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Special Issue "Circulating Tumor Cells: Pathological, Molecular and Functional Characteristics"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (29 February 2020).

Special Issue Editor

Dr. Ewa A. Grzybowska
E-Mail
Guest Editor
Cancer Center Institute, Roentgena 5, 02-781 Warsaw, Poland
Interests: circulating tumor cells; breast cancer; tumor heterogeneity; invasion
Special Issues and Collections in MDPI journals

Special Issue Information

Dear Colleagues,

Nowadays, therapeutic decisions are based overwhelmingly on the pathological and molecular characterization of the primary tumor. However, metastatic lesions can be highly heterogenous and substantially different from the primary neoplasm, which hampers the efficacy of the treatment. Moreover, the inaccessibility of metastatic foci and invasiveness of a biopsy represent challenging problems and call for some other means of secondary tumor characterization. Analysis of the circulating tumor cells (CTCs) as a surrogate for the metastatic lesion is non-invasive and allows for detailed characterization of the phenotype, genotype, heterogeneity, expression profiling and functional analysis of tumor cells. These data should have a direct impact on the diagnosis and decision-making related to adjuvant treatment by helping with patient stratification and should also provide valuable information about the biology of metastasis. This open-access Special Issue will bring together original research and review articles on CTCs, their biology, methods of enumeration and characterization, their role in metastasis and their predictive value for diagnostics and therapeutic applications. It will provide a platform to share new discoveries, approaches and technical developments in the field of CTC research and the development of the next generation of anti-metastasis treatments.

Topics of this Special Issue include, but are not limited to

  • Diagnostic and therapeutic applications of CTC research;
  • The biology of CTCs in different cancer types;
  • CTC mobilization by treatment;
  • Phenotypic/genotypic heterogeneity of CTCs;
  • Technical developments in the isolation, enumeration and characterization of CTCs;
  • Functional analysis of CTCs, including culture and CDX;
  • CTC analysis in single cell resolution;
  • Cell free DNA vs. CTC;
  • Epithelial/hybrid/mesenchymal properties of CTCs;
  • CTC clusters: biology, enumeration, role in metastasis.

Dr. Ewa A. Grzybowska
Guest Editor

Manuscript Submission Information

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Keywords

  • CTC
  • metastasis
  • tumor heterogeneity
  • invasion
  • single cell analysis
  • cancer dissemination
  • circulation
  • treatment response
  • CTC clusters

Published Papers (10 papers)

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Research

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Article
A Pilot Study for the Feasibility of Exome-Sequencing in Circulating Tumor Cells Versus Single Metastatic Biopsies in Breast Cancer
Int. J. Mol. Sci. 2020, 21(14), 4826; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21144826 - 08 Jul 2020
Viewed by 1351
Abstract
The comparison of the landscape of somatic alterations in circulating tumor cells (CTCs) versus metastases is challenging. Here, we comprehensively characterized the somatic landscape in bulk (amplified and non-amplified), spike-in breast cancer cells, CTCs, and metastases from breast cancer patients using whole-exome sequencing [...] Read more.
The comparison of the landscape of somatic alterations in circulating tumor cells (CTCs) versus metastases is challenging. Here, we comprehensively characterized the somatic landscape in bulk (amplified and non-amplified), spike-in breast cancer cells, CTCs, and metastases from breast cancer patients using whole-exome sequencing (WES). We determined the level of genomic concordance for somatic nucleotide variants (SNVs), copy number alterations (CNAs), and structural variants (SVs). The variant allele fractions (VAFs) of somatic variants were remarkably similar between amplified and non-amplified cell line samples as technical replicates. In clinical samples, a significant fraction of somatic variants had low VAFs in CTCs compared to metastases. The most frequently recurrent gene mutations in clinical samples were associated with an elevated C > T mutational signature. We found complex rearrangement patterns including intra- and inter-chromosomal rearrangements, singleton, and recurrent gene fusions, and tandem duplications. We observed high molecular discordance for somatic alterations between paired samples consistent with marked heterogeneity of the somatic landscape. The most prevalent copy number calls were focal deletion events in CTCs and metastases. Our results demonstrate the feasibility of an integrated workflow for the identification of a complete repertoire of somatic alterations and highlight the intrapatient genomic differences that occur between CTCs and metastases. Full article
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Article
Evolution of Estrogen Receptor Status from Primary Tumors to Metastasis and Serially Collected Circulating Tumor Cells
Int. J. Mol. Sci. 2020, 21(8), 2885; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21082885 - 20 Apr 2020
Cited by 1 | Viewed by 785
Abstract
Background: The estrogen receptor (ER) can change expression between primary tumor (PT) and distant metastasis (DM) in breast cancer. A tissue biopsy reflects a momentary state at one location, whereas circulating tumor cells (CTCs) reflect real-time tumor progression. We evaluated ER-status during tumor [...] Read more.
Background: The estrogen receptor (ER) can change expression between primary tumor (PT) and distant metastasis (DM) in breast cancer. A tissue biopsy reflects a momentary state at one location, whereas circulating tumor cells (CTCs) reflect real-time tumor progression. We evaluated ER-status during tumor progression from PT to DM and CTCs, and related the ER-status of CTCs to prognosis. Methods: In a study of metastatic breast cancer, blood was collected at different timepoints. After CellSearch® enrichment, CTCs were captured on DropMount slides and evaluated for ER expression at baseline (BL) and after 1 and 3 months of therapy. Comparison of the ER-status of PT, DM, and CTCs at different timepoints was performed using the McNemar test. The primary endpoint was progression-free survival (PFS). Results: Evidence of a shift from ER positivity to negativity between PT and DM was demonstrated (p = 0.019). We found strong evidence of similar shifts from PT to CTCs at different timepoints (p < 0.0001). ER-positive CTCs at 1 and 3 months were related to better prognosis. Conclusions: A shift in ER-status from PT to DM/CTCs was demonstrated. ER-positive CTCs during systemic therapy might reflect the retention of a favorable phenotype that still responds to therapy. Full article
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Article
Heterogeneity of Stemlike Circulating Tumor Cells in Invasive Breast Cancer
Int. J. Mol. Sci. 2020, 21(8), 2780; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21082780 - 16 Apr 2020
Cited by 6 | Viewed by 937
Abstract
The presence of stem and epithelial–mesenchymal-transition (EMT) features in circulating tumor cells (CTCs) determines their invasiveness, adaptability to the microenvironment, and resistance to proapoptotic signals and chemotherapy. It also allows them to fulfil the role of metastatic “seeds”. We evaluated the heterogeneity of [...] Read more.
The presence of stem and epithelial–mesenchymal-transition (EMT) features in circulating tumor cells (CTCs) determines their invasiveness, adaptability to the microenvironment, and resistance to proapoptotic signals and chemotherapy. It also allows them to fulfil the role of metastatic “seeds”. We evaluated the heterogeneity of stem CTCs by their CD44, ALDH1, and CD133 expression depending on N-cadherin expression in breast-cancer patients. A total of 38 female patients were selected for this study. CTC phenotypes were determined by flow cytometry before any type of treatment. Multiplex immunofluorescence was used for the evaluation of tumor-cell heterogeneity in primary lesions. In patients who had CD44-CD24- CTCs, a subset of cells with the expression of other stem-cell markers (CD133 and ALDH1) were detected. Expression of CD133 and/or ALDH1 may be associated with expression of N-cadherin: all populations of N-cadherin+ CTCs demonstrate stem features; in the absence of N-cadherin expression, true nonstem (CD44-CD24-CD133-ALDH1-) cells are found. The heterogeneity of stem marker expression in CTCs was observed regardless of N-cadherin expression. In our study, stromal cell-derived factor-1 (SDF-1) receptor expression in CTCs did not depend on stemlike traits, but was instead associated with N-cadherin expression. Subpopulations of tumor cells, detected both in tumors and blood, were identified. Breast cancer was characterized by pronounced interpersonal and intrapersonal heterogeneity of CTCs by the presence and combination of various stem features and N-cadherin expression. To complete the characterization of stemlike features of CTCs, we suggest the simultaneous use of the three stem markers. Full article
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Article
Optimization of rVAR2-Based Isolation of Cancer Cells in Blood for Building a Robust Assay for Clinical Detection of Circulating Tumor Cells
Int. J. Mol. Sci. 2020, 21(7), 2401; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21072401 - 31 Mar 2020
Cited by 2 | Viewed by 1169
Abstract
Early detection and monitoring of cancer progression is key to successful treatment. Therefore, much research is invested in developing technologies, enabling effective and valuable use of non-invasive liquid biopsies. This includes the detection and analysis of circulating tumor cells (CTCs) from blood samples. [...] Read more.
Early detection and monitoring of cancer progression is key to successful treatment. Therefore, much research is invested in developing technologies, enabling effective and valuable use of non-invasive liquid biopsies. This includes the detection and analysis of circulating tumor cells (CTCs) from blood samples. Recombinant malaria protein VAR2CSA (rVAR2) binds a unique chondroitin sulfate modification present on the vast majority of cancers and thereby holds promise as a near-universal tumor cell-targeting reagent to isolate CTCs from complex blood samples. This study describes a technical approach for optimizing the coupling of rVAR2 to magnetic beads and the development of a CTC isolation platform targeting a range of different cancer cell lines. We investigate both direct and indirect approaches for rVAR2-mediated bead retrieval of cancer cells and conclude that an indirect capture approach is most effective for rVAR2-based cancer cell retrieval. Full article
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Article
The Lack of Evidence for an Association between Cancer Biomarker Conversion Patterns and CTC-Status in Patients with Metastatic Breast Cancer
Int. J. Mol. Sci. 2020, 21(6), 2161; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21062161 - 21 Mar 2020
Cited by 1 | Viewed by 974
Abstract
Circulating tumor cell (CTC) detection is a prognostic factor in the metastatic breast cancer (MBC) setting. Discrepancies in primary (PT) and metastatic tumor (MT) genetic profiles are also of prognostic importance. Our study aimed to compare the CTC statuses and prognoses between those [...] Read more.
Circulating tumor cell (CTC) detection is a prognostic factor in the metastatic breast cancer (MBC) setting. Discrepancies in primary (PT) and metastatic tumor (MT) genetic profiles are also of prognostic importance. Our study aimed to compare the CTC statuses and prognoses between those with subtype stable MBCs and MBCs with specific biomarker conversions. The study enrolled 261 MBC patients, treated at the National Center for Tumor Diseases, Heidelberg, Germany in a five-year period. All underwent PT and MT biopsies and subsequent CTC enumeration before the initiation of systemic therapy. ER and HER2 statuses of the PTs and MTs were determined and progression free survivals (PFSs) and overall survivals (OSs) were recorded. We compared CTC statuses, CTC counts, PFSs and OSs between subgroups of patients with different receptor change patterns. Patients who had tumors that converted to triple negative MTs had the shortest median OSs, while HER2 expression was not associated with a shorter median OS. No significant differences in PFSs and OSs have been demonstrated by Kaplan-Meier curve comparisons in any of the subgroup analyses. CTC counts were similar in all subgroups. CTCs were comparably less frequently detected in patients with a stable HER2 expression. Similar proportions of CTC positives were observed in all other subtype change pattern subgroups, barring the aforementioned HER2 stable subgroup. The detection of CTCs was of no appreciable prognostic value in different receptor change pattern subgroups in our cohort. Full article
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Article
Analysis of Single Circulating Tumor Cells in Renal Cell Carcinoma Reveals Phenotypic Heterogeneity and Genomic Alterations Related to Progression
Int. J. Mol. Sci. 2020, 21(4), 1475; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21041475 - 21 Feb 2020
Cited by 5 | Viewed by 1225
Abstract
Circulating tumor cells (CTCs) are promising biomarkers for prognosis, therapeutic response prediction, and treatment monitoring in cancer patients. Despite its epithelial origin, renal cell carcinoma (RCC) shows low expression of epithelial markers hindering CTC-enrichment approaches exploiting epithelial cell surface proteins. In 21 blood [...] Read more.
Circulating tumor cells (CTCs) are promising biomarkers for prognosis, therapeutic response prediction, and treatment monitoring in cancer patients. Despite its epithelial origin, renal cell carcinoma (RCC) shows low expression of epithelial markers hindering CTC-enrichment approaches exploiting epithelial cell surface proteins. In 21 blood samples serially collected from 10 patients with metastatic RCC entering the TARIBO trial, we overcame this limitation using the marker-independent Parsortix™ approach for CTC-enrichment coupled with positive and negative selection with the DEPArray™ with single cell recovery and analysis for copy number alterations (CNA) by next generation sequencing NGS. Two CTC subpopulations were identified: epithelial CTC (eCTC) and non-conventional CTC (ncCTC) lacking epithelial and leukocyte markers. With a threshold ≥1CTC/10 mL of blood, the positivity rates were 28% for eCTC, 62% for ncCTCs, and 71% considering both CTC types. In two patients with detectable eCTCs at baseline, progression free survival was less than 5 months. In an index case, hierarchical structure by translational oncology (TRONCO) identified three clones among 14 CTCs collected at progression and at baseline, each containing cells with a 9p21.3loss, a well-known metastasis driving subclonal alteration. CTCs detection in RCC can be increased by marker-independent approaches, and CTC molecular characterization can allow detection of subclonal events possibly related to tumor progression. Full article
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Review

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Review
Circulating Tumor Cell Clusters: United We Stand Divided We Fall
Int. J. Mol. Sci. 2020, 21(7), 2653; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21072653 - 10 Apr 2020
Cited by 11 | Viewed by 1428
Abstract
The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their [...] Read more.
The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their being minimally invasive biomarkers has positioned them for diagnosis, prognosis, and recurrence monitoring tools. Initially, CTC clusters were out of focus, but major recent advances in the knowledge of their biogenesis and dissemination reposition them as critical actors in the pathophysiology of cancer, especially metastasis. Increasing evidence suggests that “united” CTCs, organized in clusters, resist better and carry stronger metastatic capacities than “divided” single CTCs. This review gathers recent insight on CTC cluster origin and dissemination. We will focus on their distinct molecular package necessary to resist multiple cell deaths that all circulating cells normally face. We will describe the molecular basis of their increased metastatic potential as compared to single CTCs. We will consider their clinical relevance as prognostic biomarkers. Finally, we will propose future directions for research and clinical applications in this promising topic in cancer. Full article
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Review
Tumor-Cell–Macrophage Fusion Cells as Liquid Biomarkers and Tumor Enhancers in Cancer
Int. J. Mol. Sci. 2020, 21(5), 1872; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21051872 - 09 Mar 2020
Cited by 8 | Viewed by 1656
Abstract
Although molecular mechanisms driving tumor progression have been extensively studied, the biological nature of the various populations of circulating tumor cells (CTCs) within the blood is still not well understood. Tumor cell fusion with immune cells is a longstanding hypothesis that has caught [...] Read more.
Although molecular mechanisms driving tumor progression have been extensively studied, the biological nature of the various populations of circulating tumor cells (CTCs) within the blood is still not well understood. Tumor cell fusion with immune cells is a longstanding hypothesis that has caught more attention in recent times. Specifically, fusion of tumor cells with macrophages might lead to the development of metastasis by acquiring features such as genetic and epigenetic heterogeneity, chemotherapeutic resistance, and immune tolerance. In addition to the traditional FDA-approved definition of a CTC (CD45-, EpCAM+, cytokeratins 8+, 18+ or 19+, with a DAPI+ nucleus), an additional circulating cell population has been identified as being potential fusions cells, characterized by distinct, large, polymorphonuclear cancer-associated cells with a dual epithelial and macrophage/myeloid phenotype. Artificial fusion of tumor cells with macrophages leads to migratory, invasive, and metastatic phenotypes. Further studies might investigate whether these have a potential impact on the immune response towards the cancer. In this review, the background, evidence, and potential relevance of tumor cell fusions with macrophages is discussed, along with the potential role of intercellular connections in their formation. Such fusion cells could be a key component in cancer metastasis, and therefore, evolve as a diagnostic and therapeutic target in cancer precision medicine. Full article
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Review
Heterogeneity of Circulating Tumor Cells in Breast Cancer: Identifying Metastatic Seeds
Int. J. Mol. Sci. 2020, 21(5), 1696; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21051696 - 02 Mar 2020
Cited by 5 | Viewed by 1773
Abstract
Metastasis being the main cause of breast cancer (BC) mortality represents the complex and multistage process. The entrance of tumor cells into the blood vessels and the appearance of circulating tumor cells (CTCs) seeding and colonizing distant tissues and organs are one of [...] Read more.
Metastasis being the main cause of breast cancer (BC) mortality represents the complex and multistage process. The entrance of tumor cells into the blood vessels and the appearance of circulating tumor cells (CTCs) seeding and colonizing distant tissues and organs are one of the key stages in the metastatic cascade. Like the primary tumor, CTCs are extremely heterogeneous and presented by clusters and individual cells which consist of phenotypically and genetically distinct subpopulations. However, among this diversity, only a small number of CTCs is able to survive in the bloodstream and to form metastases. The identification of the metastasis-initiating CTCs is believed to be a critical issue in developing therapeutic strategies against metastatic disease. In this review, we summarize the available literature addressing morphological, phenotypic and genetic heterogeneity of CTCs and the molecular makeup of specific subpopulations associated with BC metastasis. Special attention is paid to the need for in vitro and in vivo studies to confirm the tumorigenic and metastatic potential of metastasis-associating CTCs. Finally, we consider treatment approaches that could be effective to eradicate metastatic CTCs and to prevent metastasis. Full article
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Review
Circulating Tumor Cells in Early and Advanced Breast Cancer; Biology and Prognostic Value
Int. J. Mol. Sci. 2020, 21(5), 1671; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21051671 - 29 Feb 2020
Cited by 8 | Viewed by 1432
Abstract
Breast cancer metastasis is the leading cause of cancer deaths in women and is difficult to combat due to the long periods in which disseminated cells retain a potential to be re-activated and start the relapse. Assessing the number and molecular profile of [...] Read more.
Breast cancer metastasis is the leading cause of cancer deaths in women and is difficult to combat due to the long periods in which disseminated cells retain a potential to be re-activated and start the relapse. Assessing the number and molecular profile of circulating tumor cells (CTCs) in breast cancer patients, especially in early breast cancer, should help in identifying the possibility of relapse in time for therapeutic intervention to prevent or delay recurrence. While metastatic breast cancer is considered incurable, molecular analysis of CTCs still have a potential to define particular susceptibilities of the cells representing the current tumor burden, which may differ considerably from the cells of the primary tumor, and offer more tailored therapy to the patients. In this review we inspect the routes to metastasis and how they can be linked to specific features of CTCs, how CTC analysis may be used in therapy, and what is the current status of the research and efforts to include CTC analysis in clinical practice. Full article
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