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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

SRR2 reporter activity is a novel marker to enrich for a more tumorigenic cell subset within the CD44/CD24 populationA. FACS dot plot showing the sorting scheme of the RU CD44High/CD24Neg and RR CD44High/CD24− subsets. Percentages of gated populations from the live single cell population are reported. Cells were subsequently collected and seeded at 2500 cells/well in a 96-well Matrigel colony formation assay. Photographs were taken at 5X objective on Day 7. B. Purified RU CD44High/CD24− and RR CD44High/CD24− cell subsets as described above from MDA-MB-231 SRR2 cells were resuspended in 1:1 Matrigel/PBS. 4000 cells were injected with 200 μL of Matrigel/PBS solution subcutaneously bilaterally into 6-8 week old SCID/Beige females. Photographs depict representative tumors at Day 40. Resultant tumors were dissociated and analyzed by flow cytometry for GFP and CD44 expression. Representative 2 of 6 mice shown. C. Fresh TNBC patient tumors were dissociated, infected with the lentiviral SRR2 reporter, and assessed for GFP by flow cytometry.
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Figure 4: SRR2 reporter activity is a novel marker to enrich for a more tumorigenic cell subset within the CD44/CD24 populationA. FACS dot plot showing the sorting scheme of the RU CD44High/CD24Neg and RR CD44High/CD24− subsets. Percentages of gated populations from the live single cell population are reported. Cells were subsequently collected and seeded at 2500 cells/well in a 96-well Matrigel colony formation assay. Photographs were taken at 5X objective on Day 7. B. Purified RU CD44High/CD24− and RR CD44High/CD24− cell subsets as described above from MDA-MB-231 SRR2 cells were resuspended in 1:1 Matrigel/PBS. 4000 cells were injected with 200 μL of Matrigel/PBS solution subcutaneously bilaterally into 6-8 week old SCID/Beige females. Photographs depict representative tumors at Day 40. Resultant tumors were dissociated and analyzed by flow cytometry for GFP and CD44 expression. Representative 2 of 6 mice shown. C. Fresh TNBC patient tumors were dissociated, infected with the lentiviral SRR2 reporter, and assessed for GFP by flow cytometry.

Mentions: Next, we asked if the SRR2 reporter activity is a useful marker to isolate a more robust tumorigenic subset within the CD44High/CD24− tumor-initiating cell population [11]. RU and RR derived from MDA-MB-231 were used for these experiments. As shown in Figure 4A, within the CD44High/CD24− population, RR cells gave rise to significantly more colonies (2X) in Matrigel (Figure 4A). We then performed SCID/Beige mouse xenograft assay using RU and RR cells within the CD44High/CD24− cell population. As shown in Figure 4B, RR cells were significantly more tumorigenic in vivo, forming significantly larger tumors within 6 weeks after xenografting. Moreover, upon dissociation of the resultant xenograft tumors, we found that the tumors derived from RR cells comprised mostly GFP-negative cells and a small subset of GFPlow cells suggesting that RR gave rise to RU cells in vivo (Figure 4B). In comparison, RU cells were homogeneously GFP-negative (Figure 4B).


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)

SRR2 reporter activity is a novel marker to enrich for a more tumorigenic cell subset within the CD44/CD24 populationA. FACS dot plot showing the sorting scheme of the RU CD44High/CD24Neg and RR CD44High/CD24− subsets. Percentages of gated populations from the live single cell population are reported. Cells were subsequently collected and seeded at 2500 cells/well in a 96-well Matrigel colony formation assay. Photographs were taken at 5X objective on Day 7. B. Purified RU CD44High/CD24− and RR CD44High/CD24− cell subsets as described above from MDA-MB-231 SRR2 cells were resuspended in 1:1 Matrigel/PBS. 4000 cells were injected with 200 μL of Matrigel/PBS solution subcutaneously bilaterally into 6-8 week old SCID/Beige females. Photographs depict representative tumors at Day 40. Resultant tumors were dissociated and analyzed by flow cytometry for GFP and CD44 expression. Representative 2 of 6 mice shown. C. Fresh TNBC patient tumors were dissociated, infected with the lentiviral SRR2 reporter, and assessed for GFP by flow cytometry.
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Related In: Results  -  Collection

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Figure 4: SRR2 reporter activity is a novel marker to enrich for a more tumorigenic cell subset within the CD44/CD24 populationA. FACS dot plot showing the sorting scheme of the RU CD44High/CD24Neg and RR CD44High/CD24− subsets. Percentages of gated populations from the live single cell population are reported. Cells were subsequently collected and seeded at 2500 cells/well in a 96-well Matrigel colony formation assay. Photographs were taken at 5X objective on Day 7. B. Purified RU CD44High/CD24− and RR CD44High/CD24− cell subsets as described above from MDA-MB-231 SRR2 cells were resuspended in 1:1 Matrigel/PBS. 4000 cells were injected with 200 μL of Matrigel/PBS solution subcutaneously bilaterally into 6-8 week old SCID/Beige females. Photographs depict representative tumors at Day 40. Resultant tumors were dissociated and analyzed by flow cytometry for GFP and CD44 expression. Representative 2 of 6 mice shown. C. Fresh TNBC patient tumors were dissociated, infected with the lentiviral SRR2 reporter, and assessed for GFP by flow cytometry.
Mentions: Next, we asked if the SRR2 reporter activity is a useful marker to isolate a more robust tumorigenic subset within the CD44High/CD24− tumor-initiating cell population [11]. RU and RR derived from MDA-MB-231 were used for these experiments. As shown in Figure 4A, within the CD44High/CD24− population, RR cells gave rise to significantly more colonies (2X) in Matrigel (Figure 4A). We then performed SCID/Beige mouse xenograft assay using RU and RR cells within the CD44High/CD24− cell population. As shown in Figure 4B, RR cells were significantly more tumorigenic in vivo, forming significantly larger tumors within 6 weeks after xenografting. Moreover, upon dissociation of the resultant xenograft tumors, we found that the tumors derived from RR cells comprised mostly GFP-negative cells and a small subset of GFPlow cells suggesting that RR gave rise to RU cells in vivo (Figure 4B). In comparison, RU cells were homogeneously GFP-negative (Figure 4B).

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