3A) In contrast, KRas-G12V-activation or p53-knockout alone did

3A). In contrast, KRas-G12V-activation or p53-knockout alone did not induce tumor growth, at least within the time investigated. The chosen oncogenic setup limited the life span of mice to 5-8 weeks following electroporation due to primary tumor progression. Panobinostat price Analysis of tumor growth after electroporation by measuring the tumor area in histologic sections showed that growth

was slightly decelerated after rapid growth between days 14 and 21 (Fig. 3B). When we performed histopathologic examinations in tissue of primary tumor and the tumor margin, we could clearly identify a mass-forming, intrahepatic cholangiocarcinoma reflecting the bile-ductular type,[30] which is characterized by glandular and ductular structures intersected by extended regions of tumor stroma (Fig. 3C, upper lane). It is well known that activated KRas leads to downstream induction of the mitogen-activated protein kinase (MAPK)-pathway by phosphorylation

GS-1101 solubility dmso of Erk. Staining tumor tissue slices for phospho-Erk revealed that ductular cells are highly positive for this MAPK-pathway activation marker (Fig. 3C, lower lane). Transformed cells should also be deleted of p53 by the cotransfected Cre-recombinase. To analyze KRas expression and p53-knockout, single tumor cells were isolated from tumor tissue and then analyzed by quantitative polymerase chain reaction (qPCR). Using this approach, tumor cells can be completely separated from tumor stroma and healthy liver cells, thus allowing accurate determination of tumor-specific p53 and KRas levels. Complementary DNA (cDNA) quantification of tumor cells showed high expression of KRas, whereas p53 was absent in comparison with nontransduced liver of p53fl/fl mice or the murine tumor cell line CMT64 (p53wt/KRas-G12V) as controls (Fig. 3D). To further confirm the complete p53-knockout, genotyping of isolated tumor cells was performed showing Cre-mediated recombination of intron 1 and intron 10 (Fig. 3E, bottom lane). For comparison, the nonrecombined loxP-sites within the introns

were amplified from liver tissue of nontransduced mice as this website well as wild-type alleles from the cell line CMT64, where the corresponding PCR products are shorter (Fig. 3E, middle lanes). As internal control, an exon 11-specific PCR-fragment was detected in all analyzed samples (Fig. 3E, upper lane). At the timepoint of death due to primary tumor growth, no metastases could be detected in any other organs. However, we observed satellites that were located close to the primary tumor. Importantly, these satellites seemed to already affect the vascular system, as they could be found to infiltrate vessels located in the periportal field (Fig. 3F, left). Staining phospho-Erk in these satellite structures also showed MAPK-pathway activation (Fig. 3F, right).

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