Limits...
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

In vivotumour growth analysis of SP and non-SP (NSP) cells isolated from HK1 NPC cell line. (A) SP or NSP cell inoculation in nude mice. Distinct difference in tumour latency was observed in the lowest inoculated cell number (10 cells). (B) Limiting dilution analysis of SP and NSP tumours. Tumour-initiating cells (TICs) were similarly enriched in both SP and NSP cells (p > 0.05). (C) Representative gross morphology of tumours inoculated with (i) 1000 SP cells, (ii) 1000 NSP cells, (iii) 100 SP cells, (iv) 100 NSP cells, (v) 10 SP cells and (vi) 10 NSP cells. (D) H & E photos of tumours after inoculated into nude mice with (i) unsorted HK1 cells, (ii) SP cells and (iii) NSP cells. Tumours derived from SP and NSP cells showed similar histomorphology to unsorted HK1 cells (black arrowhead pointing to squamous carcinoma; long arrow pointing to keratinization). (iv) A SP cell derived tumour showed vascular invasion (white arrowhead pointing to the invasion of SP cells into a blood vessel). All photos at original magnification 400X.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: In vivotumour growth analysis of SP and non-SP (NSP) cells isolated from HK1 NPC cell line. (A) SP or NSP cell inoculation in nude mice. Distinct difference in tumour latency was observed in the lowest inoculated cell number (10 cells). (B) Limiting dilution analysis of SP and NSP tumours. Tumour-initiating cells (TICs) were similarly enriched in both SP and NSP cells (p > 0.05). (C) Representative gross morphology of tumours inoculated with (i) 1000 SP cells, (ii) 1000 NSP cells, (iii) 100 SP cells, (iv) 100 NSP cells, (v) 10 SP cells and (vi) 10 NSP cells. (D) H & E photos of tumours after inoculated into nude mice with (i) unsorted HK1 cells, (ii) SP cells and (iii) NSP cells. Tumours derived from SP and NSP cells showed similar histomorphology to unsorted HK1 cells (black arrowhead pointing to squamous carcinoma; long arrow pointing to keratinization). (iv) A SP cell derived tumour showed vascular invasion (white arrowhead pointing to the invasion of SP cells into a blood vessel). All photos at original magnification 400X.

Mentions: A pilot study showed that 1000 unsorted HK1 cells could form tumours in nude mice (data not shown). To determine the tumour-forming ability of SP and NSP cells, 1000, 100 and 10 cells were inoculated subcutaneously into nude mice (Figure 5A). Three out of four tumours from inoculation of 10 SP cells which were detected earlier than the first tumour from inoculation of NSP cells of the same number suggested that SP cells may have a growth advantage compared to NSP cells. The growth advantage was however lost at higher inoculations of 100 and 1000 cells. Limiting dilution analysis using the Extreme Limiting Dilution Analysis (ELDA) software showed that the estimated number of tumour-initiating cells based on the in vivo assay was not significantly different between the SP and NSP subpopulations, implying that the difference in the potential of SP and NSP cells to form tumours was not significant (Figure 5B). Both SP and NSP tumours showed similar histomorphology as seen in unsorted HK1 cells grown in mice (Figure 5C). Histologically, there was also no substantial difference between SP and NSP tumours in the degree of differentiation, stromal reaction and cell pleomorphism (Figure 5D), except for one SP tumour which showed vascular invasion (Figure 5 Div).Figure 5


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)

In vivotumour growth analysis of SP and non-SP (NSP) cells isolated from HK1 NPC cell line. (A) SP or NSP cell inoculation in nude mice. Distinct difference in tumour latency was observed in the lowest inoculated cell number (10 cells). (B) Limiting dilution analysis of SP and NSP tumours. Tumour-initiating cells (TICs) were similarly enriched in both SP and NSP cells (p > 0.05). (C) Representative gross morphology of tumours inoculated with (i) 1000 SP cells, (ii) 1000 NSP cells, (iii) 100 SP cells, (iv) 100 NSP cells, (v) 10 SP cells and (vi) 10 NSP cells. (D) H & E photos of tumours after inoculated into nude mice with (i) unsorted HK1 cells, (ii) SP cells and (iii) NSP cells. Tumours derived from SP and NSP cells showed similar histomorphology to unsorted HK1 cells (black arrowhead pointing to squamous carcinoma; long arrow pointing to keratinization). (iv) A SP cell derived tumour showed vascular invasion (white arrowhead pointing to the invasion of SP cells into a blood vessel). All photos at original magnification 400X.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: In vivotumour growth analysis of SP and non-SP (NSP) cells isolated from HK1 NPC cell line. (A) SP or NSP cell inoculation in nude mice. Distinct difference in tumour latency was observed in the lowest inoculated cell number (10 cells). (B) Limiting dilution analysis of SP and NSP tumours. Tumour-initiating cells (TICs) were similarly enriched in both SP and NSP cells (p > 0.05). (C) Representative gross morphology of tumours inoculated with (i) 1000 SP cells, (ii) 1000 NSP cells, (iii) 100 SP cells, (iv) 100 NSP cells, (v) 10 SP cells and (vi) 10 NSP cells. (D) H & E photos of tumours after inoculated into nude mice with (i) unsorted HK1 cells, (ii) SP cells and (iii) NSP cells. Tumours derived from SP and NSP cells showed similar histomorphology to unsorted HK1 cells (black arrowhead pointing to squamous carcinoma; long arrow pointing to keratinization). (iv) A SP cell derived tumour showed vascular invasion (white arrowhead pointing to the invasion of SP cells into a blood vessel). All photos at original magnification 400X.
Mentions: A pilot study showed that 1000 unsorted HK1 cells could form tumours in nude mice (data not shown). To determine the tumour-forming ability of SP and NSP cells, 1000, 100 and 10 cells were inoculated subcutaneously into nude mice (Figure 5A). Three out of four tumours from inoculation of 10 SP cells which were detected earlier than the first tumour from inoculation of NSP cells of the same number suggested that SP cells may have a growth advantage compared to NSP cells. The growth advantage was however lost at higher inoculations of 100 and 1000 cells. Limiting dilution analysis using the Extreme Limiting Dilution Analysis (ELDA) software showed that the estimated number of tumour-initiating cells based on the in vivo assay was not significantly different between the SP and NSP subpopulations, implying that the difference in the potential of SP and NSP cells to form tumours was not significant (Figure 5B). Both SP and NSP tumours showed similar histomorphology as seen in unsorted HK1 cells grown in mice (Figure 5C). Histologically, there was also no substantial difference between SP and NSP tumours in the degree of differentiation, stromal reaction and cell pleomorphism (Figure 5D), except for one SP tumour which showed vascular invasion (Figure 5 Div).Figure 5

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