Nature Biotechnology: doi: /nbt Supplementary Figure 1

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1 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.

2 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.

3 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.

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5 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 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).

6 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.

7 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.

8 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.

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10 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).

11 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.

12 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.

13 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.

14 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.

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16 Supplementary Figure 12

17 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.

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19 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.

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21 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.

22 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.