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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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Stemness-associated marker ALP increased in spheroid cells derived from ovarian cancer cell lines and patients' tissues(A) ALP activity was assessed in spheroids and aggregated counterpart human SKOV3 and CP70 cells 18 h after attachment to the culture plate. ALP activity was detected only in spheroids in both cell lines. (B) ALP activity was upregulated in spheroids from three different cell lines, compared with their differentiated forms in cells that were allowed to attach to the culture plate in standard medium for 3–5 days. The red line encircles the extended region of adhesive SR1 cells, and arrows indicate ALP-negative differentiated SR1 cells. (C) SR1 and SR2 cell lines were isolated from ascites fluids from patients with ovarian cancers. Most spheroids displayed SR2 morphology. (D–F) SR cells isolated from patients' tissue were enriched and passaged three times for further assays. (D) After adipogenesis induction, SR cells could differentiate into adipocytes and displayed oil droplets in the cell bodies. (E) Alcian blue staining confirmed that isolated SR cells showed chondrogenic morphology and displayed the chondrocyte-specific marker 21 days after the induction of chondrogenesis. (F) Isolated SR cells exhibited a neuron-like morphology 14 days after induction and expressed α−internexin 21 days after induction. Patient specimens (solid tumor)-derived SR1 (G) and SR2 (H) cells were cultured in standard culture dishes for 6 h, and ALP activity was measured. (G) ALP activity was detectable only in undifferentiated SR cells and not in differentiated cells (black arrows). The red line encircles the extended region of adhesive SR1 cells. (I, J) When cultured in standard medium under adhesion conditions, after 7 days, ALP activity decreased significantly in differentiated SR cells derived from two individual patients. The left panel shows a bright field view of differentiated SR cells before ALP activity staining; the right panel shows the region of remaining ALP activity in the differentiated SR cells (open arrow).
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Figure 4: Stemness-associated marker ALP increased in spheroid cells derived from ovarian cancer cell lines and patients' tissues(A) ALP activity was assessed in spheroids and aggregated counterpart human SKOV3 and CP70 cells 18 h after attachment to the culture plate. ALP activity was detected only in spheroids in both cell lines. (B) ALP activity was upregulated in spheroids from three different cell lines, compared with their differentiated forms in cells that were allowed to attach to the culture plate in standard medium for 3–5 days. The red line encircles the extended region of adhesive SR1 cells, and arrows indicate ALP-negative differentiated SR1 cells. (C) SR1 and SR2 cell lines were isolated from ascites fluids from patients with ovarian cancers. Most spheroids displayed SR2 morphology. (D–F) SR cells isolated from patients' tissue were enriched and passaged three times for further assays. (D) After adipogenesis induction, SR cells could differentiate into adipocytes and displayed oil droplets in the cell bodies. (E) Alcian blue staining confirmed that isolated SR cells showed chondrogenic morphology and displayed the chondrocyte-specific marker 21 days after the induction of chondrogenesis. (F) Isolated SR cells exhibited a neuron-like morphology 14 days after induction and expressed α−internexin 21 days after induction. Patient specimens (solid tumor)-derived SR1 (G) and SR2 (H) cells were cultured in standard culture dishes for 6 h, and ALP activity was measured. (G) ALP activity was detectable only in undifferentiated SR cells and not in differentiated cells (black arrows). The red line encircles the extended region of adhesive SR1 cells. (I, J) When cultured in standard medium under adhesion conditions, after 7 days, ALP activity decreased significantly in differentiated SR cells derived from two individual patients. The left panel shows a bright field view of differentiated SR cells before ALP activity staining; the right panel shows the region of remaining ALP activity in the differentiated SR cells (open arrow).

Mentions: Alkaline phosphatase is a hydrolase responsible for removing phosphate groups from nucleotides [31], proteins, and alkaloids. ALP activity can be used to test the stemness pluripotency of ESCs or embryonic carcinoma cells [32-34]. We tested whether ALP was active in ovarian cancer stem-like spheroids enriched from cell lines and patients' specimens. However, only some spheroids possessed distinct ALP activity (Figure 4A). For confirmation of ALP activity, a single SR1 spheroid was dissected, and almost every cell was ALP positive (Supplementary Information, Figure S5A and S5B); however, the ALP activity decreased in SR progenies after a prolonged period of differentiation (Figure 4B). Both types of spheres were observed in ascites harvested from different EOC patients without culturing (Figure 4C). SR1 and SR2 could also be enriched from cancer tissues after culture. In clinical specimen-derived SR1, the capacities for adipogenesis (Figure 4D), chondrogenesis (Figure 4E), and neurogenesis (Figure 4F) were confirmed, further supporting the potency of the translineage differentiation. Furthermore, the ALP activity (Figure 4G and 4H) decreased or was lost after differentiation in SR progenies derived from ascites and cancer tissues (Figure 4I and 4J, Supplementary Information, Figure S5C).


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)

Stemness-associated marker ALP increased in spheroid cells derived from ovarian cancer cell lines and patients' tissues(A) ALP activity was assessed in spheroids and aggregated counterpart human SKOV3 and CP70 cells 18 h after attachment to the culture plate. ALP activity was detected only in spheroids in both cell lines. (B) ALP activity was upregulated in spheroids from three different cell lines, compared with their differentiated forms in cells that were allowed to attach to the culture plate in standard medium for 3–5 days. The red line encircles the extended region of adhesive SR1 cells, and arrows indicate ALP-negative differentiated SR1 cells. (C) SR1 and SR2 cell lines were isolated from ascites fluids from patients with ovarian cancers. Most spheroids displayed SR2 morphology. (D–F) SR cells isolated from patients' tissue were enriched and passaged three times for further assays. (D) After adipogenesis induction, SR cells could differentiate into adipocytes and displayed oil droplets in the cell bodies. (E) Alcian blue staining confirmed that isolated SR cells showed chondrogenic morphology and displayed the chondrocyte-specific marker 21 days after the induction of chondrogenesis. (F) Isolated SR cells exhibited a neuron-like morphology 14 days after induction and expressed α−internexin 21 days after induction. Patient specimens (solid tumor)-derived SR1 (G) and SR2 (H) cells were cultured in standard culture dishes for 6 h, and ALP activity was measured. (G) ALP activity was detectable only in undifferentiated SR cells and not in differentiated cells (black arrows). The red line encircles the extended region of adhesive SR1 cells. (I, J) When cultured in standard medium under adhesion conditions, after 7 days, ALP activity decreased significantly in differentiated SR cells derived from two individual patients. The left panel shows a bright field view of differentiated SR cells before ALP activity staining; the right panel shows the region of remaining ALP activity in the differentiated SR cells (open arrow).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Stemness-associated marker ALP increased in spheroid cells derived from ovarian cancer cell lines and patients' tissues(A) ALP activity was assessed in spheroids and aggregated counterpart human SKOV3 and CP70 cells 18 h after attachment to the culture plate. ALP activity was detected only in spheroids in both cell lines. (B) ALP activity was upregulated in spheroids from three different cell lines, compared with their differentiated forms in cells that were allowed to attach to the culture plate in standard medium for 3–5 days. The red line encircles the extended region of adhesive SR1 cells, and arrows indicate ALP-negative differentiated SR1 cells. (C) SR1 and SR2 cell lines were isolated from ascites fluids from patients with ovarian cancers. Most spheroids displayed SR2 morphology. (D–F) SR cells isolated from patients' tissue were enriched and passaged three times for further assays. (D) After adipogenesis induction, SR cells could differentiate into adipocytes and displayed oil droplets in the cell bodies. (E) Alcian blue staining confirmed that isolated SR cells showed chondrogenic morphology and displayed the chondrocyte-specific marker 21 days after the induction of chondrogenesis. (F) Isolated SR cells exhibited a neuron-like morphology 14 days after induction and expressed α−internexin 21 days after induction. Patient specimens (solid tumor)-derived SR1 (G) and SR2 (H) cells were cultured in standard culture dishes for 6 h, and ALP activity was measured. (G) ALP activity was detectable only in undifferentiated SR cells and not in differentiated cells (black arrows). The red line encircles the extended region of adhesive SR1 cells. (I, J) When cultured in standard medium under adhesion conditions, after 7 days, ALP activity decreased significantly in differentiated SR cells derived from two individual patients. The left panel shows a bright field view of differentiated SR cells before ALP activity staining; the right panel shows the region of remaining ALP activity in the differentiated SR cells (open arrow).
Mentions: Alkaline phosphatase is a hydrolase responsible for removing phosphate groups from nucleotides [31], proteins, and alkaloids. ALP activity can be used to test the stemness pluripotency of ESCs or embryonic carcinoma cells [32-34]. We tested whether ALP was active in ovarian cancer stem-like spheroids enriched from cell lines and patients' specimens. However, only some spheroids possessed distinct ALP activity (Figure 4A). For confirmation of ALP activity, a single SR1 spheroid was dissected, and almost every cell was ALP positive (Supplementary Information, Figure S5A and S5B); however, the ALP activity decreased in SR progenies after a prolonged period of differentiation (Figure 4B). Both types of spheres were observed in ascites harvested from different EOC patients without culturing (Figure 4C). SR1 and SR2 could also be enriched from cancer tissues after culture. In clinical specimen-derived SR1, the capacities for adipogenesis (Figure 4D), chondrogenesis (Figure 4E), and neurogenesis (Figure 4F) were confirmed, further supporting the potency of the translineage differentiation. Furthermore, the ALP activity (Figure 4G and 4H) decreased or was lost after differentiation in SR progenies derived from ascites and cancer tissues (Figure 4I and 4J, Supplementary Information, Figure S5C).

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