Cellular & Molecular Immunology (2010) 7, 94–99 ß 2010 CSI and USTC. All rights reserved 1672-7681/10 $32.00 www.nature.com/cmi

REVIEW

Premetastatic milieu explained by TLR4 -mediated homeostatic

Yoshiro Maru

Accumulating evidence suggests that Toll-like 4 (TLR4), a sensor for danger signals, is expressed not only in immune cells, but also in resident epithelial cells, and appears to participate in tissue homeostasis. To explain the premetastatic microenvironment created by the newly discovered endogenous TLR4 ligands, I propose a hypothesis of homeostatic inflammation that includes the classical danger hypothesis. Cellular & Molecular Immunology (2010) 7, 94–99; doi:10.1038/cmi.2009.113; published online 8 February 2010

Keywords: cancer; endogenous ligand; ; TLR4

INTRODUCTION functional diversity of CD41 T cells, for example, as represented by To accomplish hematogenous metastasis, tumor cells must undergo Th17 and regulatory T cells (Tregs) in addition to the classical Th1 and detachment from their primary sites, intravasation, migration Th2 cells, demonstrated that these cells communicate with other through circulation, extravasation, resettlement and regrowth in immune cells in the mononuclear phagocyte system, including mono- metastatic organs. This complicated biological process can be partly cytes, macrophages and DCs (myeloid DCs in this text, hereafter sim- explained by the concept of epithelial mesenchymal transition, in ply called DC) that originate from bone marrow.6 which primary tumor cells lose E-cadherin-mediated contacts and In this minireview, I focus on the possible biological significance of degrade the extracellular matrix with matrix metalloproteases endogenous TLR4 ligands in tumor immunology. Because S100A8/ (MMPs) to invade the surrounding tissues.1 This migration is remin- and SAA3 expression is induced in the lungs, which are des- iscent of the chemokine-regulated mobilization of immune cells to tined to allow metastasis, I first briefly review the immunological remote tissues. We have found that metastasis-promoting chemokines events that occur in primary tumors and then extend the discussion S100A8/S100A9 (two that can form homo- or heterodimeric to metastatic sites. Finally, I explain the premetastatic microenviron- structures, which are described here as S100A8/S100A9 unless other- ment and propose the hypothesis of homeostatic inflammation.7 wise indicated) and serum amyloid A3 (SAA3) are endogenous ligands for Toll-like receptor 4 (TLR4), one of the first defense sensors for ACQUISITION OF METASTATIC POTENTIAL IN PRIMARY danger signals in the .2 S100A8 and S100A9 TUMORS were identified in 1987 in the synovial fluid of rheumatoid-arthritis Stromal expression profiles of 53 breast tumors have shown patients (reviewed in Ref. 3). Whereas knockout of the S100A8 gene is results associated with Th1-type immune responses.8 A protective role embryonic lethal at E9.5, S100A9 appears to stabilize S100A8 because of Th17 cells in antitumor immunity, in which these cells induce the knockout of S100A9 abolishes the expression of S100A8. SAA3 belongs expression of Th1-type chemokines, such as CXCL9 and CXCL10, has to the acute phase reactant SAA family. Proposed candidate receptors been shown in 201 patients.9 Another study of 104 for SAA1 and SAA2 include the receptor for advanced glycation end- ovarian tumors has shown that Treg is recruited to tumors by the products and TLR2.4 tumor-derived chemokine CCL22, acts to suppress growth and inter- Metastasis has been studied from the standpoint of intrinsic tumor feron (IFN)-c production of T cells and is associated with poor sur- cell properties and host responses, including angiogenesis and vival.10 Thus, it is likely that the that favors immune surveillance at primary sites. On the one hand, given that Th1 responses results in good antitumor immunity, and this favorable adaptive antitumor immunity of endogenous origin, in general, fails condition is affected by tumors. to work efficiently in clinical settings, much effort has been made to Immunoediting may undergo epithelial mesenchymal transition. abrogate immunoediting in tumor cells by modifying naturally occur- This event could be accompanied by a loss of tumor antigen and ring tumor antigens and manipulating dendritic cells (DCs), which MHC molecules through unknown mechanisms.11 However, that is bridge innate and adaptive immunity.5 On the other hand, there have not sufficient to explain immunosuppression. One of the major and been controversies involving the correlation between T-cell infiltra- initially proven factors involved in epithelial mesenchymal transition tion within tumor tissues and patient prognosis. The recently revealed is transforming growth factor (TGF)-b derived from mesenchymal

Department of Pharmacology, Tokyo Women’s Medical University, Tokyo, Japan Correspondence: Professor Y Maru, Department of Pharmacology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: [email protected] Received 26 November 2009; accepted 2 December 2009 Premetastatic milieu explained by homeostatic inflammation Y Maru 95

Figure 1 A simplified schematic representation of the tumor microenvironment with respect to immune cells. | -----: inhibition, r: stimulation. CSF, colony-stimulating factor; DC, ; EGF, ; IFN, interferon; LPS, ; mDC, mature dendritic cells; TAM, tumor-associated macrophage; TGF, transforming growth factor; TLR4, Toll-like receptor 4; Treg, regulatory T cell; VEGF, vascular endothelial growth factor. cells in tumor tissues or from tumor cells themselves.1 As shown in lipopolysaccharide (LPS), are thought to activate M1 macrophages, mouse experiments with T-cell-specific deletion of TGF-b receptor II, which in turn produce , including TNF-a, IL-6 and IL-12, TGF-b not only autonomously inhibits CD41 T-cell differentiation and chemokines, such as CXCL9 and CXCL10. This event establishes into Th1, but also maintains Tregs12 (Figure 1). Another tumor- a danger signal that not only mobilizes Th1 cells by the chemokines, derived immunosuppressive growth factor is vascular endothelial but also kills invading pathogens by production of NO and ROS growth factor (VEGF). Activation of VEGF receptor 2 (VEGFR2) in (Figure 1). Although detailed expression analysis of S100A8/S100A9 endothelial cells plays an essential role in their proliferation and vas- and SAA3 in primary tumor cells awaits further study, other endogen- cular permeability by promoting structures, such as fenestration in the ous TLR4 ligand candidates derived from dying tumor cells, such as endothelium, through which primary tumor cells extravasate. Tumor high-mobility group box 1 and heat shock cognate 70, or from cells almost always produce abundant VEGF. Thus, a decrease in destroyed extracellular matrix and vessels, including hyaluronan VEGF may allow T-cell infiltration into tumors. Tumor-necrosis fac- fragments and fibrin, could substitute for LPS.7 However, tumor- tor (TNF)-a-induced nuclear factor (NF)-kB activation in DC mat- associated macrophages (TAM) manifest M2 features, and are less uration was shown to be blocked by VEGFR1 on DCs.13 In addition to cytotoxic to tumor cells. M2-polarized macrophages express IL-10 VEGF, maturation of DCs is also blocked by tumor-producing colony- and CCL22, which recruits Tregs.15 TAM are frequently found in stimulating factor (CSF)-1, IL-10 and TGF-b (Figure 1). Both CSF-1 avascular areas and, therefore, express VEGF in a hypoxia inducible and IL-10 promote macrophage differentiation and growth. factor-1-dependent manner. A variety of tumor cells have been found Immature DCs and macrophages are thought to be distributed to express TLR4, which, once activated, secrete CCL20 to recruit throughout tumor tissue, but mature DCs are found in peritumorous immature DCs that show a low level of antigen presentation even after regions. Given the unique ability of DCs to present tumor antigens to TLR4 activation.16 T cells, their link to adaptive immunity is also abrogated by tumors. Then, what reinforces M2 polarization in TAM? In the mouse mam- Macrophages are functionally versatile and have a spectrum of mary tumor virus-polyoma middle T model, it was functional states between M1 and M2, whose polarization is shown shown that Th2-cell-derived IL-4 not only polarizes CD11b1Gr1- to be regulated by cytokines derived from subsets of CD41 T F4/801 TAM to M2, but also works in concert with CSF-1 to induce cells.14 Conventionally, Th1 cytokines, such as IFN-c and microbial TAM to secrete epidermal growth factor (EGF).17 Activation of EGF

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receptor in tumor cells results in CSF-1 expression, amplifying bidir- promote expression through cytokines, such as TNF-a,but ectional signaling between M2 macrophages and tumor cells. EGF also induce the expression of plasminogen activator inhibitor-1 receptor-activated tumor cells obtain a greater ability to accomplish through activating transcription factor 3.7 Therefore, TLR4 activation lung metastasis without changing VEGF expression levels and angio- by endogenous in the tumor microenvironment may facilitate genic density at primary sites. Given that both RAG-null and CD4-null coagulation and block fibrinolysis. Fibrin and fibrinogen have been mice display a decreased number of circulating tumor cells and atte- claimed to activate TLR4. We have performed similar experiments with nuated lung metastasis, cells of acquired immunity can actively par- B16 and Lewis lung carcinoma (LLC) and found that the chemokines ticipate in metastasis. Because IL-4 cannot directly activate NF-kB, S100A8/S100A9 and SAA3 are upregulated in premetastatic lungs in a whose inhibition can switch polarization from M2 to M1 by targeting primary tumor-derived growth-factor-dependent fashion, including IkB kinase-b activity,18 other factors should be involved in the polar- TNF-a, TGF-b and VEGF.24 Both chemokines were identified as strong ization and/or maintenance of the M2 status. candidates for endogenous TLR4 agonists, as judged by surface plas- Gr-11CD11b1 myeloid cells, which were initially found in tumor- mon resonance analysis. Less efficient, but appreciable binding of SAA3 bearing mice, can promote antigen-specific immunosuppressive Tregs to TLR2 was also found.2 Principally, chemokine receptor-expressing in a CD80-mediated manner.19 In the mouse ovarian surface epithelial cells migrate toward regions abundant in the corresponding chemo- cell 1D8 model of ovarian cancer, both Tregs and Gr-11CD11b1 cells kine. Serum levels of SAA3 increase in the course of primary tumor are required for the suppression of human papillomavirus type 16 development (Figure 2a). A peak concentration of 0.15 mg/ml was virus-like particle-induced release of IFN-c by CD25-depleted sple- observed 8 days after tumor challenge, which was two magnitude less nocyte cultures. In addition, they not only favor IL-10 production, but (one hundredth, ten to the minus second) than that of SAA1 in the also suppress IL-12 secretion in macrophages, providing Th2-like res- same tumor-bearing mice or in cancer patients. The numbers of ponses.20 Moreover, Gr-11CD11b1 myeloid cells directly suppress CD11b1 cells as well as endothelial cells with induced expression of T-cell immunity by Nox-2-mediated generation of ROS.21 SAA3 are also increased. Therefore, TLR4-expressing cells in the mono- Carcinoma-associated fibroblasts may also participate in cancer nuclear phagocyte system could travel to the lungs. The upregulation of immunology. These cells produce CXCL12 (stromal cell-derived SAA3 that we observed in premetastatic lungs might be mediated by the factor-1), VEGF and MMP9. CXCL12 can induce chemotaxis of vascular instability induced by the MMPs in a TNF-a-dependent man- Tregs.22 Correlation between Treg infiltration and CXCL12 expression ner. However, in our study, fibrinogen failed to induce SAA3 and has been found in .23 Classical Tregs inhibit T cells in S100A8 in macrophages and endothelial cells (Ishibashi and Maru, lymphoid organs. Although CXCL12 is expressed in bone marrow, G- unpublished results). On the contrary, S100A8/S100A9 was shown to CSF has been reported to reduce CXCL12 in bone marrow. These changes dissociate cell–cell contacts in both endothelial and epithelial cells, may mobilize Tregs from the bone-marrow reservoir to the periphery.22 promoting leukocyte infiltration.30 SAA3 was shown to induce several species of MMPs in chondrocytes.31 Whichever event comes first, vas- PRIMARY TUMORS AFFECT PREMETASTATIC TISSUES cular destabilization, if any, or upregulation of endogenous TLR4 ago- Although metastatic outgrowth of tumor cells is abrogated in CD4- nists may serve as a danger signal even before the eventual arrival of the null conditions, including the RAG knockout, which attenuated EGF true danger, metastasizing tumor cells. Injection of LLC or 3LL, a highly receptor-mediated activation of metastatic potential of primary tumor metastatic variant of LLC, into the tail vein of mice to block pulmonary cells, we still do not know the mechanisms of the last three steps in homing and spontaneous metastasis by inhibition of the S100A8– metastasis: extravasation, recruitment and regrowth in the lungs.24 SAA3–TLR4 system, respectively, demonstrates that the fourth and Less than 1% of recruited tumor cells appear to achieve regrowth. fifth steps in metastasis and, as a consequence, the final regrowth step, 2 How metastasizing tumor cells survive and start outgrowth in a given are regulated by TLR4. In metastasis induced by tumor-derived versi- organ is still an unanswered question, but this event has recently been can, TLR2 plays an essential role in driving the production of bone 32 reported to be regulated by VEGFR1 in the lungs,25 Src-mediated marrow-derived myeloid cells. In both cases, cross-talk between TLR signaling of CXCL12 in bone,26 and transcription factor HoxB9 and and the TNF receptor exists. SAA3 is capable of inducing TNF-a Lef1-dependent Wnt/Tcf signaling in brain and bone.27 Even in RAG- expression in the lungs,33 and versican-induced metastasis is abrogated null mice, normal breast cells can be recruited to the lungs without by knocking out TNF-a.32 oncogene activation.28 Experimental activation of oncogenes after TLR4 is expressed in granulocyte-macrophage progenitors through homing results in metastatic colonization. This finding suggests that differentiated myeloid cells. This differentiation usually occurs upon tumor cells may travel to potential metastatic sites at any stage of the the action of cytokines, such as G-CSF, but LPS-stimulated granulocyte- multistep malignant transformation sequence. Once regrowth starts, macrophage progenitors do not require action.33 We do not involvement of T cells that facilitate regrowth through bidirectional know the exact properties of recruited Gr-11CD11b1 myeloid cells, signaling between macrophages and regrowing tumor cells may be whose number is increased in premetastatic lungs,7 which are inhib- accomplished, as in the case of primary tumors described above. ited in TLR4 knockout mice. However, their number is increased in Premetastatic organs can be affected by primary tumors before the peripheral blood, possibly by the action of G-CSF produced from metastasizing tumor cells actually arrive. Microarray-based gene primary tumors.34 These cells are shown to make direct contact with expression analysis of premetastatic lungs of B16 melanoma-bearing CD81 T cells and render them unresponsive to antigen stimulation by mice revealed the upregulation of Angpt2, MMP3 and MMP10, but abolishing CD8 and TCR through the production of NO and ROS.35 not VEGF, with a concomitant lung-specific fibrin deposit, suggesting IFN-c and LPS were shown to sustain Gr-11CD11b1-cell expan- vascular destabilization.29 Primary tumor-derived TNF-a and TGF-b sion.36 Interestingly, the endogenous TLR4 ligand S100A9, but not were assumed to be responsible for the upregulation of these three S100A8, was revealed to induce Gr-11CD11b1-cell accumulation . In primary tumors, tumor cells express a tissue factor that and inhibit DC differentiation, as judged by cytokine- and LPS- activates the extrinsic coagulation pathway, and a fibrin deposit is mediated in vitro maturation assays of hematopoietic progenitor cells observed on the surface of TAM. LPS-stimulated TLR4 can not only from S100A9 transgenic mice.37 Conversely, transplanted tumors are

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Figure 2 (a) Serum levels of serum amyloid A3 (SAA3) in tumor-bearing mice after subcutaneous transplantation of Lewis lung carcinoma. (b) Binding assay of the membrane fraction of THP-1 with glutathione-S-transferase (GST) or GST-mouse SAA3. Binding is inhibited by an anti-mouse SAA3 (mSAA3) antibody (Ab). (c) Sensing mechanisms are represented by groups of cells, including dendritic cells (D), macrophages (M) and resident epithelial cells (E), all of which express Toll-like receptor 4 (TLR4). An endogenous ligand, such as SAA3, may be engaged in homeostasis that regulates host responses to extrinsic molecules, like lipopolysaccharide (LPS). The sensed information is passed onto immune cells (I). SAA3 is sensed before tumor cell (TC) arrival in the premetastatic milieu. This logic is reversed in the danger hypothesis, in which TC destruction releases alarmins, such as high-mobility group box 1 and heat shock cognate protein 70, which are proposed to stimulate TLR4. Since the premetastatic milieu (Pre-met) is based on the growth factor-stimulated induction of the endogenous TLR4 agonists, it is theoretically reversible provided that it is devoid of tumor cells. efficiently rejected in S100A9 knockout mice, a phenotype which was T cells may also be directly involved in premetastatic lungs. The rescued by anti-CD8 blocking antibody. Collectively, lung recruitment production of CD41 T cells is slightly increased in premetastatic lungs of TLR4-expressing cells that may be included in the Gr-11CD11b1 and is enhanced 24 h after injection of tumor cells into the tail vein of population of myeloid cells38 is likely to prepare an immunosuppres- mice (Sakurai and Maru, unpublished results). In the 4T1 breast can- sive microenvironment to allow metastasizing tumor cells to establish cer-bearing mouse model, premetastatic lungs were also shown to themselves. express CCL17 and CCL22, which mobilize CCR4-expressing Tregs

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and tumor cells to the lungs.39 Successful killing of natural killer cells 1. The autoregulatory loop: S100A8 induces the expression of SAA3, by CCR41 Tregs in vitro suggests immune escape that may also occur and SAA3 amplifies the expression of its own mRNA.2 In the in the lungs. inflammation-associated liver carcinogenesis model, S100A8/ Presence of concrete inflammation in the lungs facilitates metastasis. S100A9 was shown as a NF-kB target.46 An established ovalbumin sensitization/aerosol challenge model of 2. The negative feedback loop: both agonists activate activating asthma, combined with a metastasis assay, has shown more than three- transcription factor 3, resulting in a negative feedback on LPS- fold enhancement in metastasis that is abrogated by CD41 T-cell deple- stimulated NF-kB activation.7 tion.40 An LPS-induced danger signal emitted by either resident 3. Oscillatory activation: sustained TLR4 activation, which is macrophages or DCs results in M1-like responses to eliminate the patho- required for cytokine expression, is consistent with oscillation logical microbes. A danger signal provoked by induced expression of in NF-kB signaling due to coordinated degradation and resynth- endogenous TLR4 agonists may initially provide M1-like responses. esis of IkB proteins (conceptually reviewed in Ref. 47). Recruited immature DCs in the lungs may differentiate into mature Accumulation of distinct signaling exerted at different time points DCs, but with no antigen to present in the absence of microbes or tumor may reach a threshold for . How endogenous ligand- cells. This abortive maturation expands the field of bone marrow- induced signaling contributes to the summation of the oscillatory derived myeloid cells that respond to primary tumor-producing growth activation of NF-kB awaits further study. As expected from the pos- factors to generate more TLR4 agonists, presumably with the recruit- itive results on surface plasmon resonance analysis between purified ment of immunosuppressive Tregs. TLR4-expressing tumor cells can TLR4/MD-2 complex and SAA3, SAA3 bound to the immobilized reach the immunocompromised lungs to start colonization. membrane fraction of human monocytic cells that express TLR4; this The aforementioned idea that TLR4 favors metastasis is seemingly binding was inhibited by an anti-SAA3 antibody in a dose-dependent in disagreement with the report that the frequency of post-operation manner (Figure 2b). Pre-treatment of these cells with SAA3 followed metastasis is higher in patients that possess TLR4 with a loss-of- by LPS stimulation showed neither an increase nor a decrease in function mutation than that without.41 The report lacks information phosphorylated IkB signals in the presence of proteasome inhibitors on organotropism, and not only immune cells, but breast cancer cells (Tsukahara and Maru, unpublished results), suggesting that endogen- themselves also lack TLR4, which could result in a poor metastatic ous ligands might interfere with exogenous LPS. An anti-S100A8 microenvironment in the lung and less tumor cell mobilization. blocking antibody abrogated transepithelial migration of macro- Therefore, it is difficult to precisely interpret the results for the bio- phages and neutrophils into the alveolar space.48 In addition, logical significance of TLR4. S100A8 is capable of inducing NOS2,49 and TLR4 signaling is closely linked with ROS generation.50–52 Thus, endogenous ligand may THE CONCEPT OF HOMEOSTATIC INFLAMMATION EXPLAINS serve to balance TLR4-mediated NF-kB signaling that is sensitive to THE PREMETASTATIC MICROENVIRONMENT redox. When the radiosensitivity of different TLR4-expressing cells is con- Furthermore, in the gastrointestinal tract system, TLR4-expressing trolled in the LPS-induced asthma model, intratracheal administra- mucosal cells communicate with DCs in intestinal homeostasis. A 1 1 tion of LPS results in an increased cellular influx of Gr-1 CD11b granulocyte-macrophage progenitor comprises a common precursor 1 1 42 cells and MHC II CD11c DCs in the lungs. Production of CCL20 for macrophages and DCs that express CX3CR1.53 Antigens are 1 in a TLR4-dependent fashion in lung structural cells attracts CCR6 sampled in gastrointestinal tract by DCs that extend transepithelial cells. Upregulation of the CCL2–CCR2 system also recruits dendrites while maintaining the integrity of the epithelial barrier in a 1 1 1 CCR2 Gr-1 myeloid cells, which are precursors for CD11b DCs. CX3CR1-dependent manner. In , regulated NF-kB signal- TLR4 in lung epithelial cells was shown to be essential for triggering ing through dynamically balanced ROS production by Nox family asthma. TLR4-expressing Clara cells, which play a xenobiotic role in members may play a role in gastrointestinal tract homeostasis, which terminal bronchiolar epithelium, could be supplied from bone mar- needs to discriminate between commensal and pathogenic row when the epithelium is injured.43 Although it is hard to administer microbes.54 In dextran sodium sulfate-induced colitis, TLR4 activa- S100A8 or SAA3 preferentially in the lungs, a similar experiment to tion was shown to play a protective role in the regulation of colonic ours with LPS demonstrated SAA3 induction in the lungs (Tomita and repair.55 SAA3 is expressed in colon epithelial cells possibly as a con- Maru, unpublished results). These data suggest that TLR4 that is sequence of NF-kB signaling exerted by LPS from commensal bac- expressed not only in mononuclear phagocyte system, but also in teria.56 SAA3 induces IL-6 expression in macrophage RAW cells.33 In epithelial cells may serve as a sensor to defend against constant assaults the colitis-associated cancer model, both IL-6 and its signaling via by air-borne bacteria or chemicals in a homeostatic manner. Notably, Stat3 are required for the survival of epithelial cells.57 TLR4 knockout mice display emphysema with activation of Nox3.44 In In premetastatic lungs, endogenous TLR4 ligands are upregulated the bleomycin-induced acute lung-injury model, transepithelial without tissue destruction. This upregulation alone contributes to migration of leukocytes is impaired in TLR4/TLR2 double-knockout lung homeostasis in the absence of primary tumors. However, this mice; in this system, an endogenous TLR4 candidate hyaluronan frag- event mobilizes tumor cells from the primary site as well as a variety ment induced CXCL2 (MIP-2).45 However, before tissue destruction, of immune cells that could facilitate immune escape. Once bidirec- high molecular weight hyaluronan contributes to tissue architecture tional signal amplification between macrophages and tumor cells maintenance and repair via the TLR4/TLR2 system by regulating the starts, as exemplified by CD41 T-cell-induced EGF expression in basal activation of NF-kB. Displacement of the high molecular weight macrophages in primary tumors, metastasis is achieved to produce a form by its fragment may switch homeostasis to a danger signal. classical danger signal. These sequential events, from physiological Then, what is the role of endogenous TLR4 ligands in the lung? Both host defense, its aberration by remote control of primary tumors S100A8 and SAA3 can activate NF-kB signaling.7,24 At least three and eventually to the production of a danger signal after the arrival modes of regulation in the signaling of those endogenous TLR4 ago- and regrowth of tumor cells, are what I propose as homeostatic inflam- nists suggest their participation in homeostasis: mation (Figure 2).

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Cellular & Molecular Immunology