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SOX17 enables immune evasion of early colorectal adenomas and cancers

Nature volume 627, pages 636–645 (2024)Cite this article

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Abstract

A hallmark of cancer is the avoidance of immune destruction. This process has been primarily investigated in locally advanced or metastatic cancer1,2,3; however, much less is known about how pre-malignant or early invasive tumours evade immune detection. Here, to understand this process in early colorectal cancers (CRCs), we investigated how naive colon cancer organoids that were engineered in vitro to harbour Apc-null, KrasG12D and Trp53-null (AKP) mutations adapted to the in vivo native colonic environment. Comprehensive transcriptomic and chromatin analyses revealed that the endoderm-specifying transcription factor SOX17 became strongly upregulated in vivo. Notably, whereas SOX17 loss did not affect AKP organoid propagation in vitro, its loss markedly reduced the ability of AKP tumours to persist in vivo. The small fraction of SOX17-null tumours that grew displayed notable interferon-γ (IFNγ)-producing effector-like CD8+ T cell infiltrates in contrast to the immune-suppressive microenvironment in wild-type counterparts. Mechanistically, in both endogenous Apc-null pre-malignant adenomas and transplanted organoid-derived AKP CRCs, SOX17 suppresses the ability of tumour cells to sense and respond to IFNγ, preventing anti-tumour T cell responses. Finally, SOX17 engages a fetal intestinal programme that drives differentiation away from LGR5+ tumour cells to produce immune-evasive LGR5− tumour cells with lower expression of major histocompatibility complex class I (MHC-I). We propose that SOX17 is a transcription factor that is engaged during the early steps of colon cancer to orchestrate an immune-evasive programme that permits CRC initiation and progression.

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Fig. 1: Early in vivo epigenetic alterations of AKP CRCs converge on SOX17.
Fig. 2: SOX17 loss leads to immune-mediated rejection of colon tumours.
Fig. 3: SOX17 suppresses CD8+ T cell-mediated tumour elimination via IFNγ signalling.
Fig. 4: SOX17 suppresses IFNGR1-mediated MHC-I expression to evade CD8+ T cell-mediated tumour cell killing.
Fig. 5: SOX17 produces LGR5− cells to evade anti-tumour immunity.

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Data availability

Datasets generated in this study are available at the Gene Expression Omnibus (GEO) repository (GSE222713, token: ifylaumkxlsvvqh): including RNA-seq data (GSE222709, GSE222710 and GSE245668), ATAC-seq data (GSE222712) and single-cell RNA-seq data (GSE222711). Any additional information required to reanalyse the data reported in this paper is available upon request.

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Acknowledgements

The authors thank the Swanson Biotechnology Center at the Koch Institute, which encompasses the Flow Cytometry, Histology, Microscopy and Genomics and Bioinformatics Core facilities (NCI P30-CA14051); the Department of Comparative Medicine for mouse husbandry support; S. Holder and members of the Hope Babette Tang (1983) Histology Facility for substantial histology support; members of the Yilmaz laboratory for discussions; K. Kelley for laboratory management; L. Galoyan for administrative assistance; K. W. Wucherpfennig for reading of the manuscript; L. M. LaFave for help and advice with the ATAC-seq experiments; C. P. Concepcion for help and advice with the CUT&RUN experiments; and A. Bhutkar for help and advice for the RNA-seq and ATAC-seq analyses. N.G. is supported by Postdoctoral Fellowship for Research Abroad from Japan Society for the Promotion of Science and 1K99AG076987-01A1. S.G. is supported by the research grant of Astellas Foundation and Postdoctoral Fellowship for Research Abroad from Japan Society for the Promotion of Science. Ö.H.Y. is supported by R01CA254314, R01CA257523, R01DK126545, R01DK133919, and U54CA224068; a Pew-Stewart Trust scholar award; AFAR and Glenn Foundation for Medical Research Breakthroughs in Gerontology (BIG) Award; Kenneth Rainin Foundation Innovator Award; and a Crohn’s & Colitis Foundation Senior Research Award. Ö.H.Y. receives support from the MIT Stem Cell Initiative via Fondation MIT. J.A. is supported by R01DK132544, the Smith Family Awards Program for Excellence in Biomedical Research, the Ludwig Center at Harvard, the Ira Schneider Foundation, the Claudia Adams Barr Program in Innovative Basic Cancer Research Program, and the Innovation research Funds at DFCI. J.A. is a member of the Parker Institute for Cancer Immunotherapy and a New York Stem Cell Foundation–Robertson Investigator, and her research is funded, in part, by these two institutes. Ö.H.Y. and J.A. together are supported by the Footbridge and Traditional Bridge Koch Institute–Dana-Farber Cancer Institute awards.

Author information

Author notes

  1. These authors contributed equally: Peter M. K. Westcott, Saori Goto

  2. These authors jointly supervised this work: Judith Agudo, Ömer H. Yilmaz

Authors and Affiliations

  1. Department of Biology, The David H. Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA

    Norihiro Goto, Peter M. K. Westcott, Saori Goto, Shinya Imada, George Eng, Jonathan Braverman, Tyler Jacks & Ömer H. Yilmaz

  2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    Peter M. K. Westcott

  3. Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

    Martin S. Taylor, George Eng & Ömer H. Yilmaz

  4. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA

    Jonathan Braverman

  5. Department of Pathology, Beth Israel Deaconess Hospital and Harvard Medical School, Boston, MA, USA

    Vikram Deshpande & Ömer H. Yilmaz

  6. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA

    Judith Agudo

  7. Department of Immunology, Harvard Medical School, Boston, MA, USA

    Judith Agudo

  8. Ludwig Center at Harvard, Boston, MA, USA

    Judith Agudo

  9. Parker Institute for Cancer Immunotherapy at Dana-Farber Cancer Institute, Boston, MA, USA

    Judith Agudo

  10. New York Stem Cell Foundation–Robertson Investigator, New York, NY, USA

    Judith Agudo

  11. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Ömer H. Yilmaz

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Contributions

N.G., P.M.K.W., T.J., J.A. and Ö.H.Y. conceived the study. N.G., P.M.K.W., S.G., J.A. and Ö.H.Y. designed the experiments. N.G. performed most of the experiments. P.M.K.W. assisted with co-culture experiments of T cells and organoids and provided reagents. S.G. performed the autochthonous mouse adenoma model experiments. P.M.K.W. assisted with the RNA-seq and ATAC-seq analyses. S.I. and S.G. assisted organoid orthotopic transplantation experiments. J.B. generated mouse naive AKP organoids. G.E. established healthy human colon organoids from patients. M.S.T. and V.D. provided human histopathological samples with diagnostic information and assisted in the interpretation of histology. N.G., J.A, and Ö.H.Y wrote the paper. All the authors assisted in the interpretation of the experiments and the editing of the paper.

Corresponding authors

Correspondence to Judith Agudo or Ömer H. Yilmaz.

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Competing interests

Ö.H.Y. holds equity and is a scientific advisory board member in Ava Lifesciences and AI Proteins. Ö.H.Y. receives research support from Microbial Machines. Ö.H.Y. is a consultant for Nestle. T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific and a co-founder of Dragonfly Therapeutics and T2 Biosystems; serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics; and is president of Break Through Cancer. The laboratory of T.J. currently receives funding from Johnson & Johnson and The Lustgarten Foundation, but these funds did not support the research described in this manuscript. The other authors declare no competing interests.

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Nature thanks Ophir Klein, Sean Morrison and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 In vivo transplantation of naïve AKP organoids induces SOX17 expression.

a, Schematic of generation of mouse naïve AKP organoids by shApc and primary tumour-derived AKP organoids from immunocompetent mice by colonoscopy-based orthotopic transplantation. b, c, PCA plot (b) and volcano plot (c) of RNA-seq in naïve and primary tumour-derived AKP organoids (shApc model) from immunocompetent mice. n = 3-6 mice per group. d, Schematic of RNA-seq in bulk resected primary tumours as well as in flow cytometry-sorted tdTomato+ tumour cells that were freshly isolated from primary tumours. e, f, PCA plot (e) and volcano plot (f) of RNA-seq in naïve AKP organoids and bulk resected primary tumours. n = 3-6 mice per group. g, h, PCA plot (g) and volcano plot (h) of RNA-seq in naïve AKP organoids and flow cytometry-sorted tdTomato+ tumour cells that were freshly isolated from primary tumours. n = 3-4 mice per group.

Extended Data Fig. 2 Autochthonous tumours and transplanted tumours in immunodeficient mice show elevated SOX17 expression.

a, Autochthonous tumour models by colonoscopy-guided 4-OH-Tamoxifen injection into the colonic mucosa of Villin-CreERT2; Apc f/f; Kras LSL-G12D; P53 f/f; Rosa LSL-tdTomato (AKPVT) mice. b-d, PCA plot (b), volcano plot (c), and GSEA (d) of RNA-seq in normal colon and autochthonous AKPVT tumours. n = 4-7 mice per group. e, Schematic of generation of mouse naïve AKP organoids and mouse primary tumour-derived AKP organoids from immunodeficient mice (Rag2−/− mice) by colonoscopy-based orthotopic transplantation. f, g, PCA plot (f) and volcano plot (g) of RNA-seq in naïve and primary tumour-derived AKP organoids from immunodeficient mice. n = 3 mice per group.

Extended Data Fig. 3 Generation of human naive AKP organoids.

a, Schematic of generation of human naive AKP organoids by CRISPR-Cas9 editing. b, Representative image of human APC−/− organoids and Sanger sequencing result confirming the biallelic frameshift mutations in APC. c, Representative image of human APC−/−; P53−/− organoids and Sanger sequencing result confirming the biallelic frameshift mutations in TP53. d, e, Strategy to knock in a KRASG12D point mutation by homology directed repair (d), and PCR results confirming the correct homology directed repair (e). f, Representative image of human APC−/−; KRASG12D; P53−/− organoids and the Sanger sequencing result confirming the KRASG12D point mutation. g, Immunoblots for SOX17 in human naive AKP organoids and in vivo tumour-derived human AKP organoids.

Extended Data Fig. 4 SOX17 loss does not affect tumour growth/proliferation in immunodeficient mice.

a, IHC for SOX17 in sgScramble, sgSox17-1, and sgSox17-2 in vivo tumour-derived AKP organoids. b, Time-course images of sgScramble, sgSox17-1, and sgSox17-2 AKP organoids. 2,000 single cells in 10 µl Matrigel were plated for each well. Representative of 10 wells. Representative of three independent experiments. See also Fig. 2c for organoid growth curve. c, d, IHC for BrdU (c) and the percentage of BrdU+ cells (d) in sgScramble, sgSox17-1, and sgSox17-2 AKP organoids that were incubated for 4 hrs with 10 µM BrdU in the culture media. n = 30 organoids per group. e, Representative images and H&E staining of orthotopically transplanted sgScramble, sgSox17-1, and sgSox17-2 AKP tumours in immunocompetent mice. f, g, IHC for Ki67 (f) and quantification (g) in orthotopically transplanted sgScramble, sgSox17-1, and sgSox17-2 AKP tumours in NCG mice. n = 20 fields from 5 mice per group. h, i, IHC for Ki67 (h) and quantification (i) in orthotopically transplanted sgScramble, sgSox17-1, and sgSox17-2 AKP tumours in Rag2−/− mice. n = 20 fields from 5 mice per group. One-way ANOVA (d, g, i). Data are mean ± SD. N.S. not significant. Scale bar, 20 μm (a, c, f, h), 1000 μm (b), 500 μm (e).

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Extended Data Fig. 5 Sox17 knockdown in established AKP tumours inhibits tumour growth.

a, Constructs for Sox17 knockdown and control. b, qRT-PCR for Sox17 mRNA expression 48 hr after doxycycline administration. n = 3 organoid culture wells per group. c, Schematic of Sox17 knockdown in established AKP tumours using doxycycline-inducible system. d, Tumour formation was confirmed by colonoscopy 1 week after the transplantation. e, f, Representative images (e) and tumour size (f) 2 weeks after doxycycline administration. n = 15 mice per group. Pooled from three independent experiments. g, h, IHC for CD4 and CD8 (g) and quantification (h) in control (shRenilla) and Sox17 knockdown (shSox17) tumours. n = 20 fields from 5 mice per group. Unpaired two-tailed t-tests (b, f, h). Data are mean ± SD. *p < 0.05. Scale bar, 20 μm (g).

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Extended Data Fig. 6 Time-course analysis of scRNA-seq of immune cells in control and SOX17-null tumours.

a, b, UMAP plots of CD45+ cells in control and SOX17KO AKP tumours at 1-, 2- and 4-weeks post-transplantation. Classified by sample groups (a) and cell clusters (b). Control at 1 wk; n = 5,296 cells, control at 2 wks; n = 4,585 cells, control at 4 wks; n = 6,230 cells, SOX17KO at 1 wk; n = 7,047 cells, SOX17KO at 2 wks; n = 2,894 cells, SOX17KO at 4 wks; n = 6,207 cells, pooled from 5 mice in each group. c, Expressions of key genes used for identification of major populations in the scRNA-seq analysis of control and SOX17KO tumours.

Extended Data Fig. 7 SOX17 alters CD4+ T cell subsets in AKP CRCs.

a, b, Time-course analysis of CD45+ cells in control and SOX17KO AKP tumours at 1-, 2- and 4-weeks post-transplantation by scRNA-seq. UMAP plots (a). Bar graphs showing % of each subcluster (b). c-e, UMAP plots (c) of re-clustering of CD4+ T cell clusters (clusters 5 and 9 in Extended Data Fig. 7b). Bar graphs showing % of each subcluster (d). Dot plots of gene expressions in each subcluster (e).

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Extended Data Fig. 8 Effector Th1 CD4+ T cells contribute to the elimination of SOX17-null tumours.

a-c, IHC for SOX17 (a) and CD4 (b) and quantification (c) in sgScramble, sgSox17-1, and sgSox17-2 AKP tumours at 1-, 2- and 4-weeks post-transplantation. n = 20 fields from 5 mice per group. d, Violin plot for Ifng expression in scRNA-seq data from Extended Data Fig. 6a. e-g, Schematic (e), representative images (f), and tumour engraftment rate (g) of orthotopically transplanted SOX17KO (sgSox17-1 and sgSOX17-2) AKP tumours in isotype control- and anti-CD4 antibody-treated mice. n = 10 mice per group. Pooled from two independent experiments. h, SOX17-null tumour size in anti-CD4 antibody and anti-CD8 antibody-treated mice. n = 9-10 mice per group. Pooled from two independent experiments. i, j, IHC for CD8 (i) and quantification (j) in isotype control- and anti-CD4 antibody-treated mice. n = 20 fields from 5 mice. One-way ANOVA (c). Fisher’s exact test (g). Unpaired two-tailed t-tests (h, j). Data are mean ± SD. *p < 0.05. Scale bar, 25 μm (a), 20 μm (b, i).

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Extended Data Fig. 9 SOX17 expression augments the resistance of colon cancer cells to CD8+ T cell-mediated killing.

a-b, Sanger sequencing chromatograms and TIDE analyses for sgH2k1 clones (a) and sgB2m clones (b). c-e, Schematic (c), representative images (d), and tumour engraftment rate (e) of orthotopic transplantation of SOX17KO (sgSox17-1); sgScramble, SOX17KO; H2K1 KO (sgH2k1 clone 3), and SOX17KO; B2M KO (sgB2m clone 23) AKP organoids into the colons of immunocompetent mice. n = 7-13 mice per group. f, Schematic of co-culture experiments of SOX17OE SIINFEKL-expressing colon cancer organoids with activated OT-1 T cells. g-i, Representative images (g), and quantification of organoids (h) and CD8+ T cell numbers (i) in the co-culture experiments. n = 3 per group. j, Ifnar1 and Ifnar2 expression by RNA-seq data of control and SOX17KO AKP organoids. n = 3 per group. k, l, Flow cytometry analysis of protein surface levels of IFNAR1 (k) and quantification (l). n = 3 per group. Chi-square test (e). Unpaired two-tailed t-tests (h, I, j). One-way ANOVA (l). Data are mean ± SD. *p < 0.05. N.S. not significant. Scale bar, 500 μm (h).

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Extended Data Fig. 10 Generation of SOX17 overexpression organoids and IFNGR1 knockout organoids.

a, Constructs for constitutive SOX17 overexpression (SOX17OE). b, Immunoblots confirmed SOX17 overexpression in naïve AKP organoids transduced with a constitute SOX17OE cassette. c, Constructs for dox-inducible SOX17OE. d, Immunoblots confirmed SOX17 overexpression in naïve AKP organoids transduced with a dox-inducible SOX17OE cassette 72 h after doxycycline administration. e, qRT-PCR for Ifngr1 mRNA expression in constitutive and dox-inducible SOX17OE AKP organoids compared with controls. n = 3-6 each group. f, Sanger sequencing chromatograms for IFNGR1KO clones. g, h, Flow cytometry (g) and quantification (h) of IFNGR1 expression. n = 3 per group. i, j, Flow cytometry (i) and quantification (j) of MHC-I expressions 24 hr after IFNγ (10 ng/ml) administration in the organoids. n = 3 per group. One-way ANOVA (h). Unpaired two-tailed t-test (j). Data are mean ± SD. N.S. not significant.

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Extended Data Fig. 11 In vivo APC loss induces SOX17 expression.

a-c, Schematic (a), representative images (b), and tumour engraftment rate (c) of orthotopic transplantation of SOX17KO (sgSox17-1) and SOX17KO; CXCL10KO (sgCxcl10 clones 6 and 9) AKP organoids into immunocompetent recipient mice. n = 20 mice per group. Sanger sequencing results of CXCL10KO organoids are in Supplementary Fig. 4. d, IHC for SOX17 in Villin-CreERT2; Apc f/f mouse normal colon and adenoma. e, Schematic of generation of naïve and primary tumour-derived Apc−/− organoids. f, qRT-PCR for Sox17 mRNA expression in naïve and primary tumour-derived Apc−/− organoids. n = 3 mice per group. g, H&E staining and IHC for β-Catenin and SOX17 in Lgr5-CreERT2; Apc f/f and Lgr5-CreERT2; Apc f/f; Sox17 f/f mouse intestines after tamoxifen injections. Representative of n = 6 mice per group. h, Flow cytometry of MHC-I (H-2Kb) expression in Lgr5-GFP− and Lgr5-GFP+ cells from Lgr5-CreERT2; Apc−/− adenoma. n = 4 mice. See Fig. 5i for quantification. Chi-square test (c). Unpaired two-tailed t-test (f). Data are mean ± SD. *p < 0.05. Scale bar, 20 μm (d, g).

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Extended Data Fig. 12 SOX17 expression in human adenomas and CRCs anti-correlates with CD8+ T cell infiltration.

a, b, Representative images (a) and classification (b) of IHC for SOX17 and CD8 in adenomas from patients with (n = 8) or without (n = 11) familial adenomatous polyposis (FAP). c-e, Representative images (c) and classification (d) of IHC for SOX17 and CD8 in CRC samples at different pathologic tumour stages (pT1: n = 9, pT2: n = 10, pT3: n = 10, pT4: n = 10). Plot (e) showing the % of CD8high tumours and CD8low tumours in SOX17high/med tumours versus SOX17low/neg tumours. n = 39 cases. Tumours were classified into SOX17high (≥70%), SOX17med (<70%, ≥30%), SOX17low (<30%, >0%), and SOX17neg (0%) based on the percentage of SOX17 nuclear staining-positive cells. Tumours with ≥ 50 CD8+ cells per x20 field were classified as CD8high tumours, <50 CD8+ cells per x20 field as CD8low tumours. Scale bar, 20 μm (a, c). Fisher’s exact test (e). *p < 0.05.

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Extended Data Fig. 13 Frequency of Lgr5-GFP+ cells decreases over time in adenomas.

a, Schematic of time-course analysis of the tumours in Lgr5-CreERT2; Apc f/f mice after tamoxifen injections. b, c, IF for Lgr5-GFP and β-catenin in Lgr5-CreERT2; Apc f/f mouse intestines (b) and quantification for the percentage of Lgr5-GFP+ cells in β-catenin nuclear accumulation positive tumour cells (c). n = 30 fields from 5 mice per group. d, Schematic of anti-CD8 antibody treatment in Lgr5-CreERT2; Apc f/f mice with tamoxifen injections. e, f, IF for Lgr5-GFP (e) and quantification of Lgr5-GFP+ areas (f) in anti-CD8 or isotype antibody-treated Lgr5-CreERT2; Apc f/f mouse intestines. n = 10 fields from 5 mice per group. g, Lgr5 mRNA expression in RNA-seq of control and SOX17KO AKP organoids. n = 3 per group. h, ISH for Lgr5 in control and SOX17OE AKP tumours. Representative of n = 9-11 mice per group. i, qRT-PCR for Lgr5 mRNA expression in control and SOX17OE naïve AKP organoids. n = 3 per group. One-way ANOVA (c). Unpaired two-tailed t-test (f, g, i). Data are mean ± SEM (c) and mean ± SD (f, g, i). *p < 0.05. Scale bar, 50 μm (b, e), 20 μm (h).

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Extended Data Fig. 14 LGR5+ and LGR5- tumour cells are highly plastic and give rise to one another during tumour growth.

a, Schematic of generation of Lgr5-GFP; AKP organoids and orthotopic transplantation. b, Flow cytometry for Lgr5-GFP+ cells from Lgr5-GFP; AKP tumours and wild-type colon epithelia (negative control). Representative of n = 6 mice. c, qRT-PCR for Lgr5 mRNA expression in sorted Lgr5-GFP− and Lgr5-GFP+ cells from Lgr5-GFP; AKP tumours. n = 3 mice per group. d, e, Schematic (d) and tumour engraftment rate (e) of orthotopic transplantation of sorted Lgr5-GFP− and Lgr5-GFP+ cells into the colons of immunocompetent mice. In each mouse, 4,000 cells were orthotopically transplanted. n = 10-13 mice per group. Representative of two independent experiments. f, IF for Lgr5-GFP and SOX17 in Lgr5-GFP− cell-derived tumours and Lgr5-GFP+ cell-derived tumours. Representative of n = 6 mice per each group. Unpaired two-tailed t-test (c). Fisher’s exact test (e). Data are mean ± SD. N.S. not significant. *p < 0.05. Scale bar, 50 μm (f).

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Goto, N., Westcott, P.M.K., Goto, S. et al. SOX17 enables immune evasion of early colorectal adenomas and cancers. Nature 627, 636–645 (2024). https://doi.org/10.1038/s41586-024-07135-3

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