Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products

AbstractINTRODUCTIONCirculating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients.METHODSDLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture.RESULTSViable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture.CONCLUSIONSThe increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

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Circulating Tumor Cells in HER-2 Positive Metastatic Breast Cancer Patients Treated with Trastuzumab and Chemotherapy

The International Journal of Biological Markers ◽

10.1177/172460080902400101 ◽

2009 ◽

Vol 24 (1) ◽

pp. 1-10 ◽

Cited By ~ 4

Author(s):

Raquel A. Nunes ◽

Xiaochun Li ◽

Soonmo Peter Kang ◽

Harold Burstein ◽

Lisa Roberts ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Treatment Response ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Metastatic Breast ◽

Breast Cancer Patients ◽

Her 2

The detection of circulating tumor cells (CTCs) in peripheral blood may have important prognostic and predictive implications in breast cancer treatment. A limitation in this field has been the lack of a validated method of accurately measuring CTCs. While sensitivity has improved using RT-PCR, specificity remains a major challenge. The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2-positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Serial CTCs were measured at planned time points and clinical correlations were made. Immunomagnetic selection of circulating epithelial cells was used to address the specificity of tumor cell detection using cytokeratin 19 (CK19). In addition, the extracellular domain of the HER-2 protein (HER-2/ECD) was measured to determine if CTCs detected by CK19 accurately reflect tumor burden. The presence of CTCs at first restaging was associated with disease progression. We observed an association between CK19 and HER-2/ECD. The association of HER-2/ECD with clinical response followed a similar pattern to that seen with CK19. Finally, the absence of HER-2/ECD at best overall response and a change of HER-2/ECD from positive at baseline to negative at best overall response was associated with favorable treatment response. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients. The association between CK19 and markers of disease burden is in line with the concept that CTCs may be a reliable measure of tumor cells in the peripheral blood of patients with metastatic breast cancer. The association of CTCs at first restaging with treatment failure indicates that CTCs may have a role as surrogate markers to monitor treatment response.

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

Cancers ◽

10.3390/cancers12102784 ◽

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.

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Detection of circulating tumor cells in peripheral blood of heavily treated metastatic breast cancer patients

Breast Cancer ◽

10.1007/s12282-011-0259-4 ◽

2011 ◽

Vol 18 (3) ◽

pp. 195-202 ◽

Cited By ~ 7

Author(s):

Nahomi Tokudome ◽

Yoshinori Ito ◽

Shunji Takahashi ◽

Kokoro Kobayashi ◽

Shinichiro Taira ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Metastatic Breast ◽

Breast Cancer Patients

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Abstract 798: Label-free enrichment and detection of circulating tumor cells in metastatic breast cancer patients show 82% of cohorts have detectable targets

10.1158/1538-7445.am2017-798 ◽

2017 ◽

Author(s):

Yu-Jen Chang ◽

Chen-Lin Chen ◽

Wei-Fan Hsu ◽

Meng-Ze Li ◽

Wei-Yuan Ma ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Metastatic Breast ◽

Label Free ◽

Breast Cancer Patients ◽

Free Enrichment

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Liquid biopsy using the nanotube-CTC-chip: capture of invasive CTCs with high purity using preferential adherence in breast cancer patients

Lab on a Chip ◽

10.1039/c9lc00274j ◽

2019 ◽

Vol 19 (11) ◽

pp. 1899-1915 ◽

Cited By ~ 17

Author(s):

Masoud S. Loeian ◽

Sadegh Mehdi Aghaei ◽

Farzaneh Farhadi ◽

Veeresh Rai ◽

Hong Wei Yang ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

High Purity ◽

Peripheral Blood ◽

Liquid Biopsy ◽

Breast Cancer Patients ◽

Multiple Phenotypes

We report the development of the nanotube-CTC-chip for isolation of circulating tumor cells of multiple phenotypes from peripheral blood.

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Use of circulating tumor cells (CTC) in peripheral blood of breast cancer patients before and after adjuvant chemotherapy to predict risk for relapse: The SUCCESS trial.

Journal of Clinical Oncology ◽

10.1200/jco.2010.28.15_suppl.1003 ◽

2010 ◽

Vol 28 (15_suppl) ◽

pp. 1003-1003 ◽

Cited By ~ 44

Author(s):

B. K. Rack ◽

C. Schindlbeck ◽

U. Andergassen ◽

A. Schneeweiss ◽

T. Zwingers ◽

...

Keyword(s):

Breast Cancer ◽

Adjuvant Chemotherapy ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Breast Cancer Patients ◽

Before And After

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Morphologic variation of circulating tumor cells in metastatic breast cancer patients visualized by immunofluorescent stain pattern.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e21079 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e21079-e21079

Author(s):

Daniel Adams ◽

Stuart S Martin ◽

Monica Charpentier ◽

Olga V Makarova ◽

Peixuan Zhu ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Metastatic Breast ◽

Immunofluorescent Staining ◽

Initial Assessment ◽

Breast Cancer Patients

e21079 Background: Isolation of circulating tumor cells (CTCs) using microfiltration is a growing utility in the field of CTC detection. The microfiltration approach can be used on peripheral blood as a non-invasive liquid biopsy for cancer detection and subtyping. We present a utilization of the CellSieve microfilter to subtype CTCs based on immunofluorescent staining pattern of cytokeratin filamentation and EpCAM surface marker expression. Our initial study on CTCs in patient blood indicates that disseminated CTC populations have high rates of phenotypic heterogeneity. Further detailed molecular analysis and patient tracking of these phenotypes may lead to individualized patient assessment based on CTC characterization. Methods: 7.5 mL of whole blood collected from metastatic breast cancer patients were diluted in a fixative solution. An 8 µm CellSieve precision microfilter was placed into a filter holder and the samples were passed through the filter (~ 90 seconds). The cells captured on the filter were fixed, permeabilized, and stained with DAPI, cytokeratin (FITC), EpCAM (PE), and CD45 (Cy5). Cells without CD45 staining were classified by their morphology, nuclear integrity and the presence of cytokeratin and EpCAM staining. Results: In our initial assessment, patient samples were found to have a number of phenotypic CTC subtypes. Cytokeratin filamentation was clearly seen on a number of CTCs while other cells presented with spotted patterns, implying CTCs in various stages of apoptosis. Later stage apoptosis, with segmented nuclear signature, was also seen in various samples. Cell clusters and cells in division were DAPI positive, while EpCAM positivity was negligible, 0-3 cells/sample, correlating with established data from the CellSearch CTC assay. Conclusions: In addition to enumeration, phenotypic variation of CTCs may be a valuable tool for the personalized care of cancer patients. We have shown that individual breast cancer patients have overlapping phenotypes of CTCs circulating in their peripheral blood. We have begun categorizing patients based on these phenotypes and plan to correlate them with overall prognosis.

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