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In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis

A Corrigendum to this article was published on 08 December 2017

This article has been updated

Abstract

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.

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Figure 1: CRISPR-Cas9-based in situ Apc editing in the colon epithelium induces adenoma formation.
Figure 2: Orthotopic transplantation models of mouse- and patient-derived colorectal cancer.
Figure 3: Lgr5 cell lineage tracing and sequential mutagenesis in established orthotopic colon adenomas.

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  • 06 July 2017

    In the version of this article initially published, the initial “J” was omitted from an author's name, which should appear as Francisco J Sánchez-Rivera. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by the Howard Hughes Medical Institute (T.J., R.O.H.), NIH (K08 CA198002, J.R.; K99 CA187317, T.T.; Ö.H.Y.; R01CA211184, Ö.H.Y.; U54-CA163109, R.O.H.), the Sigrid Juselius Foundation (Ö.H.Y), the Maud Kuistila Foundation and the Hope Funds for Cancer Research (Ö.H.Y.), Department of Defense (PRCRP Career Development Award CA120198; J.R.), and the V Foundation V Scholar Award (J.R. and Ö.H.Y.), the Sidney Kimmel Scholar Award (Ö.H.Y.), the Pew-Stewart Trust Scholar Award (Ö.H.Y.), the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund (Ö.H.Y.), American Federation of Aging Research (AFAR, Ö.H.Y.), the Hope Funds for Cancer Research (T.T.), the Metastasis/Cancer Research Postdoc fellowship from the MIT Ludwig Center for Molecular Oncology Research (S.R.), the Bloodwise (UK) Visiting Fellowship Grant 14043 (A.R.), and by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. We thank T. Papagiannakopoulos for helpful discussions; Y. Soto-Feliciano for help and expertise with massively parallel sequencing; K. Bedrossian for assistance with human colorectal cancer sample collection at Tufts Medical Center; the Swanson Biotechnology Center at the Koch Institute for technical support, specifically K. Cormier and C. Condon at the Hope Babette Tang (1983) Histology Facility; S. Holder for histology support; and Y.D. Soo and the Peterson (1957) Nanotechnology Materials Core Facility for assistance with electron microscopy. L-WRN cells were a kind gift from T. Stappenbeck, Washington University.

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J.R. and T.T. performed all experiments and participated in their design and interpretation with T.J. and Ö.H.Y. J.R. and Ö.H.Y. developed and optimized the colonoscopy mucosal injection technique, with assistance from D.K. and P.K. J.R. wrote the paper with support from T.T. and Ö.H.Y. N.M.C. contributed to study design, plasmid design, lentivirus production, and mucosal injections. F.J.S.-R. contributed to plasmid and study design, and performed massively parallel sequencing. M.A., Y.K.P., R.N., R.A., X.L., D.K., K.W., S.R., and A.A. assisted with mucosal injections, mouse and human organoid derivation, molecular biology, and immunohistochemistry. A.R. assisted with humanized mouse experiments. M.A.O. designed and synthesized lipid nanoparticles for mRNA encapsulation. G.E., E.T.S., M.S.T., A.J.B., Y.S., J.Y., L.C., V.D., and L.Z. assisted with human CRC specimen collection. S.B. performed organoid qRT-PCR. A.B. performed bioinformatics analysis. R.L., J.L., J.C., P.N.T., R.O.H., and T.J. participated in interpretation of results. Ö.H.Y. supervised all aspects of the study.

Corresponding author

Correspondence to Ömer H Yilmaz.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Mucosal injection of PGK::Cre lentivirus results in recombination in crypt base stem cells and tumorigenesis in Apcfl/fl mice.

(a) Schematic and matching optical colonoscopy images of colonoscopy-guided mucosal injection. Injection of 50 μl of liquid results in a “bubble” between the epithelium and lamina propria. (b) Bioluminescence (IVIS) and tdTomato fluorescence imaging of Rosa26LSL-tdTomato/LSL-Luciferase mice (N=2) two days after mucosal delivery of adenovirus expressing Cre-recombinase (Ad5CMV::Cre, titer 300,000 TU/μl). Arrowheads indicate recombined areas. (c) EpCAM and tdTomato immunofluorescence imaging of colon sections from Rosa26LSL-tdTomato/+ mice seven days following colonoscopy-guided mucosal injection of a lentiviral vector expressing Cre recombinase (lenti-PGK::Cre, titer 100,000 TU/μl). Arrows indicate recombination in crypt base stem cells. Arrowhead shows recombination in stromal cells (N=5 mice). (d) PGK::Cre-induced tumors in Apcfl/fl mice (titer 30,000 TU/μl). Tumors are visualized with colonoscopy (dotted line), hematoxylin and eosin histology, and β-catenin immunohistochemistry. Histology images are 20X and insets are 60X (Scale bar: 200 μm). (R26: Rosa26; PGK: human phosphoglycerate kinase-1 promoter; H&E: hematoxylin and eosin; T: tumor; N: normal).

Supplementary Figure 2 In situ deletion of Apc using floxed alleles results in tumorigenesis.

(a) Tumors in Apcfl/fl mice after colonoscopy-guided mucosal injection of Ad5CMV::Cre (titer: 300,000 TU/μl). Tumorigenesis is indicated by colonoscopy, necropsy, hematoxylin and eosin (H&E) histology, and β-catenin immunohistochemistry. (b) tdTomato immunofluorescence imaging of Rosa26LSL-tdTomato;VillinCreER mice injected via colonoscopy with 100 μM 4-hydroxytamoxifen. Arrows indicate recombination in epithelial cells. (c) Tumors in the distal colons of Apcfl/fl;VillinCreER mice following injection of 100 μM 4-hydroxytamoxifen (colonoscopy, H&E staining, and β-catenin immunohistochemistry). H&E staining, biopsy of an in vivo tumor. (d) Tumorigenesis in Apcfl/fl;Lgr5eGFP-creER/+ mice after mucosal delivery of 100 μM 4-hydroxytamoxifen (colonoscopy, brightfield/GFP necropsy, H&E staining, and GFP immunofluorescence). Arrows indicate Lgr5+/GFP+ adenoma cells. Tumors are indicated with dotted lines. Histology images are 20X and insets are 60X (Scale bar: 200 μm) (tdT or tdTom:tTdTomato; R26: Rosa26; N: normal; T: tumor).

Supplementary Figure 3 Mucosal delivery of lipid nanoparticle-encapsulated Cre mRNA induces tumor formation in Apcfl/fl mice

(a) Lipid nanoparticles are composed of mRNA and an amine-containing ionizable lipid that electrostatically complexes with the negatively charged mRNA and can both help facilitate cellular uptake and endosomal escape of the mRNA to the cytoplasm. In addition, a phospholipid is added that provides structure to the lipid nanoparticle and can assist in endosomal escape. Cholesterol is added to enhance the stability and promote membrane fusion. A lipid-anchored polyethylene glycol is also added to stabilize the particles and reduce nonspecific interactions. As represented in the schematic, the LNPs assume a multi-laminar spherical shape. (b) Cryogenic transmission electron microscopy image of lipid nanoparticles (arrowhead) in a buffered solution on a lacey copper grid coated with a continuous carbon film (Scale bar: 200 nm). (c) Bioluminescence (IVIS) imaging two days after colonoscopy-guided mucosal delivery into wild-type mice with luciferase mRNA alone (CL) or lipid nanoparticle-encapsulated luciferase mRNA (LNP). Arrowhead denotes luminescence signal at mucosal injection sites. (d) EpCAM and tdTomato immunofluorescence imaging of colon sections from Rosa26LSL-tdTomato/+ mice seven days after colonoscopy-guided mucosal injection of lipid nanoparticle-encapsulated Cre mRNA (N=3). Arrows indicate recombined crypt cells, and arrowheads point to recombination in stromal cells. Injection of Cre mRNA alone did not induce recombination in Rosa26LSL-tdTomato/+ recipient colons (N=3 mice, not shown). (e) Tumors following delivery of lipid nanoparticle-encapsulated Cre mRNA to the colon mucosa of Apcfl/fl mice are demonstrated by colonoscopy (dotted line), H&E staining, and β-catenin immunohistochemistry. (PEG: polyethylene glycol; TEM: transmission electron microscopy; TdTom: TdTomato; R26: Rosa26). Histology images are 20X and insets are 60X (Scale bar: 200 μm).

Supplementary Figure 4 Analysis of mutations introduced in vivo by CRISPR-Cas9 at the Apc locus.

Tumors initiated by disruption of Apc in vivo in the colon epithelium were subjected to massively parallel sequencing of a 200 base pair (bp) genomic region comprising 100 bp on either side of the sgApc binding site. (a) Tumor containing 4 different types of Apc mutations induced by U6::sgApc-EFS::Cas9-P2A-GFP lentivirus infection in a wild-type mouse (representative results from eight analyzed tumors); (b) Representative 6-week-old and 1-year-old tumors containing multiple types of Apc mutations induced by U6::sgApc-EFS::turboRFP in Rosa26LSL-Cas9-eGFP/+;VillinCreER mice. Blue in the pie charts represents the fraction of wild-type reads, most likely arising from wild-type stroma present in the tumors. Alignments of the wild-type locus sequence and the most common mutated alleles are shown below the pie charts. The 10 most common mutant alleles among 25 six-week-old samples and five one-year-old samples are described. Locations of the guide RNA target sequence and PAM sequence (light orange highlight) are indicated. Each mutant allele is characterized by its frequency in the total pool of mutant reads or samples, the type of event (DEL: deletion, INS: insertion), event size (in bp), and impact on the coding sequence (FS: frameshift mutation; NFS: non-frameshift mutation). Deletions are indicated by red dashes and insertions by red triangles. Locations of the guide RNA target sequence, PAM sequence, and mutant allele frequencies are described.

Supplementary Figure 5 CRISPR-Cas9-based in situ Apc and Trp53 editing in the colonic epithelium induces adenoma formation.

(a) One-year-old tumors in tamoxifen-treated Rosa26LSL-Cas9-eGFP/+;VillinCreER mice that were injected under colonoscopy guidance with U6::sgApc-EFS::turboRFP lentivirus (titer: 10,000 TU/μl). Tumors were seen with white light/GFP/turboRFP fluorescence colonoscopy and necropsy. Immunofluorescence for GFP, turboRFP, and β-catenin demonstrate GFP+ adenoma (arrows) and limited turboRFP expression in stromal cells (arrowheads). (b) H&E staining of six-week-old vs. one-year-old tumors from tamoxifen-treated Rosa26LSL-Cas9-eGFP/+;VillinCreER mice that were injected with U6::sgApc-EFS::turboRFP lentivirus. (c) Colon tumors from Apcfl/fl;Rosa26LSL-Cas9-eGFP/+ mice injected under colonoscopy guidance with U6::sgTrp53-CMV::Cre lentivirus (titer: 10,000 TU/μl). Tumors were imaged by white light/GFP fluorescence colonoscopy and necropsy as well as hematoxylin and eosin (H&E) histology. (d) Tumors in tamoxifen-treated Rosa26LSL-Cas9-eGFP/+;VillinCreER mice that received mucosal delivery of hU6::sgApc-sU6::sgTrp53-EFS::turboRFP lentivirus (titer: 10,000 TU/μl). Tumors were seen with white light/GFP/turboRFP fluorescence colonoscopy and necropsy and examined with H&E staining. Histology images are 20X and insets are 60X (Scale bar: 200 μm). (tRFP: turboRFP, R26: Rosa26).

Supplementary Figure 6 Analysis of mutations introduced in vivo by CRISPR-Cas9 at the Apc and Trp53 loci.

(a) Tumors were induced in Apcfl/fl;R26LSL-Cas9-eGFP/+ mice by mucosal injection of U6::sgTrp53-CMV::Cre lentivirus, then subjected to massively parallel sequencing of a 200 base pair (bp) genomic region consisting of 100 bp on either side of the sgTrp53 binding site. (b) Mucosal delivery of hU6::sgApc-sU6::sgTrp53-EFS::turboRFP lentivirus into Rosa26LSL-Cas9-eGFP/+;VillinCreER mice treated with tamoxifen generated tumors that were then subjected to massively parallel sequencing of a 200 bp genomic region consisting of 100 bp on either side of the loci in Apc and Trp53 targeted by the respective sgRNAs. Blue in the pie charts represents the fraction of wild-type reads, most likely arising from wild-type stroma present in the tumors. Alignments of the wild-type locus sequence and the 10 most common mutated alleles in a representative sample and in three tumors are shown. Locations of the guide RNA target sequence and PAM sequence (light orange highlight) are indicated. Each mutant allele is characterized by its frequency in the total pool of mutant reads or samples, the type of event (DEL: deletion, INS: insertion), event size (in bp), and impact on the coding sequence (FS: frameshift mutation; NFS: non-frameshift mutation). Deletions are indicated by red dashes and insertions by red triangles. Locations of the guide RNA target sequence, PAM sequence, and mutant allele frequencies are described.

Supplementary Figure 7 Orthotopic engraftment of intestinal organoids.

(a) Orthotopic transplantation of small intestinal organoids derived from tamoxifen-treated Rosa26LSL-tdTomato/+;VillincreER mice. tdTomato+ organoids are visualized in vivo with white light colonoscopy and tdTomato fluorescence colonoscopy. Mice received a pulse of 5-ethynyl-2’-deoxyuridine (EdU) four hours before sacrifice. Arrowheads on immunofluorescence images indicate tdTomato+ intestinal organoids in the lamina propria of the recipient mice. Arrows point to lysozyme positive Paneth cells and EdU positive proliferating cells. Orthotopic transplantation was successful in all three mice receiving two organoid injections each. (b) Tumors in mice transplanted with Apcfl/flVillincreER intestinal organoids and treated with tamoxifen. Tumors are imaged with colonoscopy, necropsy, hematoxylin and eosin histology, and β-catenin immunohistochemistry (N=2). Tumors are indicated with dotted lines. (Scale bar: 200 μm). Dotted lines indicate tumors. (EdU: 5-ethynyl-2’-deoxyuridine; NSG: nod SCID gamma; H&E: hematoxylin and eosin).

Supplementary Figure 8 Orthotopic transplantation of murine colorectal cancer cells and organoids results in tumor formation.

(a) Tumors in C57BL/6 mice following orthotopic transplantation of a murine CRC cell line derived from a Cre-excised ApcΔ/Δ;KrasG12D/+Trp53Δ/Δ (AKP) genetically engineered tumor. Tumors are visualized with colonoscopy, hematoxylin and eosin (H&E) stain, and β-catenin immunohistochemistry. Arrow indicates invasion of the muscularis propria. (b) Wild-type C57BL/6 intestinal organoids infected with U6::sgApc-EFS::Cas9-P2A-GFP lentivirus, then selected in media without Wnt agonists. (c) sgApc intestinal organoids were subjected to massively parallel sequencing of a 200 base pair (bp) genomic region comprising 100 bp on either side of the sgRNA binding site. Locations of the guide RNA target sequence and PAM sequence (light orange highlight) are indicated. Each mutant allele is characterized by its frequency in the total pool of mutant reads, the type of event (DEL: deletion, INS: insertion), event size (in bp), and impact on the coding sequence (FS: frameshift mutation; NFS: non-frameshift mutation). Deletions are indicated by red dashes and insertions by red triangles. The ten most common mutated sequences are listed. (d) RT-PCR for selected Wnt target genes in sgApc small intestinal and colon organoids. (e) Tumors in C57BL/6 mice following orthotopic engraftment of sgApc organoids (colonoscopy, necropsy, H&E stain, and β-catenin immunohistochemistry). (f) Tumors in C57BL/6 mice transplanted with AKP colon organoids (colonoscopy, necropsy, H&E stain, β-catenin immunohistochemistry, and trichrome stain). Arrows indicate invasion of the muscularis propria. Arrowheads show desmoplastic reaction. (g) Tumors following orthotopic transplantation of Apcfl/fl;Rosa26LSL-Cas9-eGFP/+ colon organoids infected with U6::sgTrp53-CMV::Cre lentivirus (white light/GFP fluorescence colonoscopy, GFP/β-catenin immunofluorescence). Histology images are 20X and insets are 60X (Scale bar: 200 μm). Dotted lines indicate tumors. (R26: Rosa26; N: normal; T: tumor).

Supplementary Figure 9 Analysis of mutations introduced by CRISPR-Cas9 at the sgApc locus in wild-type colon organoids and organoid orthotopic transplant tumors.

(a) Wild-type colon organoids were infected with U6::sgApc-EFS::Cas9-P2A-GFP lentivirus, then subjected to massively parallel sequencing of a 200 base pair (bp) genomic region comprising 100 bp on either side of the sgApc binding site. Alignments of the wild-type locus sequence and all 18 mutant reads are shown next to the pie chart. (b) These Apc-null organoids were orthotopically transplanted into recipient NSG mice. Eight weeks after transplantation, three samples each from two tumors were sequenced at the sgApc locus, as described above. Alignments of the wild-type locus sequence and the 10 most common mutant reads are shown. (c) Summary of the 10 most common Apc mutations in the six orthotopic transplant samples from Tumors 1 and 2. Locations of the guide RNA target sequence and PAM sequence (light orange highlight) are indicated. Each mutant allele is characterized by its frequency in the total pool of mutant reads or samples, the type of event (DEL: deletion, INS: insertion), event size (in bp), and impact on the coding sequence (FS: frameshift mutation; NFS: non-frameshift mutation). Deletions are indicated by red dashes and insertions by red triangles.

Supplementary Figure 10 Analysis of mutations introduced by CRISPR-Cas9 at the Trp53 locus in Apcfl/fl;R26LSL-Cas9-eGFP/+ colon organoids and organoid orthotopic transplant tumors.

(a) Apcfl/fl;R26LSL-Cas9-eGFP/+ colon organoids were infected with U6::sgTrp53-CMV::Cre lentivirus, and then subjected to massively parallel sequencing of a 200 bp genomic region comprising 100 bp on either side of the sgTrp53 binding site. (b) ApcΔ/Δ, Trp53-edited organoids were then orthotopically transplanted into recipient NSG mice to form tumors that were similarly sequenced at the sgTrp53 locus (N=3; data from a representative tumor are shown). Blue in the pie charts represents the fraction of wild-type reads, most likely arising from wild-type stroma present in the tumors. Alignments of the wild-type locus sequence and the 10 most commonly mutated sequences are shown below the pie charts. Locations of the guide RNA target sequence and PAM sequence (light orange highlight) are described. Each mutant allele is characterized by its frequency in the total pool of mutant reads, the type of event (DEL: deletion, INS: insertion), event size (in bp), and impact on the coding sequence (FS: frameshift mutation; NFS: non-frameshift mutation). Deletions are indicated by red dashes and insertions by red triangles.

Supplementary Figure 11 Orthotopic engraftment of human colorectal cancer cell lines and patient-derived tumors.

Tumors initiated by orthotopic engraftment of (a) LS174 human CRC cells, (b) HT29 human CRC cells, and (c) patient-derived CRC (colonoscopy, necropsy, hematoxylin and eosin staining, and β-catenin immunohistochemistry). Arrows indicate invasion invasion into the muscularis propria. Histology images are 20X and insets are 60X (Scale bar: 200 μm). Dotted lines indicate tumors. (N: normal; T: tumor; NSG: nod SCID gamma; H&E: hematoxylin and eosin).

Supplementary Figure 12 Orthotopic engraftment of patient-derived organoids.

(a) Patient A colorectal cancer hematoxylin and eosin (H&E) histology and corresponding patient-derived organoids. (b) Human CDX2 in situ hybridization in tumors derived from orthotopic transplantation of Patient A CRC organoids. (c) Lynch Syndrome colorectal cancer H&E histology (characterized by a high level of microsatellite instability and tumor lymphocytic infiltration; Patient B) and corresponding tumor organoids engrafted into the flanks of recipient NSG mice (H&E stain, β-catenin immunohistochemistry). (d) Tumors derived from orthotopic transplantation of Patient B organoids (H&E histology, β-catenin immunohistochemistry). Arrows indicate invasion of the muscularis propria. (e) Liver metastasis after orthotopic transplantation of Patient B tumor organoids (H&E staining, CDX2/human Keratin20 immunohistochemistry). (f) Analysis of human CD3 T cells in Patient B-derived tumors orthotopically transplanted into NSG mice with a reconstituted human immune system (humanized NSG). These tumors and their exhibited human CD3 T cell infiltration by immunohistochemistry and flow cytometry, with enrichment for human memory T cells (CD45RO memory / CD45 RA naive ratio = 26) compared to spleen control (memory / naive ratio = 2.2). Histology images are 20X and insets are 60X (Scale bar: 200 μm). Dotted lines indicate tumors. (ISH: in situ hybridization; N: normal; T: tumor; S: stromal lymphocytes; NSG: nod SCID gamma; hKeratin20: human Keratin20; MSI-H, microsatellite instability-high).

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Supplementary Figures 1–12 and Supplementary Tables 1 and 2 (PDF 6327 kb)

Colonoscopy-guided mucosal injection.

During optical colonoscopy, a 33-gauge needle with 45-degree bevel is inserted into the working channel of the endoscopy and directed to the colonic mucosa without passing through the muscularis propria or serosa. 50-100 μl of liquid (containing virus or organoids, for example) is then rapidly injected to produce a mucosal bubble. (MPG 20682 kb)

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Roper, J., Tammela, T., Cetinbas, N. et al. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis. Nat Biotechnol 35, 569–576 (2017). https://doi.org/10.1038/nbt.3836

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