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The novel myxofibrosarcoma cell line MUG-Myx1 expresses a tumourigenic stem-like cell population with high aldehyde dehydrogenase 1 activity.

Lohberger B, Stuendl N, Wolf E, Liegl-Atzwanger B, Leithner A, Rinner B - BMC Cancer (2013)

Bottom Line: These neoplasms mainly affect patients in the sixth to eighth decades of life and the overall 5-year survival rate is 60-70%.In addition, MUG-Myx1 ALDH1(high) cells showed an upregulation of the ABC transporter ABCB1 and ABCG2; higher c-Myc, E-cadherin and SOX-2 expression; and a higher potential for tumourigenicity and proliferation levels.Furthermore, because of their tumourigenicity, the cell line is also suitable for in vivo experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, Medical University of Graz, Auenbruggerplatz 5, A-8036, Graz, Austria. birgit.lohberger@medunigraz.at.

ABSTRACT

Background: Myxofibrosarcoma comprises a spectrum of malignant neoplasms withprominent myxoid stromata, cellular pleomorphism, and distinct curvilinear vascular patterns. These neoplasms mainly affect patients in the sixth to eighth decades of life and the overall 5-year survival rate is 60-70%.

Methods: After the establishment of the novel myxofibrosarcoma cell lines MUG-Myx1, cells were characterized using short tandem repeat (STR), copy number variation (CNV), and genotype/loss-of-heterozygosity (LOH) analyses. The growth behaviour of the cells was analyzed with the xCELLigence system and an MTS assay. The tumourigenicity of MUG-Myx1 was proved in NOD/SCID mice. Additionally, a stem-like cell population with high enzymatic activity of aldehyde dehydrogenase 1 (ALDH1(high)) was isolated for the first time from myxofibrosarcoma cells using the Aldefluor® assay followed by FACS analysis.

Results: The frozen primary parental tumour tissue and the MUG-Myx1 cell line showed the same STR profile at the markers D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, D8S1179, TPOX, and FGY. Typically, myxofibrosarcoma gain and/or amplification was mapped to 7p21.3-q31.1, q31.1-q31.33, q33-q36.2, p21.3, p21.2, p14.1-q11.23, q31.33-q33, p21.2-p14.1, q11.23-q21.3, q36.2-q36.3, which, respectively are known to harbour tumour-associated genes, including TIF, BRAF, MLL3, SMO, and MET. Typically an LOH for myxofibrosarcoma on chr5 q21 was found. In addition, MUG-Myx1 ALDH1(high) cells showed an upregulation of the ABC transporter ABCB1 and ABCG2; higher c-Myc, E-cadherin and SOX-2 expression; and a higher potential for tumourigenicity and proliferation levels.

Conclusion: The new myxofibrosarcoma cell line MUG-Myx1 was established to enrich the bank of publicly available cell lines, with respect to providing comprehensive genetic and epigenetic characterization. Furthermore, because of their tumourigenicity, the cell line is also suitable for in vivo experiments.

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Characterization of the ALDH1 high subpopulation. (A) Aldehyde dehydrogenase 1 (ALDH1) expression in MUG-Myx1 cells using the Aldefluor® assay. (B) ALDH1 expression in percentage of gated cells. (C) The normalized expression levels from ABC transporter genes and stemness markers in ALDH1high cells compared to ALDH1low control cells (black bar). (D) SCID mice ALDH1low and ALDH1high tumours differed significantly in their tumour weights. (E) The IHC analysis using anti-Ki-67 proliferation marker revealed an increased proliferation level of ALDH1high as compared to ALDH1low mouse tumours.
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Figure 3: Characterization of the ALDH1 high subpopulation. (A) Aldehyde dehydrogenase 1 (ALDH1) expression in MUG-Myx1 cells using the Aldefluor® assay. (B) ALDH1 expression in percentage of gated cells. (C) The normalized expression levels from ABC transporter genes and stemness markers in ALDH1high cells compared to ALDH1low control cells (black bar). (D) SCID mice ALDH1low and ALDH1high tumours differed significantly in their tumour weights. (E) The IHC analysis using anti-Ki-67 proliferation marker revealed an increased proliferation level of ALDH1high as compared to ALDH1low mouse tumours.

Mentions: We used the Aldefluor® assay followed by FACS analysis to assess the presence and quantity of ALDH1high cell populations in the MUG-Myx1 cell line. In order to set a marker for ALDH1high cells, diethylaminobenzaldehyde (DEAB) control cells were used to ensure the accuracy of the analysis. MUG-Myx1 cells in a low passage (p17) and in a high passage (p87) (Figure 3A) were treated in the presence of the ALDH1 inhibitor DEAB or stained with Aldefluor® reagent, which are defined as ALDH1low and ALDH1high cells. Sorting experiments were performed a minimum of seven times on each passage. The amount of ALDH1high cells given on average ± SD was 6.16 ± 1.75% for the lower passage (n = 7) and 4.53 ± 1.55% for the higher passage of MUG-Myx1 (n = 8) (Figure 3B).


The novel myxofibrosarcoma cell line MUG-Myx1 expresses a tumourigenic stem-like cell population with high aldehyde dehydrogenase 1 activity.

Lohberger B, Stuendl N, Wolf E, Liegl-Atzwanger B, Leithner A, Rinner B - BMC Cancer (2013)

Characterization of the ALDH1 high subpopulation. (A) Aldehyde dehydrogenase 1 (ALDH1) expression in MUG-Myx1 cells using the Aldefluor® assay. (B) ALDH1 expression in percentage of gated cells. (C) The normalized expression levels from ABC transporter genes and stemness markers in ALDH1high cells compared to ALDH1low control cells (black bar). (D) SCID mice ALDH1low and ALDH1high tumours differed significantly in their tumour weights. (E) The IHC analysis using anti-Ki-67 proliferation marker revealed an increased proliferation level of ALDH1high as compared to ALDH1low mouse tumours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219478&req=5

Figure 3: Characterization of the ALDH1 high subpopulation. (A) Aldehyde dehydrogenase 1 (ALDH1) expression in MUG-Myx1 cells using the Aldefluor® assay. (B) ALDH1 expression in percentage of gated cells. (C) The normalized expression levels from ABC transporter genes and stemness markers in ALDH1high cells compared to ALDH1low control cells (black bar). (D) SCID mice ALDH1low and ALDH1high tumours differed significantly in their tumour weights. (E) The IHC analysis using anti-Ki-67 proliferation marker revealed an increased proliferation level of ALDH1high as compared to ALDH1low mouse tumours.
Mentions: We used the Aldefluor® assay followed by FACS analysis to assess the presence and quantity of ALDH1high cell populations in the MUG-Myx1 cell line. In order to set a marker for ALDH1high cells, diethylaminobenzaldehyde (DEAB) control cells were used to ensure the accuracy of the analysis. MUG-Myx1 cells in a low passage (p17) and in a high passage (p87) (Figure 3A) were treated in the presence of the ALDH1 inhibitor DEAB or stained with Aldefluor® reagent, which are defined as ALDH1low and ALDH1high cells. Sorting experiments were performed a minimum of seven times on each passage. The amount of ALDH1high cells given on average ± SD was 6.16 ± 1.75% for the lower passage (n = 7) and 4.53 ± 1.55% for the higher passage of MUG-Myx1 (n = 8) (Figure 3B).

Bottom Line: These neoplasms mainly affect patients in the sixth to eighth decades of life and the overall 5-year survival rate is 60-70%.In addition, MUG-Myx1 ALDH1(high) cells showed an upregulation of the ABC transporter ABCB1 and ABCG2; higher c-Myc, E-cadherin and SOX-2 expression; and a higher potential for tumourigenicity and proliferation levels.Furthermore, because of their tumourigenicity, the cell line is also suitable for in vivo experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, Medical University of Graz, Auenbruggerplatz 5, A-8036, Graz, Austria. birgit.lohberger@medunigraz.at.

ABSTRACT

Background: Myxofibrosarcoma comprises a spectrum of malignant neoplasms withprominent myxoid stromata, cellular pleomorphism, and distinct curvilinear vascular patterns. These neoplasms mainly affect patients in the sixth to eighth decades of life and the overall 5-year survival rate is 60-70%.

Methods: After the establishment of the novel myxofibrosarcoma cell lines MUG-Myx1, cells were characterized using short tandem repeat (STR), copy number variation (CNV), and genotype/loss-of-heterozygosity (LOH) analyses. The growth behaviour of the cells was analyzed with the xCELLigence system and an MTS assay. The tumourigenicity of MUG-Myx1 was proved in NOD/SCID mice. Additionally, a stem-like cell population with high enzymatic activity of aldehyde dehydrogenase 1 (ALDH1(high)) was isolated for the first time from myxofibrosarcoma cells using the Aldefluor® assay followed by FACS analysis.

Results: The frozen primary parental tumour tissue and the MUG-Myx1 cell line showed the same STR profile at the markers D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, D8S1179, TPOX, and FGY. Typically, myxofibrosarcoma gain and/or amplification was mapped to 7p21.3-q31.1, q31.1-q31.33, q33-q36.2, p21.3, p21.2, p14.1-q11.23, q31.33-q33, p21.2-p14.1, q11.23-q21.3, q36.2-q36.3, which, respectively are known to harbour tumour-associated genes, including TIF, BRAF, MLL3, SMO, and MET. Typically an LOH for myxofibrosarcoma on chr5 q21 was found. In addition, MUG-Myx1 ALDH1(high) cells showed an upregulation of the ABC transporter ABCB1 and ABCG2; higher c-Myc, E-cadherin and SOX-2 expression; and a higher potential for tumourigenicity and proliferation levels.

Conclusion: The new myxofibrosarcoma cell line MUG-Myx1 was established to enrich the bank of publicly available cell lines, with respect to providing comprehensive genetic and epigenetic characterization. Furthermore, because of their tumourigenicity, the cell line is also suitable for in vivo experiments.

Show MeSH
Related in: MedlinePlus