenges of current methods for CTC studies and promising research directions.
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Theil, Gerit, Joanna Bialek, Christine Weiß, Felix Lindner, and Paolo Fornara. "Strategies for Isolating and Propagating Circulating Tumor Cells in Men with Metastatic Prostate Cancer." Diagnostics 12, no. 2 (February 15, 2022): 497. http://dx.doi.org/10.3390/diagnostics12020497.
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Selecting a well-suited method for isolating/characterizing circulating tumor cells (CTCs) is challenging. Evaluating sensitive and specific markers for prostate cancer (PCa)-specific CTC identification and analysis is crucial. We used the CellCollector EpCAM-functionalized system (CC-EpCAM) and evaluated and developed a PCa-functionalized version (CC-PCa); we then compared CTC isolation techniques that exploit the physical and biological properties of CTCs. We established two cohorts of metastatic PCa patients (mPCa; 15 in cohort 1 and 10 in cohort 2). CTC cultivation experiments were conducted with two capturing methods (Ficoll and ScreenCell). The most sensitive detection rates and highest CTC counts were reached with the CC-PCa and ScreenCell system. Patients with ≥5 CTCs isolated with CC-EpCAM had an overall survival (OS) of 0.93 years, and patients with ≥5 CTCs isolated with CC-PCa had an OS of 1.5 years in cohort 1. Nevertheless, we observed the highest sensitivity and specificity for 24-month survival by the Ficoll with CD45 depletion and ScreenCell system with May-Grunwald Giemsa (MGG) staining. The EpCAM molecule is an essential factor related to OS for CTC isolation based on biological properties in mPCa patients. The best-suited CTC capture system is not limited to one characteristic of cells but adapted to downstream analysis.
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Francescangeli, Federica, Valentina Magri, Maria Laura De Angelis, Gianluigi De Renzi, Orietta Gandini, Ann Zeuner, Paola Gazzaniga, and Chiara Nicolazzo. "Sequential Isolation and Characterization of Single CTCs and Large CTC Clusters in Metastatic Colorectal Cancer Patients." Cancers 13, no. 24 (December 18, 2021): 6362. http://dx.doi.org/10.3390/cancers13246362.
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Circulating tumor cells (CTCs) detach from a primary tumor or its metastases and circulate in the bloodstream. The vast majority of CTCs are deemed to die into the bloodstream, with only few cells representing viable metastatic precursors. Particularly, single epithelial CTCs do not survive long in the circulation due to the loss of adhesion-dependent survival signals. In metastatic colorectal cancer, the generation of large CTC clusters is a very frequent occurrence, able to increase the aptitude of CTCs to survive in the bloodstream. Although a deepened analysis of large-sized CTC clusters might certainly offer new insights into the complexity of the metastatic cascade, most CTC isolation techniques are unfortunately not compatible with large-sized CTC clusters isolation. The inappropriateness of standard CTC isolation devices for large clusters isolation and the scarce availability of detection methods able to specifically isolate and characterize both single CTCs and CTC clusters finally prevented in-depth studies on the prognostic and predictive value of clusters in clinical practice, unlike that which has been described for single CTCs. In the present study, we validated a new sequential filtration method for the simultaneous isolation of large CTC clusters and single CTCs in patients with metastatic colorectal cancer at failure of first-line treatments. The new method might allow differential downstream analyses for single and clustered CTCs starting from a single blood draw, opening new scenarios for an ever more precise characterization of colorectal cancer metastatic cascade.
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Jiang, Xiaocheng, Keith H. K. Wong, Aimal H. Khankhel, Mahnaz Zeinali, Eduardo Reategui, Matthew J. Phillips, Xi Luo, et al. "Microfluidic isolation of platelet-covered circulating tumor cells." Lab on a Chip 17, no. 20 (2017): 3498–503. http://dx.doi.org/10.1039/c7lc00654c.
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Ahmed, Shakera, Atul Bharde, Muhammad Mosaraf Hossain, Ramendu Parial, Nusrat Jahan Nayeema, Manisha Das, Mizanur Rahman, et al. "Circulating tumor cells (CTCs) detection and isolation in different subtypes of early-stage breast cancer patients from Bangladesh." Journal of Clinical Oncology 41, no. 16_suppl (June 1, 2023): e12529-e12529. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.e12529.
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e12529 Background: Breast cancer is a highly heterogeneous pathophysiology characterized by poor outcomes. Due to the increasing incidence and disease progression rates and undefined relapse periods, reliable disease monitoring is a challenge and has remained an unmet need. Advancements in liquid biopsy have significantly enhanced our understanding of clinical oncology. CTC-based liquid biopsy is emerging as a reliable prognostic tool to predict various clinical indicators. Although extensively investigated in metastatic breast cancers, little is known about CTCs in early-stage breast cancers. CTCs with respect to different molecular subtypes of breast cancer in early-stage breast cancer patients is evaluated. Methods: In this prospective clinical trial (CMC 59.27.0000.013.19 PG.009.2022/262) 40 early-stage patients with luminal (A +B, 33.33%), HER 2 positive (12.8%), triple-negative (12.8%) and undetermined (41.07%) subtype were recruited. CTCs were isolated in 1.5 ml blood using the Drug Controller General of India approved OncoDiscover CTC test. This platform contains affinity-based magnetic nanoparticles to mediate EpCAM-based CTC isolation. CTCs were detected as CK18+, DAPI+, and CD45- cells using a fluorescence detection-based automated digital imaging platform. Results: CTCs were detected in 60 % of patients with a mean CTC count of 1 cell / 1.5ml blood. Among total positive patients, the luminal subtype was the least positive (46 %), followed by TNBC (60 %) and undetermined (62.5 %) subgroup, while all HER2-positive patients showed the presence of CTCs. Besides individual cells, CTC clusters were detected in 12.5 % of patients and they were equally distributed in luminal and HER2-positive subpopulations. When analyzed on the scale of tumor grade, grade I patients did not show the presence of CTC, while 58.33 % of grade II patients had ≥ 1 CTC. All grade III patients showed the presence of ≥ 1 CTC. CTC count was high among CTC-positive grade II patients (average 2 CTCs) and correlated well with the presence of CTC clusters in these patients. Patients who had surgical intervention had a low CTC burden compared to patients who did not have a surgical resection. 75 % of treatment naïve patients showed the presence of CTC while 58 % of patients receiving chemotherapy alone showed the presence of 1 CTC. 50 % of patients who had surgery followed by CT+RT showed the presence of 1 CTC. Conclusions: The presence of CTCs may suggest for biological progression of disease in early-stage BC patients. CTC detected in all HER2-positive patients suggested the high shedding nature of these tumors, which correlates well with their reported migratory tendency. The presence of CTCs did not show a clear correlation with the treatment regimen. However, this data is based on a single time point and needs longitudinal correlation with CTC on a larger sample size. Clinical trial information: 59.27.0000.013.19 PG.009.2022/262 .
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Liao, Chia-Jung, Chia-Hsun Hsieh, Feng-Chun Hung, Hung-Ming Wang, Wen-Pin Chou, and Min-Hsien Wu. "The Integration of a Three-Dimensional Spheroid Cell Culture Operation in a Circulating Tumor Cell (CTC) Isolation and Purification Process: A Preliminary Study of the Clinical Significance and Prognostic Role of the CTCs Isolated from the Blood Samples of Head and Neck Cancer Patients." Cancers 11, no. 6 (June 6, 2019): 783. http://dx.doi.org/10.3390/cancers11060783.
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Conventional positive and negative selection-based circulating tumor cell (CTC) isolation methods might generally ignore metastasis-relevant CTCs that underwent epithelial-to- mesenchymal transition and suffer from a low CTC purity problem, respectively. To address these issues, we previously proposed a 2-step CTC isolation method integrating a negative selection CTC isolation and subsequent spheroid cell culture. In addition to its ability to isolate CTCs, more importantly, the spheroid cell culture used could serve as a cell culture model mimicking the process of new tumor tissue formation during cancer metastasis. Therefore, it is promising not only to selectively isolate metastasis-relevant CTCs but also to test the potential of cancer metastasis and thus the prognosis of disease. To explore these issues, experiments were performed. The key findings of this study demonstrated that the method was able to harvest both epithelial (E)- and mesenchymal (M)-type CTCs without selection bias. Moreover, both the M-type CTC count and the information obtained from the multidrug resistance-associated protein 2 (MRP2) and MRP5 gene expression analysis of the CTCs isolated via the 2-step CTC isolation method might be able to serve as prognostic factors for progression-free survival in head and neck squamous cell carcinoma.
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Tretyakova, Maria S., Maxim E. Menyailo, Anastasia A. Schegoleva, Ustinia A. Bokova, Irina V. Larionova, and Evgeny V. Denisov. "Technologies for Viable Circulating Tumor Cell Isolation." International Journal of Molecular Sciences 23, no. 24 (December 15, 2022): 15979. http://dx.doi.org/10.3390/ijms232415979.
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The spread of tumor cells throughout the body by traveling through the bloodstream is a critical step in metastasis, which continues to be the main cause of cancer-related death. The detection and analysis of circulating tumor cells (CTCs) is important for understanding the biology of metastasis and the development of antimetastatic therapy. However, the isolation of CTCs is challenging due to their high heterogeneity and low representation in the bloodstream. Different isolation methods have been suggested, but most of them lead to CTC damage. However, viable CTCs are an effective source for developing preclinical models to perform drug screening and model the metastatic cascade. In this review, we summarize the available literature on methods for isolating viable CTCs based on different properties of cells. Particular attention is paid to the importance of in vitro and in vivo models obtained from CTCs. Finally, we emphasize the current limitations in CTC isolation and suggest potential solutions to overcome them.
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Xiao, Jerry, Joseph R. McGill, Kelly Stanton, Joshua D. Kassner, Sujata Choudhury, Richard Schlegel, Zuben E. Sauna, Paula R. Pohlmann, and Seema Agarwal. "Efficient Propagation of Circulating Tumor Cells: A First Step for Probing Tumor Metastasis." Cancers 12, no. 10 (September 28, 2020): 2784. http://dx.doi.org/10.3390/cancers12102784.
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Circulating tumor cells (CTCs) represent a unique population of cells that can be used to investigate the mechanistic underpinnings of metastasis. Unfortunately, current technologies designed for the isolation and capture of CTCs are inefficient. Existing literature for in vitro CTC cultures report low (6−20%) success rates. Here, we describe a new method for the isolation and culture of CTCs. Once optimized, we employed the method on 12 individual metastatic breast cancer patients and successfully established CTC cultures from all 12 samples. We demonstrate that cells propagated were of breast and epithelial origin. RNA-sequencing and pathway analysis demonstrated that CTC cultures were distinct from cells obtained from healthy donors. Finally, we observed that CTC cultures that were associated with CD45+ leukocytes demonstrated higher viability. The presence of CD45+ leukocytes significantly enhanced culture survival and suggests a re-evaluation of the methods for CTC isolation and propagation. Routine access to CTCs is a valuable resource for identifying genetic and molecular markers of metastasis, personalizing the treatment of metastatic cancer patients and developing new therapeutics to selectively target metastatic cells.
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Pastuszka, Paweł, Zdzisław Bogdanowicz, Artur Kowalik, Krzysztof Gruszczyński, and Mariusz Łapiński. "Technical aspects of circulating tumor cells (CTC) isolation using molecular sieve." Bulletin of the Military University of Technology 66, no. 1 (March 31, 2017): 41–54. http://dx.doi.org/10.5604/01.3001.0009.9483.
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The article presents the characteristics of circulating tumor cells (CTC) in the context of their enrichment. It presents the possibility of the use of molecular sieves in CTC isolation. It shows important technical factors affecting the efficiency of CTC enrichment using molecular sieves and indicates the necessity of compromise choice of these parameters. It shows 2D molecular sieve construction made within the Biomedical Engineering Centre of MUT. It presents the future directions of research which assumes the use of this construction for a molecular filter in the form of sieve stack. Keywords: molecular sieves, circulating tumor cells (CTC) enrichment, laser photolithography, laser ablation
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Descamps, Lucie, Damien Le Roy, and Anne-Laure Deman. "Microfluidic-Based Technologies for CTC Isolation: A Review of 10 Years of Intense Efforts towards Liquid Biopsy." International Journal of Molecular Sciences 23, no. 4 (February 10, 2022): 1981. http://dx.doi.org/10.3390/ijms23041981.
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The selection of circulating tumor cells (CTCs) directly from blood as a real-time liquid biopsy has received increasing attention over the past ten years, and further analysis of these cells may greatly aid in both research and clinical applications. CTC analysis could advance understandings of metastatic cascade, tumor evolution, and patient heterogeneity, as well as drug resistance. Until now, the rarity and heterogeneity of CTCs have been technical challenges to their wider use in clinical studies, but microfluidic-based isolation technologies have emerged as promising tools to address these limitations. This review provides a detailed overview of latest and leading microfluidic devices implemented for CTC isolation. In particular, this study details must-have device performances and highlights the tradeoff between recovery and purity. Finally, the review gives a report of CTC potential clinical applications that can be conducted after CTC isolation. Widespread microfluidic devices, which aim to support liquid-biopsy-based applications, will represent a paradigm shift for cancer clinical care in the near future.
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Kuvendjiska, Jasmina, Peter Bronsert, Verena Martini, Sven Lang, Martha Pitman, Jens Hoeppner, and Birte Kulemann. "Non-Metastatic Esophageal Adenocarcinoma: Circulating Tumor Cells in the Course of Multimodal Tumor Treatment." Cancers 11, no. 3 (March 21, 2019): 397. http://dx.doi.org/10.3390/cancers11030397.
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Background: Isolation of circulating tumor cells (CTC) holds the promise to improve response-prediction and personalization of cancer treatment. In this study, we test a filtration device for CTC isolation in patients with non-metastatic esophageal adenocarcinoma (EAC) within recent multimodal treatment protocols. Methods: Peripheral blood specimens were drawn from EAC patients before and after neoadjuvant chemotherapy (FLOT)/chemoradiation (CROSS) as well as after surgery. Filtration using ScreenCell® devices captured CTC for cytologic analysis. Giemsa-stained specimens were evaluated by a cytopathologist; the cut-off was 1 CTC/specimen (6 mL). Immunohistochemistry with epithelial (pan-CK) and mesenchymal markers (vimentin) was performed. Results: Morphologically diverse malignant CTCs were found in 12/20 patients in at least one blood specimen. CTCs were positive for both vimentin and pan-CK. More patients were CTC positive after neoadjuvant therapy (6/20 vs. 9/15) and CTCs per/ml increased in most of the CTC-positive patients. After surgery, 8/13 patients with available blood specimens were still CTC positive. In clinical follow-up, 5/9 patients who died were CTC-positive. Conclusions: Detection of CTC by filtration within multimodal treatment protocols of non-metastatic EAC is feasible. The rate of CTC positive findings and the quantity of CTCs changes in the course of multimodal neoadjuvant chemoradiation/chemotherapy and surgery.
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Zhang, Shuai, Yue Wang, Chaoqiang Yang, Junwen Zhu, Xiongying Ye, and Wenhui Wang. "On-chip circulating tumor cells isolation based on membrane filtration and immuno-magnetic bead clump capture." Nanotechnology and Precision Engineering 5, no. 1 (March 1, 2022): 013003. http://dx.doi.org/10.1063/10.0009560.
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Isolating rare circulating tumor cells (CTCs) from blood is critical for the downstream analysis that is important in cancer-related research, diagnosis, and medicine, and efforts are ongoing to increase the efficiency and purity of CTC isolation in microfluidics. Reported in this paper is a two-stage integrated microfluidic chip for coarse-to-fine CTC isolation from whole blood. First, blood cells are removed by filtration using a micropore-array membrane, then CTCs and other cells that are trapped in the micropores are peeled off the membrane by a novel release method based on air–liquid interfacial tension, which significantly increases the recovery rate of CTCs. The second stage is CTC capture based on an on-chip dense immuno-magnetic-bead clump, which offers high capture efficiency and purity. Both the micropore filtration and immuno-magnetic-bead capture are validated and optimized experimentally. Overall, the integrated microfluidic chip can realize a recovery rate of 85.5% and a purity of 37.8% for rare cancer cells spiked in whole blood.
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Lim, Minji, Suhyun Park, Hyoung-Oh Jeong, Sung Hee Park, Sumit Kumar, Aelee Jang, Semin Lee, Dong Uk Kim, and Yoon-Kyoung Cho. "Circulating Tumor Cell Clusters Are Cloaked with Platelets and Correlate with Poor Prognosis in Unresectable Pancreatic Cancer." Cancers 13, no. 21 (October 20, 2021): 5272. http://dx.doi.org/10.3390/cancers13215272.
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Circulating tumor cells (CTCs) are known to be heterogeneous and clustered with tumor-associated cells, such as macrophages, neutrophils, fibroblasts, and platelets. However, their molecular profile and clinical significance remain largely unknown. Thus, we aimed to perform a comprehensive gene expression analysis of single CTCs and CTC clusters in patients with pancreatic cancer and to identify their potential clinical relevance to provide personalized medicine. Epitope-independent, rapid (>3 mL of whole blood/min) isolation of single CTCs and CTC clusters was achieved from a prospective cohort of 16 patients with unresectable pancreatic cancer using a centrifugal microfluidic device. Forty-eight mRNA expressions of individual CTCs and CTC clusters were analyzed to identify pancreatic CTC phenotype. CTC clusters had a larger proportion of mesenchymal expression than single CTCs (p = 0.0004). The presence of CTC clusters positively correlated with poor prognosis (progression-free survival, p = 0.0159; overall survival, p = 0.0186). Furthermore, we found that most CTCs in these patients (90.7%) were cloaked with platelets and found the presence of a positive correlation between the increase in CTC clusters and rapid disease progression during follow-ups. Efficient CTC cluster isolation and analysis techniques will enhance the understanding of complex tumor metastasis processes and can facilitate personalized disease management.
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Zuo, Li, Wei Niu, and Anqi Li. "Isolation of Circulating Tumor Cells of Ovarian Cancer by Transferrin Immunolipid Magnetic Spheres and Its Preliminary Clinical Application." Nano LIFE 09, no. 01n02 (March 2019): 1940001. http://dx.doi.org/10.1142/s1793984419400014.
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Circulating tumor cells (CTCs) play an important role in cancer prognosis, treatment monitoring and metastasis diagnosis. However, due to the extremely low concentration of CTC in the peripheral blood, its isolation and enrichment are critical steps for early diagnosis. Herein, we used the transferrin modified lipid magnetic spheres for the isolation of ovarian cancer CTCs, and studied the relationship between the CTCs count and the clinical case parameters, prognosis of ovarian cancer. The result showed that no CTC was found in the peripheral blood of 30 patients with benign cysts, and 34 out of 46 patients with ovarian cancer were positive for CTC, with a positive rate of 73.9%. Analysis of the parameters of the clinical cases showed that the positive rate of CTC was related to the clinical stages, and that it was not significantly related to the age, histopathological types and pathological grades of patients. Of the 34 CTC-positive patients, 18 had progression-free survival, with a survival rate of 52.9%, and of the 11 CTC-negative patients, 9 had progression-free survival, with a survival rate of 81.8%. The results showed that the transferrin lipid magnetic spheres prepared in this study, could effectively isolate the CTCs in the peripheral blood of patients with ovarian cancer, that the level of CTC in ovarian cancer patients was related to its clinical stage, and that the progression-free survival of the patients with a high level of CTCs was relatively short. Therefore, this study shows that the transferrin lipid magnetic sphere can achieve effective isolation of ovarian cancer CTC, which can be used as an auxiliary diagnostic method in comprehensive diagnosis of ovarian cancer.
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McGregor, John R., Wolfram E. Samlowski, Shweta Tharkar, Sreekanth Donepudi, and Soldano Ferrone. "Isolation and expansion of circulating tumor cells (CTC) from melanoma patients using a novel cell culture technique." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 10614. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.10614.
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10614 Background: Identification of rare (>2-5) circulating tumor cells (CTC) in 7.5 ml blood by immunofluorescence assay (IFA) correlates with a poor prognosis in colon, breast, prostate and lung cancer. Changes in CTC count during treatment also predict the eventual patient progression and survival in these cancers. Existing assays do not detect melanoma CTC, however. In addition, isolation of viable CTC remains problematic. To overcome these limitations we attempted to develop novel melanoma CTC assays, using IFA and cell culture approaches. Methods: Blood samples were obtained from patients and controls following informed consent. The buffy coat (white cells + tumor) was isolated by Ficoll/Hypaque centrifugation, and split into 6 replicate cultures in proprietary TrueCells medium. After 21 days in culture, tumor colonies were counted, and stained for melanoma and leukocyte markers. Buffy coat cells from parallel blood samples were stained with a panel of CSPG4-specific mAb (a pan-melanoma marker) on ultraclean glass slides for analysis by immunofluorescence microscopy. Results: Blood samples were obtained from 16 melanoma patients, ages 28-87. Eight patients were men and 8 were women. CSPG4+ events (>2) were detected in 8/16 patients by IFA (range 0-52). In contrast, tumor cell colonies of >50 cells grew in 12 out of 16 patients with Stage 3 or 4 melanoma (range 0-1054), shown in Table. Cells isolated from CTC colonies produced melanin, stained for CSPG4 and other melanoma markers, but not for leukocyte markers. Control cultures grew no tumor colonies. Conclusions: Our pilot study shows that melanoma CTC can be identified by both IFA and cultured from blood in many patients with stage 3 or 4 melanoma. These CTC exhibited cytologic characteristics diagnostic of melanoma. The culture assay may represent a useful means of enumerating, isolating, and expanding viable melanoma CTC for further molecular study. [Table: see text]
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Liu, Zongbin, Yuqing Huang, Wenli Liang, Jing Bai, Hongtao Feng, Zhihao Fang, Geng Tian, et al. "Cascaded filter deterministic lateral displacement microchips for isolation and molecular analysis of circulating tumor cells and fusion cells." Lab on a Chip 21, no. 15 (2021): 2881–91. http://dx.doi.org/10.1039/d1lc00360g.
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We present a cascaded filter deterministic lateral displacement design to achieve one-step and label-free CTC isolation with enhanced performance, and combine CTC enrichment and single-cell RNA sequencing for molecular analysis.
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Philippron, Annouck, Lieven Depypere, Steffi Oeyen, Bram De Laere, Charlotte Vandeputte, Philippe Nafteux, Katleen De Preter, and Piet Pattyn. "Evaluation of a marker independent isolation method for circulating tumor cells in esophageal adenocarcinoma." PLOS ONE 16, no. 5 (May 7, 2021): e0251052. http://dx.doi.org/10.1371/journal.pone.0251052.
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Objective The enrichment of circulating tumor cells (CTCs) from blood provides a minimally invasive method for biomarker discovery in cancer. Longitudinal interrogation allows monitoring or prediction of therapy response, detection of minimal residual disease or progression, and determination of prognosis. Despite inherent phenotypic heterogeneity and differences in cell surface marker expression, most CTC isolation technologies typically use positive selection. This necessitates the optimization of marker-independent CTC methods, enabling the capture of heterogenous CTCs. The aim of this report is to compare a size-dependent and a marker-dependent CTC-isolation method, using spiked esophageal cells in healthy donor blood and blood from patients diagnosed with esophageal adenocarcinoma. Methods Using esophageal cancer cell lines (OE19 and OE33) spiked into blood of a healthy donor, we investigated tumor cell isolation by Parsortix post cell fixation, immunostaining and transfer to a glass slide, and benchmarked its performance against the CellSearch system. Additionally, we performed DEPArray cell sorting to infer the feasibility to select and isolate cells of interest, aiming towards downstream single-cell molecular characterization in future studies. Finally, we measured CTC prevalence by Parsortix in venous blood samples from patients with various esophageal adenocarcinoma tumor stages. Results OE19 and OE33 cells were spiked in healthy donor blood and subsequently processed using CellSearch (n = 16) or Parsortix (n = 16). Upon tumor cell enrichment and enumeration, the recovery rate ranged from 76.3 ± 23.2% to 21.3 ± 9.2% for CellSearch and Parsortix, respectively. Parsortix-enriched and stained cell fractions were successfully transferred to the DEPArray instrument with preservation of cell morphology, allowing isolation of cells of interest. Finally, despite low CTC prevalence and abundance, Parsortix detected traditional CTCs (i.e. cytokeratin+/CD45-) in 8/29 (27.6%) of patients with esophageal adenocarcinoma, of whom 50% had early stage (I-II) disease. Conclusions We refined an epitope-independent isolation workflow to study CTCs in patients with esophageal adenocarcinoma. CTC recovery using Parsortix was substantially lower compared to CellSearch when focusing on the traditional CTC phenotype with CD45-negative and cytokeratin-positive staining characteristics. Future research could determine if this method allows downstream molecular interrogation of CTCs to infer new prognostic and predictive biomarkers on a single-cell level.
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Keup, Corinna, Markus Storbeck, Siegfried Hauch, Peter Hahn, Markus Sprenger-Haussels, Mitra Tewes, Pawel Mach, Oliver Hoffmann, Rainer Kimmig, and Sabine Kasimir-Bauer. "Cell-Free DNA Variant Sequencing Using CTC-Depleted Blood for Comprehensive Liquid Biopsy Testing in Metastatic Breast Cancer." Cancers 11, no. 2 (February 18, 2019): 238. http://dx.doi.org/10.3390/cancers11020238.
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Liquid biopsy analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for personalized treatment. Since cfDNA and CTCs are considered to give additive information and blood specimens are limited, isolation of cfDNA and CTC in an “all from one tube” format is desired. We investigated whether cfDNA variant sequencing from CTC-depleted blood (CTC-depl. B; obtained after positive immunomagnetic isolation of CTCs (AdnaTest EMT-2/Stem Cell Select, QIAGEN)) impacts the results compared to cfDNA variant sequencing from matched whole blood (WB). Cell-free DNA was isolated using matched WB and CTC-depl. B from 17 hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) metastatic breast cancer patients (QIAamp MinElute ccfDNA Kit, QIAGEN). Cell-free DNA libraries were constructed (customized QIAseq Targeted DNA Panel for Illumina, QIAGEN) with integrated unique molecular indices. Sequencing (on the NextSeq 550 platform, Illumina) and data analysis (Ingenuity Variant Analysis) were performed. RNA expression in CTCs was analyzed by multimarker quantitative PCR. Cell-free DNA concentration and size distribution in the matched plasma samples were not significantly different. Seventy percent of all variants were identical in matched WB and CTC-depl. B, but 115/125 variants were exclusively found in WB/CTC-depl. B. The number of detected variants per patient and the number of exclusively detected variants per patient in only one cfDNA source did not differ between the two matched cfDNA sources. Even the characteristics of the exclusively detected cfDNA variants in either WB or CTC-depl. B were comparable. Thus, cfDNA variants from matched WB and CTC-depl. B exhibited no relevant differences, and parallel isolation of cfDNA and CTCs from only 10 mL of blood in an “all from one tube” format was feasible. Matched cfDNA mutational and CTC transcriptional analyses might empower a comprehensive liquid biopsy analysis to enhance the identification of actionable targets for individual therapy strategies.
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Lei, Kin. "A Review on Microdevices for Isolating Circulating Tumor Cells." Micromachines 11, no. 5 (May 22, 2020): 531. http://dx.doi.org/10.3390/mi11050531.
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Cancer metastasis is the primary cause of high mortality of cancer patients. Enumeration of circulating tumor cells (CTCs) in the bloodstream is a very important indicator to estimate the therapeutic outcome in various metastatic cancers. The aim of this article is to review recent developments on the CTC isolation technologies in microdevices. Based on the categories of biochemical and biophysical isolation approaches, a literature review and in-depth discussion will be included to provide an overview of this challenging topic. The current excellent developments suggest promising CTC isolation methods in order to establish a precise indicator of the therapeutic outcome of cancer patients.
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Nicolazzo, Chiara, Angela Gradilone, Flavia Loreni, Cristina Raimondi, and Paola Gazzaniga. "EpCAMlow Circulating Tumor Cells: Gold in the Waste." Disease Markers 2019 (September 17, 2019): 1–5. http://dx.doi.org/10.1155/2019/1718920.
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The CellSearch® system which is still considered the gold standard for the enumeration of circulating tumor cells (CTC) utilizes antibodies against the epithelial cell adhesion molecule (EpCAM) for CTC enrichment. Recently, CTC discarded by the CellSearch® system due to their low EpCAM expression have been isolated and analyzed. We here sought to discuss technical and biological issues concerning the isolation and characterization of EpCAMlow CTC, highlighting the enormous potential of this subpopulation discarded by CellSearch®, which might instead reveal an unexpected clinical significance in tumor types where CTC enumeration has never been validated for prognostic and predictive purpose.
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Gwak, Hogyeong, Junmoo Kim, Leila Kashefi-Kheyrabadi, Bongseop Kwak, Kyung-A. Hyun, and Hyo-Il Jung. "Progress in Circulating Tumor Cell Research Using Microfluidic Devices." Micromachines 9, no. 7 (July 14, 2018): 353. http://dx.doi.org/10.3390/mi9070353.
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Circulating tumor cells (CTCs) are a popular topic in cancer research because they can be obtained by liquid biopsy, a minimally invasive procedure with more sample accessibility than tissue biopsy, to monitor a patient’s condition. Over the past decades, CTC research has covered a wide variety of topics such as enumeration, profiling, and correlation between CTC number and patient overall survival. It is important to isolate and enrich CTCs before performing CTC analysis because CTCs in the blood stream are very rare (0–10 CTCs/mL of blood). Among the various approaches to separating CTCs, here, we review the research trends in the isolation and analysis of CTCs using microfluidics. Microfluidics provides many attractive advantages for CTC studies such as continuous sample processing to reduce target cell loss and easy integration of various functions into a chip, making “do-everything-on-a-chip” possible. However, tumor cells obtained from different sites within a tumor exhibit heterogenetic features. Thus, heterogeneous CTC profiling should be conducted at a single-cell level after isolation to guide the optimal therapeutic path. We describe the studies on single-CTC analysis based on microfluidic devices. Additionally, as a critical concern in CTC studies, we explain the use of CTCs in cancer research, despite their rarity and heterogeneity, compared with other currently emerging circulating biomarkers, including exosomes and cell-free DNA (cfDNA). Finally, the commercialization of products for CTC separation and analysis is discussed.
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Hendricks, Alexander, Burkhard Brandt, Reinhild Geisen, Katharina Dall, Christian Röder, Clemens Schafmayer, Thomas Becker, Sebastian Hinz, and Susanne Sebens. "Isolation and Enumeration of CTC in Colorectal Cancer Patients: Introduction of a Novel Cell Imaging Approach and Comparison to Cellular and Molecular Detection Techniques." Cancers 12, no. 9 (September 16, 2020): 2643. http://dx.doi.org/10.3390/cancers12092643.
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Circulating tumour cells (CTC) were proven to be prognostically relevant in cancer treatment, e.g., in colorectal cancer (CRC). This study validates a molecular detection technique through using a novel cell imaging approach for CTC detection and enumeration, in comparison to a size-based cellular and correlated the data to clinico-pathological characteristics. Overall, 57 CRC patients were recruited for this prospective study. Blood samples were analysed for CTCs by three methods: (1) Epithelial marker immunofluorescence staining combined with automated microscopy using the NYONE® cell imager; (2) isolation by size using membrane filtration with the ScreenCell® Cyto IS device and immunofluorescence staining; (3) detection by semi-quantitative Cytokeratin-20 RT-qPCR. Enumeration data were compared and correlated with clinic-pathological parameters. CTC were detected by either approach; however, with varying positivity rates: NYONE® 36.4%, ScreenCell® 100%, and PCR 80.5%. All methods revealed a positive correlation of CTC presence and higher tumour burden, which was most striking using the ScreenCell® device. Generally, no intercorrelation of CTC presence emerged amongst the applied techniques. Overall, enumeration of CTC after isolation by size demonstrated to be the most reliable strategy for the detection of CTC in CRC patients. Ongoing studies will have to unravel the prognostic value of this finding, and validate this approach in a larger cohort.
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Takagi, Hidenori, Liang Dong, Morgan D. Kuczler, Kara Lombardo, Mitsuharu Hirai, Sarah R. Amend, and Kenneth J. Pienta. "Analysis of the Circulating Tumor Cell Capture Ability of a Slit Filter-Based Method in Comparison to a Selection-Free Method in Multiple Cancer Types." International Journal of Molecular Sciences 21, no. 23 (November 27, 2020): 9031. http://dx.doi.org/10.3390/ijms21239031.
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Circulating tumor cells (CTCs) are a promising biomarker for cancer liquid biopsy. To evaluate the CTC capture bias and detection capability of the slit filter-based CTC isolation platform (CTC-FIND), we prospectively compared it head to head to a selection-free platform (AccuCyte®-CyteFinder® system). We used the two methods to determine the CTC counts, CTC positive rates, CTC size distributions, and CTC phenotypes in 36 patients with metastatic cancer. Between the two methods, the median CTC counts were not significantly different and the total counts were correlated (r = 0.63, p < 0.0001). The CTC positive rate by CTC-FIND was significantly higher than that by AccuCyte®-CyteFinder® system (91.7% vs. 66.7%, p < 0.05). The median diameter of CTCs collected by CTC-FIND was significantly larger (13.0 μm, range 5.2–52.0 vs. 10.4 μm, range 5.2–44.2, p < 0.0001). The distributions of CTC phenotypes (CK+EpCAM+, CK+EpCAM− or CK−EpCAM+) detected by both methods were similar. These results suggested that CTC-FIND can detect more CTC-positive cases but with a bias toward large size of CTCs.
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Payne, Karl, Jill Brooks, Rachel Spruce, Nikolaos Batis, Graham Taylor, Paul Nankivell, and Hisham Mehanna. "Circulating Tumour Cell Biomarkers in Head and Neck Cancer: Current Progress and Future Prospects." Cancers 11, no. 8 (August 5, 2019): 1115. http://dx.doi.org/10.3390/cancers11081115.
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Head and neck cancer (HNC) continues to carry a significant burden of disease both for patients and health services. Facilitating biomarker-led treatment decisions is critical to improve outcomes in this group and deliver therapy tailored to the individual tumour biological profile. One solution to develop such biomarkers is a liquid biopsy analysing circulating tumour cells (CTCs)—providing a non-invasive and dynamic assessment of tumour specific alterations in ‘real-time’. A major obstacle to implementing such a test is the standardisation of CTC isolation methods and subsequent down-stream analysis. Several options are available, with a recent shift in vogue from positive-selection marker-dependent isolation systems to marker-independent negative-selection techniques. HNC single-CTC characterisation, including single-cell sequencing, to identify actionable mutations and gene-expression signatures has the potential to both guide the understanding of patient tumour heterogeneity and support the adoption of personalised medicine strategies. Microfluidic approaches for isolating CTCs and cell clusters are emerging as novel technologies which can be incorporated with computational platforms to complement current diagnostic and prognostic strategies. We review the current literature to assess progress regarding CTC biomarkers in HNC and potential avenues for future translational research and clinical implementation.
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Lianidou, Evi S., and Athina Markou. "Circulating Tumor Cells in Breast Cancer: Detection Systems, Molecular Characterization, and Future Challenges." Clinical Chemistry 57, no. 9 (September 1, 2011): 1242–55. http://dx.doi.org/10.1373/clinchem.2011.165068.
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BACKGROUND Circulating tumor cell (CTC) analysis is a promising new diagnostic field for estimating the risk for metastatic relapse and metastatic progression in patients with cancer. CONTENT Different analytical systems for CTC isolation and detection have been developed as immunocytochemical and molecular assays, most including separation steps by size or biological characteristics, such as expression of epithelial- or cancer-specific markers. Recent technical advancements in CTC detection and characterization include methods based on multiplex reverse-transcription quantitative PCR and approaches based on imaging and microfilter and microchip devices. New areas of research are directed toward developing novel assays for CTC molecular characterization. QC is an important issue for CTC analysis, and standardization of micrometastatic cell detection and characterization methodologies is important for the incorporation of CTCs into prospective clinical trials to test their clinical utility. The molecular characterization of CTCs can provide important information on the molecular and biological nature of these cells, such as the status of hormone receptors and epidermal and other growth factor receptor family members, and indications of stem-cell characteristics. This information is important for the identification of therapeutic targets and resistance mechanisms in CTCs as well as for the stratification of patients and real-time monitoring of systemic therapies. SUMMARY CTC analysis can be used as a liquid biopsy approach for prognostic and predictive purposes in breast and other cancers. In this review we focus on state-of-the-art technology platforms for CTC isolation, imaging, and detection; QC of CTC analysis; and ongoing challenges for the molecular characterization of CTCs.
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Leitão, Tito Palmela, Patrícia Corredeira, Sandra Kucharczak, Margarida Rodrigues, Paulina Piairo, Carolina Rodrigues, Patrícia Alves, et al. "Clinical Validation of a Size-Based Microfluidic Device for Circulating Tumor Cell Isolation and Analysis in Renal Cell Carcinoma." International Journal of Molecular Sciences 24, no. 9 (May 7, 2023): 8404. http://dx.doi.org/10.3390/ijms24098404.
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Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0–27), 101.8 (0–255), and 3.2 (0–10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0–255), and 0.2 (0–1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.
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Ruiz-Rodríguez, Antonio J., Maria P. Molina-Vallejo, Inés Aznar-Peralta, Cristina González Puga, Inés Cañas García, Encarna González, Jose A. Lorente, M. Jose Serrano, and M. Carmen Garrido-Navas. "Deep Phenotypic Characterisation of CTCs by Combination of Microfluidic Isolation (IsoFlux) and Imaging Flow Cytometry (ImageStream)." Cancers 13, no. 24 (December 20, 2021): 6386. http://dx.doi.org/10.3390/cancers13246386.
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The isolation of circulating tumour cells (CTCs) in colorectal cancer (CRC) mostly relies on the expression of epithelial markers such as EpCAM, and phenotypic characterisation is usually performed under fluorescence microscopy with only one or two additional markers. This limits the ability to detect different CTC subpopulations based on multiple markers. The aim of this work was to develop a novel protocol combining two platforms (IsoFluxTM and ImageStream®X) to improve CTC evaluation. Cancer cell lines and peripheral blood from healthy donors were used to evaluate the efficiency of each platform independently and in combination. Peripheral blood was extracted from 16 early CRC patients (before loco-regional surgery) to demonstrate the suitability of the protocol for CTC assessment. Additionally, peripheral blood was extracted from nine patients one month after surgery to validate the utility of our protocol for identifying CTC subpopulation changes over time. Results: Our protocol had a mean recovery efficiency of 69.5% and a limit of detection of at least four cells per millilitre. We developed an analysis method to reduce noise from magnetic beads used for CTC isolation. CTCs were isolated from CRC patients with a median of 37 CTCs (IQ 13.0–85.5) at baseline. CTCs from CRC patients were significantly (p < 0.0001) larger than cytokeratin (CK)-negative cells, and patients were stratified into two groups based on BRAFV600E and PD-L1 expression on CK-positive cells. The changes observed over time included not only the number of CTCs but also their distribution into four different subpopulations defined according to BRAFV600E and PD-L1 positivity. We developed a novel protocol for semi-automatic CTC isolation and phenotypic characterisation by combining two platforms. Assessment of CTCs from early CRC patients using our protocol allowed the identification of two clusters of patients with changing phenotypes over time.
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El-Heliebi, Amin, Claudia Hille, Navya Laxman, Jessica Svedlund, Christoph Haudum, Erkan Ercan, Thomas Kroneis, et al. "In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells." Clinical Chemistry 64, no. 3 (March 1, 2018): 536–46. http://dx.doi.org/10.1373/clinchem.2017.281295.
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Abstract BACKGROUND Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1–76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.
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Szmulewitz, R. Z., A. J. Wyche, E. M. Posadas, and W. M. Stadler. "Fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) detection and characterization of circulating tumor cells (CTC) from men with castrate-resistant prostate cancer (CRPC)." Journal of Clinical Oncology 29, no. 7_suppl (March 1, 2011): 41. http://dx.doi.org/10.1200/jco.2011.29.7_suppl.41.
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41 Background: Men with progressive CRPC have identifiable CTC. The currently available technologies for isolation of these CTC are limited by requiring send out to a central lab, high cost, and relative inflexibility with regards to characterization of the CTC. We hypothesized that modern FACS techniques followed by IF could be utilized for point of care isolation and characterization of CTC from men with CRPC. Methods: Patients (pts) with progressive CRPC according to Prostate Cancer Working Group 2 criteria were eligible. 15mL of whole blood was collected in BD CPT Vacutainer tubes. The mononuclear cell layer was labeled with primary antibodies against CD45 and EpCAM conjugated to separate flours. The EpCAM+/CD45− population was sorted directly onto a chamber slide, fixed and stained for multiplex IF imaging of markers including: pan-cytokeratin (CK), PSA, and the androgen receptor (AR). Imaged cells were considered CTC if were nucleated and PSA or CK positive. The methodology would be considered feasible if ≥ 5 of the first 15 pts had detectable CTC. Results: 15 pts with progressive CRPC were accrued to the study. Of these, 14 had documented skeletal metastases and 13 had progressed through docetaxel chemotherapy. 13/15 pts had EpCAM+/CD45− events on flow cytometry; median 94 (range 0–1700). Ten patients had CTC imaged after IF staining. A median of 4 (0–25) CTC were detected, but in 7 pts with >100 events by flow, all had detectable CTC with a median 22 (10–25) (p<0.001 vs. # events <100). A total of 9 contaminating cells (nucleus but no CK or PSA) were observed. All CTC stained positive for AR with variable intensity. Conclusions: FACS followed by IF is feasible for detection of CTC from men with metastatic CRPC. There is considerable loss of CTC during the processing, however, in men with >100 EpCAM+ events, CTC can be imaged and characterized routinely with these methods. This is a promising technique for utilization in translational research given the relative flexibility, ease and cost. Patients are now being enrolled for pre-analytical validation of IF staining for multiple prostate cancer biomarkers including AR. No significant financial relationships to disclose.
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Park, Emily S., Justin P. Yan, Richard A. Ang, Jeong Hyun Lee, Xiaoyan Deng, Simon P. Duffy, Kevin Beja, et al. "Isolation and genome sequencing of individual circulating tumor cells using hydrogel encapsulation and laser capture microdissection." Lab on a Chip 18, no. 12 (2018): 1736–49. http://dx.doi.org/10.1039/c8lc00184g.
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Bai, Linling, Yimeng Du, Jiaxi Peng, Yi Liu, Yanmei Wang, Yanlian Yang, and Chen Wang. "Peptide-based isolation of circulating tumor cells by magnetic nanoparticles." J. Mater. Chem. B 2, no. 26 (2014): 4080–88. http://dx.doi.org/10.1039/c4tb00456f.
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Davis, Darren W., Christopher Neal, Vishal Gupta, Vladislava O. Melnikova, Jacky Woo, Elizabeth Brooke Somers, and Daniel O'Shannessy. "Antibody-independent ApoStream technology isolates folate receptor alpha (FRA)–positive circulating tumor cells from blood of non-small cell lung adenocarcinoma patients." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e21028-e21028. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e21028.
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e21028 Background: The ability to enrich and interrogate circulating tumor cells (CTCs) from blood may allow for the analysis of metastatic dissemination and potential use of CTCs as surrogates for monitoring drug efficacy. We developed ApoStream, a dieletrophoresis field-flow fractionation based platform, for antibody-independent CTC isolation. We demonstrated that ApoStream successfully isolates non-small cell lung adenocarcinoma (NSCLC) CTCs that express Folate Receptor alpha (FRA), a GPI-anchored receptor, which has emerged as a cancer biomarker and potential therapeutic target in multiple cancer types. Methods: ApoStream technology was used to enrich CTCs from NSCLC patients’ blood. CTC enrichment by ApoStream was compared to that of the FDA cleared CellSearch CTC kit. A multiplexed immunofluorescent assay was developed to enable CTC enumeration (Cytokeratin+/CD45-/DAPI+ cells) and analysis of FRA expression (detection by murine antibody clone 26B3) using single cell quantitative laser scanning cytometry (LSC). Results: In a side-by-side comparison with the CellSearch CTC kit, ApoStream isolated significantly higher numbers of CTCs in 9 metastatic adenocarcinoma NSCLC patients (Apostream: mean =139, range 3-487 per 7.5 mL of blood, versus CellSearch kit: mean =2, range 0-8 per 7.5 mL of blood, n= 9, p=0.041). All patients were found to be CTC-positive by ApoStream, while only 3 of 9 (33%) patients were CTC-positive based on the CellSearch kit. LSC analysis demonstrated that 8-33% of all CTCs isolated expressed FRA and that FRA expression was confined to CTCs only. No false positive CTCs and no FRA-expressing cells were isolated by ApoStream from normal donor blood (n=15). Conclusions: All NSCLC adenocarcinoma patients analyzed had FRA-positive CTCs, suggesting that FRA may play a key role in metastasis and that screening of patients with the ApoStream CTC isolation system may identify patients who could benefit from FRA-targeted therapy.
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Patel, Deep A., and Jonathan Blay. "Seeding metastases: The role and clinical utility of circulating tumour cells." Tumor Biology 43, no. 1 (October 21, 2021): 285–306. http://dx.doi.org/10.3233/tub-210001.
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Peripheral human blood is a readily-accessible source of patient material in which circulating tumour cells (CTCs) can be found. Their isolation and characterization holds the potential to provide prognostic value for various solid cancers. Enumeration of CTCs from blood is becoming a common practice in informing prognosis and may guide therapy decisions. It is further recognized that enumeration alone does not capture perspective on the heterogeneity of tumours and varying functional abilities of the CTCs to interact with the secondary microenvironment. Characterizing the isolated CTCs further, in particular assessing their functional abilities, can track molecular changes in the disease progress. As a step towards identifying a suite of functional features of CTCs that could aid in clinical decisions, developing a CTC isolation technique based on extracellular matrix (ECM) interactions may provide a more solid foundation for isolating the cells of interest. Techniques based on size, charge, density, and single biomarkers are not sufficient as they underutilize other characteristics of cancer cells. The ability of cancer cells to interact with ECM proteins presents an opportunity to utilize their full character in capturing, and also allows assessment of the features that reveal how cells might behave at secondary sites during metastasis. This article will review some common techniques and recent advances in CTC capture technologies. It will further explore the heterogeneity of the CTC population, challenges they experience in their metastatic journey, and the advantages of utilizing an ECM-based platform for CTC capture. Lastly, we will discuss how tailored ECM approaches may present an optimal platform to capture an influential heterogeneous population of CTCs.
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Kang, Haeli, Jinho Kim, Hyungseok Cho, and Ki-Ho Han. "Evaluation of Positive and Negative Methods for Isolation of Circulating Tumor Cells by Lateral Magnetophoresis." Micromachines 10, no. 6 (June 8, 2019): 386. http://dx.doi.org/10.3390/mi10060386.
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We developed an epithelial cell adhesion molecule (EpCAM)-based positive method and CD45/CD66b-based negative method for isolating circulating tumor cells (CTCs) by lateral magnetophoresis. The CTC recovery rate, white blood cell depletion rate, and purity of CTCs isolated using the positive and negative methods were analyzed using blood samples spiked with cancer cells with different expression levels of EpCAM. The aim was to assess the strengths and weaknesses of the positive and negative isolation methods for CTC-based diagnostics, prognostics, and therapeutics for cancer. The EpCAM-based positive method yielded CTCs of high purity, while the CD45/CD66b-based negative method yielded a large number of CTCs. In conclusion, the positive method shows promise for detecting somatic oncogenic mutations and the negative method shows promise for discovery of cellular and transcriptomic biomarkers of cancer.
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Chen, Yuanwen, Ping Xu, Weiqing Qiu, Hongyi Zhu, Changlin Qian, Huojian Shen, and Jie Zhang. "Gastric Cancer-Circulating Tumor Cells Isolation by Folic Acid Immunolipid Magnetic Beads and Clinical Application." Science of Advanced Materials 14, no. 6 (June 1, 2022): 1024–31. http://dx.doi.org/10.1166/sam.2022.4282.
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CTCs plays a significant role in tumor prognosis, treatment and metastasis diagnosis, but CTCs isolation and enrichment with low concentrations is a critical step in early diagnosis and needs to be studied urgently. In this study, the use of folic acid-modified lipid magnetic spheres was used to separate GC-CTCs, and the correlation between CTCs counts and parameters, indicators, and prognosis recorded in clinical patients with gastric cancer was investigated. In our study, it was found that there were 26 benign patients with no CTC detected in their peripheral blood. Among 40 gastric cancer patients, CTC was detected, that is, CTC positive in 35 cases (the positive rate was 87.5%). At the same time, by analyzing the parameters recorded in clinical patients, it was found that the CTC positive was related to clinical stage, but not with patient age, histopathological type, and pathological grade. In addition, we also confirmed that the folic acid lipid magnetic spheres prepared by us can effectively separate GC-CTCs, and confirmed that the CTCs level was correlated with clinical stage, and the degree of progression is related to the clinical stage of gastric cancer patients: Samples with high CTC detection had shorter PFS. This study proves that folic acid lipid magnetic spheres can effectively separate gastric cancer CTCs, which is a reliable auxiliary means for the comprehensive diagnosis.
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Rupp, Brittany, Sarah Owen, Harrison Ball, Kaylee Judith Smith, Valerie Gunchick, Evan T. Keller, Vaibhav Sahai, and Sunitha Nagrath. "Integrated Workflow for the Label-Free Isolation and Genomic Analysis of Single Circulating Tumor Cells in Pancreatic Cancer." International Journal of Molecular Sciences 23, no. 14 (July 16, 2022): 7852. http://dx.doi.org/10.3390/ijms23147852.
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As pancreatic cancer is the third deadliest cancer in the U.S., the ability to study genetic alterations is necessary to provide further insight into potentially targetable regions for cancer treatment. Circulating tumor cells (CTCs) represent an especially aggressive subset of cancer cells, capable of causing metastasis and progressing the disease. Here, we present the Labyrinth–DEPArray pipeline for the isolation and analysis of single CTCs. Established cell lines, patient-derived CTC cell lines and freshly isolated CTCs were recovered and sequenced to reveal single-cell copy number variations (CNVs). The resulting CNV profiles of established cell lines showed concordance with previously reported data and highlight several gains and losses of cancer-related genes such as FGFR3 and GNAS. The novel sequencing of patient-derived CTC cell lines showed gains in chromosome 8q, 10q and 17q across both CTC cell lines. The pipeline was used to process and isolate single cells from a metastatic pancreatic cancer patient revealing a gain of chromosome 1q and a loss of chromosome 5q. Overall, the Labyrinth-DEPArray pipeline offers a validated workflow combining the benefits of antigen-free CTC isolation with single cell genomic analysis.
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Gribko, Alena, Janis Stiefel, Lana Liebetanz, Sophie Madeleine Nagel, Julian Künzel, Madita Wandrey, Jan Hagemann, Roland H. Stauber, Christian Freese, and Désirée Gül. "IsoMAG—An Automated System for the Immunomagnetic Isolation of Squamous Cell Carcinoma-Derived Circulating Tumor Cells." Diagnostics 11, no. 11 (November 4, 2021): 2040. http://dx.doi.org/10.3390/diagnostics11112040.
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Background: detailed information about circulating tumor cells (CTCs) as an indicator of therapy response and cancer metastasis is crucial not only for basic research but also for diagnostics and therapeutic approaches. Here, we showcase a newly developed IsoMAG IMS system with an optimized protocol for fully automated immunomagnetic enrichment of CTCs, also revealing rare CTC subpopulations. Methods: using different squamous cell carcinoma cell lines, we developed an isolation protocol exploiting highly efficient EpCAM-targeting magnetic beads for automated CTC enrichment by the IsoMAG IMS system. By FACS analysis, we analyzed white blood contamination usually preventing further downstream analysis of enriched cells. Results: 1 µm magnetic beads with tosyl-activated hydrophobic surface properties were found to be optimal for automated CTC enrichment. More than 86.5% and 95% of spiked cancer cells were recovered from both cell culture media or human blood employing our developed protocol. In addition, contamination with white blood cells was minimized to about 1200 cells starting from 7.5 mL blood. Finally, we showed that the system is applicable for HNSCC patient samples and characterized isolated CTCs by immunostaining using a panel of tumor markers. Conclusion: Herein, we demonstrate that the IsoMAG system allows the detection and isolation of CTCs from HNSCC patient blood for disease monitoring in a fully-automated process with a significant leukocyte count reduction. Future developments seek to integrate the IsoMAG IMS system into an automated microfluidic-based isolation workflow to further facilitate single CTC detection also in clinical routine.
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Zeinali, Mina, Maggie Lee, Arthi Nadhan, Anvya Mathur, Casey Hedman, Eric Lin, Ramdane Harouaka, et al. "High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients." Cancers 12, no. 1 (January 3, 2020): 127. http://dx.doi.org/10.3390/cancers12010127.
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(1) Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers and current methods for isolation of clusters may fall short. (2) Methods: We applied an inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs/CTC clusters from patients with metastatic non-small-cell lung cancer (NSCLC). (3) Results: Using Labyrinth, CTCs (PanCK+/DAPI+/CD45−) were isolated from patients (n = 25). Heterogeneous CTC populations, including CTCs expressing epithelial (EpCAM), mesenchymal (Vimentin) or both markers were detected. CTCs were isolated from 100% of patients (417 ± 1023 CTCs/mL). EpCAM− CTCs were significantly greater than EpCAM+ CTCs. Cell clusters of ≥2 CTCs were observed in 96% of patients—of which, 75% were EpCAM−. CTCs revealed identical genetic aberrations as the primary tumor for RET, ROS1, and ALK genes using fluorescence in situ hybridization (FISH) analysis. (4) Conclusions: The Labyrinth device recovered heterogeneous CTCs in 100% and CTC clusters in 96% of patients with metastatic NSCLC. The majority of recovered CTCs/clusters were EpCAM−, suggesting that these would have been missed using traditional antibody-based capture methods.
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Adams, Daniel L., R. Katherine Alpaugh, Stuart S. Martin, Monica Charpentier, Saranya Chumsri, Massimo Cristofanilli, Diane K. Adams, et al. "Precision microfilters as an all in one system for multiplex analysis of circulating tumor cells." RSC Advances 6, no. 8 (2016): 6405–14. http://dx.doi.org/10.1039/c5ra21524b.
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Cabezas-Camarero, Santiago, Vanesa García-Barberán, Virginia De la Orden-García, Beatriz Mediero-Valeros, Isabel Díaz-Millán, Cristina Saiz-Ladera, Mariona Baliu Piqué, et al. "Comparison of circulating tumor cells and cell-free DNA in the molecular characterization of patients with head and neck cancer." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e18521-e18521. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e18521.
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e18521 Background: The role of liquid biopsy in diagnosis and therapy monitoring in patients with head and neck cancer has been much less studied compared to other cancers. Our aim was to evaluate the perfomance in the isolation and recovery for molecular characterization of circulating tumour cells (CTC) of a new immunoafinity-based method and to compare it with the molecular diagnostic yield of plasma cell-free DNA. Methods: Patients with recurrent/metastatic (RM) head and neck cancer (HNC) were enrolled prospectively. Forty mililiters (ml) of plasma were collected at one or several time-points. First blood draw was always collected before starting a new therapeutic intervention or at the time of radiologic progression. For CTC detection and isolation, either anti-EpCAM or both anti-EpCAM + anti-EGFR antibodies were used. Digital PCR and castPCR were used to study KRAS and PI3KCA mutations in non-squamous HNC. A 15-gene customized NGS panel was used to characterized both CTC and cfDNA in patients with squamous HNC. Results: Between February 2016 and October 2018, 14 patients with R/M HNC were included (n = 1 local-only disease, n = 10 local and distant disease, n = 3 distant-only disease). Squamous histology (S): n = 9. Non-squamous (NS): n = 5 (1 naso-ethmoidal intestinal-type adenocarcinoma, 1 parotid gland exadenoma pleomorfic carcinoma, 2 parotid-gland salivary duct carcinomas (SDC), 1 parotid-gland high-grade neuroendocrine carcinoma). Twenty-five CTC determinations were performed. In 5 patients serial CTC determinations were performed. Median CTC was 4 (min-max: 0-49). Median CTC among 11 CTC determinations in S-HNC was 4 (min-max: 0-49). Median CTC was 3 CTC (min-max: 0-26) among the 14 determinations performed in NS-HNC. Digital PCR unveiled mutations in CTC and in cfDNA in 2 of 4 patients tested with NS histology (KRAS, PIK3CA), with one of them being concordant for the specific mutation. NGS unveiled mutations in CTC in 7/9 patients and in cfDNA in 6/9 patients, with only one loci-concordant case between CTC and plasma. Conclusions: IsoFlux detected CTC in the majority of patients with R/M HNC, regardless of the histologic type, and allowed for molecular characterization of CTC using different techniques for mutational analysis. Both NGS and digital PCR allowed for the detection in cell-free DNA of commonly mutated genes in HNC. Liquid biopsy should be more actively studied in this disease in order to better define its role in diagnosis and therapeutic monitoring.
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Loeian, Masoud S., Sadegh Mehdi Aghaei, Farzaneh Farhadi, Veeresh Rai, Hong Wei Yang, Mark D. Johnson, Farrukh Aqil, Mounika Mandadi, Shesh N. Rai, and Balaji Panchapakesan. "Liquid biopsy using the nanotube-CTC-chip: capture of invasive CTCs with high purity using preferential adherence in breast cancer patients." Lab on a Chip 19, no. 11 (2019): 1899–915. http://dx.doi.org/10.1039/c9lc00274j.
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Zhou, Jian, Qiyue Luan, Celine Macaraniag, Arielle Guzman, Maria Mantice, Oana Danciu, Kent Hoskins, and Ian Papautsky. "Abstract 5124: Isolation of viable circulating tumor cells from peripheral and central venous blood using a rapid microfluidic biochip." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5124. http://dx.doi.org/10.1158/1538-7445.am2022-5124.
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Abstract Background: Thousands of circulating tumor cells (CTCs) may be released into the blood stream of a cancer patient each day. However, only viable CTCs can cause metastasis. These viable CTCs hold the promise of deciphering the tumor heterogeneity, uncovering the mechanism of resistances to therapies, and enabling personalized treatments. Despite the continuously mounting interest, isolation of these cells remains challenging due to the multitude of factors, including rarity of the CTCs in blood, their constant exposure to therapeutic agents, and the harsh environment in the circulation. To address this, we have developed a novel microfluidic biochip capable of CTC isolation from unprocessed whole blood. Herein, we compare isolation of CTCs from the central and peripheral blood samples of metastatic breast cancer (MBC) patients using our biochip to assess impact of blood collection location on CTC count and viability. Methods: We included 19 patients with MBC, of which 26% are white, 63% are African American, and 11% are Latino. The median age of the cohort is 58 (34-75y). All patients are undergoing therapy. A total of 36 samples (7.5 mL each) were collected from either an antecubital vein (peripheral vein) or the subcutaneous port catheter (central vein) at different time points. All samples were processed within 5 hours of blood drawn in our novel microfluidic biochip (only 40 minutes per sample, without pretreatment), followed by in situ immunostaining. CK+/EpCAM+, DAPI+ and CD45- cells were counted as CTCs. In situ live/dead assay using Calcein AM and propidium iodide was performed for some samples right after CTC isolation. Results: CTCs were detected in all the samples (36/36), with a median of 15 CTCs per sample. A >5 CTCs count was found in 86% of the samples (31/36). No correlation was found between CTC count and race/ethnicity. The CTC count in central venous samples was in the range of 6-105 (median 27), while the range was 1-45 (median 10) in the peripheral samples. The difference was found to be statistically significant (p<0.001 using ANOVA). While no correlation between CTC count and patient age was found for the overall samples, we noted a significantly higher CTC count in central venous blood of younger patients (<60y) than of senior patients (p<0.05). Live/dead assay immediately following the microfluidic isolation found live CTCs in patient samples. Conclusions: Viable CTCs were rapidly isolated and detected in MBC samples using our novel microfluidic biochip. Our results show that CTC count in blood drawn from central vein yields significantly higher numbers than from the peripheral vein, suggesting that central venous samples might be preferred for prognosis and monitoring to drug response in MBC patients. Viable CTCs may provide an opportunity to evaluate the mechanism of resistance to therapies. More studies with larger cohorts are needed to further confirm these findings. Citation Format: Jian Zhou, Qiyue Luan, Celine Macaraniag, Arielle Guzman, Maria Mantice, Oana Danciu, Kent Hoskins, Ian Papautsky. Isolation of viable circulating tumor cells from peripheral and central venous blood using a rapid microfluidic biochip [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5124.
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Ramos-Medina, Rocío, Sara López-Tarruella, María del Monte-Millán, Tatiana Massarrah, and Miguel Martín. "Technical Challenges for CTC Implementation in Breast Cancer." Cancers 13, no. 18 (September 15, 2021): 4619. http://dx.doi.org/10.3390/cancers13184619.
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Breast cancer is the most common neoplasm in women worldwide. Tissue biopsy, currently the gold standard to obtain tumor molecular information, is invasive and might be affected by tumor heterogeneity rendering it incapable to portray the complete dynamic picture by the absence of specific genetic changes during the evolution of the disease. In contrast, liquid biopsy can provide unique opportunities for real-time monitoring of disease progression, treatment response and for studying tumor heterogeneity combining the information of DNA that tumors spread in the blood (circulating tumor DNA) with CTCs analysis. In this review, we analyze the technical and biological challenges for isolation and characterization of circulating tumor cells from breast cancer patients. Circulating tumor cell (CTC) enumeration value is included in numerous clinical studies due to the prognostic’s role of these cells. Despite this, there are so many questions pending to answer. How to manage lymphocytes background, how to distinguish the CTCs subtypes or how to work with frozen samples, are some of the issues that will discuss in this review. Based on our experience, we try to address these issues and other technical limitations that should be solved to optimize the standardization of protocols, sample extraction procedures, circulating-tumor material isolation (CTCs vs. ctDNA) and the very diverse methodologies employed, aiming to consolidate the use of CTCs in the clinic. Furthermore, we think that new approaches focusing on isolation CTCs in other body fluids such as cerebrospinal or ascitic fluid are necessary to increase the opportunities of circulating tumor cells in the practice clinic as well as to study the promising role of CTC clusters and their prognostic value in metastatic breast cancer.
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Jou, Hei-Jen, Hsin-Cheng Ho, Kuan-Yeh Huang, Chen-Yang Chen, Sheng-Wen Chen, Pei-Hsuan Lo, Pin-Wen Huang, Chung-Er Huang, and Ming Chen. "Isolation of TTF-1 Positive Circulating Tumor Cells for Single-Cell Sequencing by Using an Automatic Platform Based on Microfluidic Devices." International Journal of Molecular Sciences 23, no. 23 (December 1, 2022): 15139. http://dx.doi.org/10.3390/ijms232315139.
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Single-cell sequencing provides promising information in tumor evolution and heterogeneity. Even with the recent advances in circulating tumor cell (CTC) technologies, it remains a big challenge to precisely and effectively isolate CTCs for downstream analysis. The Cell RevealTM system integrates an automatic CTC enrichment and staining machine, an AI-assisted automatic CTC scanning and identification system, and an automatic cell picking machine for CTC isolation. H1975 cell line was used for the spiking test. The identification of CTCs and the isolation of target CTCs for genetic sequencing were performed from the peripheral blood of three cancer patients, including two with lung cancer and one with both lung cancer and thyroid cancer. The spiking test revealed a mean recovery rate of 81.81% even with extremely low spiking cell counts with a linear relationship between the spiked cell counts and the recovered cell counts (Y = 0.7241 × X + 19.76, R2 = 0.9984). The three cancer patients had significantly higher TTF-1+ CTCs than healthy volunteers. All target CTCs were successfully isolated by the Cell Picker machine for a subsequent genetic analysis. Six tumor-associated mutations in four genes were detected. The present study reveals the Cell RevealTM platform can precisely identify and isolate target CTCs and then successfully perform single-cell sequencing by using commercially available genetic devices.
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Chen, Shukun, Gerlinde Tauber, Tanja Langsenlehner, Linda Maria Schmölzer, Michaela Pötscher, Sabine Riethdorf, Andra Kuske, et al. "In Vivo Detection of Circulating Tumor Cells in High-Risk Non-Metastatic Prostate Cancer Patients Undergoing Radiotherapy." Cancers 11, no. 7 (July 3, 2019): 933. http://dx.doi.org/10.3390/cancers11070933.
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High-risk non-metastatic prostate cancer (PCa) has the potential to progress into lethal disease. Treatment options are manifold but, given a lack of surrogate biomarkers, it remains unclear which treatment offers the best results. Several studies have reported circulating tumor cells (CTCs) to be a prognostic biomarker in metastatic PCa. However, few reports on CTCs in high-risk non-metastatic PCa are available. Herein, we evaluated CTC detection in high-risk non-metastatic PCa patients using the in vivo CellCollector CANCER01 (DC01) and CellSearch system. CTC counts were analyzed and compared before and after radiotherapy (two sampling time points) in 51 high-risk non-metastatic PCa patients and were further compared according to isolation technique; further, CTC counts were correlated to clinical features. Use of DC01 resulted in a significantly higher percentage of CTC-positive samples compared to CellSearch (33.7% vs. 18.6%; p = 0.024) and yielded significantly higher CTC numbers (range: 0–15 vs. 0–5; p = 0.006). Matched pair analysis of samples between two sampling time points showed no difference in CTC counts determined by both techniques. CTC counts were not correlated with clinicopathological features. In vivo enrichment using DC01 has the potential to detect CTC at a higher efficiency compared to CellSearch, suggesting that CTC is a suitable biomarker in high-risk non-metastatic PCa.
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Chang, Zhi-min, Rui Zhang, Chao Yang, Dan Shao, Yuguo Tang, Wen-fei Dong, and Zheng Wang. "Cancer–leukocyte hybrid membrane-cloaked magnetic beads for the ultrasensitive isolation, purification, and non-destructive release of circulating tumor cells." Nanoscale 12, no. 37 (2020): 19121–28. http://dx.doi.org/10.1039/d0nr04097e.
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Most of the current circulating tumor cell (CTC) isolation techniques are based on immunomagnetic beads with antibodies or aptamers that specifically target epithelial cell adhesion molecules (EpCAMs).
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Liao, Chia-Jung, Chia-Hsun Hsieh, Tzu-Keng Chiu, Yu-Xian Zhu, Hung-Ming Wang, Feng-Chun Hung, Wen-Pin Chou, and Min-Hsien Wu. "An Optically Induced Dielectrophoresis (ODEP)-Based Microfluidic System for the Isolation of High-Purity CD45neg/EpCAMneg Cells from the Blood Samples of Cancer Patients—Demonstration and Initial Exploration of the Clinical Significance of These Cells." Micromachines 9, no. 11 (October 31, 2018): 563. http://dx.doi.org/10.3390/mi9110563.
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Circulating tumour cells (CTCs) in blood circulation play an important role in cancer metastasis. CTCs are generally defined as the cells in circulating blood expressing the surface antigen EpCAM (epithelial cell adhesion molecule). Nevertheless, CTCs with a highly metastatic nature might undergo an epithelial-to-mesenchymal transition (EMT), after which their EpCAM expression is downregulated. In current CTC-related studies, however, these clinically important CTCs with high relevance to cancer metastasis could be missed due to the use of the conventional CTC isolation methodologies. To precisely explore the clinical significance of these cells (i.e., CD45neg/EpCAMneg cells), the high-purity isolation of these cells from blood samples is required. To achieve this isolation, the integration of fluorescence microscopic imaging and optically induced dielectrophoresis (ODEP)-based cell manipulation in a microfluidic system was proposed. In this study, an ODEP microfluidic system was developed. The optimal ODEP operating conditions and the performance of live CD45neg/EpCAMneg cell isolation were evaluated. The results demonstrated that the proposed system was capable of isolating live CD45neg/EpCAMneg cells with a purity as high as 100%, which is greater than the purity attainable using the existing techniques for similar tasks. As a demonstration case, the cancer-related gene expression of CD45neg/EpCAMneg cells isolated from the blood samples of healthy donors and cancer patients was successfully compared. The initial results indicate that the CD45neg/EpCAMneg nucleated cell population in the blood samples of cancer patients might contain cancer-related cells, particularly EMT-transformed CTCs, as suggested by the high detection rate of vimentin gene expression. Overall, this study presents an ODEP microfluidic system capable of simply and effectively isolating a specific, rare cell species from a cell mixture.