1. Department of Pharmacology and Cancer Center, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0723, USA
2. Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, China
3. Present addresses: Department of Applied Biochemistry, Konkuk University, 322 Danwol-dong, Chungju-City, Chungbuk 380-701, Korea (Y.K.); Department of Cancer Biology, The Scripps Research Institute, 5353 Parkside Drive, Jupiter, Florida 33458, USA (J.-L.L.).

Correspondence to: Michael Karin¹ Correspondence and requests for materials should be addressed to M.K. (Email: karinoffice@ucsd.edu).

Distant-site metastases are the leading cause of cancer-associated mortality. They depend on genetic and/or epigenetic alterations that are intrinsic to cancer cells, or extrinsic factors provided by the tumour microenvironment¹. For instance, cytokines produced by inflammatory cells can enhance metastatogenesis by repressing the metastasis suppressor maspin within primary prostate carcinoma cells⁸. Furthermore, tumour progression and metastasis positively correlate with presence of infiltrates containing myeloid and lymphoid cells^{2, 9}. It has been shown that certain carcinomas secrete factors that upregulate fibronectin and recruit vascular endothelial growth factor receptor 1 (VEGFR1)-positive haematopoietic progenitors to sites of future metastatic growth, termed the pre-metastatic niche⁷. To examine whether cancer cells secrete factors that directly activate myeloid cells to produce tumour-promoting cytokines¹⁰, we collected serum-free conditioned medium from different cancer cell lines, derived mainly from C57BL6 mice, and applied it to bone-marrow-derived macrophages (BMDM), which were assayed for production of interleukin-1 (IL-1), IL-6 and TNF-. The screen included 1C1C7 and TrampC1, which are liver and prostate cancer cell lines, respectively, with little or no metastatic activity, and two metastatic breast and lung carcinomas, 4T1 and LLC, respectively. Conditioned medium from metastatic cells, especially LLC, induced higher amounts of IL-6 and TNF- secretion than conditioned medium from non-metastatic cells (Fig. 1a). IL-1 secretion was undetectable and the conditioned medium did not contain IL-6 or TNF- (data not shown). LCM also induced expression of Il1, Il6 and Tnf messenger RNAs (mRNAs), whereas serum-free medium (SFM) and NIH3T3-conditioned medium were inactive (Fig. 1b and data not shown). We investigated the metastatogenic function of some of the LCM-induced cytokines by tail vein injection of LLC into age- and sex-matched Tnf and Il6 knockout mice and wild-type (WT) controls. Tnf^-/- mice exhibited markedly (P < 0.001) reduced mortality compared with WT mice after inoculation with 1  10⁶ LLC cells (Fig. 1c) and showed an even greater survival advantage when given a smaller LLC inoculum (Supplementary Fig. 1a). Similar differences were seen in lung tumour multiplicity (Fig. 1d). By contrast, there was little difference in survival of Il6^-/- and WT mice inoculated with 1  10⁶ LLC cells (Supplementary Fig. 1b). Thus, TNF- but not IL-6 is important for LLC metastasis.

Figure 1: Metastatic carcinomas secrete factors that induce macrophage production of TNF-, needed for lung metastasis.

a, BMDM were cultured with SFM or SFM conditioned by mouse carcinoma cells (conditioned medium), and cytokine production was measured (averages  s.d., n = 3). b, BMDM were cultured with SFM or LCM, and cytokine mRNAs were measured by quantitative PCR with reverse transcription. Fold-induction above SFM-treated cells was determined (averages  s.d., n = 3; *, P < 0.001 by Student's t test). c, Survival of WT (n = 22) and Tnf^-/- (n = 15) mice inoculated with LLC (1  10⁶ cells through the tail vein (P < 0.001; log-rank test for significance). d, Lungs of WT and Tnf^-/- mice 47 days after LLC inoculation (2  10⁵ cells). Tumour multiplicities are shown underneath (averages  s.d., n = 11, P < 0.001 by Student's t-test).

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We explored the involvement of TLR family members in sensing LCM components. BMDM from mice deficient in TLR2, TLR3, TLR4 or TLR9 or their adaptor proteins, Myd88 and TRIF (which is inactivated by the Lps2 mutation: Trif^m)⁴, were examined for production of IL-6, a convenient BMDM activation marker. LCM-induced IL-6 was fully dependent on TLR2 and Myd88 but not on TLR3, TLR4, TLR9 or TRIF (Fig. 2a). Tlr2^-/- BMDM were also defective in LCM-induced Il1 and Il6 mRNA expression and LCM did not induce anti-tumorigenic type I interferon genes (Supplementary Fig. 2a), which are readily induced after TLR3 or TLR4 engagement¹¹. TLR2 was also required for LCM-induced IL-6 and TNF- secretion by alveolar macrophages, which produced tenfold more TNF- than BMDMs (Supplementary Fig. 2b). TLR2 was required for optimal LCM-induced activation of mitogen-activated protein kinases and IB kinase or IB degradation (Fig. 2b). TLR2 uses TLR1, TLR6 or CD14 as co-receptors¹². LCM-induced IL-6 production was dependent on TLR2, TLR6 and CD14 but not on TLR1 (Fig. 2c). By contrast, the response to Pam3CSK4, a bacterial lipoprotein analogue⁴, depended on TLR2 and TLR1 but not on TLR6 and CD14. These results rule out possible contamination with bacterial lipoproteins. Furthermore, anti-mycoplasma treatment of LLC had no effect on LCM activity (data not shown).

Figure 2: LLC-secreted factor activates TLR2 to induce lung inflammation.

a, BMDM from indicated mouse strains were cultured with SFM or LCM, and IL-6 production was measured (m: mutant allele; averages  s.d., n = 3, presented as percentage of WT LCM-stimulated value). b, BMDM were treated with LCM or LPS (100 ng ml^-1). Cell lysates were examined for kinase phosphorylation (P) and IB degradation by immunoblotting. Total ERK and HSP90 are loading controls. c, BMDM from indicated mouse strains were cultured with LCM or Pam3CSK4 (1 ng ml^-1), and IL-6 production was measured (averages  s.d., n = 4). d, RNA was extracted from lungs of WT or Tlr2^-/- mice at indicated times after LLC inoculation (2  10⁵ cells). mRNAs were quantified as above and the amounts in non-inoculated WT or Tlr2^-/- lungs were given a value of 1.0 (averages  s.e.m., n = 3; *P < 0.05, **P < 0.005 (compared with WT) by Student's t-test).

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To examine the in vivo role of TLR2, we inoculated sex- and age-matched Tlr2^-/- and WT mice with 2  10⁵ LLC cells through the tail vein and measured mRNAs encoding cytokines and chemokines in their lungs. LLC-induced lung inflammation has been previously described¹³, but its mechanism was unknown. Tnf, Il1, Il6 and inflammatory chemokine mRNAs were induced 5 days after LLC inoculation in WT lungs, peaking at 9 days after inoculation (Fig. 2d). None of these mRNAs was induced in lungs of Tlr2^-/- mice, whose basal content of Mip1/Ccl4, Mip2/Cxcl2 and Kc/Cxcl1 mRNAs was higher than WT lungs (Supplementary Fig. 3). In addition, Tlr2^-/- and WT mice were subcutaneously inoculated with LLC cells and examined for lung macrophage infiltration and inflammatory cytokine gene expression days later. Although no difference was observed in primary subcutaneous tumour growth, macrophage infiltration and inflammatory cytokine gene expression were greatly reduced in Tlr2^-/- mice compared with WT mice (Supplementary Fig. 4a–c).

To investigate whether TLR2 signalling contributes to LLC metastatogenesis, we inoculated age- and sex-matched Tlr2^-/- and WT mice with dsRed-labelled LLC cells through the tail vein and examined their lungs for micrometastases. WT but not Tlr2^-/- lungs showed small clusters of DsRed-LLC cells with adjacent CD11b⁺ and CD11c⁺ myeloid cells (Fig. 3a). We also detected a few cells that carried the CD3 antigen (T cells) in micro-metastases of WT lungs (data not shown). Importantly, Tlr2^-/- mice exhibited significantly greater (P < 0.02) survival than WT mice after LLC inoculation and their lungs contained fewer and smaller tumour nodules (Fig. 3b, c). Tumour nodules in WT mice contained more CD11b⁺/Gr1⁺ inflammatory monocytes/myeloid suppressors and IL-10^high/F4/80⁺ M2 macrophages (Supplementary Fig. 5). Tlr2^-/- mice exhibited significantly fewer lung and liver tumour nodules than WT mice and a lower incidence of adrenal gland metastasis after subcutaneous implantation of LLC cells (Fig. 3d). To investigate whether TLR2 acts in bone-marrow-derived cells, we examined survival of LLC-inoculated chimaeric mice. Mice reconstituted with Tlr2^-/- bone marrow (WT/Tlr2^-/-) exhibited markedly improved (P < 0.04) survival compared with mice reconstituted with WT bone marrow (WT/WT) (Fig. 3e). WT/WT and WT/Tlr2^-/- mice were also inoculated with 2  10⁵ LLC cells followed by intraperitoneal injections of LCM or SFM. Lung and liver tumour loads were significantly higher (P < 0.05) in WT/WT mice receiving LCM than in those receiving SFM along with the LLC inoculum (Fig. 3f). The pro-metastatic effect of LCM was dependent on TLR2 activation, as little or no metastatic enhancement was seen in WT/Tlr2^-/- mice. These results strongly suggest that LCM contains TLR2-activating factors that enhance metastatogenesis.

Figure 3: TLR2 is required for metastatic growth.

a, WT and Tlr2^-/- lungs were analysed 9 days after inoculation of DsRed-LLC (2  10⁵ cells) for DsRed and myeloid cells markers (CD11b, CD11c) using fluorescence microscopy (magnification 200). b, Survival of LLC inoculated (2  10⁵ cells) WT (n = 8) and Tlr2^-/- (n = 10) mice (P < 0.02; log-rank test for significance). c, Lungs and haematoxylin-and-eosin-stained lung sections (magnification 25) 20 days after LLC inoculation. Tumour multiplicity is shown on the left (averages  s.e.m., n = 8, P < 0.001 by Student's t-test). d, Tumour multiplicities of lung and liver metastatic nodules and incidence of adrenal metastasis 17 days after primary tumour removal (averages  s.e.m., WT n = 9, Tlr2^-/- n = 6; *P < 0.05 by Student's t-test). e, Survival of WT/WT or WT/Tlr2^-/- chimaeric mice inoculated with LLC (2  10⁵ cells) 6–7 weeks after bone-marrow reconstitution (P < 0.04; log-rank test for significance; n = 6). f, Lung tumour multiplicity (left), size (middle) and liver tumour multiplicity (right) in chimaeric mice, 27 (lung) or 48 (liver) days after LLC injection (2  10⁵ cells) together with SFM or LCM (averages  s.e.m., n = 8; *P < 0.05 by Student's t-test).

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To identify the nature of these factors, we collected large amounts of LCM, separated it on a mono-Q anion exchange column and monitored column fractions for their ability to induce IL-6 in BMDM. Fractions with IL-6-inducing activity were pooled and separated on a Superdex 200 sizing column (Supplementary Fig. 6). Most of the IL-6-inducing activity eluted in a few high molecular mass (greater than 400 kDa) fractions that contained several polypeptides larger than 200 kDa. These fractions were pooled, deglycosylated and subjected to mass spectrometry, resulting in identification of several peptides derived from the extracellular matrix proteins: versican V1, laminin-1, thrombospondin 2 and procollagen type III-1 (Fig. 4a). To examine which protein accounts for induction of inflammatory cytokines, we incubated LCM with individual neutralizing antibodies before BMDM stimulation and measurement of cytokine production. Incubation of LCM with antibodies to versican, laminin-1 or procollagen type III-1, but not thrombospondin 2, reduced IL-6 and TNF- production (Supplementary Fig. 7). A control antibody to HMGB1, an inflammatory mediator released by necrotic cells¹⁴, did not inhibit cytokine induction.

Figure 4: Versican is a TLR2 agonist and metastasis-enhancing factor.

a, Identification of candidate LCM macrophage activators by mass spectrometry. Probability-based Mowse scores (upper) and tryptic peptides corresponding to the indicated proteins (lower). b, LLC were transduced with indicated shRNA lentiviruses. After selection, expression of the indicated proteins was analysed. c, Left: LLC transduced with indicated lentiviruses were injected (2  10⁵ cells) into mice (n = 8). Lung tumour multiplicity was enumerated at 20 days (averages  s.e.m., P < 0.001 by Student's t-test). Right: survival of mice inoculated with LLC transduced with control or Vers shRNAs (n = 9; *P < 0.05; log-rank test for significance). d, Lung and liver tumour multiplicities and incidence of adrenal metastasis 17 days after primary tumour removal (averages  s.e.m., n = 8 and n = 5 for mice injected with control or Vers shRNA-transduced cells, respectively; *P < 0.05 by Student's t-test). e, Upper: non-transfected and human versican (hVers) transfected P29-LLC cells were analysed for versican expression. Lower: hVers non-expressing and expressing P29-LLC cells were injected through the tail vein (n = 7). After 27 days, lung metastatic nodules were enumerated (*P < 0.05 by Student's t-test). f, Left: silver staining of purified 6  His-hVers. Right: BMDM were incubated without or with His-hVers for 20–24 h, and cytokine secretion was measured (averages  s.d., n = 3). g, Lysates of Raw264.7 cells incubated with LCM or SFM for 1 h were immunoprecipitated with versican-specific or control antibody and analysed by immunoblotting with the indicated antibodies.

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To investigate the role of these proteins in LCM-enhanced metastatogenesis, we generated stable LLC cell lines containing short hairpin RNA (shRNAs) specific to versican V1, laminin-1 or procollagen III-1. Silencing efficiency was approximately 90% or higher (Fig. 4b). The silenced cells and LLC cells transduced with a control shRNA were injected into mice and lung tumour nodules and their survival monitored. Silencing of versican V1 significantly reduced (P < 0.001) tumour multiplicity, but silencing of laminin-1 resulted only in a modest inhibition, whereas procollagen III-1 silencing slightly enhanced tumour multiplicity (Fig. 4c). Furthermore, silencing of versican V1 reduced lung nodule multiplicity fourfold (Supplementary Fig. 8a). Tumours isolated from mice inoculated with versican-silenced LLC cells displayed very low versican expression (Supplementary Fig. 8b), whereas lung tumours from LLC-inoculated mice expressed more versican than normal lung (Supplementary Fig. 8c). Importantly, mice inoculated with versican-silenced LLC cells exhibited significantly improved (P < 0.05) survival (Fig. 4c). Silencing of versican also reduced metastatic spread to lung, liver and adrenal glands in the subcutaneous implantation model (Fig. 4d). To ascertain the proinflammatory and premetastatic functions of versican, we used the low metastasic LLC variant P29-LLC¹⁵. Conditioned medium from P29-LLC did not induce IL-6 in BMDM and contained very little versican (Supplementary Fig. 9). Ectopic expression of human versican in P29-LLC cells increased lung tumour multiplicity after tail vein injection (Fig. 4e).

To examine how versican activates macrophages, we produced His-tagged human versican V1 in LLC cells and purified it on an Ni-chelate column. The purified protein induced IL-6 and TNF- production in WT but not Tlr2^-/- BMDM (Fig. 4F). We investigated whether versican interacts with TLR2 or its CD14 co-receptor. Immunoprecipitation of lysates of LCM-incubated Raw264.7 macrophages with versican-specific antibody, but not a control antibody, co-precipitated TLR2 and CD14 but not TLR4 (Fig. 4g). The versican antibody did not precipitate TLR2 unless the macrophages were first incubated with LCM.

Metastasis is the result of a complex process involving invasion of adjacent tissues, intravasation, circulatory transport, arrest at a distant site, extravasation, growth, survival and neoangiogenesis¹⁶. Bone-marrow-derived cells, such as macrophages² and haematopoietic progenitors⁷, are important participants in this process; however, how they are mobilized and activated to support metastasis is unclear. Our results indicate that versican secretion by LLC cells is necessary for metastatic spread to lung, liver and adrenal gland, a process that depends on TLR2-mediated myeloid cell activation and TNF- production. Versican is an aggregating chondroitin sulphate proteoglycan that accumulates both in tumour stroma and cancer cells^{5, 6}. It can bind hyaluronan, and both versican and hyaluronan are highly expressed in non-small-cell lung cancer (NSCLC), especially in advanced disease with high recurrence rate, whereas versican in normal lung is rather low⁶. Versican or fragments thereof enhance tumour cell migration, growth and angiogenesis, processes that are of direct relevance to metastasis¹⁷. Versican also binds to several adhesion molecules expressed by inflammatory cells and has pro-inflammatory activity¹⁸. A related extracellular matrix proteoglycan, biglycan, has been reported to activate both TLR2 and TLR4¹⁹, but our results indicate that the pro-inflammatory activities of versican rely on TLR2 but not TLR4. TLR2 recognizes Gram-positive bacteria-derived lipoteichoic acid and lipoproteins⁴. This activity mainly depends on TLR2:TLR1 dimers²⁰, but the response to versican requires TLR6 and not TLR1 as a co-receptor. Although TLR2 and versican interact, it is not clear whether this interaction is direct or depends on a versican ligand, such as hyaluronan. Indeed, hyaluronan fragments can activate macrophages through TLR2 (ref. 21), and hyaluronan accumulation and the enzyme that converts large hyaluronan polymers to smaller fragments, hyaluronidase, have been linked to metastasis²².

TLR2 on host myeloid cells and their product TNF- are important positive modulators of LLC metastatic behaviour, although neither protein influences primary tumour growth of subcutaneously implanted LLC. It appears that TNF- is one of the major pro-metastatic factors produced by host myeloid cells. TNF- can suppress the apoptosis of cancer cells and stimulate their proliferation through NF-B activation²³. In addition, by increasing vascular permeability²⁴, TNF- can enhance recruitment of leukocytes as well as intravasation and extravasation of cancer cells. We suggest that versican, its interaction with TLR2 and production of TNF- by activated myeloid cells provide potential points for anti-metastatic intervention.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

S.K. was supported by the International Human Frontier Science Program Organization (IHFSPO), the National Cancer Institute-sponsored Cancer Therapeutic Training Program and a Ruth L. Kirschstein National Research Service Award. Y.K., H.T., J.-L.L., P.D. and S.G. were supported by the California Institute of Regenerative Medicine, the Japanese Respiratory Society, the Life Science Research Foundation, IHFSPO and the Crohn's and Colitis Foundation of America, respectively. Work in the M.K. laboratory was supported by grants from the National Institutes of Health and a Littlefield-AACR grant in Metastatic Colon Cancer Research. M.K. is an American Cancer Society Research Professor. We thank S. Akira and B. Beutler for TLR and adaptor protein deficient mice, D. Zimmermann for human versican construct, R. Hoffman for DsRed-LLC cells and K. Takenaga for LLC-P29 cells. We also thank R. Gallo and K. Yamasaki for the hyaluronan-inhibiting peptides and advice, J. Varner for analysing cell migration and Santa Cruz Biotechnology for gifts of antibodies.

Author Contributions S.K., H.T., W.-W.L. and M.K. conceived the project and planned experiments and analyses, which were performed by S.K., H.T. and W.-W.L. Y.K. and J.-L.L. helped with protein purification and tail-vein injection of cancer cells and tumour analysis, respectively. P.D. and S.G. analysed M2 macrophages and tissue versican content, and effect of TNF- neutralization on lung metastasis. M.K. oversaw the entire project and wrote the manuscript with S.K.

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