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Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-κB signalling.

Rajasekhar VK, Studer L, Gerald W, Socci ND, Scher HI - Nat Commun (2011)

Bottom Line: These TICs possess stem cell characteristics and multipotency as demonstrated by in vitro sphere-formation and in vivo tumour-initiation, respectively.The cells represent an undifferentiated subtype of basal cells and can be purified from prostate tumours based on coexpression of the human pluripotent stem cell marker TRA-1-60 with CD151 and CD166.These TICs exhibit increased nuclear factor-κB activity.

View Article: PubMed Central - PubMed

Affiliation: 1] Stem Cell Center and Developmental Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. [2] Sidney Kimmel Center for Prostate and Urologic Cancers, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
Androgen depletion is a key strategy for treating human prostate cancer, but the presence of hormone-independent cells escaping treatment remains a major therapeutic challenge. Here, we identify a minor subset of stem-like human prostate tumour-initiating cells (TICs) that do not express prostate cancer markers, such as androgen receptor or prostate specific antigen. These TICs possess stem cell characteristics and multipotency as demonstrated by in vitro sphere-formation and in vivo tumour-initiation, respectively. The cells represent an undifferentiated subtype of basal cells and can be purified from prostate tumours based on coexpression of the human pluripotent stem cell marker TRA-1-60 with CD151 and CD166. Such triple-marker-positive TICs recapitulate the original parent tumour heterogeneity in serial xeno-transplantations indicating a tumour cell hierarchy in human prostate cancer development. These TICs exhibit increased nuclear factor-κB activity. These findings are important in understanding the molecular basis of human prostate cancer.

No MeSH data available.


Related in: MedlinePlus

In vivo tumour-initiation by purified human prostate TICs.(a) Limiting dilution experiments for determining percent tumour-initiation by equal numbers of sphere cells, TRA-1-60-positive cells or triple positives transplanted at OT or SC sites. Blue, 10,000 cells; green, 1,000 cells and red, 200 cells. Data represents the 4-week end-point results following transplantation. (b–h) Unless otherwise stated, 5,000 marker-positive or total tumour (unsorted control) cells were used per transplantation. (b) Representative SC tumour growth at 5 weeks following transplantation. Unsorted represents a control tumour-derived from transplantation of 2×106 total tumour cells. (c) Tumour-initiation efficiency (represented as tumour volume) of the marker-positive cells at 5 weeks following transplantation. (d) Upper panel: Dark field images of primary spheres formed by marker-positive cells. Lower panel: hematoxylin and eosin staining of tumours derived from different marker-positive tumour cells. Scale bar, 100 μm. (e) Relative levels of G1 (yellow), G2 (blue) and S (red) phases of cell cycle in the different marker-positive tumour cells. Unsorted represents total tumour cells. (f) Percent triple-marker expression in sequentially passaged OT-tumours that were initially derived from the triple-marker-positive tumour cells. Percentage triple-marker-positive cells in the parent tumour form the control. (g) Maintenance of triple-marker-positive cells in the secondary passaged tumours derived from indicated marker-specific primary tumour cells. Unsorted is as described in (b). Orange-triple-marker-positive cells and purple-triple-marker-negative cells. (h) Immunoblot analysis of whole cell extracts of the tumours derived from marker-positive tumour cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) represents the loading control. Mean±s.d., n≥3.
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f3: In vivo tumour-initiation by purified human prostate TICs.(a) Limiting dilution experiments for determining percent tumour-initiation by equal numbers of sphere cells, TRA-1-60-positive cells or triple positives transplanted at OT or SC sites. Blue, 10,000 cells; green, 1,000 cells and red, 200 cells. Data represents the 4-week end-point results following transplantation. (b–h) Unless otherwise stated, 5,000 marker-positive or total tumour (unsorted control) cells were used per transplantation. (b) Representative SC tumour growth at 5 weeks following transplantation. Unsorted represents a control tumour-derived from transplantation of 2×106 total tumour cells. (c) Tumour-initiation efficiency (represented as tumour volume) of the marker-positive cells at 5 weeks following transplantation. (d) Upper panel: Dark field images of primary spheres formed by marker-positive cells. Lower panel: hematoxylin and eosin staining of tumours derived from different marker-positive tumour cells. Scale bar, 100 μm. (e) Relative levels of G1 (yellow), G2 (blue) and S (red) phases of cell cycle in the different marker-positive tumour cells. Unsorted represents total tumour cells. (f) Percent triple-marker expression in sequentially passaged OT-tumours that were initially derived from the triple-marker-positive tumour cells. Percentage triple-marker-positive cells in the parent tumour form the control. (g) Maintenance of triple-marker-positive cells in the secondary passaged tumours derived from indicated marker-specific primary tumour cells. Unsorted is as described in (b). Orange-triple-marker-positive cells and purple-triple-marker-negative cells. (h) Immunoblot analysis of whole cell extracts of the tumours derived from marker-positive tumour cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) represents the loading control. Mean±s.d., n≥3.

Mentions: By fluorescence-activated cell sorting (FACS), we investigated novel cell surface marker expression that could facilitate prospective isolation of the TICs in the human prostate CWR22 OT-tumours. First, we confirmed the expression of a set of markers known to associate with stem-like tumour cells in other epithelial cancers, including human prostate tumours1727. They included epithelial cellular adhesion molecule (EpCAM), hyaluronic acid receptor (CD44) and integrins (α2-integrin, α6-integrin and β4-integrin; Fig. 2a). Prostate tumour cells expressing these markers displayed increased sphere-formation capacity as compared with unsorted total tumour cells or cells expressing β4-integrin (Fig. 2b). Except for CD44, the percentage of cells expressing these markers (EpCAM, α2-integrin for example) was consistently enriched over sequential passages of sphere cells. The above markers are associated with many different cell types including stromal and/or interstitial cells in the prostate and in other organs. Therefore, to facilitate precise identification of stem-like TICs, we investigated novel markers such as the tumour rejection antigen, TRA-1-60, a cell surface epitope of human embryonic, embryonal germline and teratocarcinoma stem cells28. Sphere-forming cells and a subset of tumour cells express TRA-1-60, related pluripotent stem cell markers such as TRA-1-81 and stage-specific embryonic antigens such as SSEA1, SSEA4 for example (Fig. 2a, c–f; Supplementary Fig. S2). TRA-1-60 or TRA-1-81-positive tumour cells formed spheres more efficiently than cells positive for other known markers (Fig. 2b,g), and the TRA-1-60-positive tumour cells were moderately more efficient in tumour induction than the primary sphere cells (Fig. 3a).


Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-κB signalling.

Rajasekhar VK, Studer L, Gerald W, Socci ND, Scher HI - Nat Commun (2011)

In vivo tumour-initiation by purified human prostate TICs.(a) Limiting dilution experiments for determining percent tumour-initiation by equal numbers of sphere cells, TRA-1-60-positive cells or triple positives transplanted at OT or SC sites. Blue, 10,000 cells; green, 1,000 cells and red, 200 cells. Data represents the 4-week end-point results following transplantation. (b–h) Unless otherwise stated, 5,000 marker-positive or total tumour (unsorted control) cells were used per transplantation. (b) Representative SC tumour growth at 5 weeks following transplantation. Unsorted represents a control tumour-derived from transplantation of 2×106 total tumour cells. (c) Tumour-initiation efficiency (represented as tumour volume) of the marker-positive cells at 5 weeks following transplantation. (d) Upper panel: Dark field images of primary spheres formed by marker-positive cells. Lower panel: hematoxylin and eosin staining of tumours derived from different marker-positive tumour cells. Scale bar, 100 μm. (e) Relative levels of G1 (yellow), G2 (blue) and S (red) phases of cell cycle in the different marker-positive tumour cells. Unsorted represents total tumour cells. (f) Percent triple-marker expression in sequentially passaged OT-tumours that were initially derived from the triple-marker-positive tumour cells. Percentage triple-marker-positive cells in the parent tumour form the control. (g) Maintenance of triple-marker-positive cells in the secondary passaged tumours derived from indicated marker-specific primary tumour cells. Unsorted is as described in (b). Orange-triple-marker-positive cells and purple-triple-marker-negative cells. (h) Immunoblot analysis of whole cell extracts of the tumours derived from marker-positive tumour cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) represents the loading control. Mean±s.d., n≥3.
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f3: In vivo tumour-initiation by purified human prostate TICs.(a) Limiting dilution experiments for determining percent tumour-initiation by equal numbers of sphere cells, TRA-1-60-positive cells or triple positives transplanted at OT or SC sites. Blue, 10,000 cells; green, 1,000 cells and red, 200 cells. Data represents the 4-week end-point results following transplantation. (b–h) Unless otherwise stated, 5,000 marker-positive or total tumour (unsorted control) cells were used per transplantation. (b) Representative SC tumour growth at 5 weeks following transplantation. Unsorted represents a control tumour-derived from transplantation of 2×106 total tumour cells. (c) Tumour-initiation efficiency (represented as tumour volume) of the marker-positive cells at 5 weeks following transplantation. (d) Upper panel: Dark field images of primary spheres formed by marker-positive cells. Lower panel: hematoxylin and eosin staining of tumours derived from different marker-positive tumour cells. Scale bar, 100 μm. (e) Relative levels of G1 (yellow), G2 (blue) and S (red) phases of cell cycle in the different marker-positive tumour cells. Unsorted represents total tumour cells. (f) Percent triple-marker expression in sequentially passaged OT-tumours that were initially derived from the triple-marker-positive tumour cells. Percentage triple-marker-positive cells in the parent tumour form the control. (g) Maintenance of triple-marker-positive cells in the secondary passaged tumours derived from indicated marker-specific primary tumour cells. Unsorted is as described in (b). Orange-triple-marker-positive cells and purple-triple-marker-negative cells. (h) Immunoblot analysis of whole cell extracts of the tumours derived from marker-positive tumour cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) represents the loading control. Mean±s.d., n≥3.
Mentions: By fluorescence-activated cell sorting (FACS), we investigated novel cell surface marker expression that could facilitate prospective isolation of the TICs in the human prostate CWR22 OT-tumours. First, we confirmed the expression of a set of markers known to associate with stem-like tumour cells in other epithelial cancers, including human prostate tumours1727. They included epithelial cellular adhesion molecule (EpCAM), hyaluronic acid receptor (CD44) and integrins (α2-integrin, α6-integrin and β4-integrin; Fig. 2a). Prostate tumour cells expressing these markers displayed increased sphere-formation capacity as compared with unsorted total tumour cells or cells expressing β4-integrin (Fig. 2b). Except for CD44, the percentage of cells expressing these markers (EpCAM, α2-integrin for example) was consistently enriched over sequential passages of sphere cells. The above markers are associated with many different cell types including stromal and/or interstitial cells in the prostate and in other organs. Therefore, to facilitate precise identification of stem-like TICs, we investigated novel markers such as the tumour rejection antigen, TRA-1-60, a cell surface epitope of human embryonic, embryonal germline and teratocarcinoma stem cells28. Sphere-forming cells and a subset of tumour cells express TRA-1-60, related pluripotent stem cell markers such as TRA-1-81 and stage-specific embryonic antigens such as SSEA1, SSEA4 for example (Fig. 2a, c–f; Supplementary Fig. S2). TRA-1-60 or TRA-1-81-positive tumour cells formed spheres more efficiently than cells positive for other known markers (Fig. 2b,g), and the TRA-1-60-positive tumour cells were moderately more efficient in tumour induction than the primary sphere cells (Fig. 3a).

Bottom Line: These TICs possess stem cell characteristics and multipotency as demonstrated by in vitro sphere-formation and in vivo tumour-initiation, respectively.The cells represent an undifferentiated subtype of basal cells and can be purified from prostate tumours based on coexpression of the human pluripotent stem cell marker TRA-1-60 with CD151 and CD166.These TICs exhibit increased nuclear factor-κB activity.

View Article: PubMed Central - PubMed

Affiliation: 1] Stem Cell Center and Developmental Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. [2] Sidney Kimmel Center for Prostate and Urologic Cancers, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
Androgen depletion is a key strategy for treating human prostate cancer, but the presence of hormone-independent cells escaping treatment remains a major therapeutic challenge. Here, we identify a minor subset of stem-like human prostate tumour-initiating cells (TICs) that do not express prostate cancer markers, such as androgen receptor or prostate specific antigen. These TICs possess stem cell characteristics and multipotency as demonstrated by in vitro sphere-formation and in vivo tumour-initiation, respectively. The cells represent an undifferentiated subtype of basal cells and can be purified from prostate tumours based on coexpression of the human pluripotent stem cell marker TRA-1-60 with CD151 and CD166. Such triple-marker-positive TICs recapitulate the original parent tumour heterogeneity in serial xeno-transplantations indicating a tumour cell hierarchy in human prostate cancer development. These TICs exhibit increased nuclear factor-κB activity. These findings are important in understanding the molecular basis of human prostate cancer.

No MeSH data available.


Related in: MedlinePlus