Specifications
Immunogen
The immunogen is a Red Fluorescent Protein (RFP) fusion protein corresponding to the full length amino acid sequence (234aa) derived from the mushroom polyp coral Discosoma.
Target
Alternative Names
DsRed,DsRed2,GFP like chromoprotein,red fluorescent protein,Red fluorescent protein drFP583,Red fluorescent protein eqFP611,Red fluorescent protein FP593,Red fluorescent protein zoanRFP,RFP,
Citations
Publication ()
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Oncogenic KRAS cells use Wnt signalling and cell dormancy to override homeostatic cell elimination mechanisms in adult pancreas
Beatriz Salvador-Barbero, Markella Alatsatianos, Jennifer P. Morton, Owen J. Sansom, Catherine Hogan
Applications: IF
Reactive species: Mouse
"Abstract: Epithelial tissues use homeostatic defence mechanisms to actively expel aberrant or genetically mutant cells and prevent disease. When present in healthy tissues in low numbers, we show that cells expressing cancer-causing mutations (KrasG12D, p53R172H) compete with normal cells for survival and are often eliminated. Thus, tumour initiation must require mechanisms whereby mutant cells override tissue defence mechanisms to remain in a tissue; however, the biology of these initial events is poorly understood. Here, we use an in vivo model of sporadic tumorigenesis in the adult pancreas to show that a population of KrasG12D- or p53R172H-expressing cells are never eliminated from the epithelium. Using RNA sequencing of non-eliminated populations and quantitative fluorescence imaging, we show that β-catenin-independent Wnt5a signalling, and cell dormancy are key features of surviving KrasG12D cells in vivo. We demonstrate that Wnt5a (and not Wnt3a) inhibits apical extrusion of RasV12 cells in vitro by promoting stable E-cadherin-based cell-cell adhesions at RasV12-normal cell-cell boundaries. Inhibition of Wnt5a signalling restores E-cadherin dynamics at normal-mutant boundaries and apical extrusion in vitro. RasV12 cells arrested in the cell cycle are not extruded and this is rescued when Wnt signalling is inhibited. In the pancreas, Wnt signalling, E-cadherin and β-catenin are increased at cell-cell contacts between non-eliminated KrasG12D cells and normal neighbours. Importantly, we demonstrate that active Wnt signalling is a general mechanism required to promote KrasG12D and p53R172H cell survival in vivo. Treatment with porcupine inhibitor rescues pancreas tissue defence by switching mutant cell retention to cell expulsion. Our results suggest that RAS mutant cells activate Wnt and a dormant cell state to avoid cell expulsion and to survive in the adult pancreas."
Article snippet: For β-catenin E-cadherin, anti-K-cadherin, and RFP (Creative Diagnostics) IF staining, pancreas was embedded in butyl-methyl methacrylate plastic (BMMA) under UV following dehydration and resin infiltration. Tissue was sectioned in 2µm thick slices and rehydrated. Tissue staining followed the same protocol as IF in FFPE sections. 
Figure 1. Wnt pathway is activated at normal-mutant cell-cell boundaries in vivo.
KRASG12D cells override homeostatic cell elimination mechanisms in adult pancreas via Wnt5a and cell dormancy
Salvador-Barbero, B., Alatsatianos, M., Morton, J. P., Sansom, O. J., & Hogan, C.
Applications: IF
Reactive species: Mouse
"Abstract: Background & aims: The adult pancreas protects against cancer by actively expelling genetically mutated cells. Pancreatic cancer starts with cells carrying KRAS mutations; however, it is not clear how some KRAS mutant cells override cell elimination mechanisms to survive in tissues.
Methods: An in vivo mouse model of sporadic tumorigenesis was used to induce Kras and/or Tp53 mutations in low numbers of cells in the adult pancreas. The mutant cell fate was monitored over time using quantitative fluorescence imaging. Gene signatures of noneliminated mutant cell populations were identified using bulk RNA sequencing. Differential gene expression was overlapped with publicly available datasets. Key molecular pathways were validated in murine pancreas using immunofluorescence and functionally tested using inhibitor studies in vivo and epithelial coculture systems in vitro.
Results: Although most genetically mutant cells are eliminated from the adult pancreas, a population of KRASG12D- or p53R172H-expressing cells are stably retained. Wnt5a signaling, cell dormancy, and stemness were identified as key features of surviving KrasG12D cells in vivo. Wnt5a specifically inhibits apical extrusion of RasV12 cells by promoting stable E-cadherin-based cell-cell adhesions at RasV12: normal cell-cell boundaries in vitro. In the pancreas, Wnt signaling, E-cadherin, and β-catenin are increased at cell-cell contacts between noneliminated KrasG12D cells and normal neighbors. Active Wnt signaling is a general mechanism required to promote KrasG12D and p53R172H cell retention and cell survival in vivo.
Conclusions: RAS mutant cells activate Wnt5a and cell dormancy to avoid cell expulsion and to survive in the adult pancreas."
Article snippet: For β-catenin, E-cadherin, K-cadherin, and RFP (Creative Diagnostics, 1:500) immunofluorescence staining, pancreas was embedded in butyl-methyl methacrylate plastic under UV after dehydration and resin infiltration. 
Figure 1. Representative images of endogenous RFP fluorescence. 
Figure 2. Representative images of RFP and β-catenin staining. 
Figure 3. Representative images of endogenous RFP fluorescence in KC pancreas harvested 28 days post-vehicle/WNT-974-treatment.
COA
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