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A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer

Abstract

Cancer stem cells (CSCs) have been hypothesized to represent the driving force behind tumour progression and metastasis, making them attractive cancer targets. However, conclusive experimental evidence for their functional relevance is still lacking for most malignancies. Here we show that the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) identifies intestinal CSCs in mouse tumours engineered to recapitulate the clinical progression of human colorectal cancer. We demonstrate that selective Lgr5+ cell ablation restricts primary tumour growth, but does not result in tumour regression. Instead, tumours are maintained by proliferative Lgr5 cells that continuously attempt to replenish the Lgr5+ CSC pool, leading to rapid re-initiation of tumour growth upon treatment cessation. Notably, CSCs are critical for the formation and maintenance of liver metastasis derived from colorectal cancers. Together, our data highlight distinct CSC dependencies for primary versus metastasic tumour growth, and suggest that targeting CSCs may represent a therapeutic opportunity for managing metastatic disease.

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Figure 1: Engineering, characterization and engraftment of mouse-derived intestinal tumour organoids.
Figure 2: Selective depletion of Lgr5+ cells reveals a dependency on CSCs for subcutaneous tumour growth.
Figure 3: Lgr5+ CSCs are required for the formation of liver metastasis.
Figure 4: Lgr5+ CSCs are essential for the maintenance of colon-cancer-derived liver metastasis.

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Acknowledgements

We thank M. Junttila and K. Leong for generation of the AKVL mouse model, A. Bruce for help designing schematic figures, J. Jian for tissue processing, J. Stinson and the NGS laboratory for RNA-sequencing, J. Paw, C. Poon, T. Ho and J. Borneo for flow cytometry support, members of Laboratory Animal Resources for animal care and de Sauvage laboratory members for comments on the manuscript.

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Contributions

F.d.S.M. and F.J.d.S. conceptualized the project and designed experiments. F.d.S.M. engineered and characterized all mutant organoid lines, produced concentrated lentivirus, optimized the orthotopic injection methodology and conducted bioluminescent imaging. F.d.S.M., A.V.K. and N.K. performed subcutaneous transplants, endoscopic imaging, DT treatment, tumour measurements and collected tissues. F.d.S.M. and A.V.K. conducted flow cytometry experiments. J.M.H. performed portal vein injections. G.J.P.D. modified pLKO-SHC201, cloned sgRNAs and designed pGEIL-tRFP. F.d.S.M., J.D.H., J.H., J.E.A. and E.E.S. either acquired or evaluated immunohistochemistry and/or immunofluorescence data. H.K. performed histopathological evaluation of tumour and liver tissues. Z.M. contributed next-generation sequencing data. R.P. provided bioinformatics support. F.d.S.M., G.J.P.D. and F.J.d.S. wrote the manuscript.

Corresponding author

Correspondence to Frederic J. de Sauvage.

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All authors are employees of Genentech and own Roche shares.

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Reviewer Information Nature thanks F. Greten, R. Shivdasani and the anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Validation of CRISPR–Cas9-mediated gene targeting.

a, b, Gene-targeting strategy for Trp53 (a) and Smad4 (b). The protospacer adjacent motif (PAM) is underlined on the target sequence and the allelic fraction of each haplotype is indicated. c, d, Sequence alignment of exome-sequencing reads at a representative predicted off-target site for one of the sgRNAs used to mutate either Trp53 (c) or Smad4 (d). Of note, neither single-nucleotide variants nor copy-number changes could be detected. e, Relative p21 (also known as Cdkn1a) expression in AKVL and AKVPL organoids cultured in either the presence or absence of Nutlin-3 (qPCR data was normalized to Actb; n = 2 independent AKVL clones; n = 3 independent AKVPL clones generated by 2 different sgRNAs targeting Trp53). f, Relative expression of Bax (a canonical Trp53-target gene) and Id3 (a canonical Smad4-target gene) in organoids with indicated genotype (RNA-seq expression analysis was quantile normalized and represented as log2 counts per million).

Source data

Extended Data Figure 2 Genotype affects CSC content and tumorigenic capacity.

a, Per cent tumour-initiating cells (TIC) in subcutaneous AKVPL and AKVPSL tumours estimated with the extreme limiting dilution analysis (ELDA48; n = 6 allografts per dilution; error bars represent the 95% confidence interval). b, Representative images of Lgr5-GFP+ staining of n = 4 tumours per indicated genotype. DAPI nuclear staining is in blue; Lgr5 in green. Scale bar, 20 μm. c, Per cent Lgr5-GFP+ cells in subcutaneous AKVPL and AKVPSL tumours collected three weeks after implantation (FACS data represented as mean ± s.d., n = 5 tumours per genotype). d, Per cent clonogenic cells in AKVPL and AKVPSL organoid cultures estimated with ELDA48 (n = 2 independent experiment per genotype; error bars represent the 95% confidence interval). e, Lgr5-GFP+ cell count and fluorescence intensity of in vitro organoid cultures with indicated genotype as determined by FACS. *P < 0.05, **P < 0.01, ***P < 0.001; n represents the number of independent animals.

Source data

Extended Data Figure 3 Gene expression analysis and tumorigenic potential of Lgr5+ and Lgr5 cells across organoid tumours.

a, FACS plots for GFP expression in dissociated AKV, AKVPL and AKVPSL tumours. The green and grey gates represent the sorted Lgr5+ and Lgr5 populations, respectively. b, Heatmap of Lgr5-GFP+ and Lgr5-GFP cells from n = 4 AKVPSL tumours for expression of intestinal stem cell (ISC) and canonical Wnt target genes (data are represented as mean-centred RPKM). c, QuSAGE of indicated gene signatures in Lgr5-GFP+ versus Lgr5-GFP cells from AKVPSL and AKVPL tumours (n = 4 tumours per genotype). d, Tumour-engraftment efficiency of Lgr5+ and Lgr5 cells from AKVPL and AKVPSL tumours. e, Per cent tumour-initiating cells (TICs) in Lgr5-GFP+ and Lgr5-GFP cell populations from AKVPL and AKVPSL tumours estimated with ELDA48 (n ≥ 5 grafts per dilution; error bars represent the 95% confidence interval). f, Per cent GFP+ cells in secondary tumours that originated from either 2,500 Lgr5-GFP+ or 2,500 Lgr5-GFP AKVPSL tumour cells (n = 5 grafts per cell population). *P < 0.05; **P < 0.01; ***P < 0.001.

Source data

Extended Data Figure 4 Effects of Lgr5+ cell depletion on tumour growth.

a, b, Representative sections of n = 3 AKVPL tumours per condition stained for GFP (indicating Lgr5, green), cleaved caspase 3 (CC3, red) and nuclei (DAPI, blue). White arrows indicate yellow double positive cells that are detectable as early as 6 h after initiation of DT treatment, confirming specific depletion of the Lgr5-GFP+ cell population. Scale bar, 20 μm. c, d, Dosing schedule (c) and tumour growth (d) of mice with subcutaneous AKVPL allografts in response to intermittent DT treatment (n = 8 for the saline- or n = 7 for DT-treated group). e, f, Dosing schedule (e) and tumour growth (f) of mice with subcutaneous AKVPSL allografts in response to continuous DT treatment (n = 7 for each treatment group). n represents the number of independent animals.

Source data

Extended Data Figure 5 Gene expression profiling of DT-treated tumours.

a, Gene-expression analysis of bulk AKVPL tumour cells isolated by FACS at different time points after a single dose of either saline or DT (n = 4 tumours per time point and condition). b, Venn diagram depicting the number of differentially expressed genes for the samples in a at various time points after treatment. Number of genes with increasing or decreasing expression patterns are depicted. c, QuSAGE of indicated gene signatures in DT- versus saline-treated AKVPL tumours 24 h after treatment. d, Representative images of n = 3 AKVPL tumours per condition stained for BrdU (green), cleaved caspase 3 (CC3, red) and nuclei (DAPI), revealing dynamic changes in proliferation and cell death after DT treatment for indicated length of time. Scale bar, 50 μm. n represents the number of independent animals.

Extended Data Figure 6 Association of AKVPL tumours with intestinal cell signatures during DT treatment.

a, QuSAGE of intestinal cell gene signatures23 in DT- versus saline-treated AKVPL tumours 24 h after treatment. b, Representative images of n = 3 AKVPL tumours per condition stained with Alcian blue to evaluate the number of goblet cells after DT treatment for the indicated length of time (counter stained with nuclear fast red). Scale bar, 20 μm. n represents the number of independent animals.

Extended Data Figure 7 Morphology of orthotopic tumours and liver metastasis after DT treatment.

a, Quantification of bioluminescence signal in orthotopic AKVPSL tumours from saline- (n = 17) and DT-treated (n = 15) animals before liver evaluation. Data represented as mean (lines) and individual animals (circles). b, Representative images of haematoxylin and eosin staining of orthotopic AKVPSL tumours from n = 4 animals per condition after either saline or DT treatment. Boxed areas show invasive front, which is magnified on the right; note that DT treatment did not abrogate tumour invasiveness. c, Gross morphology of representative livers from n = 4 animals with orthotopic AKVPSL tumours treated with either saline (top) or DT (bottom). White arrows indicate macro-metastases that are absent from the DT-treated cohort. d, Related to Fig. 3j; histological evaluation of livers from saline- and DT-treated mice with an orthotopic AKVPSL tumour. Black arrows indicate metastatic foci that are absent from the DT-treated cohort. Representative images are shown from n = 6 per treatment group. Scale bars, 300 μm (b, d), 2 cm (c). n represents the number of independent animals.

Source data

Extended Data Figure 8 Effect of Lgr5+ cell depletion in established liver metastasis.

a, Representative bioluminescent images of mice with established liver metastasis before either saline or DT treatment (n = 7 for saline and n = 8 for DT). Pre-tx, pre-treatment. b, Quantification of the bioluminescence signal in animals with established liver metastasis before DT or saline treatment (n = 7 for saline- and n = 8 for DT-treated group) c, Quantification of the bioluminescence signal in animals with established liver metastasis during (1 w DT) DT treatment. d, Related to Fig. 4d, e; livers from either saline- or DT-treated animals with established liver metastasis were collected one week after treatment. e, Number of RFP+ tumour cells in enzymatically dissociated livers of mice with established metastasis one week after treatment (FACS data represented as mean (lines) and individual animals (circles and squares); n = 6 per treatment group). f, Number of RFP+ tumour cells in enzymatically dissociated livers of mice with established metastasis two weeks after treatment (FACS data represented as mean (lines) and individual animals (circles); n = 7 animals per treatment group). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; n represents the number of independent animals.

Source data

Supplementary information

Supplementary Table 1

Off-target prediction for Trp53 and Smad4 sgRNAs. (XLSX 13 kb)

Supplementary Table 2

Time course analysis of transcriptional changes after DT treatment in vivo. (XLSX 126 kb)

Supplementary Table 3

Quantitative gene set analysis of expression changes in tumours after DT treatment. (XLSX 186 kb)

Supplementary Table 4

Quantitative gene set analysis of expression changes of selected gene sets. (XLSX 16 kb)

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de Sousa e Melo, F., Kurtova, A., Harnoss, J. et al. A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer. Nature 543, 676–680 (2017). https://doi.org/10.1038/nature21713

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