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Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line.

Hoe SL, Tan LP, Jamal J, Peh SC, Ng CC, Zhang WC, Ahmad M, Khoo AS - Cancer Cell Int. (2014)

Bottom Line: Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye.ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells.Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia ; Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT

Background: Side population (SP) assay identifies cells with dye/drug extrusion ability, a characteristic of stem cells. Here, we determined if SP cells exist in a verified cell line originating from recurrent nasopharyngeal carcinoma (NPC) and a xenograft established from recurrent metastatic NPC. These cells were evaluated for stem-like properties via functional assays as well as for tumourigenicity.

Methods: We used Hoechst 33342 to identify the SP from non-SP (NSP) cells in HK1 NPC cell line and xeno-284 NPC xenograft. The cells were assayed for in vitro characteristics of cancer stem cells (CSC), gene expression and tumourigenicity ability. Student's t test was used to test for significance.

Results: Five to ten percent and less than 0.5% of HK1 and xeno-284 NPC cells, respectively, were SP cells. Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye. HK1 SP cells formed more holoclones, had more aldehyde dehydrogenase (ALDH) activity, divided asymmetrically and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells. However, despite these differences in vitro, both HK1 SP and NSP cells had an overall similar tumourigenic potential in vivo.

Conclusions: HK1 SP cells were ABCG2-specific as confirmed by FTC inhibition and gene expression data. Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells. We conclude that SP assay alone is not sufficient to identify CSCs in HK1 cells. Our work also suggests the presence of a stem-cell like population among NPC cells which do not display increased tumourigenicity.

No MeSH data available.


Related in: MedlinePlus

SP subpopulation enriches for stem-cell like phenotype inin vitroassays. (A) During in vitro culture, majority of SP cells formed holoclones with individual cells clustering tightly and forming a well-defined clone border. (B) NSP cells tended to form loose clusters of meroclones or, paraclones (inset) with tiny clusters of cells situated far from each other. All photos were taken on day 9 post-sorting; original magnification 100X. (C) SP cells formed more holoclones than NSP cells during in vitro culture (p < 0.0001). (D) The gating for the ALDHbright population was set by a control tube with the addition of DEAB, an ALDH inhibitor. (E) SP cells showed higher presence of ALDHbright cells (18.08 ± 11.46%) than NSP cells (5.10 ± 3.56%) (p < 0.05).
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Fig2: SP subpopulation enriches for stem-cell like phenotype inin vitroassays. (A) During in vitro culture, majority of SP cells formed holoclones with individual cells clustering tightly and forming a well-defined clone border. (B) NSP cells tended to form loose clusters of meroclones or, paraclones (inset) with tiny clusters of cells situated far from each other. All photos were taken on day 9 post-sorting; original magnification 100X. (C) SP cells formed more holoclones than NSP cells during in vitro culture (p < 0.0001). (D) The gating for the ALDHbright population was set by a control tube with the addition of DEAB, an ALDH inhibitor. (E) SP cells showed higher presence of ALDHbright cells (18.08 ± 11.46%) than NSP cells (5.10 ± 3.56%) (p < 0.05).

Mentions: Sorted SP and NSP cells exhibited different growth patterns. After a week of in vitro culture in fully-supplemented RPMI medium, most of the SP cells grew into holoclones which formed tightly-clustered cells with well-defined clone borders (Figure 2A). Clones established by the NSP cells primarily consisted of slightly-scattered cells with irregular borders (meroclones) and/or tiny clusters of cells which did not display much growth (paraclones) (Figure 2B). Repeated experiments showed that SP cells formed more holoclones than NSP cells (p < 0.0001; Figure 2C).Figure 2


Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line.

Hoe SL, Tan LP, Jamal J, Peh SC, Ng CC, Zhang WC, Ahmad M, Khoo AS - Cancer Cell Int. (2014)

SP subpopulation enriches for stem-cell like phenotype inin vitroassays. (A) During in vitro culture, majority of SP cells formed holoclones with individual cells clustering tightly and forming a well-defined clone border. (B) NSP cells tended to form loose clusters of meroclones or, paraclones (inset) with tiny clusters of cells situated far from each other. All photos were taken on day 9 post-sorting; original magnification 100X. (C) SP cells formed more holoclones than NSP cells during in vitro culture (p < 0.0001). (D) The gating for the ALDHbright population was set by a control tube with the addition of DEAB, an ALDH inhibitor. (E) SP cells showed higher presence of ALDHbright cells (18.08 ± 11.46%) than NSP cells (5.10 ± 3.56%) (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195955&req=5

Fig2: SP subpopulation enriches for stem-cell like phenotype inin vitroassays. (A) During in vitro culture, majority of SP cells formed holoclones with individual cells clustering tightly and forming a well-defined clone border. (B) NSP cells tended to form loose clusters of meroclones or, paraclones (inset) with tiny clusters of cells situated far from each other. All photos were taken on day 9 post-sorting; original magnification 100X. (C) SP cells formed more holoclones than NSP cells during in vitro culture (p < 0.0001). (D) The gating for the ALDHbright population was set by a control tube with the addition of DEAB, an ALDH inhibitor. (E) SP cells showed higher presence of ALDHbright cells (18.08 ± 11.46%) than NSP cells (5.10 ± 3.56%) (p < 0.05).
Mentions: Sorted SP and NSP cells exhibited different growth patterns. After a week of in vitro culture in fully-supplemented RPMI medium, most of the SP cells grew into holoclones which formed tightly-clustered cells with well-defined clone borders (Figure 2A). Clones established by the NSP cells primarily consisted of slightly-scattered cells with irregular borders (meroclones) and/or tiny clusters of cells which did not display much growth (paraclones) (Figure 2B). Repeated experiments showed that SP cells formed more holoclones than NSP cells (p < 0.0001; Figure 2C).Figure 2

Bottom Line: Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye.ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells.Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia ; Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT

Background: Side population (SP) assay identifies cells with dye/drug extrusion ability, a characteristic of stem cells. Here, we determined if SP cells exist in a verified cell line originating from recurrent nasopharyngeal carcinoma (NPC) and a xenograft established from recurrent metastatic NPC. These cells were evaluated for stem-like properties via functional assays as well as for tumourigenicity.

Methods: We used Hoechst 33342 to identify the SP from non-SP (NSP) cells in HK1 NPC cell line and xeno-284 NPC xenograft. The cells were assayed for in vitro characteristics of cancer stem cells (CSC), gene expression and tumourigenicity ability. Student's t test was used to test for significance.

Results: Five to ten percent and less than 0.5% of HK1 and xeno-284 NPC cells, respectively, were SP cells. Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye. HK1 SP cells formed more holoclones, had more aldehyde dehydrogenase (ALDH) activity, divided asymmetrically and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells. However, despite these differences in vitro, both HK1 SP and NSP cells had an overall similar tumourigenic potential in vivo.

Conclusions: HK1 SP cells were ABCG2-specific as confirmed by FTC inhibition and gene expression data. Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells. We conclude that SP assay alone is not sufficient to identify CSCs in HK1 cells. Our work also suggests the presence of a stem-cell like population among NPC cells which do not display increased tumourigenicity.

No MeSH data available.


Related in: MedlinePlus