Circulating Tumor Cells Enrichment Research Articles

Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Liquid Biopsy for Detection of Pancreaticobiliary Cancers by Functional Enrichment and Immunofluorescent Profiling of Circulating Tumor Cells and Their Clusters.

Circulating tumor cells (CTCs) have historically been used for prognostication in oncology. We evaluate the performance of liquid biopsy CTC assay as a diagnostic tool in suspected pancreaticobiliary cancers (PBC). The assay utilizes functional enrichment of CTCs followed by immunofluorescent profiling of organ-specific markers. The performance of the assay was first evaluated in a multicentric case-control study of blood samples from 360 participants, including 188 PBC cases (pre-biopsy samples) and 172 healthy individuals. A subsequent prospective observational study included pre-biopsy blood samples from 88 individuals with suspicion of PBC and no prior diagnosis of cancer. CTCs were harvested using a unique functional enrichment method and used for immunofluorescent profiling for CA19.9, Maspin, EpCAM, CK, and CD45, blinded to the tissue histopathological diagnosis. TruBlood® malignant or non-malignant predictions were compared with tissue diagnoses to establish sensitivity and specificity. The test had 95.9% overall sensitivity (95% CI: 86.0-99.5%) and 92.3% specificity (95% CI: 79.13% to 98.38%) to differentiate PBC (n = 49) from benign conditions (n = 39). The high accuracy of the CTC-based TruBlood test demonstrates its potential clinical application as a diagnostic tool to assist the effective detection of PBC when tissue sampling is unviable or inconclusive.

Abstract 7504: Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients

Abstract Introduction: Anaplastic lymphoma kinase positive non-small cell lung cancer (ALK+NSCLC) represents a distinct subtype of NSCLC characterized by oncogenic ALK gene mutations leading to ALK overexpression and activation in tumor cells. ALK+ NSCLC will frequently develop mutations during treatment, leading to nonresponse and eventually cancer relapse. Therefore, there is a critical need for sensitive technologies to monitor ALK+ NSCLC disease status in patients over time. To facilitate longitudinal monitoring, there has been a rapid evolution in diagnostic technologies leveraging circulating tumor cells (CTCs) as a liquid biopsy tool. CTCs can be collected non-invasively at various intervals throughout patient treatment. Moreover, they shed from solid tumors and disseminate into the bloodstream, which makes isolating CTCs a valuable source of genomic, transcriptomic, and proteomic insights pertaining to the primary tumor. In this study, we present a comprehensive workflow for conducting multi-omic analysis on CTCs derived from ALK+ NSCLC patients. Methods: Blood samples were collected from a cohort of ALK+ NSCLC patients, and CTCs were isolated using a high throughput, label free microfluidic device, Labyrinth. Enriched CTCs were immunofluorescently stained for cytokeratin, CD45, DAPI, epithelial cell adhesion molecule (EpCAM), and vimentin markers. Heterogeneous CTC populations with hybrid epithelial/mesenchymal (EM) phenotypes are reported. Fluorescence in situ hybridization (FISH) was performed on enriched CTC samples. DNA was extracted and sequenced using a 523 gene targeted sequencing panel. Single cell RNA-seq technology was performed in parallel on CTC samples to investigate the gene expression throughout treatment. Results: The median concentration of CTCs in the ALK+ NSCLC cohort (n=33) is 137 CTCs per ml of blood, and 64% of CTCs are in an EM hybrid state. In ALK+ NSCLC patients (n=3) with an average of 4.6 visits per sample, we observed a trend of decreasing CTC concentration for patients with partial response to the treatment. The presence of ALK fusion in ALK+ NSCLC patient’s CTC is confirmed by FISH. Oncogenic mutations (JAK1, SPEN, ERBB4, NTRK3) are observed in bulk CTC samples from two NSCLC patients. Single cell RNA sequencing was performed on three ALK+ NSCLC patients; 58, 199, and 700 potential CTCs were detected with positive cytokeratin expression and no white blood cell marker expression. Conclusions: This study examined the feasibility of using CTCs for studying and monitoring the clinical status of the ALK+ NSCLC patients as well as the genomic and transcriptomic signatures within their tumors. More patients will need to be processed in order to validate our workflow and reinforce our conclusions. Citation Format: Yuru Chen, Shamileh Fouladdel, Harrison Ball, Zhaoping Qin, Albert Liu, Xu Cheng, Liwei Bao, Habib Serhan, Bryce Vandenburg, Varun Kathawate, Larua Goo, Peter Ulintz, Nathan Merrill, Aaron Udager, Angel Qin, Sofia D. Merajver, Sunitha Nagrath. Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7504.

Abstract 7499: Circulating tumor cell enumeration and protein expression by CapioCyte and Allegro Plus imaging

Abstract Radiation or chemoradiation followed by surgical resection is the current standard of therapy for many diseases. These treatments are increasingly supplemented with monoclonal antibodies, such as epidermal growth factor receptor (EGFR)-targeting and programmed death ligand 1 (PD-L1) checkpoint-blocking antibodies. The use of these therapies is typically dependent on solid tissue biopsy to confirm elevated expression of these markers in the solid tumor, which carries complications and may only be sampled at diagnosis. In contrast, a liquid biopsy may detect these markers with minimal invasiveness and at multiple points throughout treatment and recovery. Here, we describe the development of an automated and integrated workflow for circulating tumor cell (CTC) analysis. We previously demonstrated that the CapioCyte method is a highly sensitive approach for enrichment of CTCs using a combination of biomimetic rolling on recombinant E-selectin and antibody-mediated capture on a nanostructured polymer surface within a flow chamber for capture. Here we report a fully automated CapioCyte instrument for CTC purification and immunofluorescence staining using epithelial, white blood cell and nuclear makers to identify and distinguish CTCs from WBCs while also quantifying EGFR or PD-L1 presence on respective CTCs. The immunostaining conditions were developed and validated for high analytical sensitivity and specificity using cell lines and health donor white blood cells with known biomarker status. The final assay conditions were transferred to the CapioCyte for automated immunostaining. CapioCyte enriched and stained CTC samples are paired with the BioView Allegro Plus platform, an imaging and analysis solution using machine learning and customized algorithms for automated imaging and CTC candidate identification based on high-resolution Z-stack images. The reported comprehensive and automated workflow was used to enumerate CTCs in patients with non-small cell lung carcinoma (NSCLC) in a feasibility cohort. Median CTC counts from NSCLC patients was 7.5 CTC/ml (n=26, mean 51.7, range 0.3 - 406.2). Studies are on-going to expand the cohort including testing blood from head and neck cancer patients and respective EGFR and PD-L1 status. The combined and automated CapioCyte-Allegro Plus workflow shows great potential for routine quantification of CTCs and EGFR and PD-L1 status. Citation Format: Ju-heon Lim, Chassidy Johnson, Tal Kaplan, Michael J. Poellmann, Yuval Harari, Elad Kfir, Andrew Z. Wang, Seungpyo Hong. Circulating tumor cell enumeration and protein expression by CapioCyte and Allegro Plus imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7499.

Abstract 7498: Acoustophoresis enriches tumor cell clusters from patients with metastatic prostate cancer

Abstract Purpose: A subset of aggressive prostate cancers (PCa) progress to invade surrounding tissue to form distant metastases by shed of circulating tumor cells (CTCs). However, due to the scarcity of CTCs among the millions of nucleated blood cells, there is an unmet need to improve CTC isolation assays. Our technique Acoustophoresis uses standing ultrasound waves in a flow-through microfluidic chamber to discriminate cells based on their size, density, and compressibility. Method: We analyzed the effectiveness of acoustophoretic circulating tumor cell (CTC) isolation from 6 mL PFA fixed red blood cell lysed blood from 12 metastatic prostate cancer patients and 20 healthy control subjects and compared it to the golden standard CellSearch. For acoustic cell separation, cells were diluted in 12 mL PBS based buffer and separated by acoustophoresis and analyzed by imaging flow cytometry using a standardized antibody panel (EpCAM, cytokeratin, CD45, CD66b, DAPI). Generally, CTCs, and clusters migrate faster than white blood cells (WBCs) when exposed to ultrasound in the microchannel during acoustophoresis and can therefore be separated from nucleated blood cells. Major findings: Our pilot study shows that acoustophoresis finds both singlets and clusters of CTCs in patients as well as aggregates of CTCs and WBCs. In total, 67 cancer cell containing cell clusters were identified. Most identified cell clusters were small, containing less than 4 cells. In addition, acoustophoresis is a label-free separation technique, and could therefore identify additional subclasses of CTCs among the detected CTCs with e.g., EpCAM low/- expression. Further, we provide extensive data for the effect of storage time on acoustophoretic separation performance for fixed prostate cancer cell line cells and WBCs. Demonstrating that sample processing can be done up to 72 hours after blood sampling. We believe this is to date the most comprehensive study of acoustic separation of CTCs in patients and control subjects. Conclusion: Label-free acoustophoresis is a potential method for future non-invasive molecular interrogation of metastatic cancers. We believe Acoustophoresis is a promising technology for CTC enrichment with the possibility to detect new subclasses of CTCs as well as cell clusters not detectable with CellSearch. CTC-clusters has previously been proposed as a promising and potent predictor to monitor progressive disease and is associated with earlier onset of metastatic disease and poor prognosis, which makes acoustophoresis ability to identify clusters of high interest. Citation Format: Cecilia Magnusson, Per Augustsson, Andreas Lenshof, Andreas Josefsson, Anders Bjartell, Yvonne Ceder, Hans Lilja, Thomas Laurell. Acoustophoresis enriches tumor cell clusters from patients with metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7498.

Abstract 7495: Validation of microfluidic isolation of circulating pancreatic tumor cells from blood

Abstract Background. Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is the fourth leading cause of cancer-related deaths in the US. PDAC is diagnosed in the late stages, which limits treatment options and yields poor clinical outcomes. Recurrence is a significant issue for patients undergoing surgery for PDAC. Circulating tumor cells (CTCs) are thought to be a potential mediator of recurrence, facilitating the formation of secondary tumor sites after surgery. Thus, there is a need to isolate CTCs to improve understanding of disease recurrence, to ultimately improve outcomes. Herein, we aim to validate our inertial microfluidic (iMF) system for isolating pancreatic CTCs from blood. We also benchmark our iMF system to a commercially available immunomagnetic negative selection system, EasySep™, and quantify target cell recovery and cell enrichment. Methods. Our iMF system employs the differential cell sizes of CTCs from blood cells and inertial migration of such cells in a straight microchannel (150 µm × 50 µm × 24 mm) to enrich CTCs from blood. The iMF system sample flow rate used is 100 µL/min, with a 200 µL/min PBS buffer flow rate. The EasySep™ Direct Human CTC Enrichment kit targets CD2, CD14, CD16, CD19, CD45, CD61, CD66b, and Glycophorin to remove blood cells from the sample using magnetic particles, thereby separating them from CTCs. We spiked low cell concentrations of 50, 100, and 500 Hoechst-stained pancreatic carcinoma cells, PANC1, in 1 mL of healthy blood then captured using either EasySep™ or iMF. Cell enumeration was performed after centrifugation or Cytospin. Results. PANC1 cells and WBCs have average sizes of 18 µm and 8.4 µm, respectively. We characterized our iMF device to capture cells ≥ 12 µm, which yields capture of most PANC1 cells. Indeed, our iMF system achieved 82.8±19.2%, 72.5±16.3%, and 59.3±3.8% recovery for 50, 100, and 500 cell-spikes in lysed blood, respectively. For the same 50, 100, and 500 cell-spikes, the total iMF CTC yields after Cytospin were 46.3±11.8, 44.3±9.1, and 32.7±2.1%, while the total CTC yields from EasySep™ were 4.6±3, 5.3±2.1, 8.3±1%, respectively. When we processed whole blood directly, EasySep™ had lower cell recovery, 3.8±0.2, 4.6±1, and 3.3±0.4%, respectively. Our system also demonstrated 8 × higher target cell enrichment compared to EasySep™. Blood lysis before iMF separation did not drastically affect the recovery of target cells. Conclusions. We determined that our system has improved performance over the commercial isolation system, EasySep™, thereby validating the effectiveness of microfluidic isolation for PDAC. In future studies, we can then use our iMF system in CTC isolation from routine blood draws from PDAC patients undergoing surgical resection. We may then isolate real patient samples and study relevant phenotypes of PDAC CTCs which may therefore be used for identifying certain mechanisms of PDAC progression. Citation Format: Celine Macaraniag, Ifra Khan, Jian Zhou, Pier Cristoforo Giulianotti, Alain Borgeat, Gina Votta-Velis, Ian Papautsky. Validation of microfluidic isolation of circulating pancreatic tumor cells from blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7495.

Abstract 7497: Advancing multi-biomarker CTC assay and CDx development through the automated GenoCTC system

Abstract Introduction: The successful outcomes observed in clinical trials emphasize the effectiveness of therapeutic approaches relying on biomarker tests, particularly in the development of cancer drugs. The parallel development of a drug and its associated predictive biomarker is considered an ideal strategy for achieving rapid and successful drug development in precision medicine. In this context, we employ an automated circulating tumor cell (CTC) isolation platform, known as GenoCTC, to facilitate the development of biomarker-driven CTC assays, with the ultimate goal of developing companion diagnostic (CDx) tests. Method: This study employed the 5th generation GenoCTC device, a fully automated system designed for marker-based CTC enrichment using immune magnetophoresis. We confirmed the isolation of CTCs through spiking tests, utilizing cancer biomarkers such as cMET, Mesothelin (MSLN), and Claudin 3 (CLDN3). cMET is a biomarker extensively studied in various cancers, particularly non-small cell lung cancer. We previously showed the prognostic significance of cMET expressing CTCs in HR+ and HER2- metastatic breast cancer patients. MSLN and CLDN3 are tumor-associated proteins showing overexpression across diverse cancer types, with a potential for the development of antibody-based cytotoxic drugs. The GenoCTC device allows efficient separation of CTCs expressing these biomarkers, ensuring high accuracy, precision, specificity, and sensitivity. Simultaneously, therapeutic drugs directed at these markers are progressing through phase trials or preclinical studies. Conclusion: Our results show that cMET, MSLN, and CLDN3 are potential markers for the development of biomarker tests, that can improve the patient selection process for personalized therapy targeting these biomarkers. Citation Format: Chaithanya Chelakkot, Jieun Park, Chae Rin Kim, Yeonu Lee, Yu Rim Lee, Saehyung Lee, Jun Young Choi, Young Ho Moon, Yoon-La Choi, Young Kee Shin, Hun Seok Lee. Advancing multi-biomarker CTC assay and CDx development through the automated GenoCTC system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7497.

Abstract 7488: Detection of PD-L1 mRNA expression in circulating tumor cells (CTCs) from patients with metastatic NSCLC, HNSCC and melanoma using a novel highly sensitive commercially available CE-IVD kit

Abstract Background: We have previously reported the development of an RT-qPCR assay for PD-L1 expression in CTCs (Strati et al, Ann Oncol, 2017). This assay is now commercially available as Oncolipsy PD-L1 kit (CE-IVD, Pharmassist, Greece) and we report here it’s application in size-based enriched CTCs from patients with metastatic NSCLC, HNSCC and melanoma, before and during PD-1/PD-L1 blockade therapies. Patients and Methods: The Oncolipsy PD-L1 kit was analytically validated using Mini-Genes as positive controls (IDT, USA). The analytical sensitivity was tested by spiking known numbers of H1975 cells in peripheral blood (PB) samples from healthy donors (HD). In total 95 patients were enrolled in the study: 69 patients with metastatic NSCLC, 19 patients with metastatic HNSCC and 7 patients with metastatic melanoma. 40 PB samples from HD were used as a control group. 10 ml of PB was collected at baseline (V0), two months after immunotherapy (V1) and at the time of disease progression (PD) or 18 months after initiation of immunotherapy (V2). PD-L1 and B2M transcripts were quantified in cDNAs obtained from CTCs isolated with the size-based PARSORTIX (ANGLE, UK) device. A sample was considered as CTC-positive when cDNAs were positive for CK-8, CK-18 or CK-19 expression. Results: Spiking experiments have shown that 10 H1975cells/10ml PB can be detected using the kit. In CTC-positive samples, overexpression of PD-L1 was detected in 15/58(25.8%) patients at V0, in 6/34(17.6%) patients at V1 and in 3/11(27.3%) at V2. Patients with elevated expression of PD-L1 at V1 in respect to V0 and detectable CTCs had longer PFS (18.5mo vs. 13.2mo, p=0.033) and longer OS (24.8mo vs. 12.8mo, p = 0.071) after initiation of immunotherapy. Patients with elevated PD-L1 at V1 compared to V0 benefited from immunotherapy, with a longer DFS (17.8mo vs. 13.4mo) and OS (29.4mo vs. 26.9mo). Conclusions: The commercially available Oncolipsy PD-L1 kit can be used for the quantification of PD-L1 transcripts in CTCs. Expression of PD-L1 in size-based enriched CTCs can predict response to PD-1/PD-L1 blockade therapy. Acknowledgements: This study has been financially supported by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T1RCI-02935). Trial registration: ClinicalTrials.gov Identifier: NCT04490564 Citation Format: Areti Strati, Martha Zavridou, Stavroula Smilkou, Victoria Tserpeli, Eleni Tzanikou, Dimitra Stergiopoulou, Eleni Efthimiadou, Emily Tsaroucha, Amanda Psyrri, Aggeliki Sfika, Evangelos Bournakis, Ioanna Balgkouranidou, Stylianos Kakolyris, Ioannis Boukovinas, Christos Papadimitriou, Loukas Kaklamanis, Ioanna Koukli, Evi Lianidou. Detection of PD-L1 mRNA expression in circulating tumor cells (CTCs) from patients with metastatic NSCLC, HNSCC and melanoma using a novel highly sensitive commercially available CE-IVD kit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7488.

Microfluidic System Consisting of a Magnetic 3D-Printed Microchannel Filter for Isolation and Enrichment of Circulating Tumor Cells Targeted by Anti-HER2/MOF@Ferrite Core-Shell Nanostructures: A Theranostic CTC Dialysis System.

Low number of circulating tumor cells (CTCs) in the blood samples and time-consuming properties of the current CTC isolation methods for processing a small volume of blood are the biggest obstacles to CTC usage in practice. Therefore, we aimed to design a CTC dialysis system with the ability to process cancer patients' whole blood within a reasonable time. Two strategies were employed for developing this dialysis setup, including (i) synthesizing novel in situ core-shell Cu ferrites consisting of the Cu-CuFe2O4 core and the MIL-88A shell, which are targeted by the anti-HER2 antibody for the efficient targeting and trapping of CTCs; and (ii) fabricating a microfluidic system containing a three-dimensional (3D)-printed microchannel filter composed of a polycaprolactone/Fe3O4 nanoparticle composite with pore diameter less than 200 μm on which a high-voltage magnetic field is focused to enrich and isolate the magnetic nanoparticle-targeted CTCs from a large volume of blood. The system was assessed in different aspects including capturing the efficacy of the magnetic nanoparticles, CTC enrichment and isolation from large volumes of human blood, side effects on blood cells, and the viability of CTCs after isolation for further analysis. Under the optimized conditions, the CTC dialysis system exhibited more than 80% efficacy in the isolation of CTCs from blood samples. The isolated CTCs were viable and were able to proliferate. Moreover, the CTC dialysis system was safe and did not cause side effects on normal blood cells. Taken together, the designed CTC dialysis system can process a high volume of blood for efficient dual diagnostic and therapeutic purposes.

Evaluating circulating tumour cell enrichment techniques to establish an appropriate method for clinical application in glioblastomas.

Brain tumours reduce life expectancy for an average of 20 years per patient, the highest of any cancer. A third of brain tumour patients visit their GP at least five times before diagnosis and many of those are diagnosed late through emergency departments. A possible solution to this challenge is to utilise a "liquid biopsy" blood test designed for circulating tumour cells (CTCs). Such a test could be applied at a primary healthcare centre, contributing to informed decision making for diagnostic imaging referrals. Furthermore, it could also be applied at secondary health care centres for the ongoing monitoring of disease recurrence. There is increased interest in CTC enrichment methods as a potential approach for faster diagnosis and monitoring of disease progression. The aim of this review to compare four CTC enrichment methods - OncoQuick®, Screen Cell®, pluriBead® and Cell Search® - with the objective of identifying a suitable method for application in the clinical setting for the isolation of CTCs from glioblastomas.

Prognostic significance of circulating tumor cells and tumor related transcripts in small cell lung cancer: A step further to clinical implementation.

Small-cell lung cancer (SCLC) is a fatal disease with limited treatment options. Circulating tumor cells (CTCs) in liquid biopsy samples may serve as predictive and prognostic biomarkers; but the analysis of CTCs is still challenging. By using microfluidic or density gradient CTC enrichment in combination with immunofluorescent (IF) staining or qPCR of CTC-related transcripts, we achieved a 60.8% to 88.0% positivity in SCLC blood samples. Epithelial and neuroendocrine transcripts including the druggable target DLL3 were associated with shorter overall survival (OS), indicating the clinical value of these markers in terms of differential diagnosis and treatment decisions. High CTC counts and the presence of CTC duplets detected by IF staining were prognostic for OS, and thus may serve as indicators of disease progression or therapy failure. In patient samples with high CTC load detected by IF staining, a concordance of the transcripts positivity in circulating free plasma RNA and CTCs was observed. Our data emphasize the role of CTCs and CTC-related transcripts and underline the clinical value of liquid biopsy analysis in SCLC.

Prognostic evaluation of a multi-target magnetic bead-enriched circulating tumor cell-enriched identification system for colorectal cancer.

Colorectal cancer (CRC) is the third most prevalent cancer in the world. Traditional tissue biopsy cannot provide dynamic monitoring of patients' tumors or reflect the characteristics of tumors in real time because the sampling process is invasive and accompanied by risks. Circulating tumor cells (CTCs) are considered a major cause of tumor metastasis, and investigating CTCs helps to understand the biology and vulnerability of malignant tumors during hematogenous metastasis. We sequentially used epithelial cell adhesion molecule (EpCAM)-coated immunoliposomal magnetic beads (Ep-IMBs) and vimentin-coated immunoliposomal magnetic beads (Vi-IMBs) to capture and characterize CTCs from 110 CRC patients. We further constructed a Cox risk regression model, optimized the model composition using backward stepwise regression, and finally applied nomograms to show the effect of each variable on survival risk. The specificity of the CTCs enrichment and identification system was 100% and the sensitivity was 79.0%. Multivariate analysis indicated total CTC number was an important predictor for bad survival, whereas American Joint Committee on Cancer (AJCC) stage, lymph node metastasis, and carcinoembryonic antigen (CEA) level were associated with prognosis, and the risk of mortality was associated with the AJCC stage of the CRC. The CTC enrichment and identification system constructed in this research demonstrated superior accuracy. In addition, CTCs can be used as an important predictor for prognosis of patients with CRC, and the combination of other clinical predictive factors can help clinicians to better design individualized treatment regimens, which is of great clinical application value.

Biomolecules-mediated electrochemical signals of Cu2+: Y-DNA nanomachines enable homogeneous rapid one-step assay of lung cancer circulating tumor cells

This study presents a straightforward efficient technique for extracting circulating tumor cells (CTCs) and a rapid one-step electrochemical method (45 min) for detecting lung cancer A549 cells based on the specific recognition of mucin 1 using aptamers and the modulation of Cu2+ electrochemical signals by biomolecules. The CTCs separation and enrichment process can be completed within 45 min using lymphocyte separation solution (LSS), erythrocyte lysis solution (ELS), and three centrifugations. Besides, the influence of various biomolecules on Cu2+ electrochemical signals is comprehensively discussed, with DNA nanospheres selected as the medium. Three single-stranded DNA sequences were hybridized to form Y-shaped DNA (Y-DNA), creating DNA nanospheres. Upon specific capture of mucin 1 by the aptamer, most DNA nanospheres could form complexes with Cu2+ (DNA nanosphere-Cu2+), significantly reducing the concentration of free Cu2+. Our approach yielded the limit of detection (LOD) of 2 ag/mL for mucin 1 and 1 cell/mL for A549 cells. 39 clinical blood samples were used for further validation, yielding results closely correlated with pathological, computed tomography (CT) scan findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve displayed an area under the curve (AUC) value of 0.960, demonstrating 100% specificity and 93.1% sensitivity for the assay. Taken together, our findings indicate that this straightforward and efficient pretreatment and rapid, highly sensitive electrochemical assay holds great promise for liquid biopsy-based tumor detection using CTCs.

Circulating Tumor Cell Enrichment and Single-Cell Isolation Combining the CellSearch® and DEPArray™ Systems.

The analysis of circulating tumor cells (CTCs) has shown potential for detection of cancer spread, prognosis, therapeutic target selection, and monitoring of treatment response. CTCs can be obtained repeatedly by simple blood draws as so-called "liquid biopsy." Thus, they can serve as a surrogate material for primary or metastatic tissue biopsies. In addition, isolation of CTCs provides the possibility to investigate those cells which may hold the (molecular) traits responsible for metastatic progression and ultimately patient death. As such, CTCs represent a target of utmost importance in cancer research and therapy. In this chapter, we describe a workflow for the enrichment of CTCs with the FDA-cleared CellSearch® system followed by the isolation of single CTCs using the DEPArray™ technology enabling further molecular single-cell analyses.

Generally applicable circulating tumor cell enrichment and identification through a membrane glycoprotein-targeting strategy combining magnetic isolation and biological orthogonality labeling.

Although the importance of circulating tumor cells (CTCs) has been widely recognized, it is still a challenge to realize high-efficiency and accurate enrichment and identification of highly heterogeneous CTCs derived from various types of tumors in complex cancer processes. Currently, the most widely used methods follow the general idea of sequential immunoaffinitive capture and immunostaining to achieve the abovementioned goal. However, different organ/tissue origins as well as the inherent heterogeneity of CTCs would lead to the missed detection of certain CTC subtypes using such methods. Further, immunocytochemistry (ICC) immunostaining disrupts the physiological structure of cells, severely limiting the detection and application scenarios that require the participation of live cells. To address these limitations, we have developed a generally applicable strategy for the isolation and labeling of CTCs. This strategy focuses on targeting the universal characteristics of all tumor cells, specifically the abnormally expressed cell membrane glycoproteins, such as the transferrin receptor and sialic acid. Strategically, transferrin-functionalized magnetic beads (TMBs) were applied to enrich CTCs, and azide-based bioorthogonal chemistry was employed to label target CTCs. Accordingly, the membrane glycoprotein-targeting strategy achieved unbiased enrichment and labeling of broad-spectrum CTCs that were both epithelial and non-epithelial phenotypic populations with varied organ/tissue origins (MCF-7, HepG2, A549, Jurkat, and B16), with a capture efficiency of >95% and a detection limit as low as 5 cells per mL in artificial blood. In particular, our developed strategy displayed excellent specificity, and the CTCs under capture and fluorescence labelling remained with good viability and could be further cultivated and analyzed. Finally, the membrane glycoprotein-targeting strategy successfully detected and identified 33-223 CTCs from 1 mL patient blood samples.

A workflow for the enrichment, the identification, and the isolation of non-apoptotic single circulating tumor cells for RNA sequencing analysis.

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells-we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients-were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.

Recent Progress in Enhanced Cancer Diagnosis, Prognosis, and Monitoring Using a Combined Analysis of the Number of Circulating Tumor Cells (CTCs) and Other Clinical Parameters.

Analysis of circulating tumor cells (CTCs) holds promise to diagnose cancer or monitor its development. Among the methods, counting CTC numbers in blood samples could be the simplest way to implement it. Nevertheless, its clinical utility has not yet been fully accepted. The reasons could be due to the rarity and heterogeneity of CTCs in blood samples that could lead to misleading results from assays only based on single CTC counts. To address this issue, a feasible direction is to combine the CTC counts with other clinical data for analysis. Recent studies have demonstrated the use of this new strategy for early detection and prognosis evaluation of cancers, or even for the distinguishment of cancers with different stages. Overall, this approach could pave a new path to improve the technical problems in the clinical applications of CTC counting techniques. In this review, the information relevant to CTCs, including their characteristics, clinical use of CTC counting, and technologies for CTC enrichment, were first introduced. This was followed by discussing the challenges and new perspectives of CTC counting techniques for clinical applications. Finally, the advantages and the recent progress in combining CTC counts with other clinical parameters for clinical applications have been discussed.

Analytical Validation of a Spiral Microfluidic Chip with Hydrofoil-Shaped Pillars for the Enrichment of Circulating Tumor Cells.

The isolation of circulating tumor cells (CTCs) from peripheral blood with high efficiency remains a challenge hindering the utilization of CTC enrichment methods in clinical practice. Here, we propose a microfluidic channel design for the size-based hydrodynamic enrichment of CTCs from blood in an epitope-independent and high-throughput manner. The microfluidic channel comprises a spiral-shaped part followed by a widening part, incorporating successive streamlined pillars, that improves the enrichment efficiency. The design was tested against two benchmark designs, a spiral microfluidic channel and a spiral microfluidic channel followed by a widening channel without the hydrofoils, by processing 5 mL of healthy blood samples spiked with 100 MCF-7 cells. The results proved that the design with hydrofoil-shaped pillars perform significantly better in terms of recovery (recovery rate of 67.9% compared to 23.6% in spiral and 56.7% in spiral with widening section), at a cost of slightly lower white blood cell (WBC) depletion (depletion rate of 94.2% compared to 98.6% in spiral and 94.2% in spiral with widening section), at 1500 µL/min flow rate. For analytical validation, the design was further tested with A549, SKOV-3, and BT-474 cell lines, yielding recovery rates of 62.3 ± 8.4%, 71.0 ± 6.5%, and 82.9 ± 9.9%, respectively. The results are consistent with the size and deformability variation in the respective cell lines, where the increasing size and decreasing deformability affect the recovery rate in a positive manner. The analysis before and after the microfluidic chip process showed that the process does not affect cell viability.

Recent Update Roles of Magnetic Nanoparticles in Circulating Tumor Cell (CTC)/Non-CTC Separation.

Metastasis of cancer is a major cause of death worldwide. Circulating tumor cells (CTCs) are important in the metastatic process of cancer. CTCs are able to circulate in the bloodstream. Therefore, they can be used as biomarkers of metastasis. However, CTCs are rare when compared to a large number of blood cells in the blood. Many CTC detection methods have been developed to increase CTC detection efficiency. Magnetic nanoparticles (MNPs) have attracted immense attention owing to their potential medical applications. They are particularly appealing as a tool for cell separation. Because of their unique properties, MNPs are of considerable interest for the enrichment of CTCs through CTC or non-CTC separation. Herein, we review recent developments in the application of MNPs to separate CTCs or non-CTCs in samples containing CTCs. This review provides information on new approaches that can be used to detect CTCs in blood samples. The combination of MNPs with other particles for magnetic-based cell separation for CTC detection is discussed. Furthermore, different approaches for synthesizing MNPs are included in this review.

Clinical values of circulating tumor cells count in localized renal cell carcinoma.

Renal cancer is one of the most common malignant tumors of the urinary system, with distant metastasis occurring 30% of patients. Therefore, early detection and monitoring of tumor progression are of great significance in the diagnosis and treatment of renal cancer. However, current biomarkers used to diagnose, monitor recurrence and assess prognosis of renal cancer are still uncertain. Circulating tumor cells (CTCs) are tumor cells detached from the primary tumor or metastasis, invaded and existing in the peripheral blood, and are one of the most promising liquid biopsy targets because they can provide complete cell biological information. Microfluidic chip has advantages of miniaturization, high integration, and fast analysis, which has advantages in CTC separation and enrichment. In this study, 1 mL peripheral blood of each 30 patients with early localized renal cancer was collected before and 1 day after surgery. CTC enrichment was performed by microfluidic chip and CTCs were identified by immunofluorescence staining. All patients were followed up for a median of 17 months. The number of CTCs before surgery was higher than that after surgery (P<0.001), and the number was positively correlated with tumor-node-metastasis (TNM) stage and International Society of Urological Pathology (ISUP) grade. Patients in group CTC ≤2 had a longer progression-free survival (PFS) than those in group CTC ≥3 (P<0.05). Surgical treatment can remarkably reduce the number of CTCs in patients, and CTC counts can also play a role in monitoring tumor load and predicting prognosis in renal cancer.