Strategies In Redifferentiating The Ductal Phenotype Of Murine Pancreatic Tumors Into A More Acinar Phenotype Using "Organoid" Models

Background:Pancreatic cancer is currently one of the deadliest solid malignancies.Most patients are not diagnosed with pancreatic cancer,until late in their diseases,after their cancer has metastasized to other distant organs.As the most common subtype of pancreatic cancer,pancreatic ductal adenocarcinoma(PDAC)is almost invariably associated with mutations in the KRAS gene.Recent studies in genetically engineered mouse models(GEMMs)suggest that ductal neoplasia predominantly initiates from acinar cells after transdifferentiation to a ductal phenotype.Given that pathways upstream or downstream of Kras signaling might be involved in the acinar-to-ductal transdifferentiation process,we hypothesized that it might be possible to redifferentiate pancreatic intraepithelial neoplasia(PanIN)and PDAC to a more acinar phenotype by blocking selected cell signaling pathways.Epigenetic modifications are also taken into consideration in this project.Methods:First of all we established 3D PanIN organoids from the PanIN lesion tissues from Mist1CreERT2/+;LSL-KrasG12D;R26m-TmG("KCiMilst1G")mice,and PDAC organoids from the PDAC tissues of Kras+/G12D;Trp53+/LSL-R172H;Pdx1-Cre("KPC")mice.We treated murine "KCiMist1G" PanIN organoids with a broad array of compounds in vitro,including MAPK inhibitors,MEK inhibitors,GSK3β inhibitors,PI3K inhibitors,MMP inhibitors,PKC iota inhibitors,HDAC inhibitors,DNA methytransferase inhibitors,and Sirtl inhibitors.We analyzed the differentiation status of these organoids by detecting the expression of typical acinar and ductal markers using immunoblotting,immunofluorescence,and quantative polymerase chain reaction analyses.Then we selected the most effective compounds and used them to treat murine "KPC" PD AC organoids,investigating if the malignant ductal phenotype of these PD AC organoids could be redifferentiated into acinar phenotype.Finally we built up an in vivo model by orthotopically transplanting the PD AC organoids into nude mice,and checked whether GSK3β inhibitor was able to redifferentiate PD AC cells into acinar phenotype.Results:The GSK3β inhibitor lithium chloride(LiCl),the PKCi inhibitor ATM,and the MMP inhibitor GM6001 were able to increase the mRNA expression of the acinar markers Amylase,Elastase,and CPA1 in murine“KCiMist1G”PanIN organoids by a factor of two-to four-fold.This was associated with a simultaneous significant decrease in the mRNA expression of ductal markers,including CK19,HNF1B,and Muc5ac.We then performed a kinetic analysis in murine "KPC" PDAC organoids using these three compounds at day 3,day 6,and day 9.We found that LiCl significantly upregulated the mRNA expression of acinar markers including Mist1 and CPA1,and downregulated the mRNA expression of ductal markers including SOX9,CK19,HNF1B,and Muc5ac,indicating partial conversion to a more acinar phenotype.We then treated the same PD AC organoids with LiCl at 4 different concentrations(10mM,20mM,30mM and 40mM)for 6 days,and observed a dose-dependent increase in the protein expression of the acinar markers Mist1 and CPA1.Downregulation of the mRNA expression of p-catenin targeting genes Axin2 and Lefl demonstrated that LiCl inhibited the transcriptional activity of β-catenin pathway in PDAC organoids.The combinational use of HD AC inhibitor SAHA and DNA methytransferase inhibitor 5-Aza and SAHA/EX527 could upregulate the mRNA expression of acinar markers(Mist1 and CPA1)by a factor of 1.5-to 3-fold,and downregulate the expression of ductal markers.Also the combinational use of HDAC inhibitor SAHA and Sirtl inhibitor EX527 was able to increase the mRNA expression of acinar markers(Amylase,Elastase,and CPA1)by a factor of 2-to 4-fold,and decrease the expression level of ductal markers.Treating PDAC organoids with SAHA and 5-Aza increased the mRNA expression of acinar markers to an even greater level(Amylase,more than 50-fold;CPA1,more than 20-fold);and treating PD AC organoids with SAHA and EX527 enhanced the mRNA expression of Amylase to more than 10-fold,indicating partial redifferentiation to a more acinar phenotype.Finally we orthotopically transplanted the GFP-positive PD AC organoids into nude mice mimicking the sequential progression of "low stage PanIN-high stage PanIN-advanced PDAC",and found that LiCl could induce partial conversion to a more acinar phenotype from PD AC in vivo.Conclusions:Based on analyses of murine PanIN and PDAC organoids,blocking selected cell signaling pathways leads to partial redifferentiation of ductal neoplasia to a more acinar phenotype,as well as epigenetic modifications.Among the cell signaling pathways we selected,GSK3β inhibition alone,and HD AC inhibition combined with DNA methytransferase inhibition and Sirt1 inhibition have the greatest efficacy in redifferentiating murine Kras-mutant pancreatic cancer cells towards an acinar cell phenotype.GSK3β,HD AC,DNA methytransferase,and Sirtl might therefore be therapeutic targets for human PanIN and PDAC.The potential in vivo redifferentiating effects of GSK3β inhibition and epigenetic modifications in mice with established PDAC,and the underlying molecular mechanisms are currently under investigation.What’s more,PanIN/PDAC organoidss and the nude mice orthotopically transplanted with these organoidss will be promising in vitro and in vivo models for pancreatic cancer research in the upcoming future.