Platelets guide the formation of early metastatic niches

Myriam Labelle

Shahinoor Begum

Richard Hynes

Supplementary Material

Supporting Information:

Click here to view.

^{Howard Hughes Medical Institute, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139; and}

^{Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, 38105}

^{To whom correspondence may be addressed. Email:}ude.tim@senyhor or gro.edujts@ellebal.mairym.

Contributed by Richard O. Hynes, June 16, 2014 (sent for review April 24, 2014; reviewed by Yibin Kang and Michael H. Kroll)

Author contributions: M.L. and R.O.H. designed research; M.L. and S.B. performed research; M.L. analyzed data; and M.L. and R.O.H. wrote the paper.

Reviewers: Y.K., Princeton University; and M.H.K., The University of Texas MD Anderson Cancer Center.

Contributed by Richard O. Hynes, June 16, 2014 (sent for review April 24, 2014; reviewed by Yibin Kang and Michael H. Kroll)

Freely available online through the PNAS open access option.

Significance

Specialized microenvironments (or “niches”) are essential for metastasis, but how cancer cells and host cells contribute to their establishment remains poorly understood. Our study reveals that platelets and granulocytes are sequentially recruited to disseminated tumor cells to form “early metastatic niches” that promote metastatic progression. Importantly, the recruitment of granulocytes is not primarily due to tumor cell-derived signals but rather relies on platelet-derived CXCL5/7 chemokines. Prevention of granulocyte recruitment via inhibition of the CXCL5/7 receptor CXCR2, or depletion of either platelets or granulocytes inhibits metastasis, thereby uncovering a key role for platelet-to-granulocyte signaling in the establishment of metastases. Specific inhibition of platelet-to-granulocyte interactions may thus represent a valuable antimetastatic therapy in addition to cancer cell-centered treatments.

Significance

Abstract

During metastasis, host cells are recruited to disseminated tumor cells to form specialized microenvironments (“niches”) that promote metastatic progression, but the mechanisms guiding the assembly of these niches are largely unknown. Tumor cells may autonomously recruit host cells or, alternatively, host cell-to-host cell interactions may guide the formation of these prometastatic microenvironments. Here, we show that platelet-derived rather than tumor cell-derived signals are required for the rapid recruitment of granulocytes to tumor cells to form “early metastatic niches.” Granulocyte recruitment relies on the secretion of CXCL5 and CXCL7 chemokines by platelets upon contact with tumor cells. Blockade of the CXCL5/7 receptor CXCR2, or transient depletion of either platelets or granulocytes prevents the formation of early metastatic niches and significantly reduces metastatic seeding and progression. Thus, platelets recruit granulocytes and guide the formation of early metastatic niches, which are crucial for metastasis.

Abstract

Interactions between host cells and tumor cells both at the primary and metastatic sites are crucial for efficient metastasis (1 –3). At the site of metastasis, host cell–tumor cell cross talk contributes to the formation of a metastatic niche, a specialized microenvironment necessary for disease progression. Leukocytes, in particular monocytes and macrophages, have been shown to be recruited to metastatic niches and to support metastasis (4 –10). Mechanistically, macrophages secrete VEGFA that facilitates tumor cell extravasation (4). In addition, interaction of monocytes/macrophages with tumor cells via VCAM1 can prevent tumor cell apoptosis and allow reinitiation of growth at the metastatic site (8, 9). Granulocytes have also been reported to interact with tumor cells and facilitate metastatic seeding possibly by enhancing tumor cell arrest and extravasation (11 –13). However, granulocytes exposed to specific environmental stimuli can kill metastatic tumor cells, suggesting that their role in metastasis is highly context dependent (14, 15).

Platelets, which interact with tumor cells during their transit through the circulation, also enhance metastasis via multiple mechanisms (3, 16). Metastatic tumor cells can express high levels of tissue factor (TF) and adhesion molecules, such as P-selectin ligands, through which they bind to and activate platelets (16). These interactions result in the formation by platelets of a physical shield around tumor cells, which prevents attacks from natural killer (NK) cells and promotes tumor cell adhesion to the endothelium (17 –19). In addition, platelets also promote tumor cell extravasation by increasing endothelial permeability and by directly signaling to tumor cells to enhance their invasive and metastatic potential (20, 21). In particular, direct contact between platelets and tumor cells triggers the TGFβ1 and NF-κB signaling pathways in tumor cells, which induce an epithelial–mesenchymal transition and promote metastasis (20). Given their important signaling roles, platelets may influence metastasis by signaling not only to tumor cells but also to host cells forming metastatic niches. Furthermore, whether platelets and leukocytes are independently recruited by cancer cells to the site of metastasis, or whether hierarchical interactions among host cells drive the formation of metastatic niches remains unknown. Here, we define the relative roles of platelets and leukocytes during the early stages of metastatic seeding and the subsequent impact of these interactions on metastatic progression.

Click here to view.

Acknowledgments

We thank Sharon Wu and Hong Jia for technical assistance, members of the Hynes Laboratory for advice, and the microscopy and flow cytometry core facilities of the Koch Institute Swanson Biotechnology Center for technical support. This work was supported by funding from American Lebanese Syrian Associated Charities (M.L.), by the Ludwig Center for Molecular Oncology at Massachusetts Institute of Technology (MIT), by Koch Institute MIT National Cancer Institute Support Grant P30-CA14051, by National Cancer Institute Grants U54-{"type":"entrez-nucleotide","attrs":{"text":"CA126515","term_id":"35005481","term_text":"CA126515"}}CA126515/U54 {"type":"entrez-nucleotide","attrs":{"text":"CA163109","term_id":"35079181","term_text":"CA163109"}}CA163109 (principal investigator, R.O.H.), and by the Howard Hughes Medical Institute, of which R.O.H. is an investigator.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1411082111/-/DCSupplemental.

Footnotes

References

1. Zhang XH, et al Selection of bone metastasis seeds by mesenchymal signals in the primary tumor stroma. Cell. 2013;154(5):1060–1073.[Google Scholar]
2. Joyce JA, Pollard JWMicroenvironmental regulation of metastasis. Nat Rev Cancer. 2009;9(4):239–252.[Google Scholar]
3. Labelle M, Hynes ROThe initial hours of metastasis: The importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discov. 2012;2(12):1091–1099.[Google Scholar]
4. Qian BZ, et al CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature. 2011;475(7355):222–225.[Google Scholar]
5. Wolf MJ, et al Endothelial CCR2 signaling induced by colon carcinoma cells enables extravasation via the JAK2-Stat5 and p38MAPK pathway. Cancer Cell. 2012;22(1):91–105.[PubMed][Google Scholar]
6. Läubli H, Spanaus KS, Borsig LSelectin-mediated activation of endothelial cells induces expression of CCL5 and promotes metastasis through recruitment of monocytes. Blood. 2009;114(20):4583–4591.[PubMed][Google Scholar]
7. Borsig L, Wong R, Hynes RO, Varki NM, Varki ASynergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis. Proc Natl Acad Sci USA. 2002;99(4):2193–2198.[Google Scholar]
8. Chen Q, Zhang XH, Massagué JMacrophage binding to receptor VCAM-1 transmits survival signals in breast cancer cells that invade the lungs. Cancer Cell. 2011;20(4):538–549.[Google Scholar]
9. Lu X, et al VCAM-1 promotes osteolytic expansion of indolent bone micrometastasis of breast cancer by engaging α4β1-positive osteoclast progenitors. Cancer Cell. 2011;20(6):701–714.[Google Scholar]
10. Läubli H, Stevenson JL, Varki A, Varki NM, Borsig LL-selectin facilitation of metastasis involves temporal induction of Fut7-dependent ligands at sites of tumor cell arrest. Cancer Res. 2006;66(3):1536–1542.[PubMed][Google Scholar]
11. Huh SJ, Liang S, Sharma A, Dong C, Robertson GPTransiently entrapped circulating tumor cells interact with neutrophils to facilitate lung metastasis development. Cancer Res. 2010;70(14):6071–6082.[Google Scholar]
12. Slattery MJ, Dong CNeutrophils influence melanoma adhesion and migration under flow conditions. Int J Cancer. 2003;106(5):713–722.[Google Scholar]
13. Spicer JD, et al Neutrophils promote liver metastasis via Mac-1-mediated interactions with circulating tumor cells. Cancer Res. 2012;72(16):3919–3927.[PubMed][Google Scholar]
14. Granot Z, et al Tumor entrained neutrophils inhibit seeding in the premetastatic lung. Cancer Cell. 2011;20(3):300–314.[Google Scholar]
15. Fridlender ZG, et al Polarization of tumor-associated neutrophil phenotype by TGF-beta: “N1” versus “N2” TAN. Cancer Cell. 2009;16(3):183–194.[Google Scholar]
16. Gay LJ, Felding-Habermann BContribution of platelets to tumour metastasis. Nat Rev Cancer. 2011;11(2):123–134.[PubMed][Google Scholar]
17. Nieswandt B, Hafner M, Echtenacher B, Männel DNLysis of tumor cells by natural killer cells in mice is impeded by platelets. Cancer Res. 1999;59(6):1295–1300.[PubMed][Google Scholar]
18. Im JH, et al Coagulation facilitates tumor cell spreading in the pulmonary vasculature during early metastatic colony formation. Cancer Res. 2004;64(23):8613–8619.[PubMed][Google Scholar]
19. Karpatkin S, Pearlstein E, Ambrogio C, Coller BSRole of adhesive proteins in platelet tumor interaction in vitro and metastasis formation in vivo. J Clin Invest. 1988;81(4):1012–1019.[Google Scholar]
20. Labelle M, Begum S, Hynes RODirect signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell. 2011;20(5):576–590.[Google Scholar]
21. Schumacher D, Strilic B, Sivaraj KK, Wettschureck N, Offermanns SPlatelet-derived nucleotides promote tumor-cell transendothelial migration and metastasis via P2Y2 receptor. Cancer Cell. 2013;24(1):130–137.[PubMed][Google Scholar]
22. Daley JM, Thomay AA, Connolly MD, Reichner JS, Albina JEUse of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol. 2008;83(1):64–70.[PubMed][Google Scholar]
23. Kowanetz M, et al Granulocyte-colony stimulating factor promotes lung metastasis through mobilization of Ly6G^Ly6C^{granulocytes.}Proc Natl Acad Sci USA. 2010;107(50):21248–21255.[Google Scholar]
24. Hodivala-Dilke KM, et al Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J Clin Invest. 1999;103(2):229–238.[Google Scholar]
25. Kunita A, et al The platelet aggregation-inducing factor aggrus/podoplanin promotes pulmonary metastasis. Am J Pathol. 2007;170(4):1337–1347.[Google Scholar]
26. Gasic GJ, Gasic TB, Stewart CCAntimetastatic effects associated with platelet reduction. Proc Natl Acad Sci USA. 1968;61(1):46–52.[Google Scholar]
27. Camerer E, et al Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis. Blood. 2004;104(2):397–401.[PubMed][Google Scholar]
28. Gasic GJ, Koch PA, Hsu B, Gasic TB, Niewiarowski SThrombogenic activity of mouse and human tumors: Effects on platelets, coagulation, and fibrinolysis, and possible significance for metastases. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 1976;86(3):263–277.[PubMed][Google Scholar]
29. Honn KV, Tang DG, Crissman JDPlatelets and cancer metastasis: A causal relationship? Cancer Metastasis Rev. 1992;11(3-4):325–351.[PubMed][Google Scholar]
30. Gleissner CA, von Hundelshausen P, Ley KPlatelet chemokines in vascular disease. Arterioscler Thromb Vasc Biol. 2008;28(11):1920–1927.[Google Scholar]
31. Zhang C, Gadue P, Scott E, Atchison M, Poncz MActivation of the megakaryocyte-specific gene platelet basic protein (PBP) by the Ets family factor PU.1. J Biol Chem. 1997;272(42):26236–26246.[PubMed][Google Scholar]
32. Stadtmann A, Zarbock ACXCR2: From bench to bedside. Front Immunol. 2012;3:263.[Google Scholar]
33. Gil-Bernabé AM, et al Recruitment of monocytes/macrophages by tissue factor-mediated coagulation is essential for metastatic cell survival and premetastatic niche establishment in mice. Blood. 2012;119(13):3164–3175.[PubMed][Google Scholar]
34. Acharyya S, et al A CXCL1 paracrine network links cancer chemoresistance and metastasis. Cell. 2012;150(1):165–178.[Google Scholar]
35. Ferjančič Š, et al VCAM-1 and VAP-1 recruit myeloid cells that promote pulmonary metastasis in mice. Blood. 2013;121(16):3289–3297.[PubMed][Google Scholar]
36. Kawamura M, et al CXCL5, a promoter of cell proliferation, migration and invasion, is a novel serum prognostic marker in patients with colorectal cancer. Eur J Cancer. 2012;48(14):2244–2251.[PubMed][Google Scholar]
37. Teramukai S, et al Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: An analysis of Japan Multinational Trial Organisation LC00-03. Eur J Cancer. 2009;45(11):1950–1958.[PubMed][Google Scholar]
38. Lee YY, et al Pretreatment neutrophil:lymphocyte ratio as a prognostic factor in cervical carcinoma. Anticancer Res. 2012;32(4):1555–1561.[PubMed][Google Scholar]
39. Gondo T, et al Prognostic value of neutrophil-to-lymphocyte ratio and establishment of novel preoperative risk stratification model in bladder cancer patients treated with radical cystectomy. Urology. 2012;79(5):1085–1091.[PubMed][Google Scholar]
40. Chiang SF, et al Can neutrophil-to-lymphocyte ratio predict the survival of colorectal cancer patients who have received curative surgery electively? Int J Colorectal Dis. 2012;27(10):1347–1357.[PubMed][Google Scholar]
41. Proctor MJ, et al A derived neutrophil to lymphocyte ratio predicts survival in patients with cancer. Br J Cancer. 2012;107(4):695–699.[Google Scholar]
42. Perez DR, et al Blood neutrophil-to-lymphocyte ratio is prognostic in gastrointestinal stromal tumor. Ann Surg Oncol. 2013;20(2):593–599.[Google Scholar]
43. Shimada H, et al Thrombocytosis associated with poor prognosis in patients with esophageal carcinoma. J Am Coll Surg. 2004;198(5):737–741.[PubMed][Google Scholar]
44. Hwang SG, et al Impact of pretreatment thrombocytosis on blood-borne metastasis and prognosis of gastric cancer. Eur J Surg Oncol. 2012;38(7):562–567.[PubMed][Google Scholar]
45. Symbas NP, et al Poor prognosis associated with thrombocytosis in patients with renal cell carcinoma. BJU Int. 2000;86(3):203–207.[PubMed][Google Scholar]
46. Stone RL, et al Paraneoplastic thrombocytosis in ovarian cancer. N Engl J Med. 2012;366(7):610–618.[Google Scholar]
47. Hernandez E, Lavine M, Dunton CJ, Gracely E, Parker JPoor prognosis associated with thrombocytosis in patients with cervical cancer. Cancer. 1992;69(12):2975–2977.[PubMed][Google Scholar]
48. Ikeda M, et al Poor prognosis associated with thrombocytosis in patients with gastric cancer. Ann Surg Oncol. 2002;9(3):287–291.[PubMed][Google Scholar]
49. Stravodimou A, Voutsadakis IAPretreatment thrombocytosis as a prognostic factor in metastatic breast cancer. Int J Breast Cancer. 2013;2013:289563.[Google Scholar]
50. Ishizuka M, Nagata H, Takagi K, Iwasaki Y, Kubota KCombination of platelet count and neutrophil to lymphocyte ratio is a useful predictor of postoperative survival in patients with colorectal cancer. Br J Cancer. 2013;109(2):401–407.[Google Scholar]
51. Holz O, et al SCH527123, a novel CXCR2 antagonist, inhibits ozone-induced neutrophilia in healthy subjects. Eur Respir J. 2010;35(3):564–570.[PubMed][Google Scholar]
52. Yang L, et al Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1^CD11b^{myeloid cells that promote metastasis.}Cancer Cell. 2008;13(1):23–35.[Google Scholar]
53. Ijichi H, et al Inhibiting Cxcr2 disrupts tumor-stromal interactions and improves survival in a mouse model of pancreatic ductal adenocarcinoma. J Clin Invest. 2011;121(10):4106–4117.[Google Scholar]
54. Bergmeier W, Rackebrandt K, Schröder W, Zirngibl H, Nieswandt BStructural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies. Blood. 2000;95(3):886–893.[PubMed][Google Scholar]
55. Nieswandt B, Bergmeier W, Rackebrandt K, Gessner JE, Zirngibl HIdentification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice. Blood. 2000;96(7):2520–2527.[PubMed][Google Scholar]
56. Ho-Tin-Noé B, Goerge T, Cifuni SM, Duerschmied D, Wagner DDPlatelet granule secretion continuously prevents intratumor hemorrhage. Cancer Res. 2008;68(16):6851–6858.[Google Scholar]
57. Lamprecht MR, Sabatini DM, Carpenter AECellProfiler: Free, versatile software for automated biological image analysis. Biotechniques. 2007;42(1):71–75.[PubMed][Google Scholar]
58. Frenette PS, Johnson RC, Hynes RO, Wagner DDPlatelets roll on stimulated endothelium in vivo: An interaction mediated by endothelial P-selectin. Proc Natl Acad Sci USA. 1995;92(16):7450–7454.[Google Scholar]
59. Hartwell DW, et al Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses. J Cell Biol. 1998;143(4):1129–1141.[Google Scholar]