Dclk1 Defines Quiescent Pancreatic Progenitors that Promote Injury-Induced Regeneration and Tumorigenesis

Abstract

The existence of adult pancreatic progenitor cells has been debated. While some favor the concept of facultative progenitors involved in homeostasis and repair, neither a location nor markers for such cells have been defined. Using genetic lineage tracing, we show that Doublecortin-like kinase-1 (Dclk1) labels a rare population of long-lived, quiescent pancreatic cells. In vitro, Dclk1+ cells proliferate readily and sustain pancreatic organoid growth. In vivo, Dclk1+ cells are necessary for pancreatic regeneration following injury and chronic inflammation. Accordingly, their loss has detrimental effects after cerulein-induced pancreatitis. Expression of mutant Kras in Dclk1+ cells does not affect their quiescence or longevity. However, experimental pancreatitis converts Kras mutant Dclk1+ cells into potent cancer-initiating cells. As a potential effector of Kras, Dclk1 contributes functionally to the pathogenesis of pancreatic cancer. Taken together, these observations indicate that Dclk1 marks quiescent pancreatic progenitors that are candidates for the origin of pancreatic cancer.

Figure 1. Pancreatic Dclk1+ cells are largely quiescent

A–D) Recombination in small (A) and large ducts (B), terminal duct/centroacinar (C) and acinar (D) cells in Dclk1 mTmG mice 24 hours post induction with Tamoxifen (n>5 mice). Arrows indicate recombined cells. E) Quantification of recombined cells by cellular compartment. F–H) Representative Immunofluorescence (green) for Dclk1 (F), pan-Cytokeratin (G) and Amylase (H) in Dclk1 tdTom mice. Arrows indicate recombined cells staining positive. I-L) Recombination in small (I) and large ducts (J), terminal duct/centroacinar (K) and acinar (L) cells in Dclk1 mTmG mice 12 months post induction with Tamoxifen (n>5 mice). Arrows indicate recombined cells. M) Traced acinar cluster 3 months post Tamoxifen N) Quantification of EpCam⁺/Dclk1⁺ cells in Dclk1 tdTom mice at baseline and 3 months after induction with Tamoxifen. Data represented as mean ± SEM, (n=4 mice) O–R) Recombination in the acinar (O&P) and ductal compartment (Q&R) in Dclk1-Cre mTmG mice at P7 (n=3 mice).

Figure 2. Dclk1+ cells display increased proliferation potential in vitro

A) Representative photographs of single cells (Day 0) and resulting spheres (Day 3–7) isolated from Dclk1 tdTom mice B) Sphere forming ability of tdTomato+ (red) and tdTomato- (green) cells shown as spheres/cell plated (n=4). C) Number of resulting spheres from Dclk1 DTA mice cultured in the absence (green) and presence of Tamoxifen (red). Data normalized to untreated controls (n=3). D) Consecutive photographs of the same sphere isolated after in vitro induction at day 3. E) Increase in sphere size (green) and traced pixels (blue) of the sphere depicted in D. F) Experimental setup for the data depicted in G. G) Number of resulting spheres from Dclk1 DTA mice after delayed treatment with Tamoxifen (red) and control mice (green) (n=3). Data normalized to untreated controls. All data represented as mean ± SEM.

Figure 3. Dclk1+ cells are critically involved in pancreatic regeneration

A) Fluorescent images of recombined acinar cells from Dclk1 mTmG control mice (left panel) and mice 1, 3 and 7 days after treatment with cerulein. B) Increase in acinar recombination depicted as singlets (green), doublets (red) and clones of three or more cells (blue) over the course of the study. Data normalized to untreated controls. (n≥3 mice/condition) C) Fluorescent images of recombined ductal cells from Dclk1 mTmG control mice and mice 1, 3 and 7 days after treatment with cerulein. D) Increase in ductal recombination depicted as singlets (green), doublets (red) and clones of three or more cells (blue) over the course of the study. Data normalized to untreated controls (n≥3 mice/condition). E) Quantification of Ki67 positive acinar cells in the Dclk1+ (green) & Dclk1- (red) lineage in the presence (+CR) and absence (−CR) of cerulein (n≥3 mice/condition). F) Quantification of Ki67 positive ductal cells in the Dclk1+ (green) & Dclk1- (red) lineage in the presence (+CR) and absence (−CR) of cerulein (n≥3 mice/condition). G–I) Morphometric quantification of recombination and representative fluorescent images from Dclk1 mTmG mice after (G) cerulein treatment (n=6 mice), (H) pancreatic duct ligation (n=4 mice) and I) partial pancreatectomy (n=4 mice) J) Survival curve of Dclk1 DTA (red) mice induced with Tamoxifen and treated with cerulein (n=8) and controls mice (Dclk1 mTmG - blue) treated with cerulein (n=8) K) Pancreatic weight/body weight ratio at time of euthanasia. Green = WT mice without cerulein (n=6), Red: DTA mice without cerulein & Tamoxifen (n=7), Blue: DTA mice without cerulein, treated with Tamoxifen (n=3), Yellow: DTA mice treated with cerulein and Tamoxifen (n=6) Data represented as mean ± SEM L) Representative H&E sections of pancreata from control and Dclk1 DTA mice at time of euthanasia M) Pancreatitis score in Dclk1 DTA mice (red) and controls (green) 3 and 7 days after cerulein treatment. (n≥ 3 mice/condition). All data represented as mean ± SEM.

Figure 4. Dclk1 gene expression contributes to pancreatic regeneration

A) Quantification of Dclk1 mRNA expression in Pdx1-Cre (Control – green) and Pdx1-Cre Dclk1^flox/flox (Dclk1^flox/flox – red) mice (n=3/condition). Data represented as mean ± SEM & normalized to controls. B) Number of resulting spheres from Pdx1-Cre Dclk1^flox/flox (DKO) mice (red) and control mice (green) (n=4). Data normalized to controls (Pdx1-Cre mice). C) Number of resulting spheres cultured in the absence (green) and the presence of 500 nm (yellow) or 1000 nm (red) XMD 8–92 (n=3 mice). Data normalized to untreated controls. D) Number of resulting spheres cultured in the absence (green) and the presence of 0,5 µm (yellow), 2,5 µm (red), 5 µm (blue) and 10 µm (violet) LRRK2-IN-1 (n=3 mice). Data normalized to untreated controls. E) Representative photographs of H&E staining in control mice (left) and DKO mice three days post chronic cerulein treatment (n=≥4 mice). F) Pancreatitis score in DKO and controls after chronic cerulein treatment. G) Pancreatic weight/body weight ratio in DKO and controls H) Representative photographs of Ki67 IHC in DKO and control mice. I) Quantification of Ki67 staining depicted in P. J) Quantification of cleaved Caspase 3 staining. All data represented as mean ± SEM.

Figure 5. Dclk1+ cells efficiently initiate pancreatic tumorigenesis

A) Low-grade mPanIN in a Dclk1 CreERT×LSL Kras^G12D mouse 3 months post induction with Tamoxifen. Arrow indicates the lesion. B) Representative fluorescent image of a Dclk1 Kras mTmG mouse 6 months after induction with Tamoxifen. C) Histopathology of a Dclk1 Kras mouse 8 weeks post treatment with cerulein. D) Tracing of mPanINs in Dclk1 Kras mTmG mice. E) IF for GFP (green) & Dclk1 (red) in a Dclk1 KRas mTmG mouse. F) Analysis of the area affected by mPanINs in Dclk1 KRas and control mice after short delay (short - 2 weeks) and prolonged delay (long – 4–6 months) of treatment with cerulein (Cer) G) Histopathology of a Dclk1 Kras (left) and a Mist1 Kras (right) mouse 8 weeks post treatment with cerulein H) Number of mPanINs in Dclk1 Kras (green) and Mist1 Kras (red) mice at indicated time points (n ≥ 3 mice/time point) I) Number of mPanINs (Figure 5H) per cell labeled. J) Representative H&E section from a ductal lesion in Dclk1 mTmG mice induced with Tamoxifen followed by implantation of DMBA K) Representative fluorescent image of a ductal lesion in Dclk1 mTmG mice induced with Tamoxifen followed by implantation of DMBA. L) Quantification of ductal lesions labeled in Dclk1 mTmG mice implanted with DMBA (n=4). All data represented as mean ± SEM.

Figure 6. Dclk1 is important for the progression of early neoplastic lesions

A) Resulting number of spheres isolated from KC mice cultured in the absence (green) and the presence of 250 nm (yellow), 500nm (red) or 1000nm (blue) XMD 8–92. Normalized to untreated controls (n=3 mice). B) Resulting number of spheres isolated from KC mice in the absence (green) and the presence of 0,5 µm (yellow), 2,5 µm (red), 5 µm (blue) and 10 µm (violet) LRRK2-IN-1. Normalized to untreated controls (n=3 mice). C) Resulting spheres isolated from KC mice (green) and KC DKO mice (red). Data normalized to controls (n=3). D) Resulting number of spheres isolated from KC mice (green) and KC DKO mice (red) treated with EGF. Data normalized to controls (n=≥3 mice). E) H&E sections from KC and KC DKO at 32 weeks of age. F) Area covered (percentage of the area analyzed) by unaffected pancreas, fibrosis and mPanINs in KC (green) and KC DKO mice at 32 weeks of age (n=4 mice). G) H&E sections from KC and KC DKO mice 16 weeks after treatment with cerulein. H) Analysis of normal and preneoplastic pancreatic tissue in KC and KC DKO mice (n=5). I) Typical lesions in KC (left) and KC DKO mice (right) 16 weeks after treatment with cerulein. All data represented as mean ± SEM.

Figure 7. Dclk1 is a potential Kras effector protein

A) IHC for pERK in mPanINs of KC and KC DKO mice B) Quantification of pERK staining in mPanINs of KC and KC DKO mice (n=5). C) Western Blot for ERK and pERK in KC and KC DKO mice (data of two independent experiments – images cropped). D&E) Western blot for ERK and pERK in MiaPaCa2 (E) and Panc1 (F) cells upon overexpression of Dclk1 (data of two independent experiments – images cropped) F) MTT assays of human pancreatic cancer cell lines upon overexpression of Dclk1 (data of three independent experiments). All data represented as mean ± SEM.