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Autochthonous mouse melanoma and mammary tumors do not express the pluripotency genes Oct4 and Nanog.

Schreiber C, Kuch V, Umansky V, Sleeman JP - PLoS ONE (2013)

Bottom Line: The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells.However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry.Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, University Heidelberg, Mannheim, Germany. caroline.schreiber@medma.uni-heidelberg.de

ABSTRACT
The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells. In somatic cells, inappropriate expression of these genes has been associated with loss of differentiation, malignant transformation, and the acquisition of cancer stem cell-like properties. As cancer stem cells have been suggested to underlie the growth and malignancy of tumors, Oct4 and Nanog may represent therapeutic targets. Their expression could also act as a marker of the cancer stem cell population, permitting its isolation and characterisation. Nevertheless, the existence of multiple pseudogenes and isoforms of these genes has complicated the interpretation of the data that supports a role for Oct4 and Nanog in the cancer context. Here we addressed this issue using knockin mice in which IRES elements are used to allow GFP expression under the control of the endogenous Oct4 or Nanog promoters, while maintaining correct expression of the Oct4 or Nanog gene. These mice were crossed with MT/ret mice that develop melanomas, and with MMTV-PyMT mice and MMTV-Neu mice that develop mammary adenocarcinomas. We analysed the tumors that developed in these compound mice for GFP expression. In this way we could assess transcription of Oct4 and Nanog in autochthonous cancers without the complication of factors such as pseudogene expression, alternative splicing and antibody specificity. Both the Oct4 and Nanog knockin tumor-bearing mice expressed GFP in blastocysts and testes as expected. However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry. Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter. Together these data suggest that Oct4 and Nanog are not expressed in tumor cells that arise in the autochthonous cancer models studied here.

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No GFP+ subpopulation can be detected in MT/ret, MMTV-PyMT or MMTV-Neu compound tumors.(A) Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumor cells and their respective GFP−/− control tumor cells. Cells were freshly isolated from tumors, enriched for Lin- cells and analysed for GFP expression by FACS. The percentage of GFP+ cells compared to GFP negative control tumors is indicated in the gate. (B) Table summarizing the results of the FACS analyses performed. The numbers represent the mean value of the percentage of GFP+ cells of all analysed samples per tumor model, ± SEM. The total number of analysed samples per tumor model is indicated in brackets. (C) Representative FACS analysis of two Neu control tumors showing variation in background fluorescence.
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pone-0057465-g003: No GFP+ subpopulation can be detected in MT/ret, MMTV-PyMT or MMTV-Neu compound tumors.(A) Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumor cells and their respective GFP−/− control tumor cells. Cells were freshly isolated from tumors, enriched for Lin- cells and analysed for GFP expression by FACS. The percentage of GFP+ cells compared to GFP negative control tumors is indicated in the gate. (B) Table summarizing the results of the FACS analyses performed. The numbers represent the mean value of the percentage of GFP+ cells of all analysed samples per tumor model, ± SEM. The total number of analysed samples per tumor model is indicated in brackets. (C) Representative FACS analysis of two Neu control tumors showing variation in background fluorescence.

Mentions: Having established that GFP is expressed appropriately under the control of the endogenous Oct4 or Nanog promoters in the compound transgenic mice, we then used FACS analysis to investigate the presence of GFP+ tumor cells in primary tumors from Oct4GFP and NanogGFP MT/ret, MMTV-PyMT and MMTV-Neu mice. As a negative control, tumors from MT/ret, MMTV-PyMT and MMTV-Neu without a germline GFP knockin in the Nanog or Oct4 genes were used. Disaggregated tumor cells were stained with CD31, CD45.2, CD140a and Ter-119 to allow leukocytes and endothelial, erythroid and mesenchymal cells to be excluded from the analysis. Dead cells were also excluded. A gate for GFP-positive cells was set using a control tumor, and all positive events in this gate for the corresponding knockin tumor cells and additional negative control tumor cells were measured (Figure 3). We could not detect a GFP+ subpopulation of tumor cells for any of the analysed knockin tumors. Although for some mice we observed an apparent very low percentage of GFP+ tumor cells above background (Figure 3A and B), we also observed a similar distribution of GFP+ cells in the negative controls in which no GFP could be expressed (Figure 3A and 3C), suggesting that the very low percentage of apparently GFP-positive tumor cells from the Oct4GFP and NanogGFP tumors reflects variation in the background signal in the gated GFP channel.


Autochthonous mouse melanoma and mammary tumors do not express the pluripotency genes Oct4 and Nanog.

Schreiber C, Kuch V, Umansky V, Sleeman JP - PLoS ONE (2013)

No GFP+ subpopulation can be detected in MT/ret, MMTV-PyMT or MMTV-Neu compound tumors.(A) Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumor cells and their respective GFP−/− control tumor cells. Cells were freshly isolated from tumors, enriched for Lin- cells and analysed for GFP expression by FACS. The percentage of GFP+ cells compared to GFP negative control tumors is indicated in the gate. (B) Table summarizing the results of the FACS analyses performed. The numbers represent the mean value of the percentage of GFP+ cells of all analysed samples per tumor model, ± SEM. The total number of analysed samples per tumor model is indicated in brackets. (C) Representative FACS analysis of two Neu control tumors showing variation in background fluorescence.
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getmorefigures.php?uid=PMC3585372&req=5

pone-0057465-g003: No GFP+ subpopulation can be detected in MT/ret, MMTV-PyMT or MMTV-Neu compound tumors.(A) Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumor cells and their respective GFP−/− control tumor cells. Cells were freshly isolated from tumors, enriched for Lin- cells and analysed for GFP expression by FACS. The percentage of GFP+ cells compared to GFP negative control tumors is indicated in the gate. (B) Table summarizing the results of the FACS analyses performed. The numbers represent the mean value of the percentage of GFP+ cells of all analysed samples per tumor model, ± SEM. The total number of analysed samples per tumor model is indicated in brackets. (C) Representative FACS analysis of two Neu control tumors showing variation in background fluorescence.
Mentions: Having established that GFP is expressed appropriately under the control of the endogenous Oct4 or Nanog promoters in the compound transgenic mice, we then used FACS analysis to investigate the presence of GFP+ tumor cells in primary tumors from Oct4GFP and NanogGFP MT/ret, MMTV-PyMT and MMTV-Neu mice. As a negative control, tumors from MT/ret, MMTV-PyMT and MMTV-Neu without a germline GFP knockin in the Nanog or Oct4 genes were used. Disaggregated tumor cells were stained with CD31, CD45.2, CD140a and Ter-119 to allow leukocytes and endothelial, erythroid and mesenchymal cells to be excluded from the analysis. Dead cells were also excluded. A gate for GFP-positive cells was set using a control tumor, and all positive events in this gate for the corresponding knockin tumor cells and additional negative control tumor cells were measured (Figure 3). We could not detect a GFP+ subpopulation of tumor cells for any of the analysed knockin tumors. Although for some mice we observed an apparent very low percentage of GFP+ tumor cells above background (Figure 3A and B), we also observed a similar distribution of GFP+ cells in the negative controls in which no GFP could be expressed (Figure 3A and 3C), suggesting that the very low percentage of apparently GFP-positive tumor cells from the Oct4GFP and NanogGFP tumors reflects variation in the background signal in the gated GFP channel.

Bottom Line: The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells.However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry.Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, University Heidelberg, Mannheim, Germany. caroline.schreiber@medma.uni-heidelberg.de

ABSTRACT
The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells. In somatic cells, inappropriate expression of these genes has been associated with loss of differentiation, malignant transformation, and the acquisition of cancer stem cell-like properties. As cancer stem cells have been suggested to underlie the growth and malignancy of tumors, Oct4 and Nanog may represent therapeutic targets. Their expression could also act as a marker of the cancer stem cell population, permitting its isolation and characterisation. Nevertheless, the existence of multiple pseudogenes and isoforms of these genes has complicated the interpretation of the data that supports a role for Oct4 and Nanog in the cancer context. Here we addressed this issue using knockin mice in which IRES elements are used to allow GFP expression under the control of the endogenous Oct4 or Nanog promoters, while maintaining correct expression of the Oct4 or Nanog gene. These mice were crossed with MT/ret mice that develop melanomas, and with MMTV-PyMT mice and MMTV-Neu mice that develop mammary adenocarcinomas. We analysed the tumors that developed in these compound mice for GFP expression. In this way we could assess transcription of Oct4 and Nanog in autochthonous cancers without the complication of factors such as pseudogene expression, alternative splicing and antibody specificity. Both the Oct4 and Nanog knockin tumor-bearing mice expressed GFP in blastocysts and testes as expected. However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry. Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter. Together these data suggest that Oct4 and Nanog are not expressed in tumor cells that arise in the autochthonous cancer models studied here.

Show MeSH
Related in: MedlinePlus