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A new method for identifying stem-like cells in esophageal cancer cell lines.

Almanaa TN, Geusz ME, Jamasbi RJ - J Cancer (2013)

Bottom Line: This line showed significantly greater ACAM staining and higher CD44 levels than YES-2.ACAM also showed significantly higher ALDH activity in YES-2CSC than in YES-2S, a cell line that has a diminished CSC subpopulation after having survived treatment with curcumin.This study also demonstrates a new method for generating and growing tumorspheres without the growth factor supplements normally used in medium to form tumorspheres.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.

ABSTRACT
Cancer stem cells (CSCs) appear to resist chemo-radiotherapy and initiate tumor recurrence in patients. Isolation and further characterization of this subpopulation is important for targeting CSCs. Flow cytometry using Aldefluor, a fluorescent substrate of aldehyde dehydrogenase, has been used to isolate CSCs from various cancer cell lines. However, new techniques are needed to locate and identify CSCs in culture for live-cell analyses such as fluorescence microscopy without introducing artifacts during cell sorting and to observe CSC and non-CSC interactions. Previously, we characterized a distinct CSC subpopulation within human esophageal cancer cell lines (ESCC). In this study we introduce the attached-cell Aldefluor method (ACAM) to detect CSCs in ESCC cell lines (KY-5, KY-10, TE-1, TE-8, YES-1, YES-2). To validate this technique, we isolated CSCs from the YES-2 parental line using standard Aldefluor flow cytometry to create a cell line enriched in CSCs (YES-2CSC). This line showed significantly greater ACAM staining and higher CD44 levels than YES-2. ACAM also showed significantly higher ALDH activity in YES-2CSC than in YES-2S, a cell line that has a diminished CSC subpopulation after having survived treatment with curcumin. ACAM stained cells within tumorspheres made from the CSC-enriched line but not differentiating cells from the tumorspheres. This study also demonstrates a new method for generating and growing tumorspheres without the growth factor supplements normally used in medium to form tumorspheres. ACAM should be evaluated using other cancer cell lines to further substantiate its effectiveness and to characterize CSCs in culture through various imaging techniques.

No MeSH data available.


Related in: MedlinePlus

Fluorescence intensity of ALDH1 reaction product in ESCC lines. A: Fluorescence in the ESCC lines, including YES-2CSC and YES-2S, after Aldefluor was applied to attached cells growing in culture. Scale bar = 100 µm B: Histograms showing the distribution of relative ALDH activity in the ESCC lines. C: Mean intensity of all ESCC lines. YES-2CSC is enriched in CSCs, and YES-2S is CSC-depleted. Oval indicates lines that are not significantly different from each other. Asterisks indicate the significantly different YES-2 lines. D: YES-2CSC compared with YES-2 to indicate the increase in stem-like cells in YES-2CSC. ADUs = Analog-to-digital units
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Figure 2: Fluorescence intensity of ALDH1 reaction product in ESCC lines. A: Fluorescence in the ESCC lines, including YES-2CSC and YES-2S, after Aldefluor was applied to attached cells growing in culture. Scale bar = 100 µm B: Histograms showing the distribution of relative ALDH activity in the ESCC lines. C: Mean intensity of all ESCC lines. YES-2CSC is enriched in CSCs, and YES-2S is CSC-depleted. Oval indicates lines that are not significantly different from each other. Asterisks indicate the significantly different YES-2 lines. D: YES-2CSC compared with YES-2 to indicate the increase in stem-like cells in YES-2CSC. ADUs = Analog-to-digital units

Mentions: To identify CSCs in monolayer cultures, we applied Aldefluor to the attached ESCC cell lines. Fluorescence staining was detected in the cytoplasm of all eight lines, including the YES-2CSC and YES-2S cell lines as shown in Figure 2A. Measuring the staining intensity of ALDH activity of individual cells indicated significant differences among the mean intensities of the cell lines (ANOVA, F=151.968, p<0.0001) (Figure 2B). The order from high to low mean staining intensity was as follows: KY-5, KY-10, YES-2CSC, YES-1, TE-8, YES-2, TE-1, YES-2S (Figure 2C). KY-5 cells had significantly higher mean Aldeflour staining than all the other cell lines (0<0.05, Scheffe test). The range was 122 ±2.82 (YES-2S) to 320 ±6.01 (KY-5) analog-to-digital units (ADUs). There was no significant difference between KY-10, YES-2CSC, YES-1, and TE-8, whereas all of the other lines were distinctly different from each other and from this intermediate group. When YES-2 and the two YES-2-derived lines were compared, they were found to be significantly different (ANOVA, F=136.5, p<0.0001). YES-2CSC had significantly higher ALDH activity than the original YES-2 line (Figure 2D), which was in turn higher than the curcumin-surviving line YES-2S (p<0.05).


A new method for identifying stem-like cells in esophageal cancer cell lines.

Almanaa TN, Geusz ME, Jamasbi RJ - J Cancer (2013)

Fluorescence intensity of ALDH1 reaction product in ESCC lines. A: Fluorescence in the ESCC lines, including YES-2CSC and YES-2S, after Aldefluor was applied to attached cells growing in culture. Scale bar = 100 µm B: Histograms showing the distribution of relative ALDH activity in the ESCC lines. C: Mean intensity of all ESCC lines. YES-2CSC is enriched in CSCs, and YES-2S is CSC-depleted. Oval indicates lines that are not significantly different from each other. Asterisks indicate the significantly different YES-2 lines. D: YES-2CSC compared with YES-2 to indicate the increase in stem-like cells in YES-2CSC. ADUs = Analog-to-digital units
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3753528&req=5

Figure 2: Fluorescence intensity of ALDH1 reaction product in ESCC lines. A: Fluorescence in the ESCC lines, including YES-2CSC and YES-2S, after Aldefluor was applied to attached cells growing in culture. Scale bar = 100 µm B: Histograms showing the distribution of relative ALDH activity in the ESCC lines. C: Mean intensity of all ESCC lines. YES-2CSC is enriched in CSCs, and YES-2S is CSC-depleted. Oval indicates lines that are not significantly different from each other. Asterisks indicate the significantly different YES-2 lines. D: YES-2CSC compared with YES-2 to indicate the increase in stem-like cells in YES-2CSC. ADUs = Analog-to-digital units
Mentions: To identify CSCs in monolayer cultures, we applied Aldefluor to the attached ESCC cell lines. Fluorescence staining was detected in the cytoplasm of all eight lines, including the YES-2CSC and YES-2S cell lines as shown in Figure 2A. Measuring the staining intensity of ALDH activity of individual cells indicated significant differences among the mean intensities of the cell lines (ANOVA, F=151.968, p<0.0001) (Figure 2B). The order from high to low mean staining intensity was as follows: KY-5, KY-10, YES-2CSC, YES-1, TE-8, YES-2, TE-1, YES-2S (Figure 2C). KY-5 cells had significantly higher mean Aldeflour staining than all the other cell lines (0<0.05, Scheffe test). The range was 122 ±2.82 (YES-2S) to 320 ±6.01 (KY-5) analog-to-digital units (ADUs). There was no significant difference between KY-10, YES-2CSC, YES-1, and TE-8, whereas all of the other lines were distinctly different from each other and from this intermediate group. When YES-2 and the two YES-2-derived lines were compared, they were found to be significantly different (ANOVA, F=136.5, p<0.0001). YES-2CSC had significantly higher ALDH activity than the original YES-2 line (Figure 2D), which was in turn higher than the curcumin-surviving line YES-2S (p<0.05).

Bottom Line: This line showed significantly greater ACAM staining and higher CD44 levels than YES-2.ACAM also showed significantly higher ALDH activity in YES-2CSC than in YES-2S, a cell line that has a diminished CSC subpopulation after having survived treatment with curcumin.This study also demonstrates a new method for generating and growing tumorspheres without the growth factor supplements normally used in medium to form tumorspheres.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.

ABSTRACT
Cancer stem cells (CSCs) appear to resist chemo-radiotherapy and initiate tumor recurrence in patients. Isolation and further characterization of this subpopulation is important for targeting CSCs. Flow cytometry using Aldefluor, a fluorescent substrate of aldehyde dehydrogenase, has been used to isolate CSCs from various cancer cell lines. However, new techniques are needed to locate and identify CSCs in culture for live-cell analyses such as fluorescence microscopy without introducing artifacts during cell sorting and to observe CSC and non-CSC interactions. Previously, we characterized a distinct CSC subpopulation within human esophageal cancer cell lines (ESCC). In this study we introduce the attached-cell Aldefluor method (ACAM) to detect CSCs in ESCC cell lines (KY-5, KY-10, TE-1, TE-8, YES-1, YES-2). To validate this technique, we isolated CSCs from the YES-2 parental line using standard Aldefluor flow cytometry to create a cell line enriched in CSCs (YES-2CSC). This line showed significantly greater ACAM staining and higher CD44 levels than YES-2. ACAM also showed significantly higher ALDH activity in YES-2CSC than in YES-2S, a cell line that has a diminished CSC subpopulation after having survived treatment with curcumin. ACAM stained cells within tumorspheres made from the CSC-enriched line but not differentiating cells from the tumorspheres. This study also demonstrates a new method for generating and growing tumorspheres without the growth factor supplements normally used in medium to form tumorspheres. ACAM should be evaluated using other cancer cell lines to further substantiate its effectiveness and to characterize CSCs in culture through various imaging techniques.

No MeSH data available.


Related in: MedlinePlus