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MALDI imaging mass spectrometry for in situ proteomic analysis of preneoplastic lesions in pancreatic cancer.

Grüner BM, Hahne H, Mazur PK, Trajkovic-Arsic M, Maier S, Esposito I, Kalideris E, Michalski CW, Kleeff J, Rauser S, Schmid RM, Küster B, Walch A, Siveke JT - PLoS ONE (2012)

Bottom Line: Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions.Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells.In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.

View Article: PubMed Central - PubMed

Affiliation: II Medizinische Klinik, Technische Universität München, Munich, Germany.

ABSTRACT
The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosin-beta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.

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Expression analysis of TMSB4X.(A) TMSB4X mRNA is significantly increased in Ptf1a+/Cre;Kras+/G12D (CK) mice compared to wild type control (p = 0.01, n = 8 vs. 6 mice). Expression levels are normalized to wild type. Error bars indicate the standard deviations normalized to the mean of the wild type. (B) Staining for TMSB4X on tissue samples from 10–30 week old CK mice (n = 10) shows expression in PanINs (arrowhead) but not in acinar (asterisk) cells (i). High-grade mPanIN3 express TMSB4X (ii). Expression in human PanIN3 (iii) and human PDAC (iv) is also detectable. Scale bars represent 50 µm. (C) ELISA for TMSB4X from serum samples of wild type and CK mice (n = 7 vs. 14 mice). The serum concentration of TMSB4X is significantly upregulated in CK mice (p = 0.043, Wilcoxon test). (D) ELISA for TMSB4X from serum samples of PDAC and CP patients as well as healthy donors. Medians are marked by red lines.
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pone-0039424-g004: Expression analysis of TMSB4X.(A) TMSB4X mRNA is significantly increased in Ptf1a+/Cre;Kras+/G12D (CK) mice compared to wild type control (p = 0.01, n = 8 vs. 6 mice). Expression levels are normalized to wild type. Error bars indicate the standard deviations normalized to the mean of the wild type. (B) Staining for TMSB4X on tissue samples from 10–30 week old CK mice (n = 10) shows expression in PanINs (arrowhead) but not in acinar (asterisk) cells (i). High-grade mPanIN3 express TMSB4X (ii). Expression in human PanIN3 (iii) and human PDAC (iv) is also detectable. Scale bars represent 50 µm. (C) ELISA for TMSB4X from serum samples of wild type and CK mice (n = 7 vs. 14 mice). The serum concentration of TMSB4X is significantly upregulated in CK mice (p = 0.043, Wilcoxon test). (D) ELISA for TMSB4X from serum samples of PDAC and CP patients as well as healthy donors. Medians are marked by red lines.

Mentions: To investigate whether ALB1 and TMSB4X are also present on transcriptional level in tumorigenic pancreata, we isolated total pancreatic RNA from 8 Ptf1a+/Cre;Kras+/G12D and 6 wild type littermates at mixed age between 4.5 and 9 months and performed quantitative RT-PCR analysis for these two candidates. The expression of both transcripts was significantly upregulated in Ptf1a+/Cre;Kras+/G12D in comparison to wild type mice (p≤0.05, Figure 3B and 4A).


MALDI imaging mass spectrometry for in situ proteomic analysis of preneoplastic lesions in pancreatic cancer.

Grüner BM, Hahne H, Mazur PK, Trajkovic-Arsic M, Maier S, Esposito I, Kalideris E, Michalski CW, Kleeff J, Rauser S, Schmid RM, Küster B, Walch A, Siveke JT - PLoS ONE (2012)

Expression analysis of TMSB4X.(A) TMSB4X mRNA is significantly increased in Ptf1a+/Cre;Kras+/G12D (CK) mice compared to wild type control (p = 0.01, n = 8 vs. 6 mice). Expression levels are normalized to wild type. Error bars indicate the standard deviations normalized to the mean of the wild type. (B) Staining for TMSB4X on tissue samples from 10–30 week old CK mice (n = 10) shows expression in PanINs (arrowhead) but not in acinar (asterisk) cells (i). High-grade mPanIN3 express TMSB4X (ii). Expression in human PanIN3 (iii) and human PDAC (iv) is also detectable. Scale bars represent 50 µm. (C) ELISA for TMSB4X from serum samples of wild type and CK mice (n = 7 vs. 14 mice). The serum concentration of TMSB4X is significantly upregulated in CK mice (p = 0.043, Wilcoxon test). (D) ELISA for TMSB4X from serum samples of PDAC and CP patients as well as healthy donors. Medians are marked by red lines.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383687&req=5

pone-0039424-g004: Expression analysis of TMSB4X.(A) TMSB4X mRNA is significantly increased in Ptf1a+/Cre;Kras+/G12D (CK) mice compared to wild type control (p = 0.01, n = 8 vs. 6 mice). Expression levels are normalized to wild type. Error bars indicate the standard deviations normalized to the mean of the wild type. (B) Staining for TMSB4X on tissue samples from 10–30 week old CK mice (n = 10) shows expression in PanINs (arrowhead) but not in acinar (asterisk) cells (i). High-grade mPanIN3 express TMSB4X (ii). Expression in human PanIN3 (iii) and human PDAC (iv) is also detectable. Scale bars represent 50 µm. (C) ELISA for TMSB4X from serum samples of wild type and CK mice (n = 7 vs. 14 mice). The serum concentration of TMSB4X is significantly upregulated in CK mice (p = 0.043, Wilcoxon test). (D) ELISA for TMSB4X from serum samples of PDAC and CP patients as well as healthy donors. Medians are marked by red lines.
Mentions: To investigate whether ALB1 and TMSB4X are also present on transcriptional level in tumorigenic pancreata, we isolated total pancreatic RNA from 8 Ptf1a+/Cre;Kras+/G12D and 6 wild type littermates at mixed age between 4.5 and 9 months and performed quantitative RT-PCR analysis for these two candidates. The expression of both transcripts was significantly upregulated in Ptf1a+/Cre;Kras+/G12D in comparison to wild type mice (p≤0.05, Figure 3B and 4A).

Bottom Line: Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions.Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells.In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.

View Article: PubMed Central - PubMed

Affiliation: II Medizinische Klinik, Technische Universität München, Munich, Germany.

ABSTRACT
The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosin-beta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.

Show MeSH
Related in: MedlinePlus