Editorial

2 pages, 167 KiB

Open AccessEditorial

Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells

by Maria José Serrano and Jose A Lorente

Cancers 2020, 12(11), 3257; https://doi.org/10.3390/cancers12113257 - 04 Nov 2020

Viewed by 1104

From the medical and scientific point of view, we are witnessing important changes in the way we approach diseases, and consequently in the way we manage patients [...] Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

Research

Jump to: Editorial, Review, Other

13 pages, 2670 KiB

Open AccessArticle

Single-Cells Isolation and Molecular Analysis: Focus on HER2-Low CTCs in Metastatic Breast Cancer

by Paolo D’Amico, Carolina Reduzzi, Wenan Qiang, Youbin Zhang, Lorenzo Gerratana, Qiang Zhang, Andrew A. Davis, Ami N. Shah, Maroua Manai, Giuseppe Curigliano and Massimo Cristofanilli

Cancers 2022, 14(1), 79; https://doi.org/10.3390/cancers14010079 - 24 Dec 2021

Cited by 6 | Viewed by 4328

Abstract

Although the detection of CTCs expressing HER2 at low intensity (HER2-low CTCs) has been shown to have a negative prognostic value in metastatic breast cancer (MBC) patients, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges behind the selective [...] Read more.

Although the detection of CTCs expressing HER2 at low intensity (HER2-low CTCs) has been shown to have a negative prognostic value in metastatic breast cancer (MBC) patients, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges behind the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs. Four different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, and SKBR3), that are known to express HER2 at different immunohistochemistry levels (respectively classified as 0, 1+, 2+, and 3+), were spiked in healthy donor blood tubes (7.5 mL) and processed with the CellSearch^® (Menarini Silicon Biosystems, Bologna, Italy) for enrichment and the DEPArray NxT^™ for single cell selection. The HER2 signal-intensities of each cell line was compared using the nonparametric Mann–Whitney U test. The optimal cut-offs to distinguish HER2 1+ from 0 and 2+ cells were calculated performing the Receiver operating characteristic (ROC) curve. Median HER2 signal-intensities detected with the DEPArray NxT^™ were: 2.59 (0), 3.58 (1+), 5.23 (2+) and 38.37 (3+). DEPArray NxT efficiently differentiated each single cell line (p < 0.001). The area under the ROC curve was 0.69 and 0.70 (respectively 0 vs. 1+ and 1+ vs. 2+) and the optimal calculated cut-offs were 2.85 (lower) and 4.64 (upper). HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. This study will allow standardized single-cell or pooled collection of HER2-low CTCs for downstream molecular analyses. Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

► Show Figures

Figure 1

15 pages, 2183 KiB

Open AccessArticle

Deep Phenotypic Characterisation of CTCs by Combination of Microfluidic Isolation (IsoFlux) and Imaging Flow Cytometry (ImageStream)

by Antonio J. Ruiz-Rodríguez, 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

Cancers 2021, 13(24), 6386; https://doi.org/10.3390/cancers13246386 - 20 Dec 2021

Cited by 10 | Viewed by 3698

Abstract

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 [...] Read more.

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 (IsoFlux^TM 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 BRAF^V600E 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 BRAF^V600E 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. Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

► Show Figures

Graphical abstract

15 pages, 2120 KiB

Open AccessArticle

Circulating Tumor Cells Enumeration from the Portal Vein for Risk Stratification in Early Pancreatic Cancer Patients

by Javier Padillo-Ruiz, Gonzalo Suarez, Sheila Pereira, Francisco José Calero-Castro, Jose Tinoco, Luis Marin, Carmen Bernal, Carmen Cepeda-Franco, Jose Maria Alamo, Francisco Almoguera, Hada C. Macher, Paula Villanueva, Francisco José García-Fernandez, Inmaculada Gallego, Manuel Romero, Miguel Angel Gomez-Bravo, Valeria Denninghoff and María José Serrano

Cancers 2021, 13(24), 6153; https://doi.org/10.3390/cancers13246153 - 07 Dec 2021

Cited by 12 | Viewed by 3091

Abstract

Background. Effective biomarkers are needed to enable personalized medicine for pancreatic cancer patients. This study analyzes the prognostic value, in early pancreatic cancer, of single circulating tumor cell (CTC) and CTC clusters from the central venous catheter (CVC) and portal blood (PV). Methods. [...] Read more.

Background. Effective biomarkers are needed to enable personalized medicine for pancreatic cancer patients. This study analyzes the prognostic value, in early pancreatic cancer, of single circulating tumor cell (CTC) and CTC clusters from the central venous catheter (CVC) and portal blood (PV). Methods. In total, 7 mL of PV and CVC blood from 35 patients with early pancreatic cancer were analyzed. CTC were isolated using a positive immunomagnetic selection. The detection and identification of CTC were performed by immunocytochemistry (ICC) and were analyzed by Epi-fluorescence and confocal microscopy. Results. CTC and the clusters were detected both in PV and CVC. In both samples, the CTC number per cluster was higher in patients with grade three or poorly differentiated tumors (G3) than in patients with well (G1) or moderately (G2) differentiated. Patients with fewer than 185 CTC in PV exhibited a longer OS than patients with more than 185 CTC (24.5 vs. 10.0 months; p = 0.018). Similarly, patients with fewer than 15 clusters in PV showed a longer OS than patients with more than 15 clusters (19 vs. 10 months; p = 0.004). These significant correlations were not observed in CVC analyses. Conclusions. CTC presence in PV could be an important prognostic factor to predict poor prognosis in early pancreatic cancer. In addition, the number of clustered-CTC correlate to a tumor negative differentiation degree and, therefore, could be used as a diagnostic biomarker for pancreatic cancer. Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

► Show Figures

Figure 1

22 pages, 8431 KiB

Open AccessArticle

Image-Based Identification and Genomic Analysis of Single Circulating Tumor Cells in High Grade Serous Ovarian Cancer Patients

by Carolin Salmon, Janina Levermann, Rui P. L. Neves, Sven-Thorsten Liffers, Jan Dominik Kuhlmann, Paul Buderath, Rainer Kimmig and Sabine Kasimir-Bauer

Cancers 2021, 13(15), 3748; https://doi.org/10.3390/cancers13153748 - 26 Jul 2021

Cited by 4 | Viewed by 2235

Abstract

In Ovarian Cancer (OC), the analysis of single circulating tumor cells (sCTCs) might help to investigate genetic tumor evolution during the course of treatment. Since common CTC identification features failed to reliably detect CTCs in OC, we here present a workflow for their [...] Read more.

In Ovarian Cancer (OC), the analysis of single circulating tumor cells (sCTCs) might help to investigate genetic tumor evolution during the course of treatment. Since common CTC identification features failed to reliably detect CTCs in OC, we here present a workflow for their detection and genomic analysis. Blood of 13 high-grade serous primary OC patients was analyzed, using negative immunomagnetic enrichment, followed by immunofluorescence staining and imaging for Hoechst, ERCC1, CD45, CD11b and cytokeratin (CK) and sCTC sorting with the DEPArray^TM NxT. The whole genome of single cells was amplified and profiled for copy number variation (CNV). We detected: Type A-cells, epithelial (Hoechst^pos, ERCC1^pos, CD45^neg, CD11b^pos, CK^pos); Type B-cells, potentially epithelial (Hoechst^pos, ERCC1^pos, CD45^neg, CD11b^pos, CK^neg) and Type C-cells, potentially mesenchymal (Hoechst^pos, ERCC1^pos, CD45^neg, CD11b^neg, CK^neg). In total, we identified five (38.5%) patients harboring sCTCs with an altered CN profile, which were mainly Type A-cells (80%). In addition to inter-and intra-patient genomic heterogeneity, high numbers of Type B- and C-cells were identified in every patient with their aberrant character only confirmed in 6.25% and 4.76% of cases. Further identification markers and studies in the course of treatment are under way to expand sCTC analysis for the identification of tumor evolution in OC. Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

► Show Figures

Figure 1

19 pages, 2691 KiB

Open AccessArticle

Short-Term Ex Vivo Culture of CTCs from Advance Breast Cancer Patients: Clinical Implications

by Nuria Carmona-Ule, Miriam González-Conde, Carmen Abuín, Juan F. Cueva, Patricia Palacios, Rafael López-López, Clotilde Costa and Ana Belén Dávila-Ibáñez

Cancers 2021, 13(11), 2668; https://doi.org/10.3390/cancers13112668 - 28 May 2021

Cited by 20 | Viewed by 4359

Abstract

Background: Circulating tumor cells (CTC) have relevance as prognostic markers in breast cancer. However, the functional properties of CTCs or their molecular characterization have not been well-studied. Experimental models indicate that only a few cells can survive in the circulation and eventually metastasize. [...] Read more.

Background: Circulating tumor cells (CTC) have relevance as prognostic markers in breast cancer. However, the functional properties of CTCs or their molecular characterization have not been well-studied. Experimental models indicate that only a few cells can survive in the circulation and eventually metastasize. Thus, it is essential to identify these surviving cells capable of forming such metastases. Methods: We isolated viable CTCs from 50 peripheral blood samples obtained from 35 patients with advanced metastatic breast cancer using RosetteSep^TM for ex vivo culture. The CTCs were seeded and monitored on plates under low adherence conditions and with media supplemented with growth factors and Nanoemulsions. Phenotypic analysis was performed by immunofluorescence and gene expression analysis using RT-PCR and CTCs counting by the Cellsearch^® system. Results: We found that in 75% of samples the CTC cultures lasted more than 23 days, predicting a shorter Progression-Free Survival in these patients, independently of having ≥5 CTC by Cellsearch^®. We also observed that CTCs before and after culture showed a different gene expression profile. Conclusions: the cultivability of CTCs is a predictive factor. Furthermore, the subset of cells capable of growing ex vivo show stem or mesenchymal features and may represent the CTC population with metastatic potential in vivo. Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

► Show Figures

Graphical abstract

17 pages, 4146 KiB

Open AccessArticle

ESR1 NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of ESR1 Mutations in Liquid Biopsies

by Dimitra Stergiopoulou, Athina Markou, Eleni Tzanikou, Ioannis Ladas, G. Mike Makrigiorgos, Vassilis Georgoulias and Evi Lianidou

Cancers 2021, 13(3), 556; https://doi.org/10.3390/cancers13030556 - 01 Feb 2021

Cited by 9 | Viewed by 2973

Abstract

A considerable number of estrogen receptor-positive breast cancer (ER⁺ BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) [...] Read more.

A considerable number of estrogen receptor-positive breast cancer (ER⁺ BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER⁺ BrCa. The analytical specificity, analytical sensitivity and reproducibility of the assay were validated using synthetic oligos standards. We further applied the developed ESR1 NAPA assay in 13 ER⁺ BrCa primary tumour tissues, 13 non-cancerous breast tissues (mammoplasties) and 64 liquid biopsy samples: 32 EpCAM-positive cell fractions and 32 paired plasma ctDNA samples obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5-year follow-up period. Peripheral blood from 11 healthy donors (HD) was used as a control. The developed assay is highly sensitive (a detection of mutation-allelic-frequency (MAF) of 0.5% for D538G and 0.1% for Y537S, Y537C, Y537N), and highly specific (0/13 mammoplasties and 0/11 HD for all mutations). In the plasma ctDNA, ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected in five sequential ctDNA samples during the follow-up period in the same patient. In the EpCAM-isolated cell fractions, only the Y537C mutation was detected in one patient sample at the baseline. A direct comparison of the ESR1 NAPA assay with the drop-off ddPCR using 32 identical plasma ctDNA samples gave a concordance of 90.6%. We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The clinical performance of the ESR1 NAPA assay will be prospectively evaluated in a large number of well-characterized patient cohorts. Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

► Show Figures

Figure 1

Review

Jump to: Editorial, Research, Other

16 pages, 1259 KiB

Open AccessReview

Technical Challenges for CTC Implementation in Breast Cancer

by Rocío Ramos-Medina, Sara López-Tarruella, María del Monte-Millán, Tatiana Massarrah and Miguel Martín

Cancers 2021, 13(18), 4619; https://doi.org/10.3390/cancers13184619 - 15 Sep 2021

Cited by 14 | Viewed by 2476

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

► Show Figures

Figure 1

Other

Jump to: Editorial, Research, Review

11 pages, 3076 KiB

Open AccessPerspective

Biological Characterization and Clinical Relevance of Circulating Tumor Cells: Opening the Pandora’s Box of Multiple Myeloma

by Juan-José Garcés, Jesús San-Miguel and Bruno Paiva

Cancers 2022, 14(6), 1430; https://doi.org/10.3390/cancers14061430 - 10 Mar 2022

Cited by 10 | Viewed by 2502

Abstract

Bone marrow (BM) aspirates are the gold standard for patient prognostication and genetic characterization in multiple myeloma (MM). However, they represent an important limitation for periodic disease monitoring because they entail an aggressive procedure. Moreover, recent findings show that a single BM aspirate [...] Read more.

Bone marrow (BM) aspirates are the gold standard for patient prognostication and genetic characterization in multiple myeloma (MM). However, they represent an important limitation for periodic disease monitoring because they entail an aggressive procedure. Moreover, recent findings show that a single BM aspirate is unable to reflect the complex MM heterogeneity. Recent advances in flow cytometry, microfluidics, and “omics” technologies have opened Pandora’s box of MM: The detection and isolation of circulating tumor cells (CTCs) offer a promising and minimally invasive alternative for tumor assessment and metastasis study. CTCs are detectable in premalignant and active MM states, and their enumeration has strong prognostic value, to the extent that it is challenging current stratification systems. In addition, CTCs reflect with high precision both intra- and extra-medullary disease at the phenotypic, genomic, and transcriptomic levels. Despite this high resemblance between tumor clones in distinct locations, some subtle (not random) differences might shed some light on the metastatic process. Thus, it has been suggested that a hypoxic and pro-inflammatory microenvironment could induce an arrest in proliferation forcing tumor cells to recirculate. Herein, we summarize data on the characterization of MM CTCs as well as their clinical and research potential. Full article

(This article belongs to the Special Issue Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Strategies for Isolation and Phenotypic, Genetic, and Functional Characterization of Circulating Tumor Cells

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Published Papers (9 papers)

Editorial

Research

Review

Other

Further Information

Guidelines

MDPI Initiatives

Follow MDPI