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Ovarian cancer stem-like cells show induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor.

Liu KC, Yo YT, Huang RL, Wang YC, Liao YP, Huang TS, Chao TK, Lin CK, Weng SJ, Ma KH, Chang CC, Yu MH, Lai HC - Oncotarget (2013)

Bottom Line: Alkaline phosphatase (ALP) was highly expressed in SR1 spheroids, decreased in SR2 spheroids, and was absent in differentiated progenies in accordance with the loss of stemness properties.We verified that ALP can be a marker for ovarian CSLCs, and patients with greater ALP expression is related to advanced clinical stages and have a higher risk of recurrence and lower survival rate.In summary, this research provides a plastic ovarian cancer stem cell model and a new understanding of the cross-link between stem cells and cancers.This results show that ovarian CSLCs can be suppressed by levamisole.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.

ABSTRACT
Spheroid formation is one property of stem cells-such as embryo-derived or neural stem cells-that has been used for the enrichment of cancer stem-like cells (CSLCs). However, it is unclear whether CSLC-derived spheroids are heterogeneous or whether they share common embryonic stemness properties. Understanding these features might lead to novel therapeutic approaches. Ovarian carcinoma is a deadly disease of women. We identified two types of spheroids (SR1 and SR2) from ovarian cancer cell lines and patients' specimens according to their morphology. Both types expressed stemness markers and could self-renew and initiate tumors when a low number of cells were used. Only SR1 could differentiate into multiple-lineage cell types under specific induction conditions. SR1 spheroids could differentiate to SR2 spheroids through epithelial-mesenchymal transition. Alkaline phosphatase (ALP) was highly expressed in SR1 spheroids, decreased in SR2 spheroids, and was absent in differentiated progenies in accordance with the loss of stemness properties. We verified that ALP can be a marker for ovarian CSLCs, and patients with greater ALP expression is related to advanced clinical stages and have a higher risk of recurrence and lower survival rate. The ALP inhibitor, levamisole, disrupted the self-renewal of ovarian CSLCs in vitro and tumor growth in vivo. In summary, this research provides a plastic ovarian cancer stem cell model and a new understanding of the cross-link between stem cells and cancers.This results show that ovarian CSLCs can be suppressed by levamisole. Our findings demonstrated that some ovarian CSLCs may restore ALP activity, and this suggests that inhibition of ALP activity may present a new opportunity for treatment of ovarian cancer.

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Characterization of single-cell clones isolated from CP70 SR1 and SR2 spheroids(A, B) Single cell-derived SR1 and SR2 clones are referred to as SR1SC and SR2SC, respectively. The surface of SR1SC (A) was smooth regardless of the size, whereas SR2SC (B) was morula like. (C, D) Stemness-associated markers were detected in single cell-derived SR1, SR2, and SR cells 6 weeks after differentiation, and in the original cells. (C) The population of CD133+/CD44+ cells was the highest in SR1 followed by SR2, differentiated SR cells, and the original cells. (D) Expression of both the stemness-associated marker NANOG and hESC-related marker SSEA4 was increased in the order SR1, SR2, and SR differentiated cells. SSEA4 expression was lower in CP70 original cells than in their counterpart SR cells. (E) Dissociated SR1SC cells were induced to differentiate for 14 days. At least 10 types of morphology (white arrows) with a neuron-like phenotype were observed in different fields of view, whereas CP70SR2 cells differentiated only to a simple and tumor-like morphology (F). Neuron-like morphology was observed only in CP70SR1 cells (G), and expression of multiple neuronal markers was detected by immunostaining using anti-pan-neuronal antibodies (H). However, most CP70SR2 cells were dead when treated under the condition described above for 16 days (I).
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Figure 2: Characterization of single-cell clones isolated from CP70 SR1 and SR2 spheroids(A, B) Single cell-derived SR1 and SR2 clones are referred to as SR1SC and SR2SC, respectively. The surface of SR1SC (A) was smooth regardless of the size, whereas SR2SC (B) was morula like. (C, D) Stemness-associated markers were detected in single cell-derived SR1, SR2, and SR cells 6 weeks after differentiation, and in the original cells. (C) The population of CD133+/CD44+ cells was the highest in SR1 followed by SR2, differentiated SR cells, and the original cells. (D) Expression of both the stemness-associated marker NANOG and hESC-related marker SSEA4 was increased in the order SR1, SR2, and SR differentiated cells. SSEA4 expression was lower in CP70 original cells than in their counterpart SR cells. (E) Dissociated SR1SC cells were induced to differentiate for 14 days. At least 10 types of morphology (white arrows) with a neuron-like phenotype were observed in different fields of view, whereas CP70SR2 cells differentiated only to a simple and tumor-like morphology (F). Neuron-like morphology was observed only in CP70SR1 cells (G), and expression of multiple neuronal markers was detected by immunostaining using anti-pan-neuronal antibodies (H). However, most CP70SR2 cells were dead when treated under the condition described above for 16 days (I).

Mentions: A diversity of morphology was observed in the suspension cultures of original CP70 cells (Supplementary Information, Figure S1B). To assess stemness properties of single cell-derived CSLC, pure populations of CP70SR1 and SR2 were procured (Supplementary Information, Figure S1C) [18, 24]. CP70SR1 and SR2 clones derived from single cells (also known as CP70SR1SC and CP70SR2SC, respectively) were isolated by limiting dilution. The size of the spheres did not appear to affect the distinctive appearance (Figure 2A and 2B); however, self-renewal was slower for SR1 than for SR2, suggesting SR1 was more dormant than SR2 [13]. Flow cytometry was used to identify the expression of CD44 [9, 21], CD117 [9], CD133 [6, 11], NANOG, and SSEA4 [25]. There was an order of stem-like cell hierarchy between CP70SR1, SR2, differentiated SR, and original CP70 cells (Figure 2C and 2D, Supplementary Information, Figure S1D). Interestingly, CP70SR1SC branched out and exhibited at least 10 different morphologies (Figure 2E). By contrast, CP70SR2SC showed only basic tumor cell morphology (Figure 2F).


Ovarian cancer stem-like cells show induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor.

Liu KC, Yo YT, Huang RL, Wang YC, Liao YP, Huang TS, Chao TK, Lin CK, Weng SJ, Ma KH, Chang CC, Yu MH, Lai HC - Oncotarget (2013)

Characterization of single-cell clones isolated from CP70 SR1 and SR2 spheroids(A, B) Single cell-derived SR1 and SR2 clones are referred to as SR1SC and SR2SC, respectively. The surface of SR1SC (A) was smooth regardless of the size, whereas SR2SC (B) was morula like. (C, D) Stemness-associated markers were detected in single cell-derived SR1, SR2, and SR cells 6 weeks after differentiation, and in the original cells. (C) The population of CD133+/CD44+ cells was the highest in SR1 followed by SR2, differentiated SR cells, and the original cells. (D) Expression of both the stemness-associated marker NANOG and hESC-related marker SSEA4 was increased in the order SR1, SR2, and SR differentiated cells. SSEA4 expression was lower in CP70 original cells than in their counterpart SR cells. (E) Dissociated SR1SC cells were induced to differentiate for 14 days. At least 10 types of morphology (white arrows) with a neuron-like phenotype were observed in different fields of view, whereas CP70SR2 cells differentiated only to a simple and tumor-like morphology (F). Neuron-like morphology was observed only in CP70SR1 cells (G), and expression of multiple neuronal markers was detected by immunostaining using anti-pan-neuronal antibodies (H). However, most CP70SR2 cells were dead when treated under the condition described above for 16 days (I).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926833&req=5

Figure 2: Characterization of single-cell clones isolated from CP70 SR1 and SR2 spheroids(A, B) Single cell-derived SR1 and SR2 clones are referred to as SR1SC and SR2SC, respectively. The surface of SR1SC (A) was smooth regardless of the size, whereas SR2SC (B) was morula like. (C, D) Stemness-associated markers were detected in single cell-derived SR1, SR2, and SR cells 6 weeks after differentiation, and in the original cells. (C) The population of CD133+/CD44+ cells was the highest in SR1 followed by SR2, differentiated SR cells, and the original cells. (D) Expression of both the stemness-associated marker NANOG and hESC-related marker SSEA4 was increased in the order SR1, SR2, and SR differentiated cells. SSEA4 expression was lower in CP70 original cells than in their counterpart SR cells. (E) Dissociated SR1SC cells were induced to differentiate for 14 days. At least 10 types of morphology (white arrows) with a neuron-like phenotype were observed in different fields of view, whereas CP70SR2 cells differentiated only to a simple and tumor-like morphology (F). Neuron-like morphology was observed only in CP70SR1 cells (G), and expression of multiple neuronal markers was detected by immunostaining using anti-pan-neuronal antibodies (H). However, most CP70SR2 cells were dead when treated under the condition described above for 16 days (I).
Mentions: A diversity of morphology was observed in the suspension cultures of original CP70 cells (Supplementary Information, Figure S1B). To assess stemness properties of single cell-derived CSLC, pure populations of CP70SR1 and SR2 were procured (Supplementary Information, Figure S1C) [18, 24]. CP70SR1 and SR2 clones derived from single cells (also known as CP70SR1SC and CP70SR2SC, respectively) were isolated by limiting dilution. The size of the spheres did not appear to affect the distinctive appearance (Figure 2A and 2B); however, self-renewal was slower for SR1 than for SR2, suggesting SR1 was more dormant than SR2 [13]. Flow cytometry was used to identify the expression of CD44 [9, 21], CD117 [9], CD133 [6, 11], NANOG, and SSEA4 [25]. There was an order of stem-like cell hierarchy between CP70SR1, SR2, differentiated SR, and original CP70 cells (Figure 2C and 2D, Supplementary Information, Figure S1D). Interestingly, CP70SR1SC branched out and exhibited at least 10 different morphologies (Figure 2E). By contrast, CP70SR2SC showed only basic tumor cell morphology (Figure 2F).

Bottom Line: Alkaline phosphatase (ALP) was highly expressed in SR1 spheroids, decreased in SR2 spheroids, and was absent in differentiated progenies in accordance with the loss of stemness properties.We verified that ALP can be a marker for ovarian CSLCs, and patients with greater ALP expression is related to advanced clinical stages and have a higher risk of recurrence and lower survival rate.In summary, this research provides a plastic ovarian cancer stem cell model and a new understanding of the cross-link between stem cells and cancers.This results show that ovarian CSLCs can be suppressed by levamisole.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.

ABSTRACT
Spheroid formation is one property of stem cells-such as embryo-derived or neural stem cells-that has been used for the enrichment of cancer stem-like cells (CSLCs). However, it is unclear whether CSLC-derived spheroids are heterogeneous or whether they share common embryonic stemness properties. Understanding these features might lead to novel therapeutic approaches. Ovarian carcinoma is a deadly disease of women. We identified two types of spheroids (SR1 and SR2) from ovarian cancer cell lines and patients' specimens according to their morphology. Both types expressed stemness markers and could self-renew and initiate tumors when a low number of cells were used. Only SR1 could differentiate into multiple-lineage cell types under specific induction conditions. SR1 spheroids could differentiate to SR2 spheroids through epithelial-mesenchymal transition. Alkaline phosphatase (ALP) was highly expressed in SR1 spheroids, decreased in SR2 spheroids, and was absent in differentiated progenies in accordance with the loss of stemness properties. We verified that ALP can be a marker for ovarian CSLCs, and patients with greater ALP expression is related to advanced clinical stages and have a higher risk of recurrence and lower survival rate. The ALP inhibitor, levamisole, disrupted the self-renewal of ovarian CSLCs in vitro and tumor growth in vivo. In summary, this research provides a plastic ovarian cancer stem cell model and a new understanding of the cross-link between stem cells and cancers.This results show that ovarian CSLCs can be suppressed by levamisole. Our findings demonstrated that some ovarian CSLCs may restore ALP activity, and this suggests that inhibition of ALP activity may present a new opportunity for treatment of ovarian cancer.

Show MeSH
Related in: MedlinePlus