Inhibition of Tumor Growth Cell-Derived IL-6 in TLR2-Mediated a Critical Role for Mast Cells and Mast

A Critical Role for Mast Cells and Mast Cell-Derived IL-6 in TLR2-Mediated Inhibition of Tumor Growth

This information is current as Sharon A. Oldford, Ian D. Haidl, Mackenzie A. Howatt, of September 28, 2021. Carlos A. Leiva, Brent Johnston and Jean S. Marshall J Immunol 2010; 185:7067-7076; Prepublished online 1 November 2010; doi: 10.4049/jimmunol.1001137

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A Critical Role for Mast Cells and Mast Cell-Derived IL-6 in TLR2-Mediated Inhibition of Tumor Growth

Sharon A. Oldford,*,† Ian D. Haidl,*,† Mackenzie A. Howatt,*,† Carlos A. Leiva,*,† Brent Johnston,*,†,‡,x and Jean S. Marshall*,†,x

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deﬁcient KitW-sh/W-sh mice and a complementary Matrigel–tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam3CSK4)-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented

W-sh/W-sh Downloaded from protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam3CSK4 was restored in Kit mice by local reconstitution with wild-type, but not TLR2-deﬁcient, mast cells. Mast cells secrete multiple mediators after Pam3CSK4 activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, http://www.jimmunol.org/ to our knowledge, a novel mechanism of action for TLR2 agonists in vivo. The Journal of Immunology, 2010, 185: 7067–7076.

he use of TLR agonists as immunological adjuvants in play a role in allergic disease and function as sentinel cells involved the treatment of cancer has been intensively studied. TLR- in the induction and development of effective immunity (7–9). T based immunotherapies have shown promise clinically Mast cells also play a role in tumorigenesis. Abundant mast cells are for the treatment of multiple tumor types. Specifically, TLR2 closely associated with blood vessels surrounding solid tumors and agonists have been successfully used as potential therapeutics in often correlate with enhanced tumor angiogenesis and poor prog- animal models of pancreatic and brain carcinoma (1, 2) as well as nosis (10–12). Studies using mast cell-deficient KitW/KitWv or by guest on September 28, 2021 in clinical trials (3). In general, the successful mechanism of ac- KitW-sh/KitW-sh mice showed that tumor-associated mast cells are tion of TLR agonists is considered to involve dendritic cell mat- potent tumor angiogenesis promoters, enhancing tumor growth uration with enhanced tumor Ag presentation and generation of and metastasis (13–15). Although extensive studies demonstrate a Th1 immunity and CD8 antitumor effector cells (4). TLR2 ago- protumorigenic role for mast cells (reviewed in Ref. 16), antitumor nists, such as Pam3CSK4, often do not share the strong Th1 po- actions of mast cells have also been reported. In vitro studies larizing effects of other TLR agonists (5, 6). However, they retain have demonstrated mast cells can mediate direct TNF-mediated effectiveness in tumor immunotherapy models, opening up the tumor cell cytotoxicity (17, 18). Several studies have also corre- possibility that they have a distinct mechanism of action. lated high mast cell density with improved survival (19–21). The Mast cells are localized at body sites that interface with the en- conflicting reports of associations of mast cells with prognosis are vironment, such as the skin and mucous membranes, where they potentially largely dependent on effects of the local tumor microenvironment on mast cell activation status and mediator release. During infection, activation of mast cell receptors including Fcg *Dalhousie Inflammation Group, †Department of Microbiology and Immunology, receptors, complement receptors, and TLRs results in the release of ‡ x Department of Pediatrics, and Department of Pathology, Dalhousie University, Hal- preformed mediators including histamine and proteases, the syn- ifax, Nova Scotia B3H 1X5, Canada thesis of lipid mediators, and the selective production of multiple Received for publication April 8, 2010. Accepted for publication September 28, 2010. cytokines and chemokines. These mediators are important in elim- inating pathogens, recruiting other leukocytes such as neutro- This work was supported by funding from the Canadian Cancer Society Research Institute and the Canadian Institutes of Health Research. S.A.O. was supported by phils, NK cells, and T cells, increasing vascular permeability, and a Canadian Institutes of Health Research-funded Strategic Training in Health Re- in mediating proteolytic degradation at the site of infection (9). search Initiative and by the Nova Scotia Health Research Foundation. Although the role of mast cells in protecting against infection is Address correspondence and reprint requests to Dr. Jean S. Marshall, Department of well documented, the effects of activating mast cells in the context Microbiology and Immunology, Faculty of Medicine, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada. E-mail of tumorigenesis have not been substantially explored. address: [email protected] Mast cells are prevalent in the skin and are abundant at the pe- The online version of this article contains supplemental material. riphery of melanomas (12). They respond to TLR2 agonists by Abbreviations used in this paper: BMMC, bone marrow-derived mast cell; CPT, producing leukotrienes, cytokines, and chemokines, which could camptothecin; ns, not significant; PI, propidium iodide; RFU, relative fluorescent modify the local microenvironment and recruit leukocytes poten- units; SA, streptavidin; Treg, regulatory T cell; UD, undetectable. tially to limit tumor growth (22, 23). We therefore investigated the Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 role of the mast cell in the tumor growth-inhibitory effects of the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001137 7068 ACTIVATED MAST CELLS INHIBIT TUMOR GROWTH

TLR2/1 agonist Pam3CSK4 using an orthotopic model of B16.F10 blood was collected, tumors were excised and digested in 300 U/ml col- melanoma, a well used, highly aggressive, poorly immunogenic lagenase IV (Sigma-Aldrich), and cell-free supernatants were measured tumor (24). B16.F10 growth was compared in wild-type and fluorometrically (excitation 488 nm, emission 519 nm). Relative fluorescent units (RFU) were normalized to blood plasma levels. mast cell-deficient KitW-sh/W-sh mice. Although KitW-sh/W-sh mice are mainly white, they are suitable for melanoma growth studies as the Histology W-sh Kit mutation impairs long-term endogenous melanocyte survival Formalin-fixed, paraffin-embedded Matrigel–tumor sections (3 mm) were but does not affect melanoblast development or differentiation or stained with H&E. Mature erythrocyte-containing blood vessel lumens the survival of transplanted c-Kit–expressing melanocytes. Homo- were quantified in non-overlapping fields (magnification 3400) of tumor zygote adult animals consistently retain melanocytes at selected periphery, on coded sections. Mast cells were visualized using Alcian blue (pH 0.5)–safranin O staining of Carnoy’s fixed, paraffin-embedded tumor sites including the ear pinna (25, 26). Comparative growth studies in sections. W-sh/W-sh Kit and control C57BL/6 mice identified that Pam3CSK4 inhibits tumor growth in a mast cell and mast cell-derived IL-6-de- Tumor cell cytotoxicity and cell viability pendent manner. This study demonstrates that selective activation Tumor cell proliferation was evaluated by the MTT assay. Briefly, 1 3 104 of mast cells via TLR2 can reverse their tumor-promoting potential tumor cells were incubated overnight in 96-well tissue culture plates then and induce them to inhibit tumor growth. treated, in quadruplicate, for 24 h before MTT addition. Treatments included 0, 1, or 10 mg/ml Pam3CSK4,5mg/ml of the DNA topoisomerase I inhibitor camptothecin (CPT) or DMSO vehicle control (Sigma-Aldrich), Materials and Methods or supernatants from C57BL/6 or IL-62/2 BMMCs incubated for 24 h in Mice BMMC culture medium with or without 10 mg/ml Pam3CSK4. Tumor cell 6 6 apoptosis in B16.F10 (0.5 3 10 /ml)–BMMC (1 3 10 /ml) cocultures was Downloaded from C57BL/6, B6.129S6-Tnftm1Gkl/J, B6.129S2-Il6tm1Kopf/J, B6.129P2- tm1Unc W-sh W-sh/W-sh assessed using an annexin V–FITC–propidium iodide apoptosis detection Ccl3 /J, and B6.Cg-Kit /HNihrJaeBsmJ (Kit ) were pur- kit (BD Biosciences, Mississauga, Ontario, Canada) according to the man- chased from The Jackson Laboratory (Bar Harbor, ME). KitW-sh/W-sh mice tm1Kir ufacturer’s instructions. BMMCs were excluded from analysis via bio- were bred in-house from this stock. B6.129-Tlr2 /J mice were provided tinylated anti-CD45 (eBioscience, San Diego, CA) and streptavidin (SA)– by Dr. S. Akira (Osaka, Japan) and bred in-house or purchased from The allophycocyanin (BD Biosciences) staining. Jackson Laboratory. Matched groups of male or female mice were used at 8–12 wk of age and housed under pathogen-free conditions with food and Cell counts and flow cytometry water provided ad libitum. All experiments were performed according to http://www.jimmunol.org/ protocols approved by the animal research ethics board of Dalhousie Uni- Established tumors were collagenase digested as above. Matrigel tumors versity (Halifax, Nova Scotia, Canada). were digested in 2.24 U/ml Dispase (Life Technologies, Carlsbad, CA) in PBS. Overall tumor cellularity was assessed by flow cytometry of digested Generation of bone marrow-derived mast cells and local tumors with reference to a known number of 15 mm Polybead polystyrene reconstitution of mast cell-deficient mice microspheres (Polysciences, Warrington, PA). Total leukocytes were stained with biotinylated anti-CD45 (eBioscience) and SA–PerCP (BD Bio- Bone marrow-derived mast cells (BMMCs) were generated from wild-type 2/2 2/2 2/2 2/2 sciences), anti-TCRb–FITC (eBioscience) or anti-CD3–FITC (BD Bio- C57BL/6 and gene-deficient TLR2 , TNF , IL-6 , or CCL3 sciences), anti-CD8–PE (eBioscience), and anti-NK1.1-allophycocyanin . mice as previously described (27) and were used at 98% purity. BMMCs (BD Biosciences). Biotinylated rat IgG2a isotype (eBioscience) with SA– derived from selected gene-deficient animals were functionally similar to PerCP staining did not exceed 0.1% of total. Samples were gated on CD45+ control C57BL/6 BMMCs (Supplemental Fig. 1). BMMCs were cultured cells and leukocyte subsets quantified within this gate. Staining using rele- by guest on September 28, 2021 for 16–24 h in the absence of PGE2 prior to in vitro experiments. Eight- to vant isotype control Abs (eBioscience) did not exceed 0.2% of CD45+ cells. ten-week-old KitW-sh/W-sh mice were reconstituted on the dorsal surface over the right and left flanks in 2 3 2cm2 areas via a series of injections with Chemotaxis assays BMMCs (1 3 106 intradermal and 1 3 106 s.c.), which resulted in mast cell levels comparable with those of wild-type C57BL/6 mice (Supplemental C57BL/6 spleens were dispersed by pushing through a 100-mm nylon cell Fig. 2). strainer. Erythrocytes were lysed using 0.14 M ammonium chloride plus 20 mM Tris (pH 7.4). Adherent cells were removed via incubation for 20 min Established tumor growth experiments at 37˚C in 0.1% BSA in RPMI 1640. Chemotaxis of nonadherent splenocytes 5 m B16.F10 cells (1 3 10 ) (American Type Culture Collection, Manassas, VA) was performed using 24-well Transwells with a 5- m pore size (Corning, m in 100 ml PBS were injected s.c. above the right and left flanks. At days 4 Lowell, MA). Test supernatant (600 l) was added to the bottom chamber, 3 6 m and 9 (and day 14 as indicated), mice were injected s.c. at the tumor site with and 1 10 splenocytes in 100 l RPMI 1640 containing 0.1% BSA was added to the top. Chemotaxis was carried out for 2 h at 37˚C. To enumerate 3 mgPamCSK or FSL-1 (EMC Microcollections, Tu¨bingen, Germany) 3 4 total migrating cells, each well received 15-mm polybead polystyrene in 50 ml PBS or diluent control. Tumor volume (V) was determined by + + 2 microspheres (Polysciences) as an internal standard. NK1.1 , CD3 , and caliper measurements (V = L 3 W /2). After 14 to 19 d, mice were eutha- + nized, and tumors were removed and weighed. Percentage tumor reduction = CD8 cells were assessed by flow cytometry, as described earlier. Total mi- grated subsets are expressed as a percentage of the starting cell subset pop- (Pam3CSK4 treated tumor average 2 PBS treated tumor average)/PBS treated tumor average 3 100. ulation and were calculated based on absolute numbers of each cell subset. In vivo Matrigel–tumor growth experiments In vitro cell stimulation, protein array analysis, and ELISA 6 6 Growth factor-reduced Matrigel (Becton Dickinson, San Jose, CA) sus- B16.F10 cells (0.5 3 10 /ml) and/or BMMCs (1 3 10 /ml) were incubated 5 pensions containing 1.5 3 10 B16.F10 or LLC1 cells (American Type with or without 10 mg/ml Pam3CSK4, FSL-1, LPS (Sigma-Aldrich), PGN (Invivogen), and polyinosinic-polycytidylic acid (Calbiochem, San Diego, Culture Collection), with or without 10 mg/ml Pam3CSK4 (EMC Micro- collections) and/or 3 3 105 BMMCs, were prepared. All Matrigel suspen- CA) or A23187 ionophore (Sigma-Aldrich) positive control at 37˚C in sions contained 3 ng/ml murine rIL-3 (PeproTech, Rocky Hill, NJ). C57BL/ RPMI 1640 containing 10% FBS, 3 ng/ml murine rIL-3 (PeproTech), and 6 mice (8–12 wk) were injected s.c. in four sites with 0.3 or 0.15 ml sus- 100 mg/ml soybean trypsin inhibitor (Sigma-Aldrich). Cell-free super- pensions for 4 or 10 d, as indicated. Mice were euthanized, and tumors were natants were assessed using the murine Cytokine III Ab array (Raybiotech, excised and weighed. Percentage tumor reduction = (tumor + BMMC + Norcross, GA) and ELISA kits to quantify levels of TNF and IL-13 (both eBioscience), IL-6 (PeproTech), and CCL1 and CCL3 (both R&D Systems, Pam3CSK4 Matrigel average 2 tumor + BMMC Matrigel average)/tumor + BMMC Matrigel average 3 100, following subtraction of baseline Matrigel Minneapolis, MN). plug weight determined from 10-d Matrigel plugs containing BMMCs and Statistics Pam3CSK4 without tumor cells. Tumor blood volume determination Samples were analyzed using unpaired t test or non-parametric Mann– Whitney U test as indicated based on D’Agostino and Pearson omnibus Tumor-bearing mice were anesthetized and injected via the tail vein with normality test. Matrigel–tumor experiments were assessed with repeated 100 ml 25 mg/ml FITC-labeled dextran (average m.w. 2 3 106; Sigma- measures Friedman’s test and between-group comparisons assessed by Aldrich, Oakville, Ontario, Canada) in PBS. After 5-min perfusion, 20 ml Wilcoxon matched pairs test (10 d) or repeated measures ANOVA with The Journal of Immunology 7069

Dunnett’s multiple comparison post test, using B16.F10 alone as control Whereas KitW-sh/W-sh mice have been documented to be similar (4 d), based on normality tests. The p values ,0.05 were considered signifi- to their wild-type littermates with respect to myeloid and 6 cant, and all pooled data are presented as mean SEM. lymphoid cell subsets in multiple anatomical sites (30), others have demonstrated myeloid differences compared with wild-type Results C57BL/6 mice (31). To overcome any inherent defects of the TLR2 agonist treatment inhibits B16.F10 tumor growth via mast cell-deficient mouse model and assess the physiological role mast cell activation W-sh-W-sh of mast cells in Pam3CSK4-mediated effects, Kit mice The tumor-inhibitory effects of the TLR2/1 agonist Pam3CSK4 were were locally reconstituted with BMMCs, which were allowed to investigated using an orthotopic model of B16.F10 melanoma. When mature in situ prior to performing tumor growth experiments. We treated with Pam3CSK4, tumors showed a significant reduction in also extended the time course of the tumor growth experiment to W-sh/W-sh volume (73%; Fig. 1A) and cellularity (74%; Fig. 1B). Mast cells ensure the lack of effect of Pam3CSK4 in Kit mice was were detected in close proximity to blood vessels at the periphery of not attributable to delayed tumor growth kinetics (Fig. 2A). Even both Pam3CSK4-treated and control tumors (Fig. 1C). As the ability with tumor size reaching levels comparable with those of C57BL/ of mast cells to exert a protective role in tumorigenesis may be de- 6 mice, the antitumor activity of Pam3CSK4 was absent in mast pendent on an intratumoral distribution (28, 29), we assessed the cell-deficient mice (Fig. 3A). Local reconstitution with C57BL/6, 2/2 distribution of mast cells in control and Pam3CSK4-treated tumors. but not TLR2 BMMCs, restored the Pam3CSK4-mediated Mast cells were predominately found in the stroma as opposed to decrease in tumor volume (61%, Fig. 3A) and tumor weight (63%, intratumorally for both PBS-treated (2.3 6 0.5/mm2 versus 0.5 6 Fig. 3B).

2 2 Downloaded from 0.1/mm , n =6)andPam3CSK4-treated (3.3 6 0.7/mm versus 1.7 6 TLR1 and TLR2 are expressed on both murine BMMCs (32) 0.9/mm2, n = 9) tumors. The percentage of mast cells that were found and B16 melanoma (33) (see also Supplemental Fig. 3). To con- intratumorally did not significantly differ between PBS-treated and firm the specific involvement of mast cell-associated TLR2 in the Pam3CSK4-treated tumors (21 6 5% versus 28 6 11%). The pro- antitumor effects of Pam3CSK4, the ability of wild-type and tumorigenic role of mast cells has been well characterized in several TLR22/2 BMMCs to inhibit tumor growth were compared using models (14, 15). Consistent with these findings, baseline tumor a Matrigel–tumor model. Even within the context of the tumor

growth rate, in the absence of TLR2 agonist treatment, was signifi- growth-promoting Matrigel environment (34), TLR2-expressing http://www.jimmunol.org/ cantly decreased in mast cell-deficient KitW-sh/W-sh animals compared BMMCs significantly inhibited melanoma growth in the presence with that in mast cell-containing C57BL/6 animals (67%; Fig. 1D). of Pam3CSK4 (49% inhibition), whereas Pam3CSK4-activated To investigate a possible role for mast cells in Pam3CSK4-me- TLR2-deficient BMMCs did not (Fig. 3C). Pam3CSK4 or BMMCs diated tumor growth inhibition, B16.F10 tumors were treated in alone had no inhibitory effect on tumor growth as weight of mast cell-containing C57BL/6 mice or mast cell-deficient Matrigels containing B16.F10 plus BMMCs (0.295 6 0.046 g) did W-sh/W-sh W-sh/W-sh Kit mice. In Kit mice, Pam3CSK4 did not inhibit not differ from that of B16.F10 alone (0.244 6 0.028 g) or B16. tumor growth (Fig. 2A), whereas tumor growth was decreased in F10 plus Pam3CSK4 (0.250 6 0.016 g). parallel groups of mast cell-containing mice (59%; Fig. 2B), sug- The Matrigel model was also used to assess the frequency of by guest on September 28, 2021 gesting a mast cell-dependent mechanism of Pam3CSK4 action. mature blood vessels at the tumor periphery because the decreased Decreased end-point tumor weight (65%; Fig. 2C) and tumor blood tumor blood volume of Pam3CSK4-treated tumors (Fig. 2D) volume (69%; Fig. 2D) were also mast cell dependent. Enhanced suggested mast cells could inhibit tumor angiogenesis. Matrigel growth of diluent-treated control tumors was observed in C57BL/6 plugs in which B16.F10 was grown with Pam3CSK4-activated compared with KitW-sh/W-sh, further confirming the protumorigenic BMMCs had 60% fewer vessels than those that contained role of mast cells in the absence of TLR2 activation. melanoma cells together with BMMCs alone (Fig. 3D).

FIGURE 1. Pam3CSK4 treatment de- creases B16.F10 tumor growth. C57BL/ 6 mice were injected s.c. with B16.F10 melanoma cells. Tumors were treated s.c. on days 4 and 9 post-tumor injection (arrows) with Pam3CSK4 or PBS control (n = 10 tumors per group). A, Tumor volume was monitored daily. **p = 0.004. B, After 14-d growth, overall cellularity of collagenase-disrupted tumors was assessed. Right panel depicts representative diluent PBS- or

Pam3CSK4-injected tumors. *p = 0.038. C, Tumor-associated mast cells were examined in tissue sections stained with Alcian blue–safranin O (original mag- niﬁcation 3400). Adjacent tumor cell areas are indicated by black arrows. D, Mast cell-deﬁcient KitW-sh/W-sh and C57BL/6 mice were injected s.c. with B16.F10 melanoma cells (n = 10 per group), and tumor volume was followed for 16 d. Pooled data from two independent experiments: *p = 0.026. 7070 ACTIVATED MAST CELLS INHIBIT TUMOR GROWTH

CCL3, CCL5, CXCL1/2, CXCL3, and CXCL5 production (Sup- plemental Fig. 4). Such mediator production was independent of mast cell degranulation assessed via b-hexosaminidase release (Supplemental Fig. 1). The presence of melanoma cells further enhanced mast cell mediator production, as confirmed by ELISA (Fig. 5A). Elevated chemokine production by Pam3CSK4-activated mast cells suggested mast cell-mediated tumor-inhibitory effects might involve effector cell recruitment. Flow cytometry of disrupted tumors demonstrated that Pam3CSK4 treatment was associated with infiltrating CD45+ leukocytes, including CD8+TCRb+ and NK1.1+ cell subsets. B16.F10 tumors grown in Matrigel also had a marked mononuclear cell infiltration, at day 10, in the presence of Pam3CSK4 and BMMCs (Supplemental Fig. 5). To determine the involvement of TLR2-activated mast cells in early NK and T cell recruitment, we performed flow cytometry of B16.F10 tumors in Matrigel, on day 4, in the presence or absence of Pam3CSK4 and/or BMMCs. Tumor growth was significantly decreased in the presence

of BMMCs and Pam3CSK4 and was associated with signiﬁcant Downloaded from local increases in both NK and T cells (Fig. 5B). CCL3 induces recruitment of both mature NK cells (35) and activated CD8+ T cells (36, 37). To determine whether TLR2-activated mast cells directly induce the migration of NK and CD8+ T cells and speciﬁcally to assess the role of mast cell-derived CCL3, we per-

formed in vitro chemotaxis assays of C57BL/6 splenocytes using http://www.jimmunol.org/ supernatants generated from wild-type and CCL32/2 BMMCs (Fig. 5C). Pam3CSK4-activated BMMC supernatants induced signiﬁcant chemotaxis of NK1.1+ NK cells and both CD3+CD82 and CD3+ CD8+ T cell subsets (all p , 0.05). In contrast, supernatants from mast cells that could not produce CCL3 did not induce migration of NK or T cell subsets, demonstrating a role for CCL3 in mediating

FIGURE 2. Pam3CSK4 inhibition of B16.F10 tumor growth is mast cell mast cell-dependent effector cell recruitment. dependent. A and B, Parallel groups of (A) mast cell-deficient KitW-sh/W-sh or (B) C57BL/6 mice were injected s.c. with B16.F10 cells. Tumors were TLR2-activated mast cells inhibit B16.F10 tumor growth via an by guest on September 28, 2021 treated s.c. on days 4 and 9 post-tumor injection (arrows) with Pam3CSK4 IL-6–dependent mechanism or PBS diluent control (n = 8 to 12 mice per group). *p = 0.011. C, At day Pam3CSK4-activated BMMCs secreted substantial amounts of 15, end-point tumor weight (**p = 0.007; *p = 0.027) and tumor blood IL-6 (Fig. 5A). The requirement of mast cell IL-6 in tumor growth volume (**p = 0.008, *p = 0.036) were assessed. Data pooled from two inhibition by Pam3CSK4 was confirmed via lack of Pam3CSK4 independent experiments: **p = 0.007; *p = 0.027 (end-point tumor W-sh/W-sh weight). **p = 0.008; *p = 0.036 (end-point tumor blood volume). effect after local reconstitution of Kit with BMMCs derived from IL-6–deficient mice. In contrast, tumor growth was decreased in a parallel group of wild-type C57BL/6 mice (Fig. 6A). We further TLR2-activated mast cell inhibition of B16.F10 tumor growth confirmed the requirement for mast cell-secreted IL-6 using is TNF independent the Matrigel–B16.F10 model. Tumor growth was not inhibited by 2/2 Mast cells have been shown to induce tumor cytotoxicity in a TNF- Pam3CSK4 treatment in the presence of IL-6 mast cells. Fur- dependent manner (17, 18). In vitro Pam3CSK4-stimulated BMMCs thermore, tumor blood volume and the number of mature erythro- have elevated secretion of TNF (Fig. 4A). However, coculture of cyte-containing blood vessels were not decreased in the presence of 2/2 BMMCs and B16.F10 in the presence of Pam3CSK4 did not induce Pam3CSK4-activated IL-6 BMMCs (Fig. 6B), in contrast with tumor cell apoptosis (Fig. 4B). Local reconstitution of KitW-sh/W-sh results of similar studies with wild-type BMMCs. As in vitro and mice with BMMCs derived from TNF-deficient mice restored the in vivo experiments have demonstrated IL-6 can induce melanoma antitumor effect of Pam3CSK4 as indicated by significant reduc- growth inhibition (38–41), we assessed the effect of Pam3CSK4- 2/2 tions in tumor volume (57%; Fig. 4C), end-point tumor weight activated IL-6 BMMC supernatants on B16.F10 proliferation. 2/2 (122 6 30 mg versus 78 6 52 mg, p = 0.030), and tumor blood In vitro, supernatants from Pam3CSK4-activated IL-6 BMMCs volume (72.0 6 16.1 versus 42.8 6 22.1 RFU, p = 0.035), dem- did not significantly decrease B16.F10 cell proliferation (Fig. 6C). onstrating TNF from mast cells was not critical for tumor inhibi- TLR2-activated mast cells inhibit lung carcinoma growth tion. We further assessed direct antitumor effects in vitro. B16.F10 proliferation was unaffected by Pam3CSK4 treatment but was de- To ascertain whether the inhibitory effects of TLR2-activated creased by 18% in the presence of Pam3CSK4-activated C57BL/6 mast cells are unique to melanoma or can be extended to other BMMC supernatants (Fig. 4D). tumor types, we investigated the ability of Pam3CSK4-activated mast cells to inhibit the growth of the lung cancer model LLC1, at TLR2-activated mast cells recruit NK and T cell subsets day 10, using the Matrigel–tumor model system in C57BL/6 mice. To identify potentially important mast cell mediators, supernatants TLR2 expressing BMMCs significantly inhibited LLC1 growth in of BMMCs cultured with or without Pam3CSK4 and/or B16. the presence of Pam3CSK4 (58%), whereas Pam3CSK4-activated F10 cells were examined by cytokine protein array. In BMMC–B16. TLR2-deficient BMMCs did not (Fig. 7A). In contrast, Pam3CSK4 F10 cocultures, Pam3CSK4 induced IL-6, IL-13, CCL1, CCL2, or BMMCs alone had no effect on tumor growth (Fig. 7B) as weight The Journal of Immunology 7071

FIGURE 3. Pam3CSK4 activation de- creases tumor growth in a mast cell TLR2-dependent manner. B16.F10 tumors were grown for 19 d in parallel groups of mast cell-deﬁcient KitW-sh/W-sh mice or KitW-sh/W-sh mice locally reconstituted with wild-type or TLR22/2 BMMCs 6 wk prior to tumor administration. Data pooled from two independent experiments (n = 8 to 12 mice per group). Tumors were treated s.c. on days 4, 9, and 14 post-tumor injection (arrows) with

Pam3CSK4 or diluent. A, Tumor volume was assessed by caliper measurements *p = 0.019. B, At day 19, end-point tumor weight was measured. *p = 0.033. C, Downloaded from C57BL/6 mice were injected with Matri- gel (0.3 ml) containing B16.F10 cells in the presence or absence of Pam3CSK4 and/or BMMCs derived from C57BL/6 mice or TLR22/2 mice (n =10micewith C57BL/6 BMMCs, n =13micewith 2 2 TLR2 / BMMCs). Dashed line indicates http://www.jimmunol.org/ weight of Matrigel with BMMCs plus

Pam3CSK4 in the absence of tumor (0.092 6 0.008 g, n = 8). Representative Matri- gels are depicted. *p = 0.020. D, Mature blood vessels were quantiﬁed at the tumor periphery of H&E-stained sections. Data are pooled from two separate experiments (n = 7 mice). *p = 0.016. by guest on September 28, 2021

of Matrigels containing LLC1 plus BMMCs (0.236 6 0.028 g) did mice (Fig. 7E). Mast cell dependence in the antitumor effect of not differ from that of LLC1 alone (0.257 6 0.036 g) or LLC1 plus FSL-1 was confirmed via restoration of the antitumor effects of W-sh/W-sh Pam3CSK4 (0.241 6 0.019 g). In a manner comparable with the FSL-1 in Kit mice locally reconstituted with C57BL/6 B16.F10 melanoma, decreased LLC1 tumor growth in the presence BMMCs (day 15 tumor volume: 476 6 153 mm3 [PBS] versus 3 of Pam3CSK4-activated mast cells was also associated with mono- 55 6 27 mm [Pam3CSK4], n = 4). nuclear cell infiltration (Fig. 7C) and decreased angiogenesis (Fig. 7D). Discussion A TLR2/6 agonist can mediate mast cell-dependent tumor This study demonstrates a previously unrecognized mechanism for growth inhibition TLR2-based immunotherapy with a critical role for the mast cell. To assess the broader applicability of mast cell-dependent antitumor TLR2 expression on mast cells was crucial for mediating the in- responses to TLR-based immunotherapies, we evaluated the ability hibitory effects of Pam3CSK4 on melanoma growth, even though of mast cells to produce mediators in response to a panel of TLR multiple other effector cells, and the melanoma cells themselves, agonists. These studies demonstrate that at doses similar to that used are known to express TLR2. The KitW-sh/W-sh model provides a W-sh/W-sh for Pam3CSK4 in this study (10 mg/ml), the synthetic diacylated useful tool to study the role of mast cells as Kit mice are lipopeptide FSL-1 (a TLR2/6 agonist) and LPS (a TLR4 agonist) profoundly mast cell deficient in multiple anatomical sites, with induced mediator production at least 2-fold over media control, only rare mast cells detected in the skin of adult animals (30, 42). W-sh/W-sh similar to that seen with Pam3CSK4. The synthetic dsRNA analogue Restoration of the tumor-inhibitory effect of Pam3CSK4 in Kit polyinosinic-polycytidylic acid (a TLR3 agonist) and bacterial mice via local reconstitution of mast cells indicates that other po- peptidoglycan (activates TLR2/6 and other receptors) failed to in- tential differences between mast cell-deficient mice and controls duce mediator profiles from BMMCs similar to Pam3CSK4 upon (31) were not responsible for the lack of tumor growth inhibition W-sh/W-sh 24-h stimulation (Supplemental Table I). To investigate whether by Pam3CSK4 in Kit mice. TLR2-mediated activation additional TLR2 agonists could mediate mast cell-dependent anti- completely reversed the normal protumorigenic role of mast cells in tumor effects in vivo, we performed similar studies using the TLR2/ this model and instead harnessed them to initiate complex tumor- 6 agonist FSL-1. FSL-1 treatment significantly inhibited B16.F10 inhibitory effects that include recruitment of immune effector cells tumor growth in C57BL/6 mice but not in mast cell-deficient KitW-sh/W-sh and angiogenesis inhibition. 7072 ACTIVATED MAST CELLS INHIBIT TUMOR GROWTH Downloaded from

FIGURE 4. Pam3CSK4-activated mast cells decrease B16.F10 tumor growth in a TNF-independent manner. A, TNF secretion was measured by ELISA of cell-free supernatants. A23187 ionophore induction of TNF was included as a positive control at the 6-h time point (n = three experiments/group). *p ,

0.05; ***p , 0.001 (compared with media control). B, Annexin V–propidium iodide (PI) staining of B16.F10 cells cocultured with Pam3CSK4 and/or C57BL/6 BMMCs (n = 3 experiments/group). CPT was included as a positive control with DMSO vehicle control. ***p , 0.001. C, Mast cell-deﬁcient W-sh/W-sh Kit mice were locally reconstituted with TNF-deﬁcient BMMCs 2 wk prior to tumor injection then treated on days 4 and 9 (arrows) with Pam3CSK4 http://www.jimmunol.org/ (n = 12 mice from two independent experiments per group). *p = 0.012. D, B16.F10 proliferation was assessed in the presence of increasing doses of

Pam3CSK4 (pooled data from three independent experiments) and cell-free supernatants from C57BL/6 BMMCs (pooled data from 12 independent experiments) treated for 24 h with or without Pam3CSK4. CPT was included as a positive control. Data are expressed as fold change over media control for BMMC supernatants and fold change over DMSO vehicle control for CPT. ***p , 0.0001. ns, not signiﬁcant; UD, undetectable.

Although mast cells have been demonstrated to display antitumor 6 but not IL-62/2 mast cell supernatants demonstrates the tumor- activity via TNF-mediated cytotoxicity (17, 18), the direct effects of inhibitory effects of TLR2-activated mast cells are likely to be, in activated mast cells on tumor growth occurred independently of part, mediated via the direct effects of mast cell-derived IL-6. The mast cell-mediated TNF cytotoxicity and apoptosis induction. role of IL-6 in tumorigenesis is controversial and appears to be de- by guest on September 28, 2021 Pam3CSK4 did not exhibit direct antitumor effects in vitro. However, pendent on the stage of disease and tumor type (39). However, decreased tumor cell proliferation via Pam3CSK4-activated C57BL/ in vitro studies have demonstrated IL-6 is directly growth inhibitory

FIGURE 5. Pam3CSK4-activated mast cells recruit immune effector cells in a CCL3-dependent manner. A, IL-6, IL-13, CCL1, and CCL3 secretion was measured by ELISA of cell-free BMMC supernatants (n = 3 experiments). *p , 0.05; **p , 0.01. B, C57BL/6 mice were injected with Matrigel (0.15 ml) containing B16.F10 cells in the presence or absence or Pam3CSK4 and/or BMMCs derived from C57BL/6 mice (n = 6). After 4 d, Matrigels were removed and weighed and assessed by ﬂow cytometry. *p , 0.05; **p , 0.01 (compared with B16.F10 alone). C, C57BL/6 splenocyte chemotaxis was assessed in response to supernatants from C57BL/6 or CCL32/2 BMMCs (n = 3 independent experiments for C57BL/6 BMMC and n =2 independent experiments for CCL32/2 BMMCs). *p , 0.05. The Journal of Immunology 7073

FIGURE 6. Pam3CSK4-activated mast cells mediate antitumor effects via IL-6 production. A, B16.F10 tumors were grown for 19 d and treated s.c. on days 4, 9, and 14 post-tumor injection (arrows) with

Pam3CSK4 or diluent in parallel groups of mast cell- deficient KitW-sh/W-sh mice locally reconstituted with IL-6–deficient BMMCs 6 wk prior to tumor administration or of age-matched wild-type C57BL/6 mice. Data pooled from two independent experiments (n =8 to 12 mice per group). B, C57BL/6 mice were injected with Matrigel (0.3 ml) containing B16.F10 cells in the presence or absence or Pam3CSK4 and/or BMMCs derived from IL-62/2 mice (pooled data from two separate experiments, n = 10). Tumor blood volume was assessed in digested Matrigels (n = 5), and mature blood vessels were quantified at the tumor periphery of H&E-stained tissue sections (n = 5). C, B16.F10 proliferation was assessed in the presence of cell-free supernatants from IL-62/2 BMMCs (pooled data from five independent experiments) treated for 24 h with or Downloaded from without Pam3CSK4. CPT was included as a positive control. Data are expressed as fold change over media control for BMMC supernatants and fold change over DMSO vehicle control for CPT. *p = 0.030. http://www.jimmunol.org/ for several tumor cell types, including melanoma (38, 43–45). The angiogenesis assay (40). Other mast cell-derived mediators may melanoma growth-inhibitory effects of IL-6 have also been con- also suppress tumor angiogenesis. For example, Pam3CSK4-acti- firmed in vivo (40, 41). Studies in melanoma cell lines, including vated mast cells also secrete large amounts of IL-13 (Fig. 5A), B16.F10.9 cells, have demonstrated IL-6–induced tumor cell growth a potent antiangiogenic cytokine (54). inhibition is enhanced in the presence of soluble IL-6R and occurs The crucial role of mast cells for the mobilization of immune via a STAT-dependent mechanism that involves upregulation of effector cells in response to infection is well documented (7, 8). cyclin-dependent kinase inhibitors and induction of cell cycle arrest However, their potential to enhance the recruitment of effector cells (46–48). In addition to direct effects of IL-6 on tumor growth, IL-6 to tumor sites has not been previously explored. Decreased tumor by guest on September 28, 2021 may also function indirectly to inhibit tumor growth by modulating growth in the presence of Pam3CSK4-activated mast cells is asso- T cell effector function. In other models, elevated tumor IL-6 levels ciated with the early recruitment of significant numbers of both has been shown to enhance both CD4+ and CD8+ effector/regulatory NK and T cell subsets. We have demonstrated that TLR2-activated T cell (Treg) ratios and induce significant tumor reduction in vivo mast cells secrete large amounts of CCL3, which has been dem- (49). In the presence of large amounts of IL-6, activated mast cells onstrated to be chemotactic for mature NK cells (35) and activated may also modify the antitumor immune response by skewing CD8+ T cells (36, 37). Our data from in vitro chemotaxis experi- Tregs into Th17 cells (50), which have been shown to mediate ments using CCL32/2 BMMCs demonstrates a clear role for substantial antitumor effects against B16 in vivo (51). In addition to mast cell-derived CCL3 in the recruitment of NK and CD8+ and 2 possible mast cell-mediated effects on Treg function, Pam3CSK4 CD8 T cell subsets in this model. We have also identified multiple has been shown by others to directly act on Tregs to abrogate their other cytokines and chemokines produced from Pam3CSK4-acti- suppressive function and enhance effector responses in vivo (52). vated mast cells that may enhance effector cell recruitment and To assess whether Pam3CSK4-activated BMMCs might alter activation in vivo. For example, TLR2 ligand-activated BMMCs the recruitment of Tregs to tumor sites, we performed additional secrete substantial amounts of CCL1, which can induce migration 10-d Matrigel–tumor experiments. However, numbers of Tregs of NK cells via CCR8 in other systems (55). recruited to Matrigel-tumor sites were too low to allow accurate The tumor-inhibitory effects of Pam3CSK4 in vivo may also comparison (data not shown). involve activation of other TLR2-expressing immune cells. In- The antiangiogenic effect of Pam3CSK4-activated mast cells is deed, TLR2 engagement via Pam3CSK4 can enhance the pro- particularly noteworthy as the presence of mast cells correlates liferation, survival, and effector function of CD8+ T cells and with enhanced angiogenesis in melanomas (12). Further, experi- lower their threshold of activation (56). However, the lack of W-sh/W-sh mental studies using a transgenic model of skin cancer identified Pam3CSK4-mediated antitumor effects in Kit mice and that mast cells are crucial for the induction of angiogenesis in their restoration via wild-type, but not TLR2-deficient, mast cells premalignant lesions (14). Studies in mast cell-deficient KitW/KitWv demonstrates a critical role for mast cell TLR2 expression. Fur- mice, using the B16.BL6 melanoma model, demonstrated that thermore, the use of the Matrigel–tumor model system, containing delayed tumor growth kinetics in mast cell-deficient mice is at- only mast cells and tumor cells, demonstrates that although tributable to a delayed and decreased angiogenic response (13). Pam3CSK4 may later act on other TLR2-expressing immune cells, Mast cell IL-6–dependent angiogenesis suppression is consistent the mast cell is both necessary and sufficient for initiating the with previous findings that IL-6–mediated melanoma growth in- antitumor effects after TLR2 immunotherapy. This is in keeping hibition is independent of B and T cell responses (41) and that the with the role of the mast cell as a sentinel cell in initiating immune antitumor action of IL-6 is due in part to angiogenesis in- responses after pathogen recognition (9). hibition (53). Further, IL-6–transfected B16.F10 tumors exhibit Our studies of the LLC1 tumor model demonstrate that the a significant decrease in angiogenesis via an in vivo intradermal mast cell-mediated antitumor effects of TLR2 immunotherapy are 7074 ACTIVATED MAST CELLS INHIBIT TUMOR GROWTH

not unique to melanoma and that aspects of this response may be extended to additional tumor subtypes. Mast cells are found in high numbers in lung carcinoma and many other solid tumors, where their presence correlates with increased angiogenesis (57). The marked mononuclear cell inﬁltration and decreased angiogenic response in Pam3CSK4-treated LLC1 tumors indicate a similar, multifaceted, mast cell-dependent mechanism of tumor growth inhibition to that observed in the context of the melanoma model. The TLR2/6 agonist FSL-1 also induced mast cell-dependent antitumor effects on B16.F10 tumor growth. This demonstrates that mast cell-dependent tumor growth inhibition after TLR2- targeted immunotherapy is not restricted to TLR2/1 agonists and may have broader implications to other TLR-based immunotherapeutic strategies. The link between chronic inﬂammation and tumor growth and progression is well established (58). Mast cells normally play an integral role in promoting tumor growth for many solid tumors. Accordingly, it has been proposed that inhibition of mast cell

function may be an attractive therapeutic modality in the treatment Downloaded from of solid tumors (16, 59). However, others have demonstrated mast cell inﬁltration is an independent marker of improved prognosis (19–21). The distribution of mast cells within solid tumors may be a crucial factor in their ability to provide a protective role against the growth of solid tumors. Indeed, the study by Welsh et al.

(28) of non-small cell lung carcinoma and a more recent study by http://www.jimmunol.org/ Johansson et al. (29) of prostate cancer have demonstrated that intratumoral but not peritumoral mast cells independently predict improved survival. In contrast, in a large study of 4444 breast tumors, stromal mast cells independently predicted improved survival (21). In a small-scale analysis, we did not observe significant differences in mast cell distribution after Pam3CSK4 treatment. However, an accurate comparison of mast cell distribution in the presence and absence of TLR2 agonist treatment was confounded by guest on September 28, 2021 by the small size of Pam3CSK4-treated tumors. Overall, the results of the current study suggest TLR2-mediated activation of tumor-resident mast cells may represent a potential applicable immunotherapeutic strategy. The protumorigenic role of mast cells can be reversed by selective activation via TLR2 to ef- fectively turn mast cells into triggers for antitumor effector mechanisms. Insupport ofthese findings,it hasalready beendemonstrated that tumor incidence is decreased in the presence of mast cells in a TLR activator-rich intestinal microenvironment (60). Mast cells represent attractive targets for anticancer immunotherapy due to their abundance at the periphery of many solid tumors and ideal FIGURE 7. Mast cells decrease LLC1 tumor growth in a TLR2-de- location in close proximity to blood vessels. In contrast with other pendent manner and are required for B16.F10 tumor growth inhibition by immune cells, mast cells are relatively radioresistant (61), which a TLR2/6 agonist. A, C57BL/6 mice were injected with Matrigel (0.3 ml) makes them prime candidates for combined treatment modalities. containing LLC1 cells in the presence or absence or Pam3CSK4 and/or 2/2 Mast cell activation via TLR2 therefore provides a novel opportu- BMMCs derived from C57BL/6 mice or TLR2 mice (pooled data from nity to modify the tumor microenvironment as part of a regimen of three independent experiments, n = 9 C57BL/6 BMMC, n = 13 TLR22/2 successful cancer immunotherapy. BMMC). Dashed line indicates weight of Matrigel with BMMCs plus To date, TLR2-based immunotherapy has been applied clinically Pam3CSK4 in the absence of tumor (0.092 6 0.008 g, n = 8). **p = 0.008. for pancreatic cancer using the TLR2/6 agonist MALP-2 (3). B, Representative growth of LLC1 in Matrigels containing Pam3CSK4 and/ or C57BL/6 BMMCs. C, H&E-stained sections were examined for mo- Mast cell numbers are increased in pancreatic cancer and correlate nonuclear cell infiltrate (black arrows) among tumor cells (arrowheads). with increased microvessel density (62). Thus, the clinical success Original magnification 3400. D, Mature blood vessels were quantified at observed by Schmidt et al. (3) may, in part, be attributable to the tumor periphery of H&E-stained sections. Data are pooled from two action of mast cells, such as reversal of their proangiogenic prop- separate experiments (n = 7 mice). *p = 0.016. E, C57BL/6 mice or mast erties after TLR2-mediated activation or the recruitment of immune W-sh/W-sh cell-deficient Kit mice were injected s.c. with B16.F10 cells. effector cells. Future clinical trials examining TLR2-based immu- Tumors were treated s.c. on days 4 and 9 post-tumor injection (arrows) notherapy should examine the role of tumor-resident mast cells by with FSL-1 or PBS diluent control. Data pooled from four independent assessing the correlation between mast cell density, distribution, experiments (n = 26 to 28 for C57BL/6, n = 16 for KitW-sh/W-sh). ***p , 0.001. and activation status and patient outcome. The results of the current study suggest TLR2 targeted immunotherapy may prove particularly effective at mast cell dense tissue sites including the skin and lung. Furthermore, it may be important during TLR2-based The Journal of Immunology 7075 immunotherapy to ensure that other medications that interfere with 22. Burke, S. M., T. B. Issekutz, K. Mohan, P. W. Lee, M. Shmulevitz, and J. S. Marshall. 2008. Human mast cell activation with virus-associated stimuli mast cell function or survival are not being used concurrently. leads to the selective chemotaxis of natural killer cells by a CXCL8-dependent mechanism. Blood 111: 5467–5476. Acknowledgments 23. McCurdy, J. D., T. J. Olynych, L. H. Maher, and J. S. Marshall. 2003. Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of me- We thank Yisong Wei, Nong Xu, and Julie Scott for technical assistance and diator production from human mast cells. J. Immunol. 170: 1625–1629. Dr. Shizuo Akira (Osaka University, Osaka, Japan) for providing TLR2- 24. Wang, J., S. Saffold, X. Cao, J. Krauss, and W. Chen. 1998. Eliciting T cell deficient animals. immunity against poorly immunogenic tumors by immunization with dendritic cell-tumor fusion vaccines. J. Immunol. 161: 5516–5524. 25. Cable, J., I. J. Jackson, and K. P. Steel. 1995. Mutations at the W locus affect Disclosures survival of neural crest-derived melanocytes in the mouse. Mech. Dev. 50: 139– The authors have no financial conflicts of interest. 150. 26. Cable, J., D. Huszar, R. Jaenisch, and K. P. Steel. 1994. Effects of mutations at the W locus (c-kit) on inner ear pigmentation and function in the mouse. Pigment Cell Res. 7: 17–32. References 27. Tertian, G., Y. P. Yung, D. Guy-Grand, and M. A. Moore. 1981. Long-term 1. Schneider, C., T. Schmidt, C. Ziske, K. Tiemann, K. M. Lee, V. Uhlinsky, in vitro culture of murine mast cells. I. Description of a growth factor-depen- P. Behrens, T. Sauerbruch, I. G. Schmidt-Wolf, P. F. Mu¨hlradt, et al. 2004. Tu- dent culture technique. J. Immunol. 127: 788–794. mour suppression induced by the macrophage activating lipopeptide MALP-2 in 28. Welsh, T. J., R. H. Green, D. Richardson, D. A. Waller, K. J. O’Byrne, and an ultrasound guided pancreatic carcinoma mouse model. Gut 53: 355–361. P. Bradding. 2005. Macrophage and mast-cell invasion of tumor cell islets 2. Curtin, J. F., N. Liu, M. Candolfi, W. Xiong, H. Assi, K. Yagiz, M. R. Edwards, confers a marked survival advantage in non-small-cell lung cancer. J. Clin. K. S. Michelsen, K. M. Kroeger, C. Liu, et al. 2009. HMGB1 mediates en- Oncol. 23: 8959–8967. dogenous TLR2 activation and brain tumor regression. PLoS Med. 6: e10. 29. Johansson, A., S. Rudolfsson, P. Hammarsten, S. Halin, K. Pietras, J. Jones, 3. Schmidt, J., T. Welsch, D. Ja¨ger, P. F. Mu¨hlradt, M. W. Bu¨chler, and A. Ma¨rten. P. Stattin, L. Egevad, T. Granfors, P. Wikstro¨m, and A. Bergh. 2010. Mast cells 2007. Intratumoural injection of the toll-like receptor-2/6 agonist ‘macrophage- are novel independent prognostic markers in prostate cancer and represent Downloaded from activating lipopeptide-2’ in patients with pancreatic carcinoma: a phase I/II trial. a target for therapy. Am. J. Pathol. 177: 1031–1041. Br. J. Cancer 97: 598–604. 30. Grimbaldeston, M. A., C. C. Chen, A. M. Piliponsky, M. Tsai, S. Y. Tam, and 4. Welters, M. J., M. S. Bijker, S. J. van den Eeden, K. L. Franken, C. J. Melief, S. J. Galli. 2005. Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as R. Offringa, and S. H. van der Burg. 2007. Multiple CD4 and CD8 T-cell ac- a model for investigating mast cell biology in vivo. Am. J. Pathol. 167: 835–848. tivation parameters predict vaccine efficacy in vivo mediated by individual 31. Nigrovic, P. A., D. H. Gray, T. Jones, J. Hallgren, F. C. Kuo, B. Chaletzky, DC-activating agonists. Vaccine 25: 1379–1389. M. Gurish, D. Mathis, C. Benoist, and D. M. Lee. 2008. Genetic inversion in 5. Agrawal, S., A. Agrawal, B. Doughty, A. Gerwitz, J. Blenis, T. Van Dyke, and mast cell-deficient (W(sh)) mice interrupts corin and manifests as hematopoietic

B. Pulendran. 2003. Cutting edge: different Toll-like receptor agonists instruct and cardiac aberrancy. Am. J. Pathol. 173: 1693–1701. http://www.jimmunol.org/ dendritic cells to induce distinct Th responses via differential modulation of 32. Matsushima, H., N. Yamada, H. Matsue, and S. Shimada. 2004. TLR3-, TLR7-, extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos. and TLR9-mediated production of proinﬂammatory cytokines and chemokines J. Immunol. 171: 4984–4989. from murine connective tissue type skin-derived mast cells but not from bone 6. Redecke, V., H. Ha¨cker, S. K. Datta, A. Fermin, P. M. Pitha, D. H. Broide, and marrow-derived mast cells. J. Immunol. 173: 531–541. E. Raz. 2004. Cutting edge: activation of Toll-like receptor 2 induces a Th2 im- 33. Huang, B., J. Zhao, H. Li, K. L. He, Y. Chen, S. H. Chen, L. Mayer, mune response and promotes experimental asthma. J. Immunol. 172: 2739–2743. J. C. Unkeless, and H. Xiong. 2005. Toll-like receptors on tumor cells facilitate 7. Marshall, J. S. 2004. Mast-cell responses to pathogens. Nat. Rev. Immunol. 4: evasion of immune surveillance. Cancer Res. 65: 5009–5014. 787–799. 34. Noe¨l, A., M. C. De Pauw-Gillet, G. Purnell, B. Nusgens, C. M. Lapiere, and 8. Galli, S. J., M. Grimbaldeston, and M. Tsai. 2008. Immunomodulatory mast J. M. Foidart. 1993. Enhancement of tumorigenicity of human breast adeno- cells: negative, as well as positive, regulators of immunity. Nat. Rev. Immunol. 8: carcinoma cells in nude mice by Matrigel and ﬁbroblasts. Br. J. Cancer 68: 909– 478–486. 915.

9. Dawicki, W., and J. S. Marshall. 2007. New and emerging roles for mast cells in 35. Bernardini, G., G. Sciume`, D. Bosisio, S. Morrone, S. Sozzani, and A. Santoni. by guest on September 28, 2021 host defence. Curr. Opin. Immunol. 19: 31–38. 2008. CCL3 and CXCL12 regulate trafficking of mouse bone marrow NK cell 10. Takanami, I., K. Takeuchi, and M. Naruke. 2000. Mast cell density is associated subsets. Blood 111: 3626–3634. with angiogenesis and poor prognosis in pulmonary adenocarcinoma. Cancer 88: 36. Gough, M., M. Crittenden, U. Thanarajasingam, L. Sanchez-Perez, J. Thompson, 2686–2692. D. Jevremovic, and R. Vile. 2005. Gene therapy to manipulate effector T cell 11. Yano, H., M. Kinuta, H. Tateishi, Y. Nakano, S. Matsui, T. Monden, J. Okamura, trafficking to tumors for immunotherapy. J. Immunol. 174: 5766–5773. M. Sakai, and S. Okamoto. 1999. Mast cell infiltration around gastric cancer 37. Harlin, H., Y. Meng, A. C. Peterson, Y. Zha, M. Tretiakova, C. Slingluff, cells correlates with tumor angiogenesis and metastasis. Gastric Cancer 2: 26– M. McKee, and T. F. Gajewski. 2009. Chemokine expression in melanoma 32. metastases associated with CD8+ T-cell recruitment. Cancer Res. 69: 3077– 12. Ribatti, D., A. Vacca, R. Ria, A. Marzullo, B. Nico, R. Filotico, L. Roncali, and 3085. F. Dammacco. 2003. Neovascularisation, expression of fibroblast growth factor- 38. Fontaine, V., M. Mahieu, and J. Content. 1998. Interferon-gamma and 2, and mast cells with tryptase activity increase simultaneously with pathological interleukin-6 inhibit proliferation in human melanoma cells by different sig- progression in human malignant melanoma. Eur. J. Cancer 39: 666–674. nalling pathways. Melanoma Res. 8: 24–30. 13. Starkey, J. R., P. K. Crowle, and S. Taubenberger. 1988. Mast-cell-deficient W/Wv 39. Lu, C., and R. S. Kerbel. 1993. Interleukin-6 undergoes transition from paracrine mice exhibit a decreased rate of tumor angiogenesis. Int. J. Cancer 42: 48–52. growth inhibitor to autocrine stimulator during human melanoma progression. J. 14. Coussens, L. M., W. W. Raymond, G. Bergers, M. Laig-Webster, O. Behrendtsen, Cell Biol. 120: 1281–1288. Z. Werb, G. H. Caughey, and D. Hanahan. 1999. Inflammatory mast cells up- 40. Sun, W. H., R. A. Kreisle, A. W. Phillips, and W. B. Ershler. 1992. In vivo and regulate angiogenesis during squamous epithelial carcinogenesis. Genes Dev. 13: in vitro characteristics of interleukin 6-transfected B16 melanoma cells. Cancer 1382–1397. Res. 52: 5412–5415. 15. Soucek, L., E. R. Lawlor, D. Soto, K. Shchors, L. B. Swigart, and G. I. Evan. 41. Armstrong, C. A., N. Murray, M. Kennedy, S. V. Koppula, D. Tara, and 2007. Mast cells are required for angiogenesis and macroscopic expansion of J. C. Ansel. 1994. Melanoma-derived interleukin 6 inhibits in vivo melanoma Myc-induced pancreatic islet tumors. Nat. Med. 13: 1211–1218. growth. J. Invest. Dermatol. 102: 278–284. 16. Groot Kormelink, T., A. Abudukelimu, and F. A. Redegeld. 2009. Mast cells as 42. Zhou, J. S., W. Xing, D. S. Friend, K. F. Austen, and H. R. Katz. 2007. Mast cell target in cancer therapy. Curr. Pharm. Des. 15: 1868–1878. deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis. J. Exp. 17. Benyon, R. C., E. Y. Bissonnette, and A. D. Befus. 1991. Tumor necrosis factor- Med. 204: 2797–2802. alpha dependent cytotoxicity of human skin mast cells is enhanced by anti-IgE 43. Shen, W. H., J. H. Zhou, S. R. Broussard, G. G. Freund, R. Dantzer, and antibodies. J. Immunol. 147: 2253–2258. K. W. Kelley. 2002. Proinflammatory cytokines block growth of breast cancer 18. De´ry, R. E., T. J. Lin, A. D. Befus, C. D. Milne, R. Moqbel, G. Meńard, and cells by impairing signals from a growth factor receptor. Cancer Res. 62: 4746– E. Y. Bissonnette. 2000. Redundancy or cell-type-specific regulation? Tumour 4756. necrosis factor in alveolar macrophages and mast cells. Immunology 99: 427–434. 44. Lu, C., M. F. Vickers, and R. S. Kerbel. 1992. Interleukin 6: a fibroblast-derived 19. Fleischmann, A., T. Schlomm, J. Ko¨llermann, N. Sekulic, H. Huland, M. Mirlacher, growth inhibitor of human melanoma cells from early but not advanced stages of G. Sauter, R. Simon, and A. Erbersdobler. 2009. Immunological microenviron- tumor progression. Proc. Natl. Acad. Sci. USA 89: 9215–9219. ment in prostate cancer: high mast cell densities are associated with favorable tu- 45. Takizawa, H., T. Ohtoshi, K. Ohta, N. Yamashita, S. Hirohata, K. Hirai, mor characteristics and good prognosis. Prostate 69: 976–981. K. Hiramatsu, and K. Ito. 1993. Growth inhibition of human lung cancer cell 20. Hedstro¨m, G., M. Berglund, D. Molin, M. Fischer, G. Nilsson, U. Thunberg, lines by interleukin 6 in vitro: a possible role in tumor growth via an autocrine M. Book, C. Sundstro¨m, R. Rosenquist, G. Roos, et al. 2007. Mast cell in- mechanism. Cancer Res. 53: 4175–4181. filtration is a favourable prognostic factor in diffuse large B-cell lymphoma. Br. 46. Oh, J. W., A. Katz, S. Harroch, L. Eisenbach, M. Revel, and J. Chebath. 1997. J. Haematol. 138: 68–71. Unmasking by soluble IL-6 receptor of IL-6 effect on metastatic melanoma: 21. Rajput, A. B., D. A. Turbin, M. C. Cheang, D. K. Voduc, S. Leung, growth inhibition and differentiation of B16-F10.9 tumor cells. Oncogene 15: K. A. Gelmon, C. B. Gilks, and D. G. Huntsman. 2008. Stromal mast cells in 569–577. invasive breast cancer are a marker of favourable prognosis: a study of 4,444 47. Kortylewski, M., P. C. Heinrich, A. Mackiewicz, U. Schniertshauer, cases. Breast Cancer Res. Treat. 107: 249–257. U. Klingmu¨ller, K. Nakajima, T. Hirano, F. Horn, and I. Behrmann. 1999. 7076 ACTIVATED MAST CELLS INHIBIT TUMOR GROWTH

Interleukin-6 and oncostatin M-induced growth inhibition of human A375 54. Haas, C. S., M. A. Amin, J. H. Ruth, B. L. Allen, S. Ahmed, A. Pakozdi, melanoma cells is STAT-dependent and involves upregulation of the cyclin- J. M. Woods, S. Shahrara, and A. E. Koch. 2007. In vivo inhibition of angio- dependent kinase inhibitor p27/Kip1. Oncogene 18: 3742–3753. genesis by interleukin-13 gene therapy in a rat model of rheumatoid arthritis. 48. Bo¨hm, M., U. Schulte, J. O. Funk, M. Raghunath, I. Behrmann, M. Kortylewski, Arthritis Rheum. 56: 2535–2548. P. C. Heinrich, T. Kues, T. A. Luger, and T. Schwarz. 2001. Interleukin-6- 55. Inngjerdingen, M., B. Damaj, and A. A. Maghazachi. 2000. Human NK cells resistant melanoma cells exhibit reduced activation of STAT3 and lack of in- express CC chemokine receptors 4 and 8 and respond to thymus and activation- hibition of cyclin E-associated kinase activity. J. Invest. Dermatol. 117: 132– regulated chemokine, macrophage-derived chemokine, and I-309. J. Immunol. 140. 164: 4048–4054. 49. Hsieh, C. L., S. J. Liu, C. R. Shen, M. Y. Chen, S. C. Hsu, J. P. Tsai, Y. S. Li, 56. Mercier, B. C., A. Cottalorda, C. A. Coupet, J. Marvel, and N. Bonnefoy-Be´rard. C. J. Yang, P. Chong, and H. W. Chen. 2010. IL-6-transfected tumor cells 2009. TLR2 engagement on CD8 T cells enables generation of functional modulate the status of CD8(+) and CD4(+) T cells to control tumor growth. memory cells in response to a suboptimal TCR signal. J. Immunol. 182: 1860–1867. Immunobiology 215: 486–491. 57. Tomita, M., Y. Matsuzaki, and T. Onitsuka. 2000. Effect of mast cells on tumor 50. Piconese, S., G. Gri, C. Tripodo, S. Musio, A. Gorzanelli, B. Frossi, R. Pedotti, angiogenesis in lung cancer. Ann. Thorac. Surg. 69: 1686–1690. C. E. Pucillo, and M. P. Colombo. 2009. Mast cells counteract regulatory T-cell 58. Coussens, L. M., and Z. Werb. 2002. Inflammation and cancer. Nature 420: 860– suppression through interleukin-6 and OX40/OX40L axis toward Th17- 867. cell differentiation. Blood 114: 2639–2648. 59. Maltby, S., K. Khazaie, and K. M. McNagny. 2009. Mast cells in tumor growth: 51. Muranski, P., A. Boni, P. A. Antony, L. Cassard, K. R. Irvine, A. Kaiser, angiogenesis, tissue remodelling and immune-modulation. Biochim. Biophys. C. M. Paulos, D. C. Palmer, C. E. Touloukian, K. Ptak, et al. 2008. Tumor- Acta 1796: 19–26. specific Th17-polarized cells eradicate large established melanoma. Blood 112: 60. Sinnamon, M. J., K. J. Carter, L. P. Sims, B. Lafleur, B. Fingleton, and 362–373. L. M. Matrisian. 2008. A protective role of mast cells in intestinal tumorigenesis. 52. Sutmuller, R. P., M. H. den Brok, M. Kramer, E. J. Bennink, L. W. Toonen, Carcinogenesis 29: 880–886. B. J. Kullberg, L. A. Joosten, S. Akira, M. G. Netea, and G. J. Adema. 2006. 61. Soule, B. P., J. M. Brown, N. M. Kushnir-Sukhov, N. L. Simone, J. B. Mitchell, Toll-like receptor 2 controls expansion and function of regulatory T cells. J. Clin. and D. D. Metcalfe. 2007. Effects of gamma radiation on FcepsilonRI and TLR- Invest. 116: 485–494. mediated mast cell activation. J. Immunol. 179: 3276–3286. 53. Hatzi, E., C. Murphy, A. Zoephel, H. Rasmussen, L. Morbidelli, H. Ahorn, 62. Esposito, I., M. Menicagli, N. Funel, F. Bergmann, U. Boggi, F. Mosca, K. Kunisada, U. Tontsch, M. Klenk, K. Yamauchi-Takihara, et al. 2002. N-myc G. Bevilacqua, and D. Campani. 2004. Inflammatory cells contribute to the Downloaded from oncogene overexpression down-regulates IL-6; evidence that IL-6 inhibits angio- generation of an angiogenic phenotype in pancreatic ductal adenocarcinoma. J. genesis and suppresses neuroblastoma tumor growth. Oncogene 21: 3552–3561. Clin. Pathol. 57: 630–636. http://www.jimmunol.org/ by guest on September 28, 2021 50 * Media 40 ** Pam CSK ** 3 4 30 ** A23187

-hexosaminidase release 0 -/- -/- -/- β β β β C57Bl/6 IL-6 TLR2 TNF

% BMMC

Figure S1: Gene-deficient BMMC are functionally similar to wild-type C57BL/6 BMMC. All BMMC release significant levels of β-hexosaminidase in response to A23187 6 ionophore but not Pam3CSK4. BMMC (1 x 10 /mL) in modified HEPES-Tyrode’s buffer μ μ were treated with Pam3CSK4 (10 g/mL) or A23187 (0.5 M) for 20 minutes and the level of degranulation was assessed via β-hexosaminidase release. Data are pooled from three independent BMMC activations, performed in triplicate. 300 2

200

100 Mast cells/mm Mast 0 C M BMMC BMMC -/- -/- C57BL/6 L/6 BM -6 LR2 IL 57B T h + C + + /W-s h/W-sh -s W-sh W it W-sh/W-sh it K K Kit

Figure S2: Local reconstitution with BMMC restored skin mast cell numbers to levels comparable to wild-type C57BL/6 mice. 9-10 week old KitW-sh/W-sh mice were reconstituted on the dorsal surface over right and left flanks in 2 x 2 cm2 areas via a series of injections with BMMC (1 x 106 i.d. and 1 x 106 s.c.). Following 9 weeks skin samples were removed and fixed in Carnoy’s fixative. Mast cells were visualized via Alcian blue (pH 0.5) – safranin O staining. Mast cells were detected in all skin layers in all samples (N=8-12 skin samples per group). BMMC Negative B16.F10

TLR-1

TLR-2

mHPRT

Figure S3: TLR1 and TLR2 are expressed on B16.F10 melanoma cells and BMMC. Agarose gel electrophoresis of PCR products of TLR1 and TLR2 from RT-PCR of cDNA amplified from RNA isolated from B16.F10 melanoma and BMMC using RNeasy® Mini Kit and RNase-Free DNase Set (Qiagen) according to manufacturer’s protocol. Murine HPRT was used as a positive control. The negative control consisted of DNase treated BMMC RNA. Reverse transcription was performed using the SuperscriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen), according to the manufacturer’s instructions. PCR of cDNA was performed using GoTaq® Green DNA polymerase Master Mix (Promega) and primers synthesized by Sigma Genosys: mHPRT: forward 5’-CACAGGACTAGAACACCTGC-3’, reverse 5’- GCTGGTGAAAAGGACCTCT-3’; TLR1: forward 5’-GTTGTCACTGATGTCTTCAGC- 3’, reverse 5’-GCTGTACCTTAGAGAATTCTG-3’; TLR2: forward 5’- GCCTTGACCTGTCTTTCAAC-3’, reverse 5’-GGACTGATAATTCCGGAGAC-3’. Sequence specificity was confirmed by sequencing RT-PCR products (data not shown). AB B16.F10+BMMC B16.F10+BMMC+Pam3CSK4

1 5 7 8 1 5 7 8

2 46 9 2 46 9

3 3

CD B16.F10 B16.F10+Pam3CSK4

7 7

6 6

Figure S4: Pam3CSK4 activated BMMC secrete multiple mediators. Protein array analysis of supernatants of mast cells (BMMC) (1x106/mL) co-cultured for 24 hrs at 37oC with B16.F10 melanoma cells (0.5 x 106/mL) in the absence (A) or presence (B) μ of Pam3CSK4 (10 g/mL). Pam3CSK4 treatment induced secretion, at least 2-fold excess over media control, of (1) IL-6, (2) CCL2 (MCP-1), (3) CCL1 (TCA-3), (4) CCL3 (MIP-1α), (5) IL-13, (6) CXCL1/2 (MIP-2), (7) CXCL3 (KC), (8) CXCL5 (LIX) and (9) CCL5 (RANTES), as determined by spot density analysis. C-D) Protein array analysis of supernatants of B16.F10 melanoma cells (0.5 x 106/mL) cultured for 24 hrs at 37oC μ in the absence (C) or presence (D) of Pam3CSK4 (10 g/mL). Pam3CSK4 treatment induced secretion, at least 2-fold excess over media control, of (6) CXCL1/2 (MIP-2) and (7) CXCL3 (KC), as determined by spot density analysis. A PBS Pam3CSK4 + * β β β β * 12 * 1.0 0.4 + + 10 0.3

8 TCR- 6 + 0.5 0.2 4 % CD45 % NK1.1 0.1 2

0 % CD8 0.0 0.0

B16 B16+BMMC+Pam3CSK4

Figure S5. Pam3CSK4 activated mast cells recruit immune effector cells to B16.F10 tumors. A) Male C57BL/6 mice were injected s.c. with B16.F10 melanoma cells.

Tumors were treated s.c. on days 4 and 9 post tumor injection with Pam3CSK4 or PBS control (n=7 tumors per group). After 14 days growth, tumors were removed, collagenase digested and assessed by flow cytometry. Established tumors treated + !+ with Pam3CSK4 contain elevated leukocytes (*p=0.027), including CD8 TCR T cells (*p=0.019), and NK1.1+ natural killer cells (*p=0.013), compared to controls. B) Hematoxylin and eosin stained formalin-fixed tissue sections reveal a marked mononuclear cell infiltrate (dark arrows) amongst tumor cells (open arrows) in B16.F10 6 tumors (0.5 x 10 /mL) grown for 10 days in Matrigel (0.3 mL) containing Pam3CSK4 (10 μg/mL) and BMMC (1 x 106/mL) as compared to tumors alone. Supplementary Table 1: Cytokine and chemokine production by C57BL/6 BMMC cultured with a panel of TLR agonists

IL-6 (pg/mL) IL-13 (pg/mL) CCL1 (pg/mL) CCL3 (pg/mL)

Media 49.18 ± 43.99 237.7 ± 155.1 41.27 ± 27.93 29.69 ± 10.68

FSL-1 515.7 ± 125.8 1784 ± 438.0 424.1 ± 130.3 117.4 ± 35.43

PGN 74.02 ± 31.37 355.0 ± 138.7 421.3 ± 110.6 75.70 ± 37.29

LPS 2087 ± 699.5 11557 ± 4148 3885 ± 2557 1857 ± 1442

Poly(I:C) 60.23 ± 36.78 241.6 ± 168.7 35.24 ± 28.31 30.51 ± 10.08

C57BL/6 BMMC (1 x 106/mL) were cultured for 24 hours in the presence of Media or 10 μg/mL of TLR agonist (TLR2/6 - FSL-1 and PGN; TLR4 – LPS; TLR3 – Poly(I:C)). Data are presented as mean ± SEM of 3 independent experiments). Bold numbers indicate values ≥2-fold over Media control.