CDKN2A-Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome
<p>(<b>A</b>) Pedigree of a pair of siblings carrying a heterozygous <span class="html-italic">CDKN2A</span> germline variant concurrently resulting in known missense variants of the two transcripts <span class="html-italic">P16</span> and <span class="html-italic">P14</span>, NM_000077.4:c.301G>T (NP_000068.1:p.Gly101Trp) and NM_058195.3:c.344G>T (NP_478102.2:p.Arg115Leu). For clarity, this genotype will be hereafter referred to as <span class="html-italic">CDKN2A</span><sup>WT/#</sup>. In the diagram, only the amino acid change in P16 (G101W) is indicated for each relative, if known. Age at death is indicated by a d., age of onset of the disease by an o. Individual II-5 developed uterine cancer at a young age and bladder cancer at an older age. III-4 suffers from a carcinoma of the external auditory canal, whereas the cancer entities of I-1 and I-4 were unknown. Consultant probands P1 (IV-1) and P2 (IV-2) are highlighted with an arrow. If the <span class="html-italic">CDKN2A</span> variant was confirmed by genetic analysis, it is displayed in bold. The displayed age denotes the age at which cancer was diagnosed. (<b>B</b>) Schema of iPSC generation by reprogramming of <span class="html-italic">CDKN2A</span><sup>WT/#</sup> patient hair keratinocytes and subsequent differentiation to pancreatic progenitors (PPs). (<b>C</b>) Keratinocytes growing out from a hair root and an iPSC colony derived from reprogrammed keratinocytes. (<b>D</b>) IF staining confirmed expression of pluripotency markers OCT4, NANOG, and SSEA4 in the generated patient-specific iPSCs. Scale bar: 50 µm. (<b>E</b>) Sequencing of iPSCs from both donors confirming the heterozygous <span class="html-italic">CDKN2A</span> variant, chr9:g.21971058C>A (hg38). (<b>F</b>) Patient-specific iPSCs differentiated efficiently into PPs. Representative original flow cytometry plots of undifferentiated iPSCs and <span class="html-italic">CDKN2A</span><sup>WT/#</sup> P2 iPSCs differentiated for 13 days to PPs are shown next to quantification of PDX1/NKX6-1 double positive cells. <span class="html-italic">n</span> = 3; ordinary one-way ANOVA with Tukey’s multiple comparison test. (<b>G</b>) IF staining for PP marker PDX1 and NKX6-1. Scale bar: 50 µm.</p> "> Figure 2
<p>(<b>A</b>) Schema of modeling FPC in vitro. Integration of the inducible <span class="html-italic">KRAS<sup>G12D</sup> piggyBac</span> construct in <span class="html-italic">CDKN2A</span><sup>WT/#</sup> patient iPSCs followed by directed differentiation to pancreatic duct-like organoids (PDLO) and oncogene activation by doxycycline (Dox) treatment. (<b>B</b>) Left: quantification of mCherry reporter expression in d27 PDLOs established from <span class="html-italic"><span class="html-italic">KRAS<sup>G12D</sup> </span>CDKN2A</span><span class="html-italic"><sup>WT/#</sup></span> iPSCs analyzed by flow cytometry after treatment with different Dox concentrations for 9 days; <span class="html-italic">n</span> = 2; in duplicates. Right: Flow cytometry analysis of transgenic <span class="html-italic">KRAS<sup>G12D</sup> </span>expression by HA-tag staining or direct measurement of the mCherry reporter signal; <span class="html-italic">n</span> = 3; in duplicates, Mann-Whitney test. (<b>C</b>) Left: bright field images of <span class="html-italic">CDKN2A</span>-mutated iPSC-derived PDLO cultures with or without oncogene induction for 9 days. Arrowheads highlight several lumen-filled PDLO structures. Scale bar: 200 µm. Right: the number of lumen-filled organoids was significantly increased after oncogene induction; <span class="html-italic">n</span> = 3; in triplicates, Mann-Whitney test. (<b>D</b>) IF staining of the HA-tag confirms successful transgene expression of <span class="html-italic">KRAS<sup>G12D</sup> </span>after Dox stimulation in PDLOs in vitro. Arrowheads point to disorganized PDLO structures. (<b>E</b>) Loss of apical ZO-1 in <span class="html-italic">KRAS<sup>G12D</sup></span>-expressing <span class="html-italic">CDKN2A</span><sup>WT/#</sup> patient PDLOs detected by IF staining. Scale bar: 25 µm. (<b>F</b>) IF staining of Ki-67 and tight junction protein CLDN1 indicate reduced proliferation and disrupted polarization, respectively. (<b>G</b>) Organoids were quantified in a blinded manner. Structures with a clear luminal ZO-1 expression, or with at least 50% of cells showing a strong basolateral CLDN1 localization, were counted as positive organoids. PDLOs lost epithelial polarity upon <span class="html-italic">KRAS<sup>G12D</sup> </span>induction (ZO-1: Odds ratio 11.83 with 95% confidence interval 4.56–30.70; CLDN1: Odds ratio 6.24 with 95% confidence interval 2.67–14.56); <span class="html-italic">n</span> = number of organoids, IF images from two cell lines were quantified; Fisher’s exact test. CLDN1: Claudin-1; ZO-1: Zona occludens-1. Scale bar: 50 µm, if not stated differently. Experiments were performed as end point analysis 7 or 9 days after treatment with 3 µg/mL Dox. (<b>B</b>,<b>C</b>,<b>G</b>), ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 3
<p>(<b>A</b>) Flow cytometry-based cell cycle analysis by EdU staining shows a tendency to reduced S-phase and a G0/G1 arrest after 7 days of Dox-induced <span class="html-italic">KRAS<sup>G12D</sup> </span>overexpression; <span class="html-italic">n</span> = 2; in duplicates. (<b>B</b>,<b>C</b>) Analysis of cell cycle regulators confirmed (<b>B</b>) transcriptional upregulation of <span class="html-italic">P14</span> by qPCR and (<b>C</b>) an increased number of P21 positive cells measured by flow cytometry. (<b>D</b>) IF images and (<b>E</b>) respective semi-automated quantification of the DNA damage marker γH2AX. More cells were γH2AX positive upon <span class="html-italic">KRAS<sup>G12D</sup> </span>induction (Odds ratio 2.40 with 95% confidence interval 2.01–2.87). (<b>F</b>) qPCR analysis for EMT transcription factors (<span class="html-italic">SLUG, SNAIL</span>) and mesenchymal markers (<span class="html-italic">VIM, FN1, N-CAD</span>) shows the upregulation of an EMT program upon KRAS induction. (<b>G</b>–<b>I</b>) Increased expression of mesenchymal proteins in Dox-treated organoids. IF staining for (<b>G</b>) VIM and (<b>H</b>) N-CAD/E-CAD and (<b>I</b>) respective image-based quantification. Organoids were quantified in a blinded manner. Organoids were counted as VIM<sup>high</sup> when a strong VIM signal was seen in PDLOs, whereas organoids were counted as N-CAD positive when at least one cell within an organoid was N-CAD positive. PDLOs acquired mesenchymal features upon <span class="html-italic">KRAS<sup>G12D</sup> </span>induction (VIM: <span class="html-italic">P</span>-value: 0.0259, Odds ratio 3.04 with 95% confidence interval 1.23–7.52; N-CAD: P-value: 0.0007; Odds ratio 9.11 with 95% confidence interval 2.03–40.91). (<b>B</b>,<b>C</b>,<b>F</b>): end point analysis after 7 or 9 days Dox treatment; <span class="html-italic">n</span> = 3; in duplicates, (<b>B</b>,<b>F</b>) two-way ANOVA with Sidak’s multiple comparison test, (<b>C</b>) Mann-Whitney test. (<b>E</b>,<b>I</b>): Dox treatment for 7 days; <span class="html-italic">n</span> = number of organoids, IF images from two cell lines were quantified; Fisher’s exact test. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001. In all experiments, Dox was applied at a concentration of 3 µg/mL. E-CAD: E-cadherin; FN1: Fibronectin-1; N-CAD: N-cadherin; VIM: Vimentin. Scale bars: 50 µm.</p> "> Figure 4
<p>(<b>A</b>) Schema of modeling FPC in vivo. <span class="html-italic">KRAS<sup>G12D</sup> </span>was induced for 8 weeks after orthotopic transplantation of <span class="html-italic">CDKN2A</span><span class="html-italic"><sup>WT/#</sup></span> iPSC-derived PDLOs in NSG mice. HE staining of identified engraftments with and without Dox treatment. Scale bar: 500 µm (overview), 100 µm (magnification); Hg lesion: high-grade lesion. The asterisk marks a glandular region, the pound sign a highly atypical and dysplastic region of the invasive PDAC-like tumor graft. (<b>B</b>) Summary of engraftment experiments. Left: Take rate was determined by identified engraftments per transplanted mice. Reporter expression was analyzed by mCherry and HA-tag IHC staining, and grafts were classified into normal ducts, low-grade lesions (non found), high-grade lesions, and PDAC-like tumors by the observed degree of atypia, dysplasia, and invasiveness. Right: Semi-quantitative marker expression: Each engraftment was classified into no (–), weak (+), moderate (++), or strong (+++) expression. The mean expression across the engraftments of one group is depicted. (<b>C</b>) <span class="html-italic">CDKN2A<sup>WT/#</sup></span> PDLO grafts without oncogene induction formed epithelial duct-like tissue (KRT19, KRT7). Some EMT marker expression (VIM, N-CAD) was accompanied by moderate expression of CA19-9. MUC5AC was only expressed in one graft. (<b>D</b>) Two <span class="html-italic">CDKN2A<sup>WT/#</sup></span> PDLO grafts 8 weeks after oncogene induction. The heterogeneous HA-tag expression in Hg lesion 1 correlated with increased dissemination and EMT, e.g., indicated by HA-tag and VIM staining (regions marked by arrows). This graft also strongly upregulated MUC5AC and CA19-9. The second invasive tumor graft (PDAC 1) with high levels of HA-tag expression was partially dedifferentiated (KRT19, KRT7, N-CAD) and only expressed CA19-9 and MUC5AC in some regions. All scale bars: 100 µm, if not stated differently.</p> "> Figure 5
<p>(<b>A</b>–<b>D</b>) Analysis of cell cycle checkpoint control of <span class="html-italic">CDKN2A<sup>WT/#</sup></span> PDLO grafts. IHC staining for (<b>A</b>) reporter expression (mCherry), proliferation (Ki-67), and apoptosis (cleaved CASP-3), (<b>B</b>) underlying P14, P53, P21, and (<b>C</b>) P16, RB signaling. (<b>D</b>) IF analysis of γH2AX and P16/P14. (<b>E</b>–<b>H</b>) Analysis of cell cycle checkpoint control of <span class="html-italic">CDKN2A<sup>WT/#</sup></span> PDLO grafts after 8 weeks of <span class="html-italic">KRAS<sup>G12D</sup> </span>induction analogous to A-D. All scale bars: 100 µm. CASP-3: cleaved caspase 3; (p)RB: (phosphorylated) Retinoblastoma protein.</p> "> Figure 6
<p>(<b>A</b>) Schematic overview of tumor suppressive role of <span class="html-italic">CDKN2A</span>. <span class="html-italic">CDKN2A</span> encodes for two different transcripts P16 and P14, which are both involved in controlling and restricting cell cycle progression. While the activation of P14 leads to an increase in P53 and P21, the induction of P16 results in an activation of RB by inhibiting its hyper-phosphorylation. Non-canonical, P53- and RB-independent functions of P14 and P16 are not illustrated in this simplified scheme. (<b>B</b>) Partial growth restriction in the PDAC-like tumor graft of transplanted <span class="html-italic">CDKN2A<sup>WT/#</sup> KRAS<sup>G12D</sup></span> PDLOs. In the less proliferative, highly dysplastic tumor regions indicated by a pound sign, P21 was strongly upregulated, probably mainly through a P14/P53 independent mechanism, P16 was highly expressed, and RB was not hyper-phosphorylated indicating an intact cell cycle progression barrier. All scale bars: 100 µm.</p> ">
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Generation of Patient-Specific CDKN2A-Mutated Induced Pluripotent Stem Cells
2.2. CDKN2A-Mutated PDLOs Display Oncogenic Effects after KRASG12D Induction
2.3. Tumor Formation in Xenotransplantation Experiments
2.4. Checkpoint Integrity in CDKN2AWT/# PDLO Grafts
3. Discussion
4. Materials and Methods
4.1. Materials Availability
4.2. Patient Material
4.3. Reprogramming Strategy for iPSC Generation
4.4. Embryonic and Induced Pluripotent Stem Cells
4.5. All-In-One piggyBac-System and Nucleofection
4.6. Pancreatic Progenitor Differentiation
4.7. PDLO Culture
4.8. Orthotopic Transplantation of PDLOs
4.9. qPCR Experiments
4.10. Flow Cytometry
4.11. Cell Cycle Analysis
4.12. ICC Staining
4.13. Paraffin Embedding of PDLOs
4.14. Staining on Paraffin Tissue Sections
4.15. Statistical Analysis
4.15.1. Flow Cytometry and qPCR of PDLOs
4.15.2. Bright Field Image Analysis of PDLOs
4.15.3. Quantification of IF images
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Self-Designed Primer Fwd | Self-Designed Primer Rev | Quantitect |
---|---|---|---|
FN1 | - | - | QT00038024 |
HMBS | - | - | QT00494130 |
N-CAD | - | - | QT00063196 |
P14 | ttttcgtggttcacatcccg | gggcgctgcccatcat | - |
P21 | gcgccatgtcagaaccgcct | gcaggcttcctgtgggcgga | - |
SLUG | cagtgattatttccccgtatc | ccccaaagatgaggagtatc | - |
SNAIL | gctccttcgtccttctcctc | tgacatctgagtgggtctgg | - |
VIM | gacaatgcgtctctggcacgtctt | tcctccgcctcctgcaggttctt | - |
Antibody | Species | Company | Catalogue No. | Condition | Dilution |
---|---|---|---|---|---|
CA19-9 | mouse | Thermo | MA5-12421 | ST Citrate | 1:500 |
CLDN1 | rabbit | Abcam | ab15098 | ST Tris | 1:100 |
cl-CASP3 | rabbit | Cell Signaling | 9664 | ST Citrate | 1:1000 |
E-CAD | mouse | BD Bioscience | 610182 | ST Citrate | 1:1000 |
HA-tag | rabbit | Cell Signaling | 3724 | ST Citrate | 1:500 |
Ki-67 | mouse | Dako | M7240 | ST Citrate | 1:200 |
Ki-67 | rabbit | Invitrogen | MA5-14520 | ST Citrate | 1:100 |
KRT19 (IF) | mouse | Dako | M0888 | ST Citrate | 1:100 |
KRT19 (IHC) | mouse | Dako | M0888 | Pronase | 1:100 |
KRT7 | mouse | Dako | M7018 | Pronase | 1:200 |
mCherry | rabbit | Abcam | ab167453 | No AGR | 1:500 |
MUC5AC | mouse | Santa Cruz | sc-33667 | ST Citrate | 1:100 |
N-CAD (IF) | rabbit | Cell Signaling | 13116 | ST Citrate | 1:100 |
N-CAD (IHC) | rabbit | Cell Signaling | 13116 | ST Tris | 1:100 |
P14 | mouse | Cell Signaling | 2407S | ST Citrate | 1:50 |
P16 | rabbit | Abcam | ab108349 | ST Citrate | 1:400 |
P21 | rabbit | Abcam | ab109520 | ST Citrate | 1:300 |
P53 | mouse | Santa Cruz | sc-47698 | ST Citrate | 1:100 |
pRB | rabbit | Cell Signaling | 8516 | ST Citrate | 1:200 |
RB | mouse | Cell Signaling | 9309 | ST Citrate | 1:400 |
VIM | rabbit | Cell Signaling | 5741S | ST Citrate | 1:500 |
ZO-1 | mouse | Thermo | 33-9100 | ST Citrate | 1:500 |
γH2AX | rabbit | Cell Signaling | 9718 | ST Citrate | 1:400 |
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Merkle, J.; Breunig, M.; Schmid, M.; Allgöwer, C.; Krüger, J.; Melzer, M.K.; Bens, S.; Siebert, R.; Perkhofer, L.; Azoitei, N.; et al. CDKN2A-Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome. Cancers 2021, 13, 5139. https://doi.org/10.3390/cancers13205139
Merkle J, Breunig M, Schmid M, Allgöwer C, Krüger J, Melzer MK, Bens S, Siebert R, Perkhofer L, Azoitei N, et al. CDKN2A-Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome. Cancers. 2021; 13(20):5139. https://doi.org/10.3390/cancers13205139
Chicago/Turabian StyleMerkle, Jessica, Markus Breunig, Maximilian Schmid, Chantal Allgöwer, Jana Krüger, Michael K. Melzer, Susanne Bens, Reiner Siebert, Lukas Perkhofer, Ninel Azoitei, and et al. 2021. "CDKN2A-Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome" Cancers 13, no. 20: 5139. https://doi.org/10.3390/cancers13205139