Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

Keywords: CRISPR; genome editing; mouse model; pancreatic cancer.

Figure 1.

Pancreatic retrograde ductal injection of Adeno-Cre or Lenti-Cre induces widespread recombination and initiates the development of PanINs and ductal adenocarcinoma. (A) Diagram of the pancreatic retrograde ductal injection procedure. (B) Images of retrograde ductal injection of a blue dye highlight the distribution of the injected fluid and the expansion of the injected tissue. Bars, 3 mm. (Pan) Pancreas; (Duo) duodenum. (C) Retrograde ductal Ad-Cre-injected R26^LSL-Tom (T) mice (n = 3) have widespread infection of the pancreas. Mice were analyzed 7 d after infection. Bars, 3 mm. (Pan) Pancreas; (St) stomach. (D,E) Retrograde ductal Ad-Cre-injected Kras^LSL-G12D/+;R26^LSL-Tom (KT) mice (n = 4) develop PanINs (6–7 mo after tumor initiation). Bars: D, 200 μm; E, 50 μm. (AB) Alcian blue. (F) Quantification of pancreatic lesions in Ad-Cre-infected KT mice 6–7 mo after tumor initiation. (G,H) At early time points, retrograde ductal Ad-Cre-infected Kras^LSL-G12D/+;p53^flox/flox;R26^LSL-Tom (KPT) mice (n = 4) have widespread Tomato^positive PanINs throughout the pancreas. Bars: G, 100 μm; H, 50 μm. (I,J) Retrograde ductal Ad-Cre-injected KPT mice (n = 19) develop PDAC. The yellow arrowhead indicates the enlarged gallbladder consistent with biliary obstruction. Black arrowheads indicate tumors. Bars: I, 3 mm; J, 50 μm. (K,L) Retrograde ductal Lenti-Cre-injected KPT mice develop Tomato^positive PanIN lesions (3 mo after infection). Bars: K, 2 mm; L, 50 μm. (M,N) Retrograde ductal Lenti-Cre-injected KPT mice develop Tomato^positive PDAC. A moderately differentiated CK19^positive PDAC is shown. Bars: M, 3 mm; N, 100 μm.

Figure 2.

Diverse expansion potential and metastatic ability of viral-Cre-initiated PDAC. (A) DTCs can be detected in the peritoneal cavity of Ad-Cre-infected KPT mice. Viable (DAPI^negative), lineage-negative (CD31, CD45, Ter119, F4/80)^negative cells are shown. (B,C) Retrograde ductal Ad-Cre tumor initiation in KPT mice leads to widespread metastasis. A dotted outline of liver (Liv) lobes is shown in B. (C) Representative histology of liver metastasis. Bars: B, 3 mm; C, 100 μm. (D) Quantification of DTCs and metastases in Ad-Cre-infected KT (10⁹ IU Ad-Cre, 6 mo) and KPT (10⁷–10⁸ IU Ad-Cre, 3–7 mo) mice. Data represent the number of mice with the indicated lesion/total number of mice of that genotype. For DTCs, the range of the total DTC number is shown in parentheses. “PerC” indicates metastases in the peritoneal cavity. (E,F) KPT mice with Lenti-Cre-induced PDAC can develop multifocal, multiorgan metastatic disease. (Liv) Liver. Bars: E, 2 mm; F, 100 μm. (G) Quantification of DTCs and metastases in Lenti-Cre-infected KT and KPT mice. Data are represented as in D. (H–K) Injection of Ad-Cre (H,I) or Lenti-Cre (J,K) into Kras^LSL-G12D/+;p53^flox/flox;R26^Mot/Mot (KP;R26^Mot/Mot) mice initiates diverse color labeling of early lesions (H), few of which progress into tumors (I). (J,K) A single, clonally derived, late time point primary tumor (J) seeded multiple liver metastases (K). (Duo) Duodenum; (Liv) liver. Bars, 3 mm.

Figure 3.

CRISPR/Cas9 enables in vivo genetic alteration in pancreatic cancer. (A) Schematic of the Cre-regulated Cas9 allele. (CAGGS) Cytomegalovirus immediate–early enhancer/chicken β-actin promoter; (hSpCas9) human codon-optimized Streptococcus pyogenes Cas9; (NLS) nuclear localization signal. (B) PCR genotyping of H11^LSL-Cas9 mice. (C) Efficient Cre-mediated recombination of the LSL cassette into a single LoxP site (1LoxP) in H11^LSL-Cas9 cells in vitro. These primers do not recognize the H11^wt locus. (D) Cre-regulated expression of Flag-tagged Cas9 protein in fibroblasts derived from H11^LSL-Cas9 mice. Hsp90 shows equal loading. (E) Genomic cleavage detection assay for the targeted region of Lkb1 in Lenti-sgLkb1/Cre-infected Tomato^positive fibroblasts from KT and KT;H11^LSL-Cas9/+ mice in vitro. (F–J) Representative light and fluorescent images of the pancreata from KT (n = 5), KT;H11^LSL-Cas9/+ (n = 3), and KT;Lkb1^flox/flox (n = 3) mice 2–3 mo after retrograde ductal injection of Lenti-sgLkb1/Cre as well as from KT;H11^LSL-Cas9/+ mice infected with nontargeting Lenti-sgNT/Cre (n = 5) or Lenti-sgNeo/Cre, which targets the neomycin resistance gene (n = 2). (G) Lenti-sgLkb1/Cre infection of KT;H11^LSL-Cas9/+ mice generates macroscopic Tomato^positive pancreatic tumors. Lenti-sgLkb1/Cre infection of control KT mice (F), Lenti-sgNT/Cre infection of KT;H11^LSL-Cas9/+ mice (I), and Lenti-sgNeo/Cre infection of KT;H11^LSL-Cas9/+ mice (J) generated only microscopic Tomato^positive areas, while Lenti-sgLkb1/Cre infection of KT;Lkb1^flox/flox mice induced macroscopic tumors (H). Bar, 3 mm. (Duo) Duodenum; (P) pancreas.

Figure 4.

Cas9-mediated targeting of Lkb1 in the pancreas promotes tumor growth. (A) Control Lenti-sgLkb1/Cre-infected KT mice (n= 5) have small clusters of Tomato^positive cells and very rare ADMs and PanINs. (B,C) KT;H11^LSL-Cas9/+ mice infected with Lenti-sgNT/Cre (n = 5) or Lenti-sgNeo/Cre (n = 2) do not develop tumors. (D,E) Lenti-sgLkb1/Cre-infected KT;H11^LSL-Cas9/+ mice (n = 3) develop Tomato^positive cystic tumors that are indistinguishable from the tumors that form in KT;Lkb1^flox/flox mice (n = 3). Bars: A-E, left column, 300 μm; A-E, right two columns, 30 μm. (F) Lenti-sgLkb1/Cre-infected KT;H11^LSL-Cas9/+ mice have greater tumor area than infected control mice. Each dot represents a mouse, and the bar represents the mean. Note the split scale.

Figure 5.

Cas9-mediated targeting leads to the formation of pancreatic tumors harboring deleterious Lkb1 mutations and lacking Lkb1 protein. (A) Genomic cleavage detection assay detected indels in the targeted Lkb1 locus in Tomato^positive cells isolated from Lenti-sgLkb1/Cre-infected KT;H11^LSL-Cas9/+ mice. (Som) Somatic cells. (B) Eight mutated Lkb1 alleles present in a single, oligoclonal, Tomato^positive tumor mass from a Lenti-sgLkb1/Cre-infected KT;H11^LSL-Cas9/+ mouse. The targeted Lkb1 locus, sgLkb1 sequence, and protospacer adjacent motif (PAM) are shown. (C) All observed indels in oligoclonal tumor masses create frameshift mutations in Lkb1. (D) Tumors in Lenti-sgLkb1/Cre-infected KT;H11^LSL-Cas9/+ mice lack Lkb1 protein. Positive control (pos) PDAC from an Ad-Cre-infected KP mouse and negative control (neg) tumor derived from a Lenti-sgLkb1/Cre-infected KT;Lkb1^flox/flox mouse are shown. Bar, 50 μm. (E) Neoplastic cells from the pancreas of Lenti-sgLkb1/Cre-infected KT;H11^LSL-Cas9/+ mice lack Lkb1 protein. Hsp90 shows equal loading. KPT pancreatic cancer cells and a whole KT;H11^+/+ pancreas were positive controls.