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Detection of circulating tumor cell subpopulations in patients with head and neck squamous cell carcinoma (HNSCC).

Weller P, Nel I, Hassenkamp P, Gauler T, Schlueter A, Lang S, Dountsop P, Hoffmann AC, Lehnerdt G - PLoS ONE (2014)

Bottom Line: Individual cell type profiles were analyzed.We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-.We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

ABSTRACT

Background: Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples.

Methods: 20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed.

Results: We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-. We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1).

Conclusions: This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.

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Related in: MedlinePlus

Basic principle of CTC isolation.1. Leucosep tube: 2. Separation media; 3. Whole blood/PBMNC mixture; 4. Plasma; 5. Separation media after centrifugation; 6. Erythocytes; 7. Buffy coat incl. CTCs; Cell suspension containing CTCs for Cellspin and multi-immunofluorescence staining. Before and after curative resection, mononuclear cells and CTC were isolated from peripheral venous blood using density gradient centrifugation. Cell suspensions were carefully spun onto glass slides. The presence of CTC was verified by immunofluorescence staining against pan-CK (epithelial), N-cadherin (mesenchymal), CD133 (stemcell-like); counterstaining with CD45 (hematopoietic) and DAPI (nucleus). CTC and hematopoietic cells were enumerated, normalized and individual cell type profiles were analyzed and correlated to therapeutic outcome in 10 patients.
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pone-0113706-g001: Basic principle of CTC isolation.1. Leucosep tube: 2. Separation media; 3. Whole blood/PBMNC mixture; 4. Plasma; 5. Separation media after centrifugation; 6. Erythocytes; 7. Buffy coat incl. CTCs; Cell suspension containing CTCs for Cellspin and multi-immunofluorescence staining. Before and after curative resection, mononuclear cells and CTC were isolated from peripheral venous blood using density gradient centrifugation. Cell suspensions were carefully spun onto glass slides. The presence of CTC was verified by immunofluorescence staining against pan-CK (epithelial), N-cadherin (mesenchymal), CD133 (stemcell-like); counterstaining with CD45 (hematopoietic) and DAPI (nucleus). CTC and hematopoietic cells were enumerated, normalized and individual cell type profiles were analyzed and correlated to therapeutic outcome in 10 patients.

Mentions: 20 ml citrated peripheral venous blood were drawn from HNSCC patients directly before and immediately after curative tumor resection and processed within 24 h after collection. Blood sample preparation was done as described previously [17], [19]. Briefly, 20 ml of blood were diluted with 10 ml PBS and carefully layered into a Leucosep tube containing 16 ml Ficoll-Paque (GE-Healthcare) below a porous barrier. After buoyant density gradient centrifugation (1600×g, 20°C, 20 min) the interphase consisting of peripheral blood mononuclear cells (PBMNC) and CTC was removed and washed. The cell suspension was spun onto 2–4 glass slides per sample containing a maximum of 6×106 cells per slide using the Cell Spin II centrifuge (Tharmac, Waldsolms, Germany), air-dried and subsequently fixated with 96% Ethanol. Slides were stored at 4°C until subjected to immunocytochemical staining (Fig. 1). The presence of CTC was verified by multi-immunofluorescence staining against pan-CK (epithelial), N-cadherin (mesenchymal), CD133 (stem-cell-like), counterstaining with CD45 (hematopoietic) and DAPI (nucleus). CTC and hematopoietic cells were enumerated, normalized and expressed as number of CTC per 1000 PBMNC. Individual cell type profiles were analyzed and correlated to therapeutic outcome in 10 patients.


Detection of circulating tumor cell subpopulations in patients with head and neck squamous cell carcinoma (HNSCC).

Weller P, Nel I, Hassenkamp P, Gauler T, Schlueter A, Lang S, Dountsop P, Hoffmann AC, Lehnerdt G - PLoS ONE (2014)

Basic principle of CTC isolation.1. Leucosep tube: 2. Separation media; 3. Whole blood/PBMNC mixture; 4. Plasma; 5. Separation media after centrifugation; 6. Erythocytes; 7. Buffy coat incl. CTCs; Cell suspension containing CTCs for Cellspin and multi-immunofluorescence staining. Before and after curative resection, mononuclear cells and CTC were isolated from peripheral venous blood using density gradient centrifugation. Cell suspensions were carefully spun onto glass slides. The presence of CTC was verified by immunofluorescence staining against pan-CK (epithelial), N-cadherin (mesenchymal), CD133 (stemcell-like); counterstaining with CD45 (hematopoietic) and DAPI (nucleus). CTC and hematopoietic cells were enumerated, normalized and individual cell type profiles were analyzed and correlated to therapeutic outcome in 10 patients.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257624&req=5

pone-0113706-g001: Basic principle of CTC isolation.1. Leucosep tube: 2. Separation media; 3. Whole blood/PBMNC mixture; 4. Plasma; 5. Separation media after centrifugation; 6. Erythocytes; 7. Buffy coat incl. CTCs; Cell suspension containing CTCs for Cellspin and multi-immunofluorescence staining. Before and after curative resection, mononuclear cells and CTC were isolated from peripheral venous blood using density gradient centrifugation. Cell suspensions were carefully spun onto glass slides. The presence of CTC was verified by immunofluorescence staining against pan-CK (epithelial), N-cadherin (mesenchymal), CD133 (stemcell-like); counterstaining with CD45 (hematopoietic) and DAPI (nucleus). CTC and hematopoietic cells were enumerated, normalized and individual cell type profiles were analyzed and correlated to therapeutic outcome in 10 patients.
Mentions: 20 ml citrated peripheral venous blood were drawn from HNSCC patients directly before and immediately after curative tumor resection and processed within 24 h after collection. Blood sample preparation was done as described previously [17], [19]. Briefly, 20 ml of blood were diluted with 10 ml PBS and carefully layered into a Leucosep tube containing 16 ml Ficoll-Paque (GE-Healthcare) below a porous barrier. After buoyant density gradient centrifugation (1600×g, 20°C, 20 min) the interphase consisting of peripheral blood mononuclear cells (PBMNC) and CTC was removed and washed. The cell suspension was spun onto 2–4 glass slides per sample containing a maximum of 6×106 cells per slide using the Cell Spin II centrifuge (Tharmac, Waldsolms, Germany), air-dried and subsequently fixated with 96% Ethanol. Slides were stored at 4°C until subjected to immunocytochemical staining (Fig. 1). The presence of CTC was verified by multi-immunofluorescence staining against pan-CK (epithelial), N-cadherin (mesenchymal), CD133 (stem-cell-like), counterstaining with CD45 (hematopoietic) and DAPI (nucleus). CTC and hematopoietic cells were enumerated, normalized and expressed as number of CTC per 1000 PBMNC. Individual cell type profiles were analyzed and correlated to therapeutic outcome in 10 patients.

Bottom Line: Individual cell type profiles were analyzed.We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-.We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

ABSTRACT

Background: Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples.

Methods: 20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed.

Results: We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-. We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1).

Conclusions: This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.

Show MeSH
Related in: MedlinePlus