Organoid models of human and mouse ductal pancreatic cancer

Abstract

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Figure 1. Oncogenic Kras^G12D expression in pancreatic ductal organoids is sufficient to induce pre-invasive neoplasms

(A) Hematoxylin and eosin (H&E) staining of murine pancreatic tissue used to prepare organoids (top). Arrows indicate mouse normal or PanIN ductal structures. Ducts embedded in Matrigel immediately following isolation (middle) and organoids 3 days post-isolation (bottom). Arrowheads mark isolated ducts and growing organoids. Scale bars, 50 μm. (B) Immunoblots for Kras, pan-Ras, Kras-GTP by RBD-GST pull-down and Tubulin in mouse normal (mN) and mPanIN (mP) organoids. PCR confirmation of Cre-mediated recombination of the Kras^LSL-G12D allele (bottom). (C) qRT-PCR of ductal (Pdx1, Ck19, Sox9, Hnf6), acinar (Ptf1a, Cpa1, Amy), and endocrine (Ngn3, Chga, Ins2) lineage markers in mN and mP organoids. Means of 3 biological replicates are shown. Error bars depict standard error of the means (SEMs). Values were normalized to mouse normal pancreas. (D) qRT-PCR of genes indicative of PanIN lesions (Muc5ac, Muc6, Tff1, Klf4) in mN and mP organoids. Values were normalized to mN organoids. Means of 3 biological replicates are shown. Error bars depict SEMs. **: p < 0.01 by two-tailed Student’s t-test. (E) H&E, Alcian Blue staining, and immunohistochemistry (IHC) of orthotopic, syngeneic transplants of GFP-transduced mN and mP organoids. Scale bars, 200 μm. (F) Immunoblots for Kras, pan-Ras, Kras-GTP by RBD-GST pull-down and Tubulin in Kras^+/LSL-G12D organoids transduced with adenoviral-Cre (Ad-Cre) or adenoviral-blank (Ad-Bl). PCR confirmation of Cre-mediated recombination of the Kras^LSL-G12D allele (bottom). (G) H&E, Alcian Blue staining, and IHC of orthotopic syngeneic transplants of organoids transduced with Ad-Bl (Kras^+/LSL-G12D; R26-LSL-YFP) and Ad-Cre (Kras^+/LSL-G12D; R26-YFP) 2 weeks post-transplant. Scale bars, 200 μm. See also Figure S1 and Table S1.

Figure 2. Modeling murine PDA progression with tumor- and metastasis-derived organoids

(A) H&E staining of murine tissue from which tumor and metastasis organoids were derived (top). Arrowhead indicates metastasis. Scale bars, 50 μm. Digested murine tissues embedded in Matrigel immediately following isolation (middle) and organoids 3 days post-isolation (bottom). Scale bars, 200 μm. (B) Immunoblots of selected signaling effectors, Kras-GTP and Ras-GTP by RBD-GST pull-down, and Tubulin. PCR confirmation of Kras^LSL-G12D recombination in mP, mT, and mM organoids (bottom). (C) H&E staining of tumors and metastases (met) derived from mT organoid orthotopic transplants. Scale bars, 200 μm (top) and 50 μm (bottom). (D) Loss of heterozygosity of the wild-type Trp53 allele determined by PCR (top) and immunoblot analysis of Trp53, Smad4, and p16, and Tubulin. mM3L: derived from a liver metastasis. (E) Karyotypes of organoids and monolayer (2D) cell lines. See also Figure S2 and Table S2.

Figure 3. Human pancreatic ductal organoids recapitulate features of normal and neoplastic ducts

(A) Representative images (top) and H&E staining (middle) of human organoid cultures established from: normal tissues (hN1-2), resected primary tumors (hT1-2), a resected metastatic lung lesion (hM1) and a fine-needle aspiration biopsy of a metastatic lesion (hFNA2). H&E staining of the resected tissues from which the organoids were derived (bottom). Scale bars, 500 μm (top), 250 μm (middle), and 500 μm (bottom). (B) Representative images of hN and hT organoids cultured for two weeks (1 passage) in human complete media, or in human complete media lacking the indicated factors. Scale bars, 500 μm. (C) Number of passages hN and hT organoids could be propagated in the absence of the indicated factors. (D) Targeted sequencing analysis of human organoids. Genes altered in more than one sample and/or known to be mutated in PDA are shown. If multiple mutations were found in a gene, only one mutation per gene is shown. Color key for the type of genetic alterations is shown. Met indicates organoids derived from metastatic samples. See also Figure S3, Table S3 and S4

Figure 4. Molecular characterization and orthotopic transplantation of human organoids

(A) qRT-PCR of pancreas lineage markers in hN (n = 3) and hT (n = 4) organoids. Mean expression levels were normalized to total pancreas. Error bars depict SEMs. (B) Karyotyping of human organoids (2 hN, 2hT) at the indicated passages (P). (C) CA19-9 and actin levels in hN, hT, or hM organoids. The solid line indicates non-congruent lanes. (D) H&E, Alcian Blue staining and IHC of orthotopic hT2 transplants and the primary tumor. Scale bars, 200 μm (top two panels) and 50 μm (lower two panels). See also Figure S4 and Table S4D

Figure 5. Gene expression analysis of murine organoids reveals genetic changes correlated with pancreatic cancer progression

(A) Principal component analysis of gene expression data for mN, mP, and mT organoids. (B) The number of genes differentially expressed (DESeq adjusted p value < 0.05) among mN (n = 7), mP (n = 6), and mT (n = 6) organoids. (C) Heatmap showing relative expression levels using Z-score normalization among mN, mP, and mT organoids. Color key of Z-score is shown. (D) Venn diagrams show overlap of genes significantly differentially expressed in mP and mT relative to mN organoids. The p values for overlaps were determined by two-tailed Fisher’s Exact test. (E) Genes with the largest fold-changes in mP or mT relative to mN organoids. (F) qRT-PCR validation of mN, mP and mT organoid gene expression changes. Values were normalized to mean levels in mN organoids. n = 8 mN, 7 mP, and 8 mT organoid cultures. Error bars show SEMs. *, **, ***, ns: p < 0.05, 0.01, 0.001, or not significant by two-tailed Student’s t-test. See also Figure S5 and Table S5.

Figure 6. Proteomic profiling of murine organoids uncovers molecular pathways linked to pancreatic cancer progression

(A) Protein expression changes by iTRAQ proteomic analysis of murine organoids. Both unique protein isoforms and protein isoforms encoded by the same gene are included (adjusted p value < 0.1 by linear regression analysis). (B) Heatmap of unique protein isoforms that differ (adjusted p value < 0.05) among mN, mP, and mT organoids. Color key of the Z-score is shown. (C) Venn diagrams showing overlaps between proteins differentially expressed (p < 0.05) in mP and mT relative to mN organoids. p values for overlaps were determined by two-tailed Fisher’s Exact test. (D) Venn diagrams showing overlaps between genes and proteins found differentially expressed by RNA-seq and proteomic analyses (adjusted p < 0.05). p values for the overlaps were determined by two-tailed Fisher’s Exact test. (E) Molecular pathways found enriched by GSEA analysis of RNA-seq and proteomic data. Normalized enrichment scores (NESs), p and q values are shown. (F) Heatmap showing relative gene expression levels of nucleoporins in mN, mP, and mT organoids determined by RNA-seq. Color key of the Z-score is shown. See also Figure S6, Table S6 and S7.

Figure 7. Increased levels of ACSM3, NT5E and GCNT3 correlate with mouse and human PDA progression

(A) IHC analysis of 14 candidate genes in mouse adjacent normal ducts, mPanlN and mPDA. Differential expression is indicated as: - (negative), + (weak), ++ (moderate) or +++ (strong). (B) IHC analysis of ACSM3, NT5E and GCNT3 in mouse normal ducts, mPanlN and mPDA tissues. Arrow indicates adjacent normal ducts in mPanlN tissues. Arrowhead indicates mPanlN or mPDA. Scale bars, 50 μm. (C) IHC analysis of 7 candidate genes in human normal pancreas, hT orthotopic transplants and PDA tissues. Differential expression is indicated as: - (negative), + (weak), ++ (moderate) or +++ (strong). Only the ductal component of the normal pancreas was scored. (D) IHC analysis of ACSM3, NT5E and GCNT3 in human normal pancreas and PDA tissues. Arrow indicates normal ducts and arrowhead indicates PDA. Scale bars, 50 μm. See also Figure S7.