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

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 system). 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 outcome. 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.

[1]  G S Stein,et al.  Targeted inhibition of mitochondrial Hsp90 suppresses localised and metastatic prostate cancer growth in a genetic mouse model of disease , 2011, British Journal of Cancer.

[2]  Simone Cuff,et al.  Antigen Specificity Determines the Pro- or Antitumoral Nature of CD8+ T Cells , 2009, The Journal of Immunology.

[3]  Cheng Luo,et al.  Regulation of Histone Acetylation in the Nucleus by Sphingosine-1-Phosphate , 2009, Science.

[4]  J. Allison,et al.  Combination Immunotherapy of B16 Melanoma Using Anti–Cytotoxic T Lymphocyte–Associated Antigen 4 (Ctla-4) and Granulocyte/Macrophage Colony-Stimulating Factor (Gm-Csf)-Producing Vaccines Induces Rejection of Subcutaneous and Metastatic Tumors Accompanied by Autoimmune Depigmentation , 1999, The Journal of experimental medicine.

[5]  Damian Smedley,et al.  Genome-wide Generation and Systematic Phenotyping of Knockout Mice Reveals New Roles for Many Genes , 2013, Cell.

[6]  Masaru Ishii,et al.  The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice. , 2012, The Journal of clinical investigation.

[7]  Ying Xu,et al.  Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning , 2009, The Journal of experimental medicine.

[8]  Tasuku Honjo,et al.  PD-L1/B7H-1 Inhibits the Effector Phase of Tumor Rejection by T Cell Receptor (TCR) Transgenic CD8+ T Cells , 2004, Cancer Research.

[9]  Yannick Jacques,et al.  Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor , 2007, Nature Immunology.

[10]  Josef M. Penninger,et al.  p110γ and p110δ Phosphoinositide 3-Kinase Signaling Pathways Synergize to Control Development and Functions of Murine NK Cells , 2007 .

[11]  Sarah Spiegel,et al.  Spns2, a transporter of phosphorylated sphingoid bases, regulates their blood and lymph levels, and the lymphatic network , 2013, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[12]  Åsa K. Björklund,et al.  Smart-seq2 for sensitive full-length transcriptome profiling in single cells , 2013, Nature Methods.

[13]  Sarah Spiegel,et al.  Export of sphingosine-1-phosphate and cancer progression , 2014, Journal of Lipid Research.

[14]  Andrea M Hussong,et al.  Integrative data analysis: the simultaneous analysis of multiple data sets. , 2009, Psychological methods.

[15]  D. Schadendorf,et al.  Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma , 2014, Nature.

[16]  T. Hla,et al.  Sphingosine‐1‐phosphate receptor 1 signalling in T cells: trafficking and beyond , 2014, Immunology.

[17]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[18]  T. Waldmann,et al.  Requirement for IRF-1 in the microenvironment supporting development of natural killer cells , 1998, Nature.

[19]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[20]  David J. Adams,et al.  Impact of Temporal Variation on Design and Analysis of Mouse Knockout Phenotyping Studies , 2014, PloS one.

[21]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[22]  S. Mehrotra,et al.  Communication between host organism and cancer cells is transduced by systemic sphingosine kinase 1/sphingosine 1-phosphate signalling to regulate tumour metastasis , 2012, EMBO molecular medicine.

[23]  A. Morris,et al.  The transporter Spns2 is required for secretion of lymph but not plasma sphingosine-1-phosphate. , 2012, Cell reports.

[24]  Deepta Bhattacharya,et al.  Spontaneous mutation of the Dock2 gene in Irf5−/− mice complicates interpretation of type I interferon production and antibody responses , 2012, Proceedings of the National Academy of Sciences.

[25]  Jennifer P Morton,et al.  P-Rex1 is required for efficient melanoblast migration and melanoma metastasis. , 2011, Nature communications.

[26]  Yuan Cao,et al.  Functional and molecular characterisation of EO771.LMB tumours, a new C57BL/6-mouse-derived model of spontaneously metastatic mammary cancer , 2015, Disease Models & Mechanisms.

[27]  Klaus Rajewsky,et al.  A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain gene , 1991, Nature.

[28]  W L Stanford,et al.  Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration. , 2000, Science.

[29]  Anneliese O. Speak,et al.  Gray platelet syndrome: proinflammatory megakaryocytes and α-granule loss cause myelofibrosis and confer metastasis resistance in mice. , 2014, Blood.

[30]  E. Chiang,et al.  Correction of Defects Responsible for Impaired Qa-2 Class Ib MHC Expression on Melanoma Cells Protects Mice from Tumor Growth 1 , 2003, The Journal of Immunology.

[31]  Y. Hochberg.  A sharper Bonferroni procedure for multiple tests of significance , 1988 .

[32]  Ying Xu,et al.  Lymphocyte Sequestration Through S1P Lyase Inhibition and Disruption of S1P Gradients , 2005, Science.

[33]  Jing Chen,et al.  The Role of Sphingosine-1-Phosphate Transporter Spns2 in Immune System Function , 2012, The Journal of Immunology.

[34]  I. Cuthill,et al.  Reporting : The ARRIVE Guidelines for Reporting Animal Research , 2010 .

[35]  Hiroki Tanaka,et al.  The role of nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species in the acquisition of metastatic ability of tumor cells. , 2006, The American journal of pathology.

[36]  Hideo Negishi,et al.  IRF-7 is the master regulator of type-I interferon-dependent immune responses , 2005, Nature.

[37]  Åsa K. Björklund,et al.  Full-length RNA-seq from single cells using Smart-seq2 , 2014, Nature Protocols.

[38]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[39]  Brent A Wilkerson,et al.  The role of sphingosine-1-phosphate in endothelial barrier function. , 2014, Biochimica et biophysica acta.

[40]  Yiling Lu,et al.  Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple tumor lineages. , 2006, Cancer cell.

[41]  Ajit Varki,et al.  Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[42]  Takashi Murakami,et al.  CD39/ENTPD1 expression by CD4+Foxp3+ regulatory T cells promotes hepatic metastatic tumor growth in mice. , 2010, Gastroenterology.

[43]  V. Stewart,et al.  RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement , 1992, Cell.

[44]  Ronald Naumann,et al.  An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase , 2010, Genesis.

[45]  G. Naumov,et al.  Critical steps in hematogenous metastasis: an overview. , 2001, Surgical oncology clinics of North America.

[46]  Hongbo Chi,et al.  S1P1-mTOR axis directs the reciprocal differentiation of TH1 and regulatory T cells , 2010, Nature Immunology.

[47]  D. Quail,et al.  Microenvironmental regulation of tumor progression and metastasis , 2014 .

[48]  He Xiao,et al.  The sphingosine-1-phosphate receptor agonist FTY720 and its phosphorylated form affect the function of CD4+CD25+ T cells in vitro. , 2012, International journal of molecular medicine.