Genome-wide in vivo screen identifies novel host regulators of metastatic colonization

Abstract

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment (‘host’, which includes stromal cells and the immune system1). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth (‘colonization’) being critical in determining metastatic outcome2. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

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Figure 1: Identification of microenvironmental regulators of metastatic colonization of the lung.
Figure 2: Ability of Spns2-deficient mice to regulate metastatic colonization.
Figure 3: Characterization of the lymphocyte composition and phenotype in Spns2-deficient mice.
Figure 4: Lymphocyte regulation of metastatic colonization in Spns2-deficient mice.

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Acknowledgements

This work was supported by grants from Cancer Research UK (D.J.A. and O.J.S.), the Wellcome Trust (WT098051), Combat Cancer (D.J.A.), the European Research Council (311301 COLONCAN to O.J.S. and A.D.C.), National Institutes of Health U54HG004028 (N.A.K.), and Department of Defense BCRP Program Award W81XWH-14-1-0086 (S.S.). T.T. was funded by project A27N in the SFB854, and T.B. was funded in part by an EMBO Long-Term Fellowship (ALTF 945-2015) and the European Commission (Marie Curie Action LTFCOFUND2013, GA-2013-609409). We thank J. Allegood for sphingolipid analyses and acknowledge the VCU Lipidomics Core, which is supported in part by funding from the National Institutes of Health–National Cancer Institute (NIH–NCI) Cancer Center Support Grant P30CA016059, V. Iyer (Wellcome Trust Sanger Institute) for bioinformatics analysis, and members of the Wellcome Trust Sanger Institute Research Support Facility for their care of the mice.

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L.v.d.W. devised and implemented the pulmonary metastasis screen, performing all the primary screen, confirmation and characterization studies. M.J.A. analysed the histopathological sections. A.D.C. and O.J.S. performed and analysed the intrasplenic B16-F10 assays. T.B. and T.T. performed and analysed the spontaneous metastasis assay. H.W.-J. and N.G. managed mouse breeding and were responsible for issuing phenotyping cohorts. M.D.C.V.-H., T.V., I.C.M. and K.W. performed the RNA-seq analysis. D.G. and E.R. genotyped the mice and performed gene expression analysis. S.C., A.G., E.T. and E.L.C. performed additional phenotypic characterization. The Sanger Mouse Genetics Project generated and phenotyped the mice as part of a primary phenotyping pipeline. S.S. oversaw the lipidomic analysis and provided input to the project and the manuscript. A.O.S. devised, performed and analysed the immunophenotyping assays. L.v.d.W., A.O.S. and D.J.A. led the project. L.v.d.W., A.O.S. and D.J.A. wrote the manuscript with contributions from all authors.

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Correspondence to Louise van der Weyden or David J. Adams.

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The authors declare no competing financial interests.

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Reviewer Information: Nature thanks C. Ghajar and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Lists of participants and their affiliations appear in the Supplementary Information

Extended data figures and tables

Extended Data Figure 1 Molecular function of 810 mutant mouse lines screened and phenotypic characterization members of the interferon regulatory factor (Irf) family.

a, Molecular function Gene Ontology annotation of the 810 mutant mouse lines screened as detailed in Methods. b, Experimental metastasis assay using B16-F10 cells in Irf1tm1a/tm1a, Irf5tm1e/tm1e, Irf7tm1a/tm1a and concurrent control female mice. Shown are representative data from two (Irf5), four (Irf1) or six (Irf7) independent experiments. Symbols represent individual mice with a horizontal bar at the mean. P values are from a Mann–Whitney test. c–f, Representative photographs showing B16-F10 metastatic colonies on the (c) lungs of +/+ and Irf1tm1a/tm1a mice and (d–f) the presence of extra-pulmonary metastases in Irf1tm1a/tm1a mice (tissues from three mice shown).

Extended Data Figure 2 Spontaneous pulmonary metastases and primary tumour growth in Spns2 mice.

a, Size measurements of spontaneous pulmonary HCmel12–mCherry melanoma cell metastases of male mice with representative fluorescent images (lines indicate the edge of the lungs); n = 10 per genotype, horizontal bars represent mean (of 50 individual metastases counted per genotype) (one-way ANOVA with blocking factor of experiment, cumulative results of two independent experiments shown). b, Survival curve of +/+ and tm1a/tm1a male mice (n = 10 per genotype) in a spontaneous metastasis assay using HCmel12–mCherry cells (log-rank test (Mantel–Cox), cumulative results of two independent experiments shown). c, Growth of subcutaneously administered B16-BL6 cells in +/+ (four male, five female) and tm1a/tm1a (five male, one female) mice. Symbols represent mean ± s.e.m. with a two-tailed unpaired t-test with Welch’s correction used to compare the area under the curve. d, Incidence of cancer in aged (>40 weeks) +/+ (n = 15; 4 males, 11 females) and tm1a/tm1a (n = 18; 5 males, 13 females) mice. Statistical analysis was performed using a Fisher’s exact test. Source data

Extended Data Figure 3 Phenotyping of the serum and lungs of Spns2 mice.

Sphingoid base levels in the (a) serum (+/+, n = 5; tm1a/tm1a, n = 4) and (b) lungs (+/+, n = 6; tm1a/tm1a, n = 5) of male mice; data are mean ± s.e.m., multiple two-tailed unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%. Sph, sphingosine; DHSph, dihydrosphingosine; S1P, sphingosine-1-phosphate; DHS1P, dihydrosphingosine-1-phosphate. c, Micrograms of extravasated Evans blue dye in the lungs of +/+ and tm1a/tm1a male mice. d, Number of CFSE-labelled B16-F10 cells present in the lungs of female mice 90 min after administration. e, Levels of apoptosis in B16-F10–mCherry cells 12 h after administration to male mice. Shown are representative data from three independent experiments, with symbols representing individual mice. P values are indicated from two-tailed unpaired t-test with Welch’s correction (ce).

Extended Data Figure 4 Phenotypic characterization of the haematopoietic system of Spns2 mice.

ac, The numbers of erythrocytes and platelets, monocytes, granulocytes and lymphocyte subsets present in the blood of naive +/+ and tm1a/tm1a female mice (multiple two-tailed unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%; data shown are representative of three independent experiments). d, Analysis of lymphocyte subsets in the liver of naive +/+ and tm1a/tm1a female mice (multiple two-tailed unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%; data shown are representative of three independent experiments). e, f, T- and B-lymphocyte numbers in the blood of male naive (unstimulated) bone marrow chimaeras (unpaired two-tailed t-test with Welch’s correction; data shown are representative of two independent experiments). Symbols represent individual mice; horizontal bars represent mean.

Extended Data Figure 5 Characterization of the phenotype of lymphatic endothelial cell Spns2 deficient mice.

a, b, Sphingoid base levels in the (a) serum or (b) lung of control and Spns2tm1c/tm1c; Lyve1cre/+ male mice (data are mean ± s.e.m., control n = 11, Spns2tm1c/tm1c; Lyve1cre/+ n = 10, multiple two-tailed unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%). Sph, sphingosine; DH-Sph, dihydrosphingosine; S1P, sphingosine-1-phosphate; DH-S1P, dihydrosphingosine-1-phosphate. c, Lymphocyte subsets in the spleen, lymph node, lung and liver of +/+ and tm1a/tm1a male mice (symbols represent individual mice, horizontal bars represent mean, multiple two-tailed unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%; data shown are representative of three independent experiments). d, Experimental metastasis assay using MC-38 cells in control (n = 9) and Spns2tm1c/tm1c; Lyve1cre/+ (n = 5) in female mice. Data shown are mean ± s.e.m., Mann–Whitney test, representative of three independent experiments.

Extended Data Figure 6 T cell subsets in the lungs of Spns2 mice.

The proportion of T cell subsets present in the lungs of naive +/+ and tm1a/tm1a female mice (a, b, e) and control and Spns2tm1c/tm1c; Lyve1cre/+ male mice (c, d, f). Data are shown as percentage of parent CD4+ and CD8+ T cells (a, c, e, f) or percentage of CD45+ alive lung cells present (b, d). Symbols represent individual mice with horizontal bar at the mean. P values are indicated from two-tailed unpaired t-test adjusted by the Holm–Šídák method with α set to 5%. Data shown are representative of three independent experiments.

Extended Data Figure 7 T cell subsets in the liver of Spns2 mice.

The proportion of T cell subsets present in the liver of naive +/+ versus tm1a/tm1a female mice and control versus Spns2tm1c/tm1c; Lyve1cre/+ male mice. Data are shown as percentage of parent CD4+ and CD8+ T cells (a, c, e, f) or percentage of CD45+ alive liver cells present (b, d). Symbols represent individual mice; statistical analysis used multiple two-tailed unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%, with * indicating a P value not considered significant after correcting for multiple testing. Data shown are representative of three independent experiments.

Extended Data Figure 8 Phenotyping of Spns2 lungs.

a, b, Ex vivo re-stimulation (PMA/ionomycin) of pulmonary leukocytes from B16-F10-stimulated +/+ and tm1a/tm1a female mice (two-tailed unpaired t-test adjusted by the Holm–Šídák method with α set to 5%). c, Measurement of IFN-γ in lungs of MC-38-stimulated +/+ and tm1a/tm1a male mice (two-tailed unpaired t-test with Welch’s correction). d, e, The proportion of NK cell subsets present in the lungs of naive +/+ versus tm1a/tm1a female mice (d) and control versus Spns2tm1c/tm1c; Lyve1cre/+ male mice (e) (multiple two-tailed unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%). Symbols represent individual mice, horizontal bars represent mean; data shown are representative of three independent experiments.

Extended Data Figure 9 Studies in T- and B-cell-deficient mice.

a, Measurement of lymphocyte subsets in the blood of +/+ and Rag2−/− mice (multiple two-tailed unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%). b, Experimental metastasis assay using B16-F10 cells in +/+ and Rag2−/− female mice (Mann–Whitney test). Symbols represent individual mice, horizontal bars represent mean; data shown are representative of three independent experiments.

Extended Data Figure 10 Characterization of the leukocyte composition and phenotype in DOP-treated mice.

ad, The number of leukocytes and T cell subsets present in the lungs of B16-F10-dosed glucose- or DOP-treated wild-type male mice presented as the percentages of viable CD45+ lung leukocytes (a, c) or parent CD4+ or CD8+ T cells (b, d) (multiple unpaired t-tests with P value adjusted by the Holm–Šídák method with α set to 5%). e, Experimental metastasis assay in B16-F10 dosed glucose- or DOP-treated wild-type female mice (Mann–Whitney test). Symbols represent individual mice, horizontal bars represent mean; data shown are representative of two independent experiments.

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van der Weyden, L., Arends, M., Campbell, A. et al. Genome-wide in vivo screen identifies novel host regulators of metastatic colonization. Nature 541, 233–236 (2017). https://doi.org/10.1038/nature20792

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