scholarly journals Pre-Analytical and Analytical Variables of Label-Independent Enrichment and Automated Detection of Circulating Tumor Cells in Cancer Patients

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 442 ◽  
Author(s):  
Claudia Koch ◽  
Simon A. Joosse ◽  
Svenja Schneegans ◽  
Okka J. W. Wilken ◽  
Melanie Janning ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Tumor Cell Line ◽  
Response To Therapy ◽  
Clinical Samples ◽  
Blood Collection ◽  
Multiple Parameters ◽  
Healthy Donors

Circulating tumor cells (CTCs) are promising tools for risk prediction and the monitoring of response to therapy in cancer patients. Within the EU/IMI CANCER-ID consortium, we validated CTC enrichment systems for future inclusion into clinical trials. Due to the known heterogeneity of markers expressed on CTCs, we tested the Parsortix® system (ANGLE plc) which enables label-independent CTC enrichment from whole blood based on increased size and deformability of these tumor cells compared to leukocytes. We performed extensive comparisons both with spiked-in blood models (i.e., MDA-MB-468 tumor cell line cells spiked at very low concentration into blood from healthy donors) and validated the protocol on actual clinical samples from breast, lung, and gastrointestinal cancer patients to define optimal conditions for CTC enrichment. Multiple parameters including cassette gap, separation pressure, and cell fixatives were compared in parallel. Also, the compatibility of blood collection tubes with whole genome amplification of isolated tumor cells was demonstrated and we furthermore established a workflow for semi-automated CTC detection using a quantitative cell imager. The established workflow will contribute to supporting the use of size-based CTC enrichment platforms in clinical trials testing the clinical validity and utility of CTCs for personalized medicine.

scholarly journals The Mechanical Fingerprint of Circulating Tumor Cells (CTCs) in Breast Cancer Patients

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1119
Author(s):  
Ivonne Nel ◽  
Erik W. Morawetz ◽  
Dimitrij Tschodu ◽  
Josef A. Käs ◽  
Bahriye Aktas
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Hematopoietic Cells ◽  
Surrogate Marker ◽  
Clinical Samples ◽  
Breast Cancer Patients ◽  
Shape Restoration

Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

scholarly journals Circulating Tumor Cells in Gastrointestinal Malignancies: Current Techniques and Clinical Implications

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Georg Lurje ◽  
Marc Schiesser ◽  
Andreas Claudius Hoffmann ◽  
Paul Magnus Schneider
Keyword(s):  
Colorectal Cancer ◽  
Clinical Outcome ◽  
Disease Progression ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Response To Therapy ◽  
Clinical Implications ◽  
Gastrointestinal Malignancies ◽  
Progression Of Disease

Since their introduction more than 50 years by Engell, circulating tumor cells (CTCs) have been evaluated in cancer patients and their detection has been correlated with clinical outcome, in esophageal, gastric, and colorectal cancer. With the availability of refined technologies, the identification of CTCs from peripheral blood is emerging as a useful tool for the detection of malignancy, monitoring disease progression, and measuring response to therapy. However, increasing evidence suggests a variety of factors to be responsible for disease progression. The analysis of a single CTC marker is therefore unlikely to accurately predict progression of disease with sufficient resolution and reproducibility. Here we discuss the current concept of CTCs, summarize the available techniques for their detection and characterization, and aim to provide a comprehensive update on the clinical implications of CTCs in gastrointestinal (GI) malignancies.

scholarly journals Efficient Propagation of Circulating Tumor Cells: A First Step for Probing Tumor Metastasis

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2784
Author(s):  
Jerry Xiao ◽  
Joseph R. McGill ◽  
Kelly Stanton ◽  
Joshua D. Kassner ◽  
Sujata Choudhury ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Metastatic Cancer ◽  
Metastatic Breast ◽  
Success Rates ◽  
Breast Cancer Patients ◽  
Metastatic Cells ◽  
Healthy Donors

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.

scholarly journals Self-Seeding Microwells to Isolate and Assess the Viability of Single Circulating Tumor Cells

2019 ◽  
Vol 20 (3) ◽  
pp. 477 ◽  
Author(s):  
Kiki Andree ◽  
Fikri Abali ◽  
Lisa Oomens ◽  
Fiona Passanha ◽  
Joska Broekmaat ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Cultured Cells ◽  
Metastatic Breast ◽  
Cancer Cell Line ◽  
Blood Collection ◽  
Leukocyte Depletion ◽  
Breast And Prostate Cancer ◽  
Mcf 7

The availability of viable tumor cells could significantly improve the disease management of cancer patients. Here we developed and evaluated a method using self-seeding microwells to obtain single circulating tumor cells (CTC) and assess their potential to expand. Conditions were optimized using cells from the breast cancer cell line MCF-7 and blood from healthy volunteers collected in EDTA blood collection tubes. 43% of the MCF-7 cells (nucleus+, Ethidium homodimer-1-, Calcein AM+, α-EpCAM+, α-CD45-) spiked into 7.5 mL of blood could be recovered with 67% viability and these could be further expanded. The same procedure tested in metastatic breast and prostate cancer patients resulted in a CTC recovery of only 0–5% as compared with CTC counts obtained with the CellSearch® system. Viability of the detected CTC ranged from 0–36%. Cell losses could be mainly contributed to the smaller size and greater flexibility of CTC as compared to cultured cells from cell lines and loss during leukocyte depletion prior to cell seeding. Although CTC losses can be reduced by fixation, to obtain viable CTC no fixatives can be used and pore size in the bottom of microwells will need to be reduced, filtration conditions adapted and pre-enrichment improved to reduce CTC losses.

Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) and white blood cells (WBCs) in muscle invasive and metastatic bladder cancer patients.

2016 ◽  
Vol 34 (2_suppl) ◽  
pp. 446-446 ◽  
Author(s):  
Archana Anantharaman ◽  
Terence W. Friedlander ◽  
David Lu ◽  
Rachel Krupa ◽  
Gayatri Premasekharan ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Checkpoint Inhibitors ◽  
White Blood Cells ◽  
Response To Therapy ◽  
Therapeutic Monitoring ◽  
Healthy Donors ◽  
Algorithmic Analysis ◽  
Muscle Invasive

446 Background: Recent studies indicate that PD-1 and PD-L1 checkpoint inhibitors have activity in chemotherapy refractory patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer. PD-L1 expression on tumor cells or lymphocytes may correlate with response to therapy. To identify potential patients who may benefit from PD-1/PD-L1 targeted immunotherapeutics, we utilized a non-invasive blood test to evaluate PD-L1 protein expression in CTCs and WBCs of bladder cancer patients. Methods: Whole blood from 20 patients with MIBC or mBCa were collected and shipped to Epic Sciences. All nucleated cells were plated onto glass slides and subjected to immunofluorescent (IF) staining and CTC identification using scanners and algorithmic analysis. CTCs, defined as traditional (CK+ CD45- w/ intact DAPI+ nuclei and morphologically distinct) or CK- (CK-, CD45-, DAPI+, intact and distinct) were identified. PD-L1 biomarker characterization was assessed by IF staining. UroVysion FISH testing was used to assess genomic abnormalities in a subset of patient samples. Additionally, WBCs (CD45+ cells) were assessed for PD-L1 expression, relative to healthy controls. Results: Eighty percent of patients had mBCa and 20% had MIBC, 70% percent were men, and themedian age was 67 years, (range 43 to 88). Traditional CTCs were detected in 11/20 (55%) patients. Seven out of 20 (35%) patients had PD-L1+ cells, 4 of these patients had exclusively CK-/PD-L1+ CTCs, a subset of which were confirmed as malignant via FISH. CK- CTCs were detected in 14/20 (70%) patients. Five patients had greater than 4-fold PD-L1 positivity in WBCs as compared to healthy donors. Conclusions: Patients with MIBC and mBCa patients have detectable, heterogeneous CTCs with a population of CK-/PD-L1+ CTCs. Utilization of a liquid biopsy to identify patients with PD-L1+ CTCs and PD-L1+ WBCs may enable patient selection or short term therapeutic monitoring for measuring therapeutic efficacy. Further work will investigate association of PD-L1+ CTCs and WBCs with clinical response to PD-1 checkpoint immunotherapy.

Use of circulating tumor cells in prospective clinical trials for NSCLC patients – standardization of the pre-analytical conditions

2018 ◽  
Vol 56 (6) ◽  
pp. 980-989 ◽  
Author(s):  
Marius Ilie ◽  
Véronique Hofman ◽  
Sylvie Leroy ◽  
Charlotte Cohen ◽  
Simon Heeke ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Stage Iv ◽  
High Yield ◽  
Blood Collection ◽  
Anaplastic Lymphoma ◽  
Trophoblastic Cells ◽  
Prognosis And Prediction ◽  
Nsclc Patients

Abstract Background: Circulating tumor cells (CTCs) hold potential for noninvasive diagnosis, prognosis and prediction testing in non-small cell lung cancer (NSCLC) patients. Minimizing degradation or loss of CTCs is pivotal for detection and profiling of the low abundance and fragile CTCs, particularly in clinical trials. We prospectively investigated (NCT02372448) whether a new blood collection device performed better compared to commonly used K3EDTA tubes, when subjected to long-term sample storage. Methods: Blood samples were drawn into K3EDTA and blood collection tubes (BCT) (Streck), and filtered by the Isolation by SizE of Tumor/Trophoblastic Cells (ISET® system), for CTC detection in two study populations of NSCLC patients; the training set of 14 patients with stage II/IV NSCLC, and the validation set of 36 patients with stage IV NSCLC). MET expression was evaluated by immunocytochemistry (ICC) and anaplastic lymphoma kinase (ALK) gene rearrangement by break-apart fluorescence in situ hybridization (FISH) on ISET-enriched CTCs. Results: Blood processed after 24 h and 48 h in BCT tubes showed stable CTCs counts and integrity, whereas CTCs in K3EDTA tubes showed an altered morphology in all patients. CTCs recovered in BCT or K3EDTA tubes at 24 and 48 h were evaluable by ICC for MET expression and by FISH for ALK rearrangement. Conclusions: The BCT tubes gave a high yield and preserved the integrity of CTCs after 24 and 48 h of storage at room temperature, which facilitate their molecular characterization in NSCLC patients entering clinical trials.

Programmed death-ligand 1 characterization of circulating tumor cells and white blood cells in muscle-invasive and metastatic bladder cancer patients.

2015 ◽  
Vol 33 (7_suppl) ◽  
pp. 353-353
Author(s):  
Terence W. Friedlander ◽  
David Lu ◽  
Rachel Krupa ◽  
Gayatri Premasekharan ◽  
Christopher J. Welty ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Blood Cells ◽  
White Blood Cells ◽  
Response To Therapy ◽  
Programmed Death Ligand 1 ◽  
Programmed Death ◽  
Muscle Invasive

353 Background: Muscle invasive(MIBC) and metastatic (mBCa) bladder cancer patients have few options to extend survival. Recent studies have shown that PD-1 and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity even in chemotherapy refractory patients and it has been proposed that PD-L1 expression on tumors or lymphocytes may correlate with response to therapy. To identify potential patients who may benefit from PD-1/PD-L1 targeted immunotherapeutics, we utilized a non-invasive, real-time blood test for PD-L1 protein expression in circulating tumor cells (CTCs) and white blood cells (WBCs) of bladder cancer patients. Methods: Twelve blood samples from unique patients with MIBC or mBCa were collected and shipped to Epic Sciences. All nucleated cells were plated onto glass slides and subjected to IF staining and CTC identification by fluorescent scanners and algorithmic analysis. CTCs, defined as traditional (CK+ CD45- w/ intact DAPI nuclei and morphologically distinct) or CK- (CK-, CD45-, intact and distinct) were identified. PD-L1 biomarker characterization was assessed by IF staining, and UroVysion FISH testing was used to assess genomic abnormalities in a subset of patient samples. Additionally, WBCs (CD45+ cells) were assessed for PD-L1 expression. Results: Traditional CTCs were detected in 6/12 (50%) patients. 3/12 (25%) patients had PD-L1+ cells, 2 of these patients were exclusively CK-/PD-L1+ CTCs, which were confirmed as cancer via FISH. CK- CTCs were detected in 83% (10/12) patients. 5 patients had greater than 4 fold PD-L1 positivity in WBCs as compared to healthy donor controls. Conclusions: MIBC and mBCa patients have detectable CTCs with a high frequency of CK-/PD-L1+ CTCs. Utilization of a liquid biopsy to identify patients with PD-L1+ CTCs and PD-L1+ WBCs may enable both patient selection or short term therapeutic monitoring for measuring pharmacodynamics to ensure therapy effectiveness. Further studies are planned to investigate association of PD-L1+ CTCs and WBCs with response to PD-1 and PD-L1 checkpoint immunotherapy.

scholarly journals Acoustic separation of circulating tumor cells

2015 ◽  
Vol 112 (16) ◽  
pp. 4970-4975 ◽  
Author(s):  
Peng Li ◽  
Zhangming Mao ◽  
Zhangli Peng ◽  
Lanlan Zhou ◽  
Yuchao Chen ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Peripheral Blood ◽  
Cancer Biology ◽  
Cancer Metastasis ◽  
Surface Acoustic Waves ◽  
Clinical Samples ◽  
Label Free ◽  
Blood Samples

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state.

scholarly journals Red blood cells protein profile is modified in breast cancer patients

2022 ◽  
Author(s):  
Thais Pereira-Viega ◽  
Susana B. Bravo ◽  
Antonio Gomez-Tato ◽  
Celso Yáñez-Gómez ◽  
Carmen Abuín ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Red Blood Cells ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Blood Cells ◽  
Metastatic Breast ◽  
Potential Contribution ◽  
Breast Cancer Patients ◽  
Healthy Donors

Metastasis is the primary cause of death for most breast cancer patients who succumb to the disease. During the haematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are micro-environmental and systemic processes contributing to cancer regulation. We have published that Red Blood Cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic breast cancer patients. Although the principal known role of RBCs is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 breast cancer patients, compared with 33 healthy donors. RBCs from breast cancer patients showed a different proteomic profile compared to healthy donors. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Besides, LAMP2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.

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