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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

A subset of human prostate TICs exhibits sphere-forming potential and multipotency.Unless stated otherwise, human prostate CWR22 OT-tumours were used. (a) Phase contrast images of primary sphere formation by the total tumour cells. Scale bar, 100 μm. (b) Time course of primary sphere formation (sphere number per 8×105 total tumour cells plated, n=4). (c) Time course of primary sphere growth (average number of cells per primary sphere, n=4). (d) Efficiency of sequential derivation of primary (blue circle), secondary (green square) and tertiary spheres (black triangle) determined as a function of equal numbers of starting cells from total tumour, primary spheres and secondary spheres, respectively; n=4. (e) Limiting dilution experiments determining tumour-initiation efficiency (% tumour induction) at the orthotopic (blue, tumour cells; red, primary sphere cells) and subcutaneous (black, tumour cells; green, primary sphere cells) sites. Data represents the 4-week end-point results following transplantation. Mean±s.d. (n=4). (f, g) CWR22 OT-tumour (parent tumour), the tumour cell-derived primary spheres (spheres), the sphere-derived OT-tumour (sphere tumour) and human patient primary prostate tumour (primary tumour) were used for comparative immunohistochemistry (IHC). Scale bar, 50 μm. (f) Comparative IHC for expression of differentiated prostate cell markers (AR, PSA and MET) in primary spheres versus tumours. (g) Comparative IHC for expression of markers for epithelial cells (E-cadherin) versus differentiated cells in the tumour, namely neuroendocrine cells (synaptophysin), myoepithelial cells (smooth muscle actin, SMA), and mesenchymal cells (vimentin). (h) Western blots of whole cell extracts of the parent tumour, primary spheres and sphere tumour analyzing the expression of markers for prostate basal cell (CK5), luminal cell (AR, PSA, NKX3.1, CK8 and CK18), primary prostate cancer cell (MET, αB-crystallin and maspin), putative stem cell (Musashi-1, MSH-1), basal epithelial stem cell (SOX9), cell polarity/differentiation (ZO-1) and loading control (glyceraldehyde 3-phosphate dehydrogenase, GAPDH).
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f1: A subset of human prostate TICs exhibits sphere-forming potential and multipotency.Unless stated otherwise, human prostate CWR22 OT-tumours were used. (a) Phase contrast images of primary sphere formation by the total tumour cells. Scale bar, 100 μm. (b) Time course of primary sphere formation (sphere number per 8×105 total tumour cells plated, n=4). (c) Time course of primary sphere growth (average number of cells per primary sphere, n=4). (d) Efficiency of sequential derivation of primary (blue circle), secondary (green square) and tertiary spheres (black triangle) determined as a function of equal numbers of starting cells from total tumour, primary spheres and secondary spheres, respectively; n=4. (e) Limiting dilution experiments determining tumour-initiation efficiency (% tumour induction) at the orthotopic (blue, tumour cells; red, primary sphere cells) and subcutaneous (black, tumour cells; green, primary sphere cells) sites. Data represents the 4-week end-point results following transplantation. Mean±s.d. (n=4). (f, g) CWR22 OT-tumour (parent tumour), the tumour cell-derived primary spheres (spheres), the sphere-derived OT-tumour (sphere tumour) and human patient primary prostate tumour (primary tumour) were used for comparative immunohistochemistry (IHC). Scale bar, 50 μm. (f) Comparative IHC for expression of differentiated prostate cell markers (AR, PSA and MET) in primary spheres versus tumours. (g) Comparative IHC for expression of markers for epithelial cells (E-cadherin) versus differentiated cells in the tumour, namely neuroendocrine cells (synaptophysin), myoepithelial cells (smooth muscle actin, SMA), and mesenchymal cells (vimentin). (h) Western blots of whole cell extracts of the parent tumour, primary spheres and sphere tumour analyzing the expression of markers for prostate basal cell (CK5), luminal cell (AR, PSA, NKX3.1, CK8 and CK18), primary prostate cancer cell (MET, αB-crystallin and maspin), putative stem cell (Musashi-1, MSH-1), basal epithelial stem cell (SOX9), cell polarity/differentiation (ZO-1) and loading control (glyceraldehyde 3-phosphate dehydrogenase, GAPDH).

Mentions: However, the sphere formation efficiency was very low (1–2 spheres/2,500–5,000 total tumour cells) as determined by limiting dilution assays (Fig. 1a). A minor subset of human patient primary prostate tumour cells also had sphere-forming ability albeit with even lower efficiency (about tenfold less). We observed a gradual increase in the total number of primary spheres formed over time for a given number of total tumour cells (Fig. 1b), sphere growth as determined by the increase in number of cells per sphere (Fig. 1c) and sphere-forming ability of serially passaged sphere cells (about 5- and 25-fold higher in secondary and tertiary sphere formation assays, respectively; Fig. 1d). These data demonstrate differential sphere-forming abilities among various cell types in the tumour and rule out simple cell aggregation as a primary mechanism of sphere formation. Quantitative analysis of tumour-initiation efficiency through limiting dilution experiments revealed that tumour-initiation with primary sphere-cells was at least 100-times more efficient than with the total tumour cells (Fig. 1e). Moreover, tumour initiation at OT versus sub-cutaneous (SC)-sites was about ten-times more efficient. Unless otherwise stated, we used OT-tumour-initiation assays in all further experiments.


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)

A subset of human prostate TICs exhibits sphere-forming potential and multipotency.Unless stated otherwise, human prostate CWR22 OT-tumours were used. (a) Phase contrast images of primary sphere formation by the total tumour cells. Scale bar, 100 μm. (b) Time course of primary sphere formation (sphere number per 8×105 total tumour cells plated, n=4). (c) Time course of primary sphere growth (average number of cells per primary sphere, n=4). (d) Efficiency of sequential derivation of primary (blue circle), secondary (green square) and tertiary spheres (black triangle) determined as a function of equal numbers of starting cells from total tumour, primary spheres and secondary spheres, respectively; n=4. (e) Limiting dilution experiments determining tumour-initiation efficiency (% tumour induction) at the orthotopic (blue, tumour cells; red, primary sphere cells) and subcutaneous (black, tumour cells; green, primary sphere cells) sites. Data represents the 4-week end-point results following transplantation. Mean±s.d. (n=4). (f, g) CWR22 OT-tumour (parent tumour), the tumour cell-derived primary spheres (spheres), the sphere-derived OT-tumour (sphere tumour) and human patient primary prostate tumour (primary tumour) were used for comparative immunohistochemistry (IHC). Scale bar, 50 μm. (f) Comparative IHC for expression of differentiated prostate cell markers (AR, PSA and MET) in primary spheres versus tumours. (g) Comparative IHC for expression of markers for epithelial cells (E-cadherin) versus differentiated cells in the tumour, namely neuroendocrine cells (synaptophysin), myoepithelial cells (smooth muscle actin, SMA), and mesenchymal cells (vimentin). (h) Western blots of whole cell extracts of the parent tumour, primary spheres and sphere tumour analyzing the expression of markers for prostate basal cell (CK5), luminal cell (AR, PSA, NKX3.1, CK8 and CK18), primary prostate cancer cell (MET, αB-crystallin and maspin), putative stem cell (Musashi-1, MSH-1), basal epithelial stem cell (SOX9), cell polarity/differentiation (ZO-1) and loading control (glyceraldehyde 3-phosphate dehydrogenase, GAPDH).
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f1: A subset of human prostate TICs exhibits sphere-forming potential and multipotency.Unless stated otherwise, human prostate CWR22 OT-tumours were used. (a) Phase contrast images of primary sphere formation by the total tumour cells. Scale bar, 100 μm. (b) Time course of primary sphere formation (sphere number per 8×105 total tumour cells plated, n=4). (c) Time course of primary sphere growth (average number of cells per primary sphere, n=4). (d) Efficiency of sequential derivation of primary (blue circle), secondary (green square) and tertiary spheres (black triangle) determined as a function of equal numbers of starting cells from total tumour, primary spheres and secondary spheres, respectively; n=4. (e) Limiting dilution experiments determining tumour-initiation efficiency (% tumour induction) at the orthotopic (blue, tumour cells; red, primary sphere cells) and subcutaneous (black, tumour cells; green, primary sphere cells) sites. Data represents the 4-week end-point results following transplantation. Mean±s.d. (n=4). (f, g) CWR22 OT-tumour (parent tumour), the tumour cell-derived primary spheres (spheres), the sphere-derived OT-tumour (sphere tumour) and human patient primary prostate tumour (primary tumour) were used for comparative immunohistochemistry (IHC). Scale bar, 50 μm. (f) Comparative IHC for expression of differentiated prostate cell markers (AR, PSA and MET) in primary spheres versus tumours. (g) Comparative IHC for expression of markers for epithelial cells (E-cadherin) versus differentiated cells in the tumour, namely neuroendocrine cells (synaptophysin), myoepithelial cells (smooth muscle actin, SMA), and mesenchymal cells (vimentin). (h) Western blots of whole cell extracts of the parent tumour, primary spheres and sphere tumour analyzing the expression of markers for prostate basal cell (CK5), luminal cell (AR, PSA, NKX3.1, CK8 and CK18), primary prostate cancer cell (MET, αB-crystallin and maspin), putative stem cell (Musashi-1, MSH-1), basal epithelial stem cell (SOX9), cell polarity/differentiation (ZO-1) and loading control (glyceraldehyde 3-phosphate dehydrogenase, GAPDH).
Mentions: However, the sphere formation efficiency was very low (1–2 spheres/2,500–5,000 total tumour cells) as determined by limiting dilution assays (Fig. 1a). A minor subset of human patient primary prostate tumour cells also had sphere-forming ability albeit with even lower efficiency (about tenfold less). We observed a gradual increase in the total number of primary spheres formed over time for a given number of total tumour cells (Fig. 1b), sphere growth as determined by the increase in number of cells per sphere (Fig. 1c) and sphere-forming ability of serially passaged sphere cells (about 5- and 25-fold higher in secondary and tertiary sphere formation assays, respectively; Fig. 1d). These data demonstrate differential sphere-forming abilities among various cell types in the tumour and rule out simple cell aggregation as a primary mechanism of sphere formation. Quantitative analysis of tumour-initiation efficiency through limiting dilution experiments revealed that tumour-initiation with primary sphere-cells was at least 100-times more efficient than with the total tumour cells (Fig. 1e). Moreover, tumour initiation at OT versus sub-cutaneous (SC)-sites was about ten-times more efficient. Unless otherwise stated, we used OT-tumour-initiation assays in all further experiments.

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