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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

Selection of detected CTC subtypes using multi-immunofluorescence staining.As described above we detected a variety of CTC subtypes. N-cadherin and CD133 were stained on separate slides, respectively, together with CK, CD45 and DAPI. Pseudo colours were used to depict CTC subtypes: N-cadherin (mesenchymal,yellow); CD133 (stem-cell-like, yellow), pan-cytokeratin (CK; epithelial, red), CD45 (hematopoietic, green) and DAPI (nucleus, blue). We were able to detect cells with epithelial and mesenchymal properties like N-cad+/CK+/CD45low, as well as cells with mesenchymal features such as N-cad+/CK−/CD45low. We also observed cells showing mixed characteristics like CD133+/CK+/CD45+ and cells with epithelial characteristics such as CD133−/CK+/CD45− cells. More CTC subtypes were detected, but are not shown in this figure.
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pone-0113706-g002: Selection of detected CTC subtypes using multi-immunofluorescence staining.As described above we detected a variety of CTC subtypes. N-cadherin and CD133 were stained on separate slides, respectively, together with CK, CD45 and DAPI. Pseudo colours were used to depict CTC subtypes: N-cadherin (mesenchymal,yellow); CD133 (stem-cell-like, yellow), pan-cytokeratin (CK; epithelial, red), CD45 (hematopoietic, green) and DAPI (nucleus, blue). We were able to detect cells with epithelial and mesenchymal properties like N-cad+/CK+/CD45low, as well as cells with mesenchymal features such as N-cad+/CK−/CD45low. We also observed cells showing mixed characteristics like CD133+/CK+/CD45+ and cells with epithelial characteristics such as CD133−/CK+/CD45− cells. More CTC subtypes were detected, but are not shown in this figure.

Mentions: For the investigation of cellular subtypes a multi-staining method was required in order to detect various epithelial, mesenchymal, stem cell-like and hematopoietic characteristics. Therefore, we used multi-immunofluorescence staining for CTC-subtype detection. In HNSCC blood samples we observed cells with mesenchymal features such as N-cadherin+/CK−/CD45− and cells with epithelial properties like CK+/N-cadherin−/CD45− and CK+/CD133−/CD45− and cells with both characteristics like CK+/N-cadherin+/CD45−. We also detected cells that stained positive for CD133 which is believed to be a cancer stem cell marker and found subtypes such as CD133+/CK−/CD45− and CD133+/CK+/CD45− cells. In addition, we found cells that stained positive for potential markers of CTC and CD45 such as N-cadherin−/CK+/CD45low, N-cadherin+/CK−/CD45low N-cadherin+/CK+/CD45low as well as CD133+/CK+/CD45+, CD133−/CK+/CD45low and CD133+/CK−/CD45lowcells (Fig. 2). CTC were enumerated and CTC profiles of each patient were examined. We scored the total amount of N-cadherin-positive, CK-positive and CD133-positive cells, and calculated a ratio of mesenchymal to epithelial cells and stem cell-like to epithelial cells, respectively, after density gradient centrifugation. We normalized the enumerated potential CTC against the total PBMNC number detected in the DAPI channel in each visual field and calculated the number of CTC per 1000 PBMNC. Analysis of samples from healthy donors revealed the following cut-off values for false positive events per 1000 PBMNC: 0.07 CD133+/CK+ cells; 0.07 CD133+/CK− cells; 0.01 N-cadherin+/CK+ cells; 0.03 N-cadherin+/CK− cells and a total of 0.6 CK+ cells.


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)

Selection of detected CTC subtypes using multi-immunofluorescence staining.As described above we detected a variety of CTC subtypes. N-cadherin and CD133 were stained on separate slides, respectively, together with CK, CD45 and DAPI. Pseudo colours were used to depict CTC subtypes: N-cadherin (mesenchymal,yellow); CD133 (stem-cell-like, yellow), pan-cytokeratin (CK; epithelial, red), CD45 (hematopoietic, green) and DAPI (nucleus, blue). We were able to detect cells with epithelial and mesenchymal properties like N-cad+/CK+/CD45low, as well as cells with mesenchymal features such as N-cad+/CK−/CD45low. We also observed cells showing mixed characteristics like CD133+/CK+/CD45+ and cells with epithelial characteristics such as CD133−/CK+/CD45− cells. More CTC subtypes were detected, but are not shown in this figure.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113706-g002: Selection of detected CTC subtypes using multi-immunofluorescence staining.As described above we detected a variety of CTC subtypes. N-cadherin and CD133 were stained on separate slides, respectively, together with CK, CD45 and DAPI. Pseudo colours were used to depict CTC subtypes: N-cadherin (mesenchymal,yellow); CD133 (stem-cell-like, yellow), pan-cytokeratin (CK; epithelial, red), CD45 (hematopoietic, green) and DAPI (nucleus, blue). We were able to detect cells with epithelial and mesenchymal properties like N-cad+/CK+/CD45low, as well as cells with mesenchymal features such as N-cad+/CK−/CD45low. We also observed cells showing mixed characteristics like CD133+/CK+/CD45+ and cells with epithelial characteristics such as CD133−/CK+/CD45− cells. More CTC subtypes were detected, but are not shown in this figure.
Mentions: For the investigation of cellular subtypes a multi-staining method was required in order to detect various epithelial, mesenchymal, stem cell-like and hematopoietic characteristics. Therefore, we used multi-immunofluorescence staining for CTC-subtype detection. In HNSCC blood samples we observed cells with mesenchymal features such as N-cadherin+/CK−/CD45− and cells with epithelial properties like CK+/N-cadherin−/CD45− and CK+/CD133−/CD45− and cells with both characteristics like CK+/N-cadherin+/CD45−. We also detected cells that stained positive for CD133 which is believed to be a cancer stem cell marker and found subtypes such as CD133+/CK−/CD45− and CD133+/CK+/CD45− cells. In addition, we found cells that stained positive for potential markers of CTC and CD45 such as N-cadherin−/CK+/CD45low, N-cadherin+/CK−/CD45low N-cadherin+/CK+/CD45low as well as CD133+/CK+/CD45+, CD133−/CK+/CD45low and CD133+/CK−/CD45lowcells (Fig. 2). CTC were enumerated and CTC profiles of each patient were examined. We scored the total amount of N-cadherin-positive, CK-positive and CD133-positive cells, and calculated a ratio of mesenchymal to epithelial cells and stem cell-like to epithelial cells, respectively, after density gradient centrifugation. We normalized the enumerated potential CTC against the total PBMNC number detected in the DAPI channel in each visual field and calculated the number of CTC per 1000 PBMNC. Analysis of samples from healthy donors revealed the following cut-off values for false positive events per 1000 PBMNC: 0.07 CD133+/CK+ cells; 0.07 CD133+/CK− cells; 0.01 N-cadherin+/CK+ cells; 0.03 N-cadherin+/CK− cells and a total of 0.6 CK+ cells.

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