Selective assessment of low- and high-grade PanINs elucidated strong and diffuse expression of gastrokines in low-grade PanINs, whereas only focal expression in high-grade PanINs (Fig

Selective assessment of low- and high-grade PanINs elucidated strong and diffuse expression of gastrokines in low-grade PanINs, whereas only focal expression in high-grade PanINs (Fig. proteins were absent in healthy pancreas and invasive malignancy, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis. gene; however, signaling pathways driving the progression of precursor and invasive lesions are not yet fully comprehended [6, 7]. The gene driven KC ((KC) mice (https://www.ncbi.nlm.nih.gov/geo/ ID; GSE164620). In the pancreas of 4, 10, or 17 weeks aged KC mice, we observed prominent continuous upregulation of and (Fig. ?(Fig.1A),1A), paralleling to the age of the mice. Given that we detected gastrokines in the pancreas of KC mice, we further characterized gastrokine expression in the pancreas of KC mice and in human pancreatic biopsies. Open in a separate windows Fig. 1 Gastrokine expression in pre-malignant lesions of KC mice and in human pancreas samples.A Selected genes from the microarray analysis of KC mice (at 4, 10, and 17 weeks of age). B Temporal analysis of and transcripts in KC mice by qRT-PCR (one-way ANOVA). C H&E, GKN1, and GKN2 staining in KC mice (scale bars 50?m). Acini and ADM visualized in 10 weeks aged; low- and high-grade PanINs in 9 months aged and PDAC in 12 months aged KC mice. D and mRNA transcripts from patients undergoing pancreatic surgery due to lesions in the pancreas compared to healthy control pancreas. Expression levels are reported as dCT (40-Ct). (2-test malignancy: 12.53? ?3.84, 29.77? Mmp17 ?3.84; adenoma: 7.79? ?3.84, 10.92? ?3.85) E H&E, GKN1, and Omeprazole GKN2 immunohistochemistry in human low-grade PanINs and PDAC (scale bar 100?m). We assessed the pancreas from KC mice at different time points (4, 10, 17, 26, and 52 weeks) using quantitative (qPCR) and qualitative (IHC) methods. Similar to the microarray data, qPCR analysis showed marked upregulation of gastrokines in the mouse pancreas as early as 4 weeks, gradually increasing over time (Fig. ?(Fig.1B).1B). This increase paralleled with the progression of pre-malignant lesions in KC mice (Supplementary Fig. 1A). IHC analysis showed co-expression of GKN1 and GKN2 in PanIN lesions, with strong positivity in the cytoplasm of dysplastic epithelium (Fig. ?(Fig.1C,1C, Supplementary Fig. 1B). Further, we could confirm a correlation of GKNs expression with corresponding transcription factor NKX6.3 in PanIN lesions of KC mice (Supplementary Fig. 1C). Selective assessment of low- and high-grade PanINs elucidated strong and diffuse expression of gastrokines in low-grade PanINs, whereas only focal expression in high-grade PanINs (Fig. ?(Fig.1C).1C). Interestingly, GKNs were not detectable in normal pancreas, ADM, and invasive carcinoma (Fig. ?(Fig.1C1C). Clinical value for gastrokine proteins within pancreatic lesions of mice seemed relevant when we reevaluated an old data set from a comprehensive gene expression repository of human PanIN lesions via aRNA-longSAGE analysis. In this analysis, individual single-cell pools from microdissected PanIN-1B, PanIN-2, and PanIN-3 cells were compared with pooled normal pancreatic ductal cells. A Venn diagram (Supplementary Fig. 2A) illustrates the overlaps of differentially expressed genes among the PanIN grades. In this analysis, GKN1 expression showed prominent specificity in early PanIN lesions (62% and 17% of PanIN-1B and ?2 lesions, respectively) as it was not observed in acinar cells, PanIN-3 cells, or in PDAC cells (Supplementary Fig. 2B). expression was also confirmed by qRT-PCR on microdissected samples from individual patients made up of cells of normal acini, PanINs, and PDAC (Supplementary Fig. 2C). In this data set, was not detected due to the limited number of expression tags available Omeprazole at that time. To validate further aRNA-longSAGE data and to detect expression was observed in and around samples of other pancreatic lesions (e.g., SCA, SPN, or MCN), in lesions of non-pancreatic origin (e.g., metastasis), and in healthy pancreas tissue. In contrast, in and Omeprazole around PDAC samples, there was a frequent co-expression of and expression; among them, 41% co-expressed and expression. Similar.