Limits...
Triple negative breast cancers comprise a highly tumorigenic cell subpopulation detectable by its high responsiveness to a Sox2 regulatory region 2 (SRR2) reporter.

Jung K, Gupta N, Wang P, Lewis JT, Gopal K, Wu F, Ye X, Alshareef A, Abdulkarim BS, Douglas DN, Kneteman NM, Lai R - Oncotarget (2015)

Bottom Line: We have recently described a novel phenotypic dichotomy within estrogen receptor-positive breast cancer cells; the cell subset responsive to a Sox2 regulatory region (SRR2) reporter (RR cells) are significantly more tumorigenic than the reporter unresponsive (RU) cells.Third, within the CD44(High)/CD24- tumor-initiating cell population derived from MDA-MB-231, RR cells were significantly more tumorigenic than RU cells in an in vivo SCID/Beige xenograft mouse model.To conclude, we described a novel phenotypic heterogeneity within TNBC, and the SRR2 reporter responsiveness is a useful marker for identifying a highly tumorigenic cell subset within the CD44(High)/CD24-tumor-initiating cell population.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
We have recently described a novel phenotypic dichotomy within estrogen receptor-positive breast cancer cells; the cell subset responsive to a Sox2 regulatory region (SRR2) reporter (RR cells) are significantly more tumorigenic than the reporter unresponsive (RU) cells. Here, we report that a similar phenomenon also exists in triple negative breast cancer (TNBC), with RR cells more tumorigenic than RU cells. First, examination of all 3 TNBC cell lines stably infected with the SRR2 reporter revealed the presence of a cell subset exhibiting reporter activity. Second, RU and RR cells purified by flow cytometry showed that RR cells expressed higher levels of CD44, generated more spheres in a limiting dilution mammosphere formation assay, and formed larger and more complex structures in Matrigel. Third, within the CD44(High)/CD24- tumor-initiating cell population derived from MDA-MB-231, RR cells were significantly more tumorigenic than RU cells in an in vivo SCID/Beige xenograft mouse model. Examination of 4 TNBC tumors from patients also revealed the presence of a RR cell subset, ranging from 1.1-3.8%. To conclude, we described a novel phenotypic heterogeneity within TNBC, and the SRR2 reporter responsiveness is a useful marker for identifying a highly tumorigenic cell subset within the CD44(High)/CD24-tumor-initiating cell population.

No MeSH data available.


Related in: MedlinePlus

RR cells exhibit higher CD44 expression, enhanced capacities for colony formation in vitro and higher frequency of mammosphere-forming cellsA. Flow cytometry analyses of MDA-MB-231 and SUM149 Unsorted SRR2 cells stained with CD44-APC. Cells were gated on the highest and lowest 10 to 20% GFP expression and analyzed for CD44-APC levels. B. Results for Matrigel colony formation assay, conventional mammosphere assay, and soft agar assay of untreated MDA-MB-231 RU and RR cells are shown. 2500 cells/well are seeded into a 96-well Matrigel colony formation assay and colonies are counted from photographs taken on Day 7. Photographs of Matrigel multi-cell colonies were stained with phalloidin and imaged by high content screening imaging microscopy. 10,000 cells/well are seeded into a 6-well mammosphere assay and counted on Day 7. 10,000 cells/well are seeded into a 24-well soft agar assay and counted on Day 28. C. Extreme limiting dilution analyses statistics and graphical depiction of results are shown of a limiting dilution mammosphere assay in a 96-well plate format. Cells were seeded in 10 seeding densities ranging from 1 to 1000 cells/well in 6 replicates each. D. MTS 2-dimensional proliferation assay quantification of untreated ER− RU and RR cells seeded at 2000 cells/well. 20 μL of MTS reagent is added with fresh media 2 hours prior to taking absorbance reading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496361&req=5

Figure 3: RR cells exhibit higher CD44 expression, enhanced capacities for colony formation in vitro and higher frequency of mammosphere-forming cellsA. Flow cytometry analyses of MDA-MB-231 and SUM149 Unsorted SRR2 cells stained with CD44-APC. Cells were gated on the highest and lowest 10 to 20% GFP expression and analyzed for CD44-APC levels. B. Results for Matrigel colony formation assay, conventional mammosphere assay, and soft agar assay of untreated MDA-MB-231 RU and RR cells are shown. 2500 cells/well are seeded into a 96-well Matrigel colony formation assay and colonies are counted from photographs taken on Day 7. Photographs of Matrigel multi-cell colonies were stained with phalloidin and imaged by high content screening imaging microscopy. 10,000 cells/well are seeded into a 6-well mammosphere assay and counted on Day 7. 10,000 cells/well are seeded into a 24-well soft agar assay and counted on Day 28. C. Extreme limiting dilution analyses statistics and graphical depiction of results are shown of a limiting dilution mammosphere assay in a 96-well plate format. Cells were seeded in 10 seeding densities ranging from 1 to 1000 cells/well in 6 replicates each. D. MTS 2-dimensional proliferation assay quantification of untreated ER− RU and RR cells seeded at 2000 cells/well. 20 μL of MTS reagent is added with fresh media 2 hours prior to taking absorbance reading.

Mentions: Using the established purified RU and RR cell clones derived from MDA-MB-231 and SUM149, we assessed the biological significance of the differential responsiveness to the SRR2 reporter. As shown in Figure 3A, CD44 is 2-fold higher in RR cells as compared to RU cells. In a Matrigel colony formation assay, we found that RR cells formed significantly more colonies (1.5X) than RU cells did; furthermore, the colonies formed by RR resulted in more complex multi-cellular structures, with a greater number of multi-cellular extensions protruding from the colonies into the Matrigel (Figure 3B). Compared to RU cells, RR cells also formed significantly more spheres (1.5X) in a mammosphere assay, and significantly more colonies (1.5X) in a soft agar assay (Figure 3B).


Triple negative breast cancers comprise a highly tumorigenic cell subpopulation detectable by its high responsiveness to a Sox2 regulatory region 2 (SRR2) reporter.

Jung K, Gupta N, Wang P, Lewis JT, Gopal K, Wu F, Ye X, Alshareef A, Abdulkarim BS, Douglas DN, Kneteman NM, Lai R - Oncotarget (2015)

RR cells exhibit higher CD44 expression, enhanced capacities for colony formation in vitro and higher frequency of mammosphere-forming cellsA. Flow cytometry analyses of MDA-MB-231 and SUM149 Unsorted SRR2 cells stained with CD44-APC. Cells were gated on the highest and lowest 10 to 20% GFP expression and analyzed for CD44-APC levels. B. Results for Matrigel colony formation assay, conventional mammosphere assay, and soft agar assay of untreated MDA-MB-231 RU and RR cells are shown. 2500 cells/well are seeded into a 96-well Matrigel colony formation assay and colonies are counted from photographs taken on Day 7. Photographs of Matrigel multi-cell colonies were stained with phalloidin and imaged by high content screening imaging microscopy. 10,000 cells/well are seeded into a 6-well mammosphere assay and counted on Day 7. 10,000 cells/well are seeded into a 24-well soft agar assay and counted on Day 28. C. Extreme limiting dilution analyses statistics and graphical depiction of results are shown of a limiting dilution mammosphere assay in a 96-well plate format. Cells were seeded in 10 seeding densities ranging from 1 to 1000 cells/well in 6 replicates each. D. MTS 2-dimensional proliferation assay quantification of untreated ER− RU and RR cells seeded at 2000 cells/well. 20 μL of MTS reagent is added with fresh media 2 hours prior to taking absorbance reading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496361&req=5

Figure 3: RR cells exhibit higher CD44 expression, enhanced capacities for colony formation in vitro and higher frequency of mammosphere-forming cellsA. Flow cytometry analyses of MDA-MB-231 and SUM149 Unsorted SRR2 cells stained with CD44-APC. Cells were gated on the highest and lowest 10 to 20% GFP expression and analyzed for CD44-APC levels. B. Results for Matrigel colony formation assay, conventional mammosphere assay, and soft agar assay of untreated MDA-MB-231 RU and RR cells are shown. 2500 cells/well are seeded into a 96-well Matrigel colony formation assay and colonies are counted from photographs taken on Day 7. Photographs of Matrigel multi-cell colonies were stained with phalloidin and imaged by high content screening imaging microscopy. 10,000 cells/well are seeded into a 6-well mammosphere assay and counted on Day 7. 10,000 cells/well are seeded into a 24-well soft agar assay and counted on Day 28. C. Extreme limiting dilution analyses statistics and graphical depiction of results are shown of a limiting dilution mammosphere assay in a 96-well plate format. Cells were seeded in 10 seeding densities ranging from 1 to 1000 cells/well in 6 replicates each. D. MTS 2-dimensional proliferation assay quantification of untreated ER− RU and RR cells seeded at 2000 cells/well. 20 μL of MTS reagent is added with fresh media 2 hours prior to taking absorbance reading.
Mentions: Using the established purified RU and RR cell clones derived from MDA-MB-231 and SUM149, we assessed the biological significance of the differential responsiveness to the SRR2 reporter. As shown in Figure 3A, CD44 is 2-fold higher in RR cells as compared to RU cells. In a Matrigel colony formation assay, we found that RR cells formed significantly more colonies (1.5X) than RU cells did; furthermore, the colonies formed by RR resulted in more complex multi-cellular structures, with a greater number of multi-cellular extensions protruding from the colonies into the Matrigel (Figure 3B). Compared to RU cells, RR cells also formed significantly more spheres (1.5X) in a mammosphere assay, and significantly more colonies (1.5X) in a soft agar assay (Figure 3B).

Bottom Line: We have recently described a novel phenotypic dichotomy within estrogen receptor-positive breast cancer cells; the cell subset responsive to a Sox2 regulatory region (SRR2) reporter (RR cells) are significantly more tumorigenic than the reporter unresponsive (RU) cells.Third, within the CD44(High)/CD24- tumor-initiating cell population derived from MDA-MB-231, RR cells were significantly more tumorigenic than RU cells in an in vivo SCID/Beige xenograft mouse model.To conclude, we described a novel phenotypic heterogeneity within TNBC, and the SRR2 reporter responsiveness is a useful marker for identifying a highly tumorigenic cell subset within the CD44(High)/CD24-tumor-initiating cell population.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
We have recently described a novel phenotypic dichotomy within estrogen receptor-positive breast cancer cells; the cell subset responsive to a Sox2 regulatory region (SRR2) reporter (RR cells) are significantly more tumorigenic than the reporter unresponsive (RU) cells. Here, we report that a similar phenomenon also exists in triple negative breast cancer (TNBC), with RR cells more tumorigenic than RU cells. First, examination of all 3 TNBC cell lines stably infected with the SRR2 reporter revealed the presence of a cell subset exhibiting reporter activity. Second, RU and RR cells purified by flow cytometry showed that RR cells expressed higher levels of CD44, generated more spheres in a limiting dilution mammosphere formation assay, and formed larger and more complex structures in Matrigel. Third, within the CD44(High)/CD24- tumor-initiating cell population derived from MDA-MB-231, RR cells were significantly more tumorigenic than RU cells in an in vivo SCID/Beige xenograft mouse model. Examination of 4 TNBC tumors from patients also revealed the presence of a RR cell subset, ranging from 1.1-3.8%. To conclude, we described a novel phenotypic heterogeneity within TNBC, and the SRR2 reporter responsiveness is a useful marker for identifying a highly tumorigenic cell subset within the CD44(High)/CD24-tumor-initiating cell population.

No MeSH data available.


Related in: MedlinePlus