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A rectal cancer organoid platform to study individual responses to chemoradiation

Nature Medicine volume 25, pages 1607–1614 (2019)Cite this article

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Abstract

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients’ tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.

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Fig. 1: Preservation of RC histopathology and mutational fingerprint in tumoroids.
Fig. 2: Clinically relevant responses to chemotherapy and radiation in rectal cancer tumoroids ex vivo.
Fig. 3: Establishment of an endoluminal RC assay in mice.
Fig. 4: The endoluminal rectal cancer model can be used to reflect patient-specific chemoresistance and chemosensitivity.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. The data for the RC tumoroids on the cBioPortal will be made publicly available on publication.

Code availability

The FACETS algorithm was used to determine allele specific copy number and cancer cell fraction of each mutation in tumoroids and their respective tumors46. Access to this code is available upon reasonable request.

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Acknowledgements

We thank our patients for giving consent and thereby allowing us access to their tissues for use in this study. We thank B. Carver for critical review of the manuscript as it developed. We thank A. Jungbluth for initial discussion on tissue fixation and immunostaining of the murine rectal tissues. We thank S. Chandarlapaty for discussions regarding the chemoresistance assays and cetuximab resistance assay. We thank L. Diaz for critical comments as this work developed. We thank N. Kemeny for advice, guidance and encouragement as this project developed. We thank N. Fan, S. Fujisawa, X. Liu, M. Turkekul, E. Chan and the Molecular Cytology Core for expert assistance and critical feedback in the design and execution of tissue embedding, immunostaining and processing of tissues, in addition to microscopy assistance. We thank the MSK Molecular Core Cytology Facility for critical technical assistance in performing tissue sections, immunohistochemical stains, scans and analysis (Institutional Core Grant Number P30CA008748). We thank R. Shah for initial discussions on set-up of the MSK-IMPACT experiments. We thank H. Park and L. Lee for assistance with preparation and processing of tumor and tumoroid specimens for IHC and immunofluorescence. We thank P. Watson of Memorial Sloan Kettering for sharing the Ubc-eGFP-Luc vector. We thank M. Gonzalez and R. Andrade for their work curating patient tissue and slides for analyses. We also thank members of the Sawyers laboratory for critical review, lively discussion, and helpful comments on this work as it developed. In addition, we thank R. Beauchamp and J. Goldenring for constructive criticism and comments in the development and presentation of this work. The authors thank J. Novak of Memorial Sloan Kettering for editorial assistance. This work was supported in part by the NIH/NCI Cancer Center Support Grant P30 CA008748. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R25CA020449. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.J.S. was supported by NIH/NCI grant 5R01-CA182551-04 and the MSK Colorectal Cancer Research Center. J.J.S. is also supported by the American Society of Colon and Rectal Surgeons Career Development Award, the Joel J. Roslyn Faculty Research Award, the American Society of Colon and Rectal Surgeons Limited Project Grant, the MSK Department of Surgery Junior Faculty Award and the John Wasserman Colon and Rectal Cancer Fund. J.J.S. is also supported by the Colorectal Cancer Alliance and the Chris4Life Research Award. The work was funded in part by a Stand Up to Cancer (SU2C) Colorectal Cancer Dream Team Translational Research Grant (Grant Number: SU2C: AACR-DR22-17) (J.J.S., A.C., L.E.D., K.G.). Stand Up to Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association of Cancer Research, the scientific partner of SU2C. J.J.S. is partly supported by a loan repayment grant from the NIH via the National Cancer Institute for this work. J.J.S. is also supported in part by funding from the Howard Hughes Medical Institute via C.L.S. P.B.P and M.A. are funded in part by gifts from Corinne Berezuk, Michael Stieber, and Patrick A. Gerschel. C.L.S. is an investigator of the Howard Hughes Medical Institute. This project was supported by National Institutes of Health grants CA155169, CA193837, CA224079, CA092629, CA160001, CA008748 and Starr Cancer Consortium grant I10-0062. K.G. was supported by National Institutes of Health grants K08-CA230213 and T32-CA009207, and by an American Cancer Society Postdoctoral Fellowship, AACR Basic Cancer Research Fellowship, Conquer Cancer Foundation of ASCO Young Investigator Award, Shulamit Katzman Endowed Postdoctoral Research Fellowship, and a SU2C Colorectal Cancer Dream Team Translational Research Grant (SU2C: AACR-DR22-17). J.M. is supported by National Institutes of Health grants CA94060 and CA12924, and by the MSKCC Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center. S.W.L., L.E.D. and K.P.O. are supported by grants from the NIH (U54 OD020355-01) and by the Starr Cancer Consortium (I8-A8-030). S.W.L. is an investigator of the Howard Hughes Medical Institute. K.P.O. was supported by an F30 Award from the NIH/NCI (1CA200110-01A1) and 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. L.E.D. is supported by a Stand Up to Cancer Colorectal Cancer Dream Team Translational Research Grant (Grant Number: SU2C: AACR-DR22-17) and was supported by a K22 Career Development Award from the NCI/NIH (CA 181280-01). P.B.R. is supported by the Memorial Sloan Kettering Cancer Center Imaging and Radiation Sciences Program. P.B.R. is also supported in part by a K12 Paul Calebresi Career Development Award for Clinical Oncology (K12 CA184746) and an NIH loan repayment program award (LRP).

Author information

Author notes

  1. These authors contributed equally: Karuna Ganesh, Chao Wu, Charles L. Sawyers, J. Joshua Smith.

Authors and Affiliations

  1. Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Karuna Ganesh & Richard N. Kolesnick

  2. Gastrointestinal Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Karuna Ganesh, Andrea Cercek, Leonard B. Saltz & Rona Yaeger

  3. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Chao Wu, Wouter R. Karthaus, Charles L. Sawyers & J. Joshua Smith

  4. Colorectal Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Chao Wu, Bryan C. Szeglin, Charles-Etienne Gabriel Sauvé, Mohammad Adileh, Isaac Wasserman, Michael R. Marco, Francisco Sanchez-Vega, Seo-Hyun Choi, Raphael Pelossof, Emmanouil Pappou, Arthur Elghouayel, James S. Strong, Chin-Tung Chen, Jennifer W. Harris, Martin R. Weiser, Garrett M. Nash, Jose G. Guillem, Iris H. Wei, Philip B. Paty, Julio Garcia-Aguilar & J. Joshua Smith

  5. Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Kevin P. O’Rourke, Joan Massagué & Scott W. Lowe

  6. Weill Cornell Medicine/Rockefeller University/Sloan Kettering Tri-Institutional MD–PhD Program, New York, NY, USA

    Kevin P. O’Rourke

  7. Albert Einstein College of Medicine, Bronx, NY, USA

    Bryan C. Szeglin

  8. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Youyun Zheng, Maha Shady, Helen H. Won, Peter Ntiamoah, Michael F. Berger & Jinru Shia

  9. Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Youyun Zheng, Maha Shady, Helen H. Won & Michael F. Berger

  10. Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Amanda S. Kim & Paul B. Romesser

  11. Computational Oncology Service, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Francisco Sanchez-Vega

  12. Molecular Cytology Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Afsar Barlas & Katia O. Manova-Todorova

  13. Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Harini Veeraraghavan

  14. Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Eduardo J. Ortiz & Iva Petkovska

  15. Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Jessica A. Lavery & Sujata Patil

  16. Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA

    Ronald P. DeMatteo

  17. Department of Public Health Sciences, Sylvestor Comprehensive Cancer Center, Miami, FL, USA

    Xi Chen

  18. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, University of Medical Center, Utrecht, The Netherlands

    Hans Clevers

  19. Gastrointestinal Pathology, Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Jinru Shia

  20. Sandra and Edward Meyer Cancer Center, Departments of Medicine and Biochemistry, Weill Cornell Medicine, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA

    Lukas E. Dow

Authors
  1. Karuna Ganesh
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  2. Chao Wu
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  3. Kevin P. O’Rourke
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  4. Bryan C. Szeglin
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  6. Charles-Etienne Gabriel Sauvé
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  8. Isaac Wasserman
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  9. Michael R. Marco
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  10. Amanda S. Kim
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  11. Maha Shady
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  13. Wouter R. Karthaus
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  14. Helen H. Won
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  19. Emmanouil Pappou
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  20. Arthur Elghouayel
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  21. James S. Strong
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  22. Chin-Tung Chen
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  23. Jennifer W. Harris
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  24. Martin R. Weiser
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  25. Garrett M. Nash
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  26. Jose G. Guillem
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  27. Iris H. Wei
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  28. Richard N. Kolesnick
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  29. Harini Veeraraghavan
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  30. Eduardo J. Ortiz
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  31. Iva Petkovska
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  32. Andrea Cercek
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  33. Katia O. Manova-Todorova
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  34. Leonard B. Saltz
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  35. Jessica A. Lavery
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  36. Ronald P. DeMatteo
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  37. Joan Massagué
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  38. Philip B. Paty
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  39. Rona Yaeger
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  40. Xi Chen
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  41. Sujata Patil
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  42. Hans Clevers
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  43. Michael F. Berger
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  44. Scott W. Lowe
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  45. Jinru Shia
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  46. Paul B. Romesser
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  47. Lukas E. Dow
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  48. Julio Garcia-Aguilar
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  49. Charles L. Sawyers
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  50. J. Joshua Smith
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Contributions

J.J.S. conceived the initial idea behind this work in concert with K.G., C.W., C.L.S., and J.G.-A., and edited and wrote the paper with K.G., C.W., B.C.S. and C.L.S. C.W., K.G., M.A., K.P.O., B.C.S., P.B.R., A.S.K. and J.J.S. performed the experiments and collected the data. J.J.S., C.W., C.L.S., B.C.S., C.-E.G.S. and K.G. made final edits, figures and completed the paper. K.G., K.P.O., S.W.L. and L.E.D. provided the initial technical expertise to complete this work and assisted in editing the paper, along with P.B.R. W.R.K. and H.C. provided initial expertise and critical input on the derivation of the RC tumoroids, along with critical input on the final figures and methods. P.B.R. and A.S.K. completed the radiation biology experiments. P.B.P., M.A., and R.N.K. completed initial pilot ex vivo radiation experiments that formed important preliminary data for the currently displayed radiation work. I.P., E.J.O. and H.V. made substantial contributions to interpreting and gathering radiographic data and images. I.W., R.P., M.R.M., A.E., J.S.S., J.S. and B.C.S. made substantial contributions to obtaining and interpreting the data in Figs. 2 and 3. M.S., Y.Z., F.S.-V., H.H.W. and R.P. made substantial contributions ito either obtaining or interpreting the data in Fig. 1 and Extended Data Fig. 6. S.-H.C., C.-T.C. and J.W.H. played critical roles in the collection, assessment, analysis, and execution of the experiments completed in Figs. 1, 3 and 4. J.A.L., S.P. and X.C. reviewed the data and advised the biostatistical and logistic regression aspects of this work. A.B., P.N. and K.O.M.-T. had critical roles in the characterization of the model with histopathologic and immunochemical expertise. J.S. performed the histopathologic review of tumoroid and patient H&Es. M.R.W., R.P.D., G.M.N., J.G.G., A.C., E.P., I.H.W., P.B.P., J.G.-A., L.B.S., J.M., and R.Y. contributed patients, critical clinical information, and critiqued and edited the paper. M.F.B. supervised M.S., H.H.W., and Y.Z., and conceived the MSK-IMPACT experiments and data interpretation with J.J.S. S.W.L., J.G.-A., and C.L.S. mentored J.J.S. and provided critical input, resources, critique, and oversight of this work.

Corresponding authors

Correspondence to Charles L. Sawyers or J. Joshua Smith.

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Competing interests

J.J.S. has received travel support from Intuitive Surgical Inc. and has served as a clinical advisor for Guardant Health, Inc. C.L.S. serves on the Board of Directors of Novartis, is a co-founder of ORIC Pharm and co-inventor of enzalutamide and apalutamide. He is a science advisor to Agios, Beigene, Blueprint, Column Group, Foghorn, Housey Pharma, Nextech, KSQ Therapeutics, Petra and PMV Pharma. He was a co-founder of Seragon, purchased by Genentech/Roche in 2014. J.M. is a science advisor and owns company stock in Scholar Rock. H.C. is an inventor on several patents related to organoid technology. S.W.L. is a co-founder and scientific advisory board member for ORIC Pharmaceuticals, Blueprint and Mirimus. He also serves on the scientific advisory board for Constellation, Petra and PMV and has recently served as a consultant for Forma, Boehringer Ingelheim and Aileron. J.G.-A. has received support from Medtronic (honorarium for consultancy with Medtronic), Johnson & Johnson (honorarium for delivering a talk) and Intuitive Surgical (honorarium for participating in a webinar by Intuitive Surgical). P.B.R. has received honorarium from Corning to discuss 3D cell culture techniques), has served as a consultant for AstraZeneca and is a consultant for EMD Serono for work on radiation sensitizers. R.N.K. is a cofounder of Ceramedix Holding, LLC. He also has patents unrelated to this work: R.N.K. (US7195775B1, US7850984B2, and US10052387B2), R.N.K. (US8562993B2, US9592238B2, US20150216971A1, and US20170335014A1) and R.N.K. (US20170333413A1 and US20180015183A1). K.P.O. has received an honorarium from Merck for discussing organoid platforms.

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Peer review information Javier Carmona was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Rectal cancer tumoroid derivation and patient characteristics.

The diagram shows the outcome of attempts to derive tumoroids from 84 rectal cancer (RC) tumor samples from 58 individual patients with RC. 65 RC tumoroids from 41 patients (77%) were successfully derived. For the 19 failed derivations, the points of failure are shown. Demographics from each group are displayed (RAS status (WT or mutant (MUT)), neoadjuvant therapy, metastatic status at derivation, location of the primary tumor (middle/distal or upper rectum), sex, and age). Two patients were mismatch-repair deficient (not shown).

Extended Data Fig. 2 Preservation of rectal cancer histopathology in tumoroids.

a, Gross resected rectal specimen from which the first RC tumoroid line (RC-MSK-001) was derived, and representative brightfield microscopy of the tumoroid in 3D culture 2 months after processing. Lower panels show hematoxylin and eosin (H&E) staining of the patient tumor (bottom left panel) and the derived RC-MSK-001 tumoroid (bottom right panel) in 3D culture. Scale bars, 50 μm. b, Hoechst and MitoTracker stains of a representative section of the RC-MSK-001 tumoroid demonstrate the luminal and glandular structure. Scale bars, 20 µm. c, Perineal recurrence of the original RC-MSK-001 tumor and the derived tumoroid (RC-MSK-001PR) are shown with H&E staining. Scale bar, 50 μm. d, H&E comparison of 32 additional tumoroid cell lines as noted with the corresponding primary tumor from which they were derived. Scale bars, 50 μm. All representative images are from one patient-specific tumor-to-tumoroid derivation.

Extended Data Fig. 3 Tumoroids preserve both architecture, cytology and colorectal-specific staining patterns of the primary tumors from which they were derived.

a, Examples of architecture preservation in tumoroids and primary tumors. Scale bars, 50 μm. b, Examples of cytological preservation in specific tumoroids. Scale bars: low magnification 50 μm; high magnification inset, 10 μm. Both architecture and cytology features were identified by a gastrointestinal pathologist. c, CDX2 and β-catenin were quantified by both presence and intensity of stain on a 0–3 scale. Presence is defined as the percentage of cells with staining: 0 = 0%, 1 = <30%, 2 = 30–60%, 3 = >60%. Intensity defines the strength of staining: 0 = none, 1 = weak, 2 = moderate, 3 = strong. Examples for both CDX2 and β-catenin are displayed. Intensity of staining is assessed exclusively within the nuclear compartment. Scale bar, 20 μm. d, The presence and intensity of each tumoroid is shown graphically according to the key. Cohen’s 𝛋 was used to assess similarity in score between matched primary and tumoroid samples: β-catenin presence score, 𝛋 = 0.51, P = 0.0021; β-catenin intensity score, 𝛋 = 0.63, P = 0.00034; CDX2 presence score, 𝛋 = 0.45, P = 0.0037; CDX2 intensity score, 𝛋 = 0.527, P = 0.00042. All representative images are from one patient-specific tumor-to-tumoroid derivation.

Extended Data Fig. 4 Conservation of enterocyte markers.

Eleven tumoroids are compared to their respective primary tumors for Alcian blue, CK20, CDX2, MUC-2, E-cadherin, and β-catenin staining. For immunofluorescent staining: E-cadherin (green), DAPI (blue). See Fig. 1b for another example of RC-MSK-001 Alcian blue, CK20, and CDX2 comparisons. Scale bars, 50 μm. All representative images are from one patient-specific tumor-to-tumoroid derivation.

Extended Data Fig. 5 Comparison of nuclear mismatch repair proteins between patient and tumoroid samples.

Immunohistochemistry of the nuclear mismatch repair (MMR) proteins MSH2, MSH6, MLH1, and PMS2. The presence of each protein is assessed by nuclear staining verified by pathologic analysis with (+) indicating present and (–) indicating absent staining. a, Displayed are two MMR-proficient tumoroids, RC-MSK-001 and RC-MSK-002. b, Displayed are two MMR-deficient tumors. RC-MSK-031 is deficient in MSH2 and MSH6. RC-MSK-034 is deficient in MLH1 and PMS2. Scale bars, 50 μm. All representative images are from one patient-specific tumor-to-tumoroid derivation.

Extended Data Fig. 6 The mutational fingerprint in derived RC tumoroids.

a, The mutational fingerprint of 31 RC tumoroids for the most common alterations, as determined by MSK-IMPACT, are displayed. The frequency of alteration is noted along with the type of genetic alteration relative to truncating mutation, in-frame mutation, missense mutation or splice-site alterations (as noted by the color code). b, Example of a tumoroid (RC-MSK-003) with complete conservation of mutations between the tumoroid and the primary tumor from which it was derived. c, Example of a tumoroid (RC-MSK-004) with conservation of driver mutations and the addition of two secondary mutations noted in the tumoroid in culture only. d, Percentage of concordance between tumoroid and tumor among mutations predicted to be oncogenic overall and by each patient. The mutations represented are those annotated by OncoKB10 as oncogenic or likely oncogenic in each tumoroid and tumor pair. e, All mutations called in the MSK-IMPACT sequencing of tumoroids and primary tumors are shown. The numbers of mutations are displayed with regard to each gene (by column) and each tumoroid and tumor pair (by row). Mutations are colored by concordance status.

Extended Data Fig. 7 Swimmer’s plot of each patient treated with 5-FU-based therapy whose tumoroid has been analyzed for chemosensitivity ex vivo.

Data are displayed from top to bottom by descending AUC calculated from the 5-FU dose-response experiments presented in Fig. 2a. The blue areas denote progression-free survival (PFS) intervals from treatment start date, as indicated above. Seven of the nine patients have progressed, with the current status of the two patients who have not progressed indicated (RC-MSK-039 and RC-MSK-025). All patients were treated with FOLFOX, with the exception of RC-MSK-003 (capecitabine (oral 5-FU prodrug) + oxaliplatin + bevacizumab) and RC-MSK-023 (FOLFIRI: 5-FU + leucovorin + irinotecan). Four patients for whom ex vivo chemosensitivity data are presented in Fig. 2a are not shown because they did not receive 5-FU-based therapy.

Extended Data Fig. 8 Resistance to a targeted anti-epidermal growth factor receptor therapy, cetuximab, in KRAS-mutant compared with KRAS-WT tumoroids.

Resistance to cetuximab is demonstrated in KRAS-mutant RC tumoroids (blue) compared with a KRAS-WT tumoroids (orange). Dose range was used as shown and percentage of live cells is displayed for each tumoroid. Results are from two independent experiments done in technical quadruplicate; mean ± s.e.m.

Extended Data Fig. 9 Demonstration of endorectally implanted human rectal cancer.

a, The RC-MSK-001 endoluminal mouse biopsy represented in Fig. 3a shows serially sectioned and stained hEpCAM; collagen IV; merged with DAPI. n = 5 mice scale bars: 200 µm; inset, 50 µm. b, Colon from an unimplanted NSG mouse as control for a. Scale bars, 200 µm. c,d, 12-week endoscopy of a mouse transplanted with RC-MSK-001 (n = 5) or RC-MSK-002 (n = 7) tumoroids. e,f, Distinct staining was noted for human and mouse EpCAM for RC-MSK-001 (n = 5) and RC-MSK-002 (n = 7) engrafted NSG mice. Scale bars: 500 μm; inset, 50 μm. g, RC-MSK-001 tumoroids labeled with GFP and viewed by brightfield (left), intravital GFP imaging (middle; endoscopically (right). Scale bars, 100 µm. h, Invasive rectal tumor after RC-MSK-001 tumoroid implantation (n = 7 mice) stained for H&E, GFP (IHC), and IF (hEpCAM, mEpCAM, and hKi67, each merged with DAPI). i, Independent experiment similar to Fig. 3c of one male NSG mouse euthanized at 22 weeks post-transplantation. Implanted rectal tumor, H&E, and IF (hEpCAM, mEpCAM, DAPI) showing engraftment and invasion of human tumoroids. Scale bars: H&E, 500 μm; IF, 100 μm. j–l, RC-MSK-001 endorectal tumor 16 weeks post-transplantation (n = 3 mice). H&E demonstrates invasion at the junction between the columnar and squamous epithelium of anorectal junction. j,k, H&E; l, DAPI + hEpCAM + collagen IV; scale bars are as follows: j, 1,000 μm; k,l: 400 μm; insets, 100 μm. m, Liver metastasis in an independent experiment (see Fig. 3d) after rectal transplantation in a male NSG mouse euthanized at 36 weeks. Liver metastasis shows poorly differentiated histology. Scale bars for H&E: 1,000 μm, 500 μm, 100 μm. n, Axial and coronal CT images of liver metastases in the corresponding patient (arrowheads) discussed in m. o, Human-specific Alu qPCR demonstrates that the metastases in Fig. 3d and current panel m arose from implanted human tumoroids (Results are based on three independent RNA isolates). Mean ± s.d. Staining: hEpCAM (green), hKi67 (green), mEpCAM (red), Collagen IV (red) and DAPI (blue).

Extended Data Fig. 10 Histopathologic conservation of glandular architecture in the endoluminally implanted RC tumoroids.

H&E images are shown for the RC-MSK-008, RC-MSK-002, RC-MSK-023 and RC-MSK-001 tumoroid lines. Left panels display the primary patient tumor from which the tumoroid was derived once per patient. Middle panels display the tumoroids in 3D culture. Right panels display the engrafted tumoroids within the mouse rectum following endoluminal transplantation. The number of mice engrafted with indicated tumoroids is 8, 7, 8 and 5 (top to bottom). The H&E photomicrographs demonstrate histopathologic conservation of glandular features as noted in the human adenocarcinomas from which they were derived. Scale bar, 50 μm.

Supplementary information

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Reporting Summary

Supplementary Table 1

Supplementary Table 1

Supplementary Video 1

Endoscopic video of tumor following 200,000 cells from tumoroids injected in NOD scid gamma mice rectum

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Ganesh, K., Wu, C., O’Rourke, K.P. et al. A rectal cancer organoid platform to study individual responses to chemoradiation. Nat Med 25, 1607–1614 (2019). https://doi.org/10.1038/s41591-019-0584-2

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