Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer

ABSTRACT Colorectal cancer (CRC) is a leading cause of death in the developed world, yet facile preclinical models that mimic the natural stages of CRC progression are lacking. Through the

orthotopic engraftment of colon organoids we describe a broadly usable immunocompetent CRC model that recapitulates the entire adenoma–adenocarcinoma–metastasis axis _in vivo_. The

engraftment procedure takes less than 5 minutes, shows efficient tumor engraftment in two-thirds of mice, and can be achieved using organoids derived from genetically engineered mouse models

(GEMMs), wild-type organoids engineered _ex vivo_, or from patient-derived human CRC organoids. In this model, we describe the genotype and time-dependent progression of CRCs from

adenocarcinoma (6 weeks), to local disseminated disease (11–12 weeks), and spontaneous metastasis (>20 weeks). Further, we use the system to show that loss of dysregulated Wnt signaling

is critical for the progression of disseminated CRCs. Thus, our approach provides a fast and flexible means to produce tailored CRC mouse models for genetic studies and pre-clinical

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BEING VIEWED BY OTHERS GASTROINTESTINAL CANCER ORGANOIDS—APPLICATIONS IN BASIC AND TRANSLATIONAL CANCER RESEARCH Article Open access 18 October 2021 XENTURION IS A POPULATION-LEVEL

MULTIDIMENSIONAL RESOURCE OF XENOGRAFTS AND TUMOROIDS FROM METASTATIC COLORECTAL CANCER PATIENTS Article Open access 29 August 2024 INDUCIBLE MOUSE MODELS OF COLON CANCER FOR THE ANALYSIS OF

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Download references ACKNOWLEDGEMENTS We thank D. Grace, S. Tian, and M. Taylor for technical assistance with animal colonies, other members of the Lowe laboratory for advice and discussions,

J. Shia for assistance with histopathology, M. Gollub for assistance with interpreting MRI studies, and C. LeKaye, M. Lupu, and D. Winkleman for their technical support. We also thank

members of the Englander Institute for Precision Medicine Organoid Platform, T. McNary, Y. Churakova, and C. Cheung. This work was supported by grants from the Starr Cancer Consortium

(I7-A771, to M.A.R. and H.B.; and I8-A8-030 to. S.W.L. and L.E.D.), the Department of Defense (PC121341; to H.B.), and a Damon Runyon Cancer Research Foundation-Gordon Family Clinical

Investigator Award (CI-67-13; to H.B.). This work was supported by grants from the NIH (U54 OD020355-01, R01 CA195787-01 and P30 CA008748). K.P.O'R. is supported by an F30 Award from

the NIH/NCI (1CA200110-01A1). T.B. was supported by the MSKCC Single-Cell Sequencing Initiative, The William and Joyce O'Neil Research Fund. K.P.O'R. and E.M.S. were supported by a

Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM07739 to the Weill

Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program. P.B.R. is supported by a K12 Paul Calebresi Career Development Award for Clinical Oncology (CA 187069). L.E.D. was

supported by a K22 Career Development Award from the NCI/NIH (CA 181280-01). Animal imaging studies were supported by the NIH Small-Animal Imaging Research Program (SAIRP), R24 CA83084; NIH

Center Grant, P30 CA08748; NIH Prostate SPORE, P50-CA92629. S.W.L. is the Geoffrey Beene Chair of Cancer Biology and an Investigator of the Howard Hughes Medical Institute. AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Weill Cornell Medicine/Rockefeller University/Sloan Kettering Tri-Institutional MD-PhD Program, New York, New York, USA Kevin P O'Rourke & 

Emma M Schatoff * Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA Kevin P O'Rourke, Evangelia Loizou, Geulah Livshits, Timour

Baslan, Eusebio Manchado, Janelle Simon, Paul B Romesser & Scott W Lowe * Weill Cornell Graduate School of Medical Sciences, New York, New York, USA Evangelia Loizou & Teng Han *

Hematology & Medical Oncology Division, Department of Medicine, Meyer Cancer Center, Weill Cornell Medicine, New York, New York, USA Emma M Schatoff, Benjamin Leach, Teng Han, Chantal

Pauli, Himisha Beltran, Mark A Rubin & Lukas E Dow * Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, New York, USA Paul B Romesser * Caryl and Israel Englander

Institute for Precision Medicine, Weill Cornell Medicine, New York, New York, USA Chantal Pauli, Himisha Beltran & Mark A Rubin * Howard Hughes Medical Institute, Memorial Sloan

Kettering Cancer Center, New York, New York, USA Scott W Lowe Authors * Kevin P O'Rourke View author publications You can also search for this author inPubMed Google Scholar * Evangelia

Loizou View author publications You can also search for this author inPubMed Google Scholar * Geulah Livshits View author publications You can also search for this author inPubMed Google

Scholar * Emma M Schatoff View author publications You can also search for this author inPubMed Google Scholar * Timour Baslan View author publications You can also search for this author

inPubMed Google Scholar * Eusebio Manchado View author publications You can also search for this author inPubMed Google Scholar * Janelle Simon View author publications You can also search

for this author inPubMed Google Scholar * Paul B Romesser View author publications You can also search for this author inPubMed Google Scholar * Benjamin Leach View author publications You

can also search for this author inPubMed Google Scholar * Teng Han View author publications You can also search for this author inPubMed Google Scholar * Chantal Pauli View author

publications You can also search for this author inPubMed Google Scholar * Himisha Beltran View author publications You can also search for this author inPubMed Google Scholar * Mark A Rubin

View author publications You can also search for this author inPubMed Google Scholar * Lukas E Dow View author publications You can also search for this author inPubMed Google Scholar *

Scott W Lowe View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.P.O'R., L.E.D., and S.W.L. conceived the project. K.P.O'R.,

designed, performed and analyzed experiments, and wrote the paper. E.L., G.L., E.M.S., T.B., E.M., J.S., P.R., B.L., T.H., C.P., H.B., and M.A.R. provided reagents, performed or analyzed

experiments. L.E.D. and S.W.L supervised experiments, analyzed data, and wrote the paper. CORRESPONDING AUTHORS Correspondence to Lukas E Dow or Scott W Lowe. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing financial interests. INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 OVERVIEW OF THE TRANSPLANT PROCEDURE AND SUPPLEMENTARY DATA TO

FIGURE 1. a. Under isofluorane anesthesia, the lumen of the colon is washed by a gentle PBS enema. b. Using a p200 pipette, 50 μL of organoids are pipetted into the lumen of the colon over

30 seconds. c. Using a p10 pipette, 4 μL of VetBond Tissue Adhesive is placed over the anal canal to seal it. d. Fluorescence endoscopy (top) and gross dissection images in bright field

(middle) and GFP fluorescence (bottom) of a colon from an engrafted animal maintained ON Dox for 54 weeks after transplantation. White arrows mark the GFP+ adenoma. e. Kaplan-Meier survival

curves of _shApc.3374_ engrafted mice, maintained ON or OFF Dox, as compared to our previously published _shApc.3364_ GEMM1. A significant difference (P=.0023) in survival between

_shApc.3374_ GEMMs maintained On Dox (orange) and _shApc.3374_ Transplants maintained On Dox (blue), was calculated by a Log-Rank (Mantel-Cox) test. f. Schematic depicting the treatment

protocol to test Apc restoration in long-lived engrafted _shApc_ colon stem cells (top), and gross dissection images of Apc-restored engrafted mucosa (for corresponding histology see Fig.

1B, bottom row). Scale bars are 5mm. SUPPLEMENTARY FIGURE 2 ESTABLISHING AND CREDENTIALING ORGANOIDS FROM A NEWLY GENERATED _SHAPC_ GEMM. a. Schematic depicting a newly developed transgenic

mouse harboring a unique Apc shRNA (TG-Apc.8745e) that, upon tamoxifen (4-OHT) and Dox administration, developed colon adenomas in a manner consistent with previous results1. b. H&E

stain of a TG-Apc.8745e tumor 13.1 weeks post 4-OHT/Dox administration. Scale bar is 200 μm. c. Bright field image of organoids harvested from (TG-Apc.8745e) that are grown in Dox in and the

absence of Wnt supplementation. Scale bar is 200 μm. d. Immunofluorescent stains of proliferation (EdU, Red), colonic differentiation (Krt20, Green) and nuclei (Dapi, Blue) of _shApc.8745e_

organoids grown ON Dox (Top, “+DOX/Apc OFF”) or 4 days after Dox withdrawal (Bottom, “-Dox/Apc On”). Scale bars are 50 μm. e. qRT-PCR analysis of mRNA harvested from _shAPC.8745e_ organoids

grown ON Dox (+Dox) or 4 days after Dox withdrawal (-Dox), for markers of Wnt activation (Myc, Axin2, Fzd7) and colon differentiation (Krt20). Error bars are standard deviation of 3

technical replicates. SUPPLEMENTARY FIGURE 3 COMPARISON OF APC SILENCED AND APC-RESTORED TUMORS IN THE _SHAPC.8745E_ TRANSPLANT MODEL. a. H&E and immunofluorescent stains of an

_shApc.8745e_ transplant maintained ON DOX for 40 weeks. The left most image is a low magnification image of an axial section of the entire colon, with a black arrow indicating the normal

host mucosa, two black arrowheads indicating the borders of the adenoma, and a black box indicating where the higher magnification H&E (next panel) and immunofluorescent images were

acquired. b. H&E and immunofluorescent images (Dapi, Ki67, GFP, Krt20) of a transplant that was maintained on dox for 35 days, then taken off dox for 6 weeks, and then pulsed with dox

for 2 days to allow for detection of the engraftment by GFP signal. The white box indicates the region of the image presented in high magnifications (bottom) demonstrating normal functioning

host (GFP negative) and grafted Apc-restored (GFP positive) mucosa side-by-side in the tissue. Scale bars are 200 μm. SUPPLEMENTARY FIGURE 4 _EX VIVO_ ENGINEERING OF MURINE COLON ORGANOIDS.

a. Sanger-sequencing analysis of the _Apc_ locus targeted by the sgApc-CC vector in Control (non-transfected organoids) (top), and transfected organoids (bottom). b. Top, PCR genotyping of

the _LSL-Kras__G12D_ allele from DNA extracted from non-transfected (Lane 1) organoids and sgApc-CC-transfected (Lane 2). Lane 1 shows the expected unrecombined PCR products for the

_LSL-KRAS__G12D_ allele (327bp) and the WT allele (452 bp). Lane 2 shows the expected recombined _KRAS__G12D_ product (492 bp) and WT product (452 bp). Bottom, PCR genotyping of the

_p53__loxp/loxp_ alleles from DNA extracted from non-transfected (Lane 1) and sgApc-CC-transfected (Lane 2) organoids. Lane 1 shows the expected unrecombined PCR product for the

_p53__loxp/loxp_ alleles (370bp). Lane 2 shows the expected recombined _p53_-/- product (612 bp). A known background band appears around 400-420 bp, which is indicated by an asterisk. c.

Control transfected (pMaxGFP) and sgApc transfected organoids either in the px330 backbone (middle) or Cas9-P2A-Cre “CC” backbone (right) cultured in complete growth media (Wnt3a, Egf,

Noggin, R-spondin, “WENR,” top) or selected in media lacking Wnt growth factor (ENR, bottom). d. Control transfected (pMaxGFP) or sgApc-CC transfected organoids grown in either complete

growth media (WENR, top) or media containing 10 uM Nutlin (WENR + Nut, bottom). e Schematic depiction of the protocol used to generate _Apc__mut_/ _p53__mut_ organoids via one-step

multi-allelic genome editing. f. sgApc/sgp53-CC transfected cells grown in complete growth media (“WENR,” top), or selected in complete growth media supplemented with 10 uM Nutlin (WENR +

Nut, middle), and growth media lacking Wnt or Rspo (“-Wnt, -Rspo,” bottom). Scale bars are 200 μm. g. T7 Endonuclease mutation detection of transfected colon organoids showed the expected

generation of indels at the _p53_ locus. h. Sanger-sequencing analysis of the _Apc_ locus targeted by the sgApc/sgp53-CC vector in Control (non-transfected) (top), and transfected cells

(bottom). SUPPLEMENTARY FIGURE 5 CHARACTERIZATION OF BLOOD CELL POPULATIONS IN DSS TREATED ANIMALS. Schematic illustrating the treatment and collection of mice for immunophenotyping and CBC

analysis. Peripheral blood (b) and spleens (c) were collected from mice at sequential time points and analyzed for the presence of each cell surface marker. d. Peripheral blood was collected

from mice at the corresponding time point and samples were analyzed using the Hemavet 950FS. WBC: White blood cells, LY: Lymphocytes, NE: Neutrophils, MO: Monocytes, BAE: Basophiles, EO:

Eosinophils, RBC: Red blood cells. PLT: platelets. Dot plots are presented with summary statistics that represent the mean value of each cell population as quantified by FACS from five mice

(n=5), ± SD. Significance was determined using unpaired two-tailed t-test. SUPPLEMENTARY FIGURE 6 ENDOSCOPIES AND MATCHED HISTOLOGIES OF TUMORS HARVESTED FROM THE IMMUNOCOMPETENT CRC MODEL.

C57Bl/6 _Apc__mut__/Kras__G12D__/p53__mut_ engrafted organoids examined by endoscopy (left) 5 weeks post transplant, and prepared as axial cross-sections for histological examination by

H&E staining (7 weeks post-transplant, right). Scale bars are 1 mm throughout. SUPPLEMENTARY FIGURE 7 DISEASE STAGING BY MRI, LYMPHOVASCULAR INVASION AND SURVIVAL ANALYSIS OF THE

IMMUNOCOMPETENT CRC MODEL. a. Colon endoscopy (also shown in Fig. 2) of a C57Bl/6 _Apc__mut__/Kras__G12D__/p53__mut_ transplant, 16 weeks after infusion of cells. b. MRI of the same tumor at

16 weeks (top) and 20 weeks (bottom) post transplant, displaying progressive tumor invasion into pericolorectal tissue. There is a locally invasive partially circumferential non-obstructing

lesion involving the distal colon with tumor penetration through the serosal lining of the colon involving the pericolorectal fat. The animal is oriented Dorsal (D) top, Ventral (V) bottom,

and left (L) left, right (R) right. White arrows point to areas of local tumor infiltration through the muscularis propria and serosal lining of the colon. Scale bars are 5 mm. c.

Histochemical (H&E) stains imaged at low magnification (top) and high magnification (bottom) of a C57Bl/6 _Apc__mut__/Kras__G12D__/p53__mut_ tumor 16.5 weeks post transplant. The white

arrow indicates the region depicted in the high magnification image, and the black arrow points to a nest of tumor cells that occupy the lumen of a vessel inside the primary tumor. Scale

bars are 200 μm. D. Kaplan-Meier survival curve of transgenic triple mutant animals (from Dow et al. Cell, 2015), labeled, “_shApc/Kras__G12D__/p53__mut_ GEMM” (blue line), and Foxn1nu/nu

animals that received transplants derived from triple transgenic animals, labeled as, “_shApc/Kras__G12D__/p53__mut_ Transplants” (red line), and C57Bl/6 animals that were engrafted with

‘C57Bl/6 _Apc__mut__/Kras__G12D__/p53__mut_’ cells (green line). Note that shApc triple mutant transplants are collated data from lines 4-6 of Table 1. The significant difference (p=0.03) in

survival is noted between comparable immunocompetent models: triple mutant GEMMs (blue) and triple mutant transplants (green), is shown as calculated by a Log-Rank (Mantel-Cox) test.

SUPPLEMENTARY FIGURE 8 IDENTIFICATION OF PRIMARY, REGIONALLY DISSEMINATED, AND METASTASIZED TUMORS IN THE IMMUNOCOMPETENT MOUSE MODEL OF CRC. Histochemical (H&E), immunohistochemical

(Krt20, Ki67), and immunofluorescence (Villin-Red, Non-Phosphorylated Beta-Catenin-Green, Dapi-Blue) stains of a primary tumor engraftment (a), regional dissemination to a lymph node (b),

and metastasis to the liver 21.4 weeks post transplant (c). SUPPLEMENTARY FIGURE 9 IMMUNOFLUORESCENT STAINING AND PCR GENOTYPING CONFIRM LIVER METASTASIS ORIGINATED FROM THE PRIMARY

ORTHOTOPIC TUMOR. A. Immunofluorescent images from a colorectal liver metastasis, stained with the intestine-specific marker, Villin. High magnification images (right) show apical

concentration of Villin within glandular epithelium of the metastasis. Red arrows indicate autofluorescent signal from red blood cells and background fluorescence in liver hepatocytes. Note,

fluorescent signal in hepatocytes in not localized. B. PCR detection of engineered (Cre-dependent) Kras and p53 loci in wildtype cells, _LSL-Kras_/_p53flox/flox_ organoids (pre-Cre),

_KrasG12D/p53-/-_ organoids (post-Cre), and genomic DNA from microdissected primary tumor and liver metastasis. Detection of the Cre-recombined _Kras_ and _p53_ alleles demonstrates that the

liver met is derived from organoids engrafted in the colon. Wildtype bands are present at higher frequency in the tumor tissue due to the presence of stromal and immune cells in the

dissected tumor. SUPPLEMENTARY FIGURE 10 STEPWISE PROGRESSION OF CRC DISEASE IN THE ORTHOTOPIC TRANSPLANT MODEL. Timing and anatomic staging of CRC disease in the C57Bl6/J mice transplanted

with syngeneic C57Bl/6 _Apc__mut__/Kras__G12D__/p53__mut_ organoids. Livers of the six mice that were analyzed after 16 weeks were inspected macroscopically for surface metastases, and we

performed comprehensive liver sectioning and staining on three of those six animals. SUPPLEMENTARY FIGURE 11 COMPARISON BETWEEN 2D TISSUE CULTURE AND 3D ORGANOID ORTHOTOPIC ENGRAFTMENT

MODELS. a. Bright field images of triple mutant colon organoids grown in 3-D conditions or as a 2-D cell line. b. Endoscopies of animals transplanted with 3-D organoids (left) or 2-D cell

lines (right) at 6 and 5 weeks post transplant, respectively. c. H&E stained sections of tumors harvested from mice transplanted with 3-D triple mutant organoids (top) or 2-D cell lines

(bottom). d. Kaplan-Meyer curve illustrating overall survival of animals transplanted with either 3-D organoids (red, n=21, which also appears in Supplementary Fig. 12) or 2-D cell lines

(blue, n=19). The significant difference (p<0.0001) in survival is shown as calculated by a Log-Rank (Mantel-Cox) test. e. Disease staging analysis of animals transplanted with either 2-D

cell lines or 3-D organoids at early time points (5-7 weeks). f. Copy number analysis of freshly derived triple mutant 3-D organoids or 2-D cell lines. SUPPLEMENTARY FIGURE 12 DISTANT

METASTASIS GENERATION BY VESSEL SEEDING OF ENGINEERED ORGANOIDS. Shown are whole slide scans from C57Bl/6 animals injected in the splenic vein (top left), tail vein (top right) or directly

into the liver (bottom left and right), with _Apc__mut__/Kras__G12D__/p53__mut_ organoids. SUPPLEMENTARY FIGURE 13 HISTOLOGICAL CHARACTERIZATION OF TUMORS ARISING AT DISTANT SITES FOLLOWING

VESSEL SEEDING. a. Low magnification (2.5x) and high magnification (10x) images of H&E stained section from splenic injections (top), tail vein injections (second row), and liver

injections (3rd and 4th rows). b. Results from 3 injections performed in 5 animals each. “Avg Tumor Size” is the average of cross-sectional measurements of histology specimens that are

presented as whole slide scans in Supplementary Fig. 13. Scale bars are 200μm throughout. SUPPLEMENTARY FIGURE 14 EVALUATION OF APC RESTORATION IN A METASTATIC MODEL OF CRC BY VESSEL SEEDING

OF ENGINEERED ORGANOIDS. a. Schematic representation of the generation and transduction of _shApc/Kras__G12D__/p53__mut__/MNIL-shSmad4_ or _MNIL–shRen_ organoids. b. Immunoblot of Smad4

protein levels in _shRen.713_ (Control) and _shSmad4.591_ organoids. c. Bright field images of shRen.713 (shControl) and shSmad4 organoids grown in ENRWD (Egf, Noggin, R-spondin, Wnt3a, Dox)

(left) or supplemented with 10 ng/ml TGF-Beta (ENRWD+T) (right). Scale bars are 200 μm. d. Bioluminescence imaging of animals 4 weeks post splenic injection with shSmad4 cells. e.

Quantification of luciferase signal 13 weeks after splenic injection of _shApc/Kras__G12D__/p53__R127H__/-MNIL-Ren713_ organoids (blue) and _shApc/Kras__G12D__/p53__R127H__/-MNIL-Smad4.591_

organoids (red) that were maintained on dox for 3 weeks and then randomized into Dox ON and Dox OFF groups. f. A slide scan of H&E stained

_shApc/Kras__G12D__/p53__R127H__/-MNIL-Smad4.591_ tumors that were harvested 10 weeks after splenic injection and maintained on doxycycline. g. Tumor derived organoids were generated from

gross liver tumors (left panels), examined by H&E staining, and grown as organoids under the same protocols for wild type colon growth. h. qRTPCR analysis on mRNA extracted from

organoids maintained ON Dox, or 4 days OFF dox. Samples are uninjected parental _shApc/Kras__G12D__/p53__R172H__/- /MNIL-Smad4_ organoid line, or tumor derived organoids from two different

tumors that arose from splenic injections of the parental line. SUPPLEMENTARY FIGURE 15 ENGRAFTMENT OF HUMAN CRC ORGANOIDS INTO IMMUNOCOMPROMISED MICE. a. H&E stained section of a

patient-derived CRC that was obtained from a primary tumor. b. Serial endoscopies of engrafted human CRC tumors and H&E stains of tumors at 8 weeks post transplant. C. Clinical

information related to the patient derived CRC organoid lines that were established and orthotopically engrafted. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary

Figures 1–15 (PDF 4747 kb) SUPPLEMENTARY TABLE 1 Summary of the orthotopic engraftment approaches. (XLSX 10 kb) SUPPLEMENTARY VIDEO Organoid transplant procedure (MP4 116615 kb) RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE O'Rourke, K., Loizou, E., Livshits, G. _et al._ Transplantation of engineered organoids enables rapid

generation of metastatic mouse models of colorectal cancer. _Nat Biotechnol_ 35, 577–582 (2017). https://doi.org/10.1038/nbt.3837 Download citation * Received: 08 July 2016 * Accepted: 24

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