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Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator ΔNp63α.

Portillo-Lara R, Alvarez MM - PLoS ONE (2015)

Bottom Line: This phenotype is concordant to that of CSCs in vivo.We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology.This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Monterrey, Nuevo León, México.

ABSTRACT

Background: Cancer stem cells (CSC) drive prostate cancer tumor survival and metastasis. Nevertheless, the development of specific therapies against CSCs is hindered by the scarcity of these cells in prostate tissues. Suspension culture systems have been reported to enrich CSCs in primary cultures and cell lines. However, the molecular mechanisms underlying this phenomenon have not been fully explored.

Methodology/principal findings: We describe a prostasphere assay for the enrichment of CD133+ CSCs in four commercial PCa cell lines: 22Rv1, DU145, LNCaP, and PC3. Overexpression of CD133, as determined by flow cytometric analysis, correlated with an increased clonogenic, chemoresistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Gene expression profiling was then carried out using the Cancer Reference panel and the nCounter system from NanoString Technologies. This analysis revealed several upregulated transcripts that can be further explored as potential diagnostic markers or therapeutic targets. Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures.

Conclusions/significance: We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology. This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

No MeSH data available.


Related in: MedlinePlus

Morphology and Sphere-forming efficiency are cell line dependent.A) Morphological characterization of primary prostaspheres grown in hPCM-PLUS. All PCa cell lines formed discernable spheres by day 4 of culture. 22Rv1 and DU145 spheres consist of smaller and more tightly packed together cells. LNCaP and PC3 cells appear larger and more loosely organized. Scale bar = 100 μm. B) Sphere-forming efficiency (SFE) of prostasphere cultures. PCa cells were seeded at a density of 1x103 cells/ml in 6-well ultra-low attachment plates in hPCM-PLUS and incubated for 10 days. The resulting prostaspheres could be serially passaged in vitro for up to three generations (G1-G3). SFE of spheres increased consistently in all PCa cell lines with each generation. No further amplification was attempted at this point. Three independent experiments were carried out, each one in triplicate. Scale bar = 100 μm. (*p < 0.05).
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pone.0130118.g002: Morphology and Sphere-forming efficiency are cell line dependent.A) Morphological characterization of primary prostaspheres grown in hPCM-PLUS. All PCa cell lines formed discernable spheres by day 4 of culture. 22Rv1 and DU145 spheres consist of smaller and more tightly packed together cells. LNCaP and PC3 cells appear larger and more loosely organized. Scale bar = 100 μm. B) Sphere-forming efficiency (SFE) of prostasphere cultures. PCa cells were seeded at a density of 1x103 cells/ml in 6-well ultra-low attachment plates in hPCM-PLUS and incubated for 10 days. The resulting prostaspheres could be serially passaged in vitro for up to three generations (G1-G3). SFE of spheres increased consistently in all PCa cell lines with each generation. No further amplification was attempted at this point. Three independent experiments were carried out, each one in triplicate. Scale bar = 100 μm. (*p < 0.05).

Mentions: Tumorsphere forming ability was evaluated using a seeding density of 2x104 cells/ml in 6-well ultra-low attachment plates containing 2 mL hPCM-PLUS medium. All four PCa cell lines formed spherical colonies by day 4 of culture. Parental adherent cultures were propagated simultaneously at a maximum confluence level of 80%. Day 12 tumorspheres and monolayers (Fig 1A) were dissociated and analyzed for the expression of the CSC-associated biomarkers CD133 and CD44. Expression of CD44 was detected heterogeneously at high levels in PC3 and DU145 cells, whereas minimum to no detectable expression was observed in 22Rv1 and LNCaP cells (S1A Fig). Fig 1B shows that CD133 was observed consistently at extremely low levels (< 0.5%) in all cell lines. The panel also shows that in contrast to parental cultures, the percentage of CD133+ events increased significantly (p < 0.05) in all prostasphere cultures of 22Rv1 (0.067%±0.058% to 4.267%±1.419%), DU145 (0.433%±0.252% to 2.433%±0.709%), LNCaP (0.033%±0.058% to 4.033%±1.966%), and PC3 (0.133%±0.058% to 4.467%±1.358%) cells. Prostaspheres quickly reached diameters > 100 μm by day 12 of culture (Fig 2A). The 22Rv1 and DU145 cells formed compact and round-shaped spheres with well delimited borders, whereas the LNCaP and particularly the PC3 cells formed more irregular structures consisting of larger individual cells. The sphere-forming efficiency (SFE) was evaluated for first- (Day 10), second- (Day 20), and third- (Day 30) generation spheres. SFE invariably increased (p < 0.05) with each new generation in all cell lines (Fig 2B). Interestingly, direct observation of PC3 and DU145 cells grown in suspension revealed further budding from prostaspheres at this point (S1B Fig). This observation suggests the potential formation of branching structures from established spheres.


Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator ΔNp63α.

Portillo-Lara R, Alvarez MM - PLoS ONE (2015)

Morphology and Sphere-forming efficiency are cell line dependent.A) Morphological characterization of primary prostaspheres grown in hPCM-PLUS. All PCa cell lines formed discernable spheres by day 4 of culture. 22Rv1 and DU145 spheres consist of smaller and more tightly packed together cells. LNCaP and PC3 cells appear larger and more loosely organized. Scale bar = 100 μm. B) Sphere-forming efficiency (SFE) of prostasphere cultures. PCa cells were seeded at a density of 1x103 cells/ml in 6-well ultra-low attachment plates in hPCM-PLUS and incubated for 10 days. The resulting prostaspheres could be serially passaged in vitro for up to three generations (G1-G3). SFE of spheres increased consistently in all PCa cell lines with each generation. No further amplification was attempted at this point. Three independent experiments were carried out, each one in triplicate. Scale bar = 100 μm. (*p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4481544&req=5

pone.0130118.g002: Morphology and Sphere-forming efficiency are cell line dependent.A) Morphological characterization of primary prostaspheres grown in hPCM-PLUS. All PCa cell lines formed discernable spheres by day 4 of culture. 22Rv1 and DU145 spheres consist of smaller and more tightly packed together cells. LNCaP and PC3 cells appear larger and more loosely organized. Scale bar = 100 μm. B) Sphere-forming efficiency (SFE) of prostasphere cultures. PCa cells were seeded at a density of 1x103 cells/ml in 6-well ultra-low attachment plates in hPCM-PLUS and incubated for 10 days. The resulting prostaspheres could be serially passaged in vitro for up to three generations (G1-G3). SFE of spheres increased consistently in all PCa cell lines with each generation. No further amplification was attempted at this point. Three independent experiments were carried out, each one in triplicate. Scale bar = 100 μm. (*p < 0.05).
Mentions: Tumorsphere forming ability was evaluated using a seeding density of 2x104 cells/ml in 6-well ultra-low attachment plates containing 2 mL hPCM-PLUS medium. All four PCa cell lines formed spherical colonies by day 4 of culture. Parental adherent cultures were propagated simultaneously at a maximum confluence level of 80%. Day 12 tumorspheres and monolayers (Fig 1A) were dissociated and analyzed for the expression of the CSC-associated biomarkers CD133 and CD44. Expression of CD44 was detected heterogeneously at high levels in PC3 and DU145 cells, whereas minimum to no detectable expression was observed in 22Rv1 and LNCaP cells (S1A Fig). Fig 1B shows that CD133 was observed consistently at extremely low levels (< 0.5%) in all cell lines. The panel also shows that in contrast to parental cultures, the percentage of CD133+ events increased significantly (p < 0.05) in all prostasphere cultures of 22Rv1 (0.067%±0.058% to 4.267%±1.419%), DU145 (0.433%±0.252% to 2.433%±0.709%), LNCaP (0.033%±0.058% to 4.033%±1.966%), and PC3 (0.133%±0.058% to 4.467%±1.358%) cells. Prostaspheres quickly reached diameters > 100 μm by day 12 of culture (Fig 2A). The 22Rv1 and DU145 cells formed compact and round-shaped spheres with well delimited borders, whereas the LNCaP and particularly the PC3 cells formed more irregular structures consisting of larger individual cells. The sphere-forming efficiency (SFE) was evaluated for first- (Day 10), second- (Day 20), and third- (Day 30) generation spheres. SFE invariably increased (p < 0.05) with each new generation in all cell lines (Fig 2B). Interestingly, direct observation of PC3 and DU145 cells grown in suspension revealed further budding from prostaspheres at this point (S1B Fig). This observation suggests the potential formation of branching structures from established spheres.

Bottom Line: This phenotype is concordant to that of CSCs in vivo.We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology.This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Monterrey, Nuevo León, México.

ABSTRACT

Background: Cancer stem cells (CSC) drive prostate cancer tumor survival and metastasis. Nevertheless, the development of specific therapies against CSCs is hindered by the scarcity of these cells in prostate tissues. Suspension culture systems have been reported to enrich CSCs in primary cultures and cell lines. However, the molecular mechanisms underlying this phenomenon have not been fully explored.

Methodology/principal findings: We describe a prostasphere assay for the enrichment of CD133+ CSCs in four commercial PCa cell lines: 22Rv1, DU145, LNCaP, and PC3. Overexpression of CD133, as determined by flow cytometric analysis, correlated with an increased clonogenic, chemoresistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Gene expression profiling was then carried out using the Cancer Reference panel and the nCounter system from NanoString Technologies. This analysis revealed several upregulated transcripts that can be further explored as potential diagnostic markers or therapeutic targets. Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures.

Conclusions/significance: We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology. This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

No MeSH data available.


Related in: MedlinePlus