Pancreatic Ductal Organoids React Kras Dependent to the Removal of Tumor Suppressive Roadblocks

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is still the Achilles heel in modern oncology, with an increasing incidence accompanied by a persisting high mortality. The developmental process of PDAC is thought to be stepwise via precursor lesions and sequential accumulation of mutations. Thereby, current sequencing studies recapitulate this genetic heterogeneity in PDAC and show besides a handful of driver mutations (KRAS, TP53) a plethora of passenger mutations that allow to define subtypes. However, modeling the mutations of interest and their effects is still challenging. Interestingly, organoids have the potential to recapitulate in vitro, the in vivo characteristics of the tissue they originate from. Here, we could establish and develop tools allowing us to isolate, culture, and genetically modify ductal mouse organoids. Transferred to known effectors in the IPMN-PDAC sequence, we could reveal significantly increased proliferative and self-renewal capacities for PTEN and RNF43 deficiency in the context of oncogenic KRAS^G12D in mouse pancreatic organoids. Overall, we were able to obtain promising data centering ductal organoids in the focus of future PDAC research.

Figure 1

Pancreas mouse organoids exhibit ductal origin. (a) Organoid size and numbers are depending on the age of the host, with decreased numbers and size when generated from eight compared to two months old WT mice. Organoids shown are low passages 5-7 (40x magnification). (b) Organoids are positive for CK19, FOXA2, and SOX9 (200x magnification). (c) Relative gene expression levels of ductal markers (CK19 and SOX9) and lack of expression of acinar and islet cell markers amylase and PDX1. Gene expression is normalized to HMBS. (d) Ki-67 immunofluorescence (200x magnification). (e) 49.7% of the cells are positive for Ki-67 in WT organoids.

Figure 2

Pten and Rnf43 loss supports ductal features in wild-type organoid cultures. (a) Representative pictures of organoids with shRNA knockdown of Pten (WT-shPten) or Rnf43 (WT-shRnf43) compared to WT scramble control after six days of culture (40x magnification). (b) Significantly decreased organoid growth rate after knockdown of Rnf43 (p = 0.0071). (c) RNF43 deficiency significantly increases the diameter of ductal organoids compared to WT scramble (p ≤ 0.0001). (d) Immunofluorescence stainings of shRNA-mediated knockdown Pten and Rnf43 organoids reveal the expression of FOXA2 and CK19 (400x magnification). (e) The knockdown of Pten (WT-shPten blue bar) significantly increases the expression of CK19 (p ≤ 0.0001) and SOX9 (p ≤ 0.0001) and for CK19 (p = 0.001) in case of Rnf43 (WT-shRnf43 red bar) knockdown compared to WT scramble (black bar). (f) Ki-67 immunostaining analyses of shRNA-mediated knockdown of Rnf43 and Pten in WT organoids (400x magnification). (g) Immunohistochemistry revealed no differences in the percentage of Ki-67-positive cells in WT-shPten (blue bar) or WT-shRnf43 (red bar) compared to the scramble control (black bar). For statistical analysis, two-tailed Student's t-test was used. p < 0.05 was considered to be statistically significant. Error bars represent the standard errors of the mean.

Figure 3

Oncogenic Kras fosters proliferation in PTEN- and RNF43-deficient ductal organoids. (a) Doxycycline-inducible KRAS^G12D (pLIX-403-KRAS^G12D) and control organoids are morphologically similar after six days of culture (40x magnification). (b) KRAS^G12D expression can be induced by doxycycline application in the pLIX-403-KRAS^G12D organoids. (c, d) KRAS^G12D induction does not change the number of organoids but significantly the size compared to control (p ≤ 0.0001). (e, f) In total, 57.6% of the cells are positive for Ki-67 (400x magnification). (g) Representative pictures of KC organoids with shRNA knockdown of Pten (KC-shPten) or Rnf43 (KC-shRnf43) compared to KC scramble control after eight days of culture (40x magnification). (h) Significant differences are observed in the number of organoids for KC-shPten (p = 0.0276) and KC-shRnf43 (p = 0.0234) organoids compared to control. (i) The diameter of KC organoids is significantly increased after knockdown of Pten (p < 0.0001) and Rnf43 (p < 0.0001). (j) Immunofluorescence staining of FOXA2 and CK19 in KC organoids with either shRNA-mediated knockdown of Pten (KC-shPten) or Rnf43 (KC-shRnf43) (400x magnification). (k) Relative gene expression levels are significantly elevated for CK19 (p = 0.0004) and PDX1 (p = 0.0006) in KC-shPten (blue bar) and for PDX1 (p = 0.037) in KC-shRnf43 (red bar) compared to KC scramble (black bar). Amylase is not expressed. For statistical analysis, two-tailed Student's t-test was used. p < 0.05 was considered to be statistically significant. Error bars represent the standard errors of the mean.