BRAFInhibitionStimulatesMelanoma-Associated Macrophages …Cancer Therapy: Preclinical...

Cancer Therapy: Preclinical

BRAF Inhibition Stimulates Melanoma-AssociatedMacrophages to Drive Tumor GrowthTao Wang1, Min Xiao1, Yingbin Ge2, Clemens Krepler1, Eric Belser1, Alfonso Lopez-Coral3,Xiaowei Xu4, Gao Zhang1, Rikka Azuma5, Qin Liu1, Rui Liu5, Ling Li1, Ravi K. Amaravadi6,7,Wei Xu7, Giorgos Karakousis7,8, Tara C. Gangadhar6,7, Lynn M. Schuchter6,7, Melissa Lieu9,Sanika Khare10, Molly B. Halloran1, Meenhard Herlyn1, and Russel E. Kaufman1

Abstract

Purpose: To investigate the roles of melanoma-associatedmacrophages in melanoma resistance to BRAF inhibitors(BRAFi).

Experimental Design: An in vitro macrophage and mela-noma cell coculture system was used to investigate whethermacrophages play a role in melanoma resistance to BRAFi.The effects of macrophages in tumor resistance were examin-ed by proliferation assay, cell death assay, and Westernblot analyses. Furthermore, two mouse preclinical modelswere used to validate whether targeting macrophages canincrease the antitumor activity of BRAFi. Finally, the numberof macrophages in melanoma tissues was examined byimmunohistochemistry.

Results: We demonstrate that in BRAF-mutant melanomas,BRAFi paradoxically activate themitogen-activated protein kinase(MAPK) pathway in macrophages to produce VEGF, which reac-tivates the MAPK pathway and stimulates cell growth in mela-noma cells. Blocking the MAPK pathway or VEGF signaling thenreversesmacrophage-mediated resistance. Targetingmacrophagesincreases the antitumor activity of BRAFi in mouse and humantumor models. The presence of macrophages in melanomaspredicts early relapse after therapy.

Conclusions: Our findings demonstrate that macrophagesplay a critical role in melanoma resistance to BRAFi, suggestingthat targeting macrophages will benefit patients with BRAF-mutant melanoma. Clin Cancer Res; 21(7); 1652–64. �2015 AACR.

IntroductionBRAFV600E/K mutations are present in around 40% to 50%

melanomas. Targeted therapy with small-molecule BRAF inhibi-tors (BRAFi), such as vemurafenib or dabrafenib, has improvedoverall survival in patients with advanced BRAF-mutant melano-mas (1–4). However, most patients relapse within several

months. Acquired resistance has been attributed to both geneticand/or epigenetic changes in tumor cells after treatment withBRAFi. Analyses of melanomas that have acquired resistance toBRAFi frequently have demonstrated reactivation of the mitogen-activated protein kinase (MAPK) pathway via new mutations,such as BRAF amplification and emerging splice variants (5),NRAS mutation (6), MEK1 mutation (7), or through activationof alternative survival pathways involving MAPK and phospha-tidylinositol 3-kinase/protein kinase B (PI3K/AKT; refs. 8, 9),which are essential for cell growth and survival. Of note, somemelanomas that carry an activatingBRAFmutation are resistant toBRAFi, possibly due to genetic and epigenetic heterogeneity ofcancer cells. Overall, approximately 50% of patients with mela-noma do not have significant responses to BRAFi (1, 4). Themechanisms underlying this intrinsic resistance of cancer cells toBRAFi remain poorly understood.

Melanomas that do not have an activating BRAF mutation aretypically unresponsive to BRAFi. It is of particular interest thatpatients treated with BRAFi often develop secondary cutaneousnonmelanoma tumors, suspected to be due to BRAFi induction ofsignaling pathways in precancerous skin cells. Although small-molecule inhibitors (SMI) may inhibit the desired targets intumor cells, they may also paradoxically activate the same path-ways in malignant and nonmalignant cells. For example, someAKT or mTOR inhibitors can activate the PI3K/AKT pathway intumor cells; this paradoxical activation blunts their antitumorefficacy and contributes to tumor cell resistance to AKT/mTORinhibitors (10–12). In melanoma, BRAFi activate the MAPKpathway in BRAF wild-type and NRAS-mutant tumor cells via aRAS-dependent, CRAF activation mechanism (13–15). Also,

1Molecular and Cellular Oncogenesis Program, The Wistar Institute,Philadelphia, Pennsylvania. 2Department of Physiology, Nanjing Med-ical University, Nanjing, China. 3Graduate Program, The Catholic Uni-versity of America,Washington, District of Columbia. 4Department ofPathology and Laboratory Medicine, University of Pennsylvania, Phi-ladelphia, Pennsylvania. 5UndergraduateProgram,Universityof Penn-sylvania, Philadelphia, Pennsylvania. 6Department of Medicine, Uni-versity of Pennsylvania, Philadelphia, Pennsylvania. 7Abramson Can-cer Center, University of Pennsylvania, Philadelphia, Pennsylvania.8Department of Surgery, University of Pennsylvania, Philadelphia,Pennsylvania. 9Undergraduate Program, University of the Sciences,Philadelphia, Pennsylvania. 10Biotechnology School of Engineeringand Applied Sciences, University of Pennsylvania, Philadelphia,Pennsylvania.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address for T.Wang: Center for DrugEvaluation andResearch, Food andDrug Administration, Silver Spring, Maryland.

Corresponding Authors: TaoWang, Food and Drug Administration, 10903 NewHampshire Avenue, Building 72/Room 2329, Silver Spring, MD 20903. Phone:240-402-9392; Fax: 215-898-0980; E-mail: [email protected]; MeenhardHerlyn, [email protected]; and Russel E. Kaufman, [email protected]

doi: 10.1158/1078-0432.CCR-14-1554

�2015 American Association for Cancer Research.

ClinicalCancerResearch

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increased numbers of phospho-ERK (pERK)–positive cells in thekeratinocyte compartment of skin are observed in BRAFi-treatedmice. Accordingly, paradoxical activation of the MAPK pathwayby BRAFi results in squamous-cell carcinomas in some patientstreated with BRAFi (16). To date, there has been no systematicanalysis of signaling pathways in normal cell types that areactivated by BRAFi (13). The biologic consequences andmechan-isms of this paradoxical activation of signaling pathways by SMIsand their contribution to cell growth and survival, aswell as tumorcell resistance to targeted therapy, are not well defined, especiallyin nonmalignant cells.

There is evidence that the tumormicroenvironment contributesto tumor cell resistance to anticancer therapy. Although somestudies suggested that themacrophage, amajor component of thetumor microenvironment, contributes to tumor cell resistance toanticancer therapies, including chemotherapy, radiotherapy, andimmune therapy (17, 18), other studies suggest thatmacrophagesincrease the antitumor activity of anticancer therapies (19, 20).However, most studies have not addressed the direct effects ofmacrophages on tumor cell growth in the presence of anticancertherapies, especially targeted therapy with SMIs.

Macrophages are the most abundant inflammatory cells inmelanomas (21), and the number of infiltrating macrophages,as well as the levels of macrophage-produced factors, inverselycorrelates with patients' outcome in both early and late stages ofmelanoma (22–24). Melanoma-associated macrophages pro-duce a plethora of growth factors, cytokines, chemokines, extra-cellular matrix, and proteinases, which play critical roles inmelanoma initiation, angiogenesis, growth, metastasis, andimmune suppression (25–29). However, the role ofmacrophagesin melanoma resistance to BRAFi remains poorly defined.

Therefore, we examined the roles of macrophages in melano-mas with resistance to BRAFi, and identified a uniquemechanismfor resistance by using a human macrophage and melanoma cellcoculture system. We further validated our in vitro findings inmouse melanoma models and patients' tumor samples.

Materials and MethodsCell culture

1205Lu and 451Lumelanoma cell lines were developed by ourlaboratory. A375 and SK-MEL-28 were from the ATCC. The

detailed information of cell lines can be found at: http://www.wistar.org/lab/meenhard-herlyn-dvm-dsc. Melanoma cells werecultured in melanoma medium supplemented with 2% fetalbovine serum(FBS) as described previously (28). Formacrophageand melanoma coculture experiments, melanoma cells werecocultured with respective macrophages that were differentiatedfrom monocytes using melanoma-conditioned media derivedfrom the above four melanoma cell lines as described previously(28). For Figs. 4 and 5 and Supplementary Fig S8, macrophageswere differentiated from monocytes using 1205Lu melanoma-conditioned media.

ReagentsPLX4720 and lenvatinib were from Selleck. Dabrafenib was

from ChemieTek. GW2580 and gefitinib were from LC Labora-tories. PD173074 was from Abcam. Recombinant human VEGF,anti-human VEGF blocking mAb, phospho-VEGFR1 (Y1213),phycoerythrin-conjugated anti-human VEGFR1, VEGFR2,VEGR3, and neuropilin-1 were from R&D Systems. pERK, pAKT,pNF-kB, pCRAF, pARF, pSTAT3, pVEGFR1, pp38, total ERK,CRAF, AKT, STAT3, PDGFRb, Rab11, HSP90, and Vinculin werefrom Cell Signaling Technology. Corning Transwells (pore size,0.4 mm) were from Fisher Scientific for coculture experiments.

Proliferation assayThe macrophage and melanoma coculture system was set up

as described in Fig. 1A. For coculturing melanoma cells withmacrophages, 2 � 104 melanoma cells were seeded in 24-wellplates and incubated for 18 hours. Macrophages (4 � 104) werethen added to the collagen I (1.1 mg/mL)-precoated Transwell.Indicated concentrations of various inhibitors, growth factors,and antibodies were then added to the coculture system andincubated for 3 days.

For macrophage proliferation, after monocytes had differenti-ated into macrophages, cells in 2% FBS melanoma media wereseeded into96-well plates and incubated for 3days in thepresenceof the indicated concentrations of inhibitors and blocking anti-bodies. Cell proliferation was assayed using the WST-1 prolifer-ation kit according to themanufacturer's instructions (Roche). Allexperiments were performed at least in triplicate.

ImmunoblottingMelanoma cells were cultured as described for the proliferation

assay for 6 and 18 hours and harvested for immunoblotting.Formacrophages, aftermonocytes had differentiated tomacro-

phages, cells in 2%FBSmelanomamedia were seeded into 15-mLtubes. Macrophages were incubated for the indicated times inthe presence of the indicated concentrations of BRAFi. Immuno-blotting was performed as described previously (28).

siRNA transfectionThe MEK1 and MEK2 siRNA and negative control siRNA were

from Thermo Scientific. After 24-hour transfection, 1205Lu andA375 cells were harvested and transferred to 6-well plates andincubated for another 24 hours. Cells were then cocultured withmacrophages and treated with the indicated concentration ofPLX4720 for 18 and 72 hours, respectively. Cells were harvestedfor immunoblotting with the indicated antibodies (18-hourcoculture) and flow cytometric analysis (3-day coculture). Pro-liferation assay was performed with WST-1 assay.

Translational Relevance

Targeted cancer therapy is intended to affect specific path-ways in cancer cells. Our results demonstrate that targetedtherapies, such as the BRAF inhibitor (BRAFi) vemurafenibused for BRAF-mutant melanomas, have potent effects onmacrophages in the tumor, which contribute to tumor resis-tance against the targeted therapy. Tumor macrophagesexposed to BRAFi paradoxically activate themitogen-activatedprotein kinase (MAPK) pathway that is intended to be sup-pressed and then produce a potent growth-promoting factor,VEGF, which stimulates tumor cell growth. The activation ofnontumor cells, such as macrophages, in the tumor microen-vironment by targeted therapies represents a novelmechanismfor drug resistance that must be considered in developingtargeted cancer therapies.

Macrophages Confer BRAF Inhibitor Resistance

www.aacrjournals.org Clin Cancer Res; 21(7) April 1, 2015 1653




RAS activationFor basal-level RAS activity, after macrophage differentiation,

cells in 2% FBS melanoma media were incubated for an addi-

tional 0.5 hours. Cells were harvested for ELISA measurementaccording to the manufacturer's instructions (cat# 17-497 fromMillipore).

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Figure 1.Macrophages are essential for melanoma cell growth under BRAF inhibition. A, the macrophage (Mph) and melanoma cell coculture system. Melanoma cellsare seeded on the bottom of cell culture plates. To mimic the tumor microenvironment, a layer of collagen I (Col.I) is coated on the Transwell (pore size: 0.4 mm).Macrophages are then seeded on the collagen I layer. B, 1205Lu and A375 melanoma cells were cocultured in the presence or absence of macrophages withthe indicated concentration of PLX4720 for 3 days. Cell growth was determined using the WST-1 assay. Relative growth was calculated as the ratio oftreated cells to untreated cells (without Mph coculture) at each dose. Data shown are mean � SD (n ¼ 4). �� , P < 0.001. C, cells were treated same as in B. Celldeath was determined by flow cytometry using Annexin V and 7-AAD staining. Representative images from one of six independent experiments are shown(left). Relative percentage of cell death represents the ratio between dead cells with macrophages versus without macrophages in the presence of PLX4720(right). �� , P < 0.001. D, cells were treated same as in B. Cells were stained with propidium iodide, and were analyzed for cell-cycle progression by flowcytometry. The percentage of cells in the sub-G1 and G0/G1 populations is shown. Representative images from one of four (1205Lu) and three (A375)independent experiments are shown (left). Relative percentage of cell death represents the ratio between dead cells with macrophages versus withoutmacrophages in the presence of PLX4720 (right). � , P < 0.05, �� , P < 0.01. See also Supplementary Fig. S1.

Wang et al.

Clin Cancer Res; 21(7) April 1, 2015 Clinical Cancer Research1654




Luminex assayThe production of growth factors and cytokines from macro-

phage media was measured by Luminex assay according to themanufacturer's instructions (Bio-Rad; ref. 28).

ImmunohistochemistryFor CD163 and Ki67 staining, double stains were performed

sequentially on a Leica Bond instrument using the Bond Poly-mer Refine Detection System (for Ki67) and the Bond PolymerRefine Red Detection System (for CD163). Heat-induced epi-tope retrieval was done for 20 minutes with ER1 solution (LeicaMicrosystems). The tissues were first stained with an anti-Ki67antibody (1:20 dilution; DakoM7240). Tissues were thenstained with an anti-CD163 Ab (1:50; Leica NCL). For quan-tification of CD163-positive cells, CD163-positive cells werecounted in 10 randomly selected fields (�600 magnifications)for each tumor sample. Two independent investigators evalu-ated the sections.

For mouse tissues, formalin-fixed, paraffin-embedded mousemelanoma tumor tissues were deparaffinized and subjected toantigen retrieval as described previously (28). The tissues werethen incubated with the following antibodies: anti-Ki67 (NovusBiologicals), anti-F4/80 (Abcam), CD11b, CD31, and pERK (Epi-tomics). After incubation with the primary antibody overnight at4�C, horseradish peroxidase–conjugated donkey anti-mouse,donkey anti-rabbit, or donkey anti-rat IgG (1:200; Jackson Immu-noResearch) was used. Slides were subsequently incubated for5 minutes in 3,30-diaminobenzidine (Invitrogen) and counter-stained with Haemalaun. For quantification of Ki67-positivecells, cells were counted in six randomly selected fields (�400magnifications) for each tumor sample (n ¼ 4 for each group).Two independent investigators evaluated sections.

ELISA analysisMacrophages were stimulated with PLX4720 (3 mmol/L) for

3 days. Media were harvested, and VEGF production was deter-mined by ELISA according to the manufacturer's instructions(R&D Systems).

Flow cytometric analysisFor the cell death assay, treated melanoma tissues or macro-

phages were stained with R-phycoerythrin-conjugated Annexin Vand 7-amino-actinomycin D (7-AAD) according to the manu-facturer's protocol (BD Biosciences). Cell death was quantifiedusing a Becton Dickinson FACScan cytometer.

For cell-cycle analysis, cells were fixed in 75% ethanol at�20�Covernight andwashedwith cold PBS, treatedwith 100-mg RNase A(Sigma), and stained with 50-mg propidium iodide (Roche).

Measurement of VEGF production was determined by intra-cellular staining according to the manufacturer's protocol (BDBiosciences). After monocytes had differentiated into macro-phages, cells in 2% FBS melanoma media were incubated for4 hours in the presence of the indicated concentration ofPLX4720 and GolgiPlug. After cells were washed with FACSbuffer, intracellular staining was performed with an R-phyco-erythrin-conjugated anti-VEGF mAb according to the manu-facturer's instructions (R&D Systems).

For FACS analysis of peritonealmacrophages, 10mLof cold PBSwas intraperitoneally injected into the mice after they were eutha-nized. Cells were harvested and the numbers of macrophages were

counted with a hemocytometer. An anti-mouse F4/80 (BD Bios-ciences) was used to analyze the percentage of macrophages. AllFACS data were analyzed with the FlowJo software (TreeStar).

Animal studiesAll studies were conducted under an approved IACUC proto-

col. For human xenografts, 7-week-old BALB/c female nude mice(National Cancer Institute, Bethesda, MD) were injected subcuta-neously with 1 � 106 1205Lu cells in 50% Matrigel (BD Bios-ciences) in both flanks. For mouse tumor growth, 7-week-oldC57BL/6 female mice (National Cancer Institute) were injectedsubcutaneously with 1�105 murine Yumm1.7 cells (BRAFV600E

mutant, PTEN null; gift of Dr. Marcus Bosenberg, Yale MedicalSchool, New Haven, CT). When tumors reached volumes ofapproximately 100 mm3 (1205Lu) and 180 mm3 (Yumm1.7),mice were randomly divided into four groups, with 5 animals pergroup. GW2580 was dissolved in 0.5% hydroxypropylmethylcel-lulose (Sigma-Aldrich) and0.1%Tween80, andwasdosedorally at160 mg/kg once daily. PLX4720 was dissolved in 5% DMSO,1% methylcellulose in distilled water, and animals were dosedorally at 25 mg/kg twice per day. Tumor volumes were measuredevery 3 days using a digital caliber and were calculated using theequationV¼ 0.5� L�W2.Mouse tumorswereweighed aftermicewere euthanized.

Patient SamplesFormalin-fixed, paraffin-embedded human melanoma tumor

slides (Supplementary Table S1) were from The University ofPennsylvania (Philadelphia, PA) under an approved InstitutionalReview Board protocol.

Statistical analysisPaired two-tailed t tests were performed to compare the differ-

ences in cell growth measurements between two experimentalconditions of specific cell line samples. Two-way ANOVA wasused to determine the effect of treatment groups with multipleconcentrations of inhibitors.

ResultsMacrophages confer melanoma resistance to BRAF inhibition

Macrophages can play critical roles in tumor cell resistance toanticancer therapies. We investigated whether macrophages con-fer melanoma resistance to BRAFi. We developed a model systemthat resembles the tumor microenvironment. In a Transwellcoculture system(30),we coculturedmelanoma cellswithhumanmacrophages that were differentiated frommonocytes withmod-ified melanoma conditioned media. Under those experimentalconditions, cells resemble human tumor-associated macro-phages, both phenotypically and functionally (28, 31). We thenexposed the cocultured cells to BRAFi (Fig. 1A).MutantBRAFV600E

melanoma cell lines, including 1205Lu, A375, SK-MEL-28, and451Lu cells, when cultured alone, were sensitive to PLX4720, ananalog of vemurafenib. However, whenmacrophages were addedto the cocultures, the cells of all four lines became resistant toPLX4720 (Fig. 1B and Supplementary Fig. S1A and S1B) asdemonstrated by increased proliferation with the WST-1 prolif-eration assay. Macrophages also promoted cell growth in thepresence of PLX4720 and when maintained in direct cell–cellcontact with tumor cells (Supplementary Fig. S1C). These dataindicate that growth factors produced by macrophages stimulate






macrophage-mediated drug resistance. Cell death assays byAnnexin V and 7-AAD staining indicate that macrophages protectmelanoma cells from PLX4720-induced cell death, includingapoptosis (Annexin V–positive and 7-AAD-negative) andnecrosis(Annexin V–positive and 7-AAD-positive; Fig. 1C and Supple-mentary Fig. S1D). Furthermore, flow cytometry cell-cycle anal-yses using propidium iodide staining confirmed that the percent-age of the sub-G1 population (apoptotic and necrotic cells) in1205Lu and A375 melanoma cells was significantly lower in thepresence than absence of macrophages (Fig. 1D). Of note, macro-phages do not have any effect on melanoma cell G2–M phase,suggesting that the highdosesweused (10mmol/L for 1205Lu and2 mmol/L for A375) may mainly protect tumor cells from BRAFi-induced cell death, but not promote tumor cell growth. Similarresults were obtained when another FDA-approved BRAFi, dab-rafenib, was used (Supplementary Fig. S1E and S1F). Our data

demonstrate that macrophages reduce the effects of BRAFi onmelanoma cells.

Macrophages activate the MAPK pathway in melanoma cellswhen exposed to BRAFi

Reactivation of theMAPKpathway and activation of alternativesurvival signaling pathways, such as the AKT pathway, are dem-onstrated mechanisms for melanoma cell resistance to BRAFi(9, 32–34). Because there is an abundance of macrophages inmelanomas (35), we examined whether macrophages contributeto activation of these signaling pathways in the presence of BRAFi.Addition of macrophages to the coculture system resulted in astrong increase in ERKphosphorylation in both 1205Lu andA375melanoma cell lines after 6-hour treatment with PLX4720 (Sup-plementary Fig. S2A). Reactivation of the MAPK pathway wasmaintained after 18 hours of coculture (Fig. 2A), but changes

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Figure 2.Macrophages confer melanomaresistance to BRAFi via activation ofthe MAPK pathway. A, macrophagesactivate the MAPK pathway inmelanoma cells when PLX4720 ispresent. 1205Lu and A375 melanomacells were cocultured with or withoutmacrophages in the presence orabsence of the indicated concentrationof PLX4720 for 18 hours, andmelanoma cells were harvested forimmunoblotting with the indicatedantibodies. HSP90 was used as aloading control. B, 1205Lu and A375melanoma cells were transfected withMEK1/2 siRNA and negative controlsiRNA and incubated for 48 hours.Melanoma cells were then coculturedwith or without macrophages in thepresence or absence of the indicatedconcentration of PLX4720 (3 mmol/Lfor 1205Lu, 2 mmol/L for A375) for3 days. Cell growth was determinedby the WST-1 assay as in (Fig. 1B).Data shown are mean � SD (n ¼ 4).�� , P < 0.001. C, cells were treatedsame as in B. Cell death wasdetermined as in Fig. 1C.Representative images from one offour independent experiments areshown (left). Relative percentage ofcell death represents the ratio betweendead cells with PLX4720 plus MEK1/2siRNA treatment versus PLX4720withoutMEK1/2 siRNA treatment in thepresence or absence of macrophages(right). � , P < 0.05; �� , P < 0.001. D,1205Lu and A375 melanoma cells weretransfected with MEK1/2 and controlsiRNA for 48 hours. Melanoma cellswere then cocultured with or withoutmacrophages in the presence orabsence of PLX4720 (2 mmol/L for1205Lu, 1 mmol/L for A375) for 18 hours.Cells were harvested forimmunoblotting with the indicatedantibodies. �� , P < 0.01. See alsoSupplementary Fig. S2.

Wang et al.





were not seen in other important melanoma survival signalingcomponents, such as AKT, NF-kB, CRAF, and ARAF (Supplemen-tary Fig. S2A and S2B). Activation of STAT3 signaling and upre-gulation of PDGFRb expression can confer melanoma resistanceto BRAFi (6, 36); however, we observed that macrophages neitheractivated STAT3 signaling nor upregulated PDGFRb expression inthe presence of PLX4720 (Supplementary Fig. S2C). Becauseactivation of the MAPK pathway by macrophages occurs as earlyas 6 hours, it is unlikely that resistance is due to new geneticchanges in the melanoma cells.

To determine the mechanism of macrophage-mediated mel-anoma growth promotion in the presence of BRAFi, we blockedthe MAPK pathway at the level of ERK signaling with combinedMEK1 and MEK2 siRNA. We found that MEK1/MEK2 knock-down significantly, but not completely, decreased macrophage-mediated cell growth and death-protecting effects in both1205Lu and A375 cells in the presence of PLX4720 (Fig. 2Band C). Accordingly, MEK1/MEK2 knockdown diminishedmacrophage-mediated increase in ERK activation of melanomacells (Fig. 2D). Collectively, these data demonstrate that macro-phages confer resistance to BRAFi in melanoma cells at leastpartially via reactivation of the MAPK pathway.

VEGF confers macrophage-mediated resistance to BRAFiBecause we were using the Transwell coculture system, which

allows macrophage-derived factors to stimulate melanoma cellgrowth without direct cell–cell contact, we sought to identifythe mechanisms by which soluble factors confer macrophage-mediated resistance. Many factors can rescue BRAFi-inducedcell growth inhibition, including epidermal growth factor (EGF),fibroblast growth factor 2 (FGF2), and hepatocyte growth fac-tor (HGF; refs. 37–40). Of note, fibroblast-derived HGF con-fers melanoma resistance to BRAFi through activation of bothMAPK and PI3K pathways (39). We used specific inhibitors ofthese pathways—the EGF pathway by gefitinib, an inhibitoragainst EGF receptor; the HGF pathway by AMG208, an inhib-itor against HGF receptor, c-MET; and the FGF2 pathway byPD173074, an inhibitor against FGF receptor 1—and demon-strated that they did not reverse the macrophage-mediatedgrowth-promoting effects in 1205Lu cells (SupplementaryFig. S3A), indicating that other growth factors play a role inmacrophage-mediated resistance.

To identify any growth factor involved in activation of theMAPK pathway induced by macrophages, we conducted a Lumi-nex assay to analyze production of multiple growth factorsproduced by macrophages, including M-CSF, CXCL1, IL6, TNFa,platelet-derived growth factor (PDGF), and VEGF (Supplemen-tary Fig. S3B), which activate the MAPK pathway in cells thatcontain receptors to these factors. We found that only VEGF, butnot other growth factors such as CXCL1, EGF, IL8, M-CSF, TNFa,and PDGF, rescued melanoma cells from PLX4720-induced cellgrowth inhibition and cell death (Fig. 3A and B and Supplemen-tary Figs. S3C, S4A, and S4B). VEGF produced similar effects indabrafenib-treated melanoma cells (Supplementary Fig. S4C andS4D). Accordingly, VEGF activated the MAPK pathway in both1205Lu and A375 cells (Fig. 3C), which is similar to the effectsseen in melanoma cells treated with PLX4720 in the presence ofmacrophages, although the effect of VEGF was less pronounced.

In addition to its proangiogenic effect, VEGF has direct effectson melanoma cell growth and survival (41–44). Melanoma celllines and melanoma cells in primary human melanoma tissues

are reported to express three receptors for VEGF, VEGFR1, 2, 3, aswell as another VEGFR,Neuropilin-1 (45–48).We also found that1205LuandA375melanoma cells express VEGF receptors, includ-ing VEGFR1, 2, 3, neuropilin-1, and a coreceptor of VEGF,integrin-b3 (Supplementary Fig. S5A). In our experiments,macro-phages did not significantly increase expression of individualVEGFR (Supplementary Fig. S5B).

Blockade of VEGF signalingwith a pan-VEGF receptor inhibitor(VEGFR1, 2, and 3), lenvatinib, a compound currently beingtested in clinical trials for patients with melanoma, significantlyreversed macrophage-mediated cell growth (Fig. 3D), and mod-erately reversed macrophage-mediated anti-cell death effects(Fig. 3E), as well as blocked macrophage-mediated reactivationof ERK signaling (Fig. 3F). Similar effects were observed usinganother pan-VEGF receptor inhibitor (VEGFR1, 2, and 3), briva-nib alaninate (Supplementary Fig. S6). Because lenvatinib andbrivanib alaninate also target other tyrosine kinases, wewanted toconfirm whether VEGF specifically contributes to macrophage-mediated BRAF resistance and used anti-VEGF mAbs to blockVEGF signaling. Indeed, anti-VEGF antibodies significantly re-versedmacrophage-mediated resistance (Supplementary Fig. S7).Collectively, these data demonstrate that VEGF confers macro-phage-mediated resistance to BRAFi.

BRAF inhibition paradoxically activates the MAPK pathway inmacrophages

Having shown that BRAFi affect signaling pathways in mela-nomas by the induction of macrophage-derived VEGF, we deter-mined the process by which BRAFi produce this effect on macro-phages. We first examined the change in activity of signalingpathways of macrophages exposed to BRAFi. An earlier reporthad shown that PLX4720 modestly activated the MAPK pathwayin BRAF wild-type melanoma cells (13). Our examination of thispathway in macrophages demonstrated that PLX4720 stronglyactivated the MAPK pathway (Fig. 4A). Activation of the MAPKpathway occurred as early as 30minutes after PLX4720 treatment.In particular, we observed an increase in phosphorylation ofCRAF. This is of significance because CRAF expression is requiredfor BRAFi-induced paradoxical activation of theMAPKpathway inBRAF wild-type melanoma cells (Fig. 4A). We observed a similareffect with dabrafenib (Supplementary Fig. S8A). PLX4720 treat-ment significantly increased activation of theMAPK pathway, butdecreased pERK signaling at higher dose (10 mmol/L), which issimilar to the effect of BRAFi on BRAF wild-type cancer cells(13, 15). In wild-type BRAF melanoma cells, BRAFi activate theMAPK pathway only when there is a high level of RAS activity.This then appears to promote CRAF signaling and growth of BRAFwild-type melanoma cells. We hypothesized that, like BRAF wild-type cancer cells, macrophages have a high basal level of endog-enous RAS activity to activate the MAPK pathway upon BRAFitreatment.We therefore analyzed the RAS activity inmacrophagesdifferentiated from monocytes obtained from three differentdonors. We then compared RAS levels in macrophages withexpression in 1205Lu and A375 cells. ELISA analyses demonstrat-ed a 2- to 4-fold higher level of endogenous RAS activity inmacrophages compared with BRAF-mutant melanoma cell lines(Fig. 4B). The RAS activity levels in macrophages were similar tothose observed in BRAF wild-type and NRAS-mutant melanomacells (49). Therefore, the relatively high endogenous levels ofRAS activity in macrophages might be sufficient to initiate acti-vation of downstream signaling by CRAF in the presence of






BRAFi (Fig. 4A). PLX4720 treatment significantly increased RASactivity in macrophages, as well as other RAS downstream sig-nalingmolecules, such as p38 (Fig. 4C and D). Together, our datasuggest that activation of the MAPK pathway by BRAFi in macro-

phages is possible due to high endogenous RAS activity in macro-phages. Of note, the activation of the MAPK pathway in macro-phages by BRAFi is independent of the BRAF mutation status intumor cells, suggesting that this is a more general phenomenon.

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Figure 3.VEGF confers macrophage-mediated melanoma resistance to BRAFi. A, VEGF rescues PLX4720-induced melanoma growth inhibition in the presence ofPLX4720. 1205Lu and A375 melanoma cells were cocultured with or without VEGF (10 ng/mL) in the presence or absence of PLX4720 (3 mmol/L) for3 days. Cell growth was determined by the WST-1 assay as in Fig. 1B. Data shown are mean � SD (n ¼ 4). �� , P < 0.001. B, cells were treated same as in A. Celldeath was determined as in Fig. 1C. Representative images from one of four (1205Lu) and three (A375) independent experiments are shown (left).Relative percentage of cell death represents the ratio between dead cells with VEGF versus without VEGF in the presence of PLX4720 (right). � , P < 0.05;�� , P < 0.01. C, VEGF increases activation of the MAPK pathway. 1205Lu and A375 cells were cocultured in the presence or absence of VEGF (10 ng/mL)and/or PLX4720 (1 mmol/L) for 18 hours. Cells were harvested for immunoblotting with the indicated antibodies. D, lenvatinib reverses macrophage-mediated melanoma resistance to PLX4720. 1205Lu and A375 cells were cocultured with or without macrophages in the presence or absence of PLX4720(3 mmol/L) and/or lenvatinib (10 mmol/L) for 3 days, cell growth was then determined by the WST-1 assay as in Fig. 1B. Data shown are mean � SD (n ¼ 4).�� , P < 0.001. E, lenvatinib reverses the macrophage-mediated anti-cell death effect. 1205Lu cells were treated as in D. Cell death was determined asin Fig. 1C. Representative images from one of three independent experiments are shown (left). Relative percentage of cell death represents the ratiobetween dead cells with PLX4720 plus lenvatinib versus with PLX4720 alone in the presence or absence of macrophages. � , P < 0.05. F, lenvatinib reversesthe macrophage-mediated activation of the MAPK pathway. 1205Lu and A375 cells were treated as in D but with 18-hour treatment. Cells were thenharvested for immunoblotting with the indicated antibodies. See also Supplementary Figs. S3–S7.

Wang et al.





BRAF inhibition promotes macrophage growth and survivalThe MAPK pathway is critical for normal macrophage growth

and survival. We therefore explored the biologic consequence

of BRAFi-induced paradoxical activation of the MAPK path-way in macrophages. PLX4720 treatment promoted macrophagegrowth (Fig. 4E), and increased the expression of a proliferation

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Figure 4.BRAF inhibition paradoxically activates the MAPK pathway to elicit potent biologic responses in macrophages. A, BRAF inhibition induces activation of the MAPKpathway in macrophages. Macrophages were treated with the indicated concentration of PLX4720 for 2 hours and were then harvested for immunoblottingwith the indicated antibodies. Rab11 was used as a loading control. B, macrophages have a high basal level of RAS activation. Cell lysates from 1205Lu and A375melanoma cells, as well as macrophages that were differentiated from three different donors were harvested, and analyzed for RAS activation by ELISA. C,macrophages (black and white bars) were treated with PLX4720 (3 mmol/L) for 2 and 5 minutes. Cells were lysed and analyzed for RAS activation by ELISA (n¼ 4).�� , P <0.001. D,macrophageswere treatedwith the indicated concentration of PLX4720 for 2 hours andwere then harvested for immunoblottingwith the indicatedantibodies. HSP90 was used as a loading control. E, PLX4720 promotes macrophage growth. Macrophages were treated with the indicated concentration ofPLX4720 for 3 days. Cell growth was determined by the WST-1 assay as in Fig. 1B. Data shown are mean � SD (n ¼ 4). �� , P < 0.001. F, PLX4720 increasesthe expression of PCNA. Macrophages were treated with PLX4720 (3 mmol/L) for the indicated time. Cells were harvested for immunoblotting with theindicated antibodies. G, PLX4720 protects macrophage from cell death. Macrophages were treated with PLX4720 (3 mmol/L) for 3 days, and cell death wasdetermined as in Fig. 1C. Representative images from one of four independent experiments are shown (left). Relative percentage of cell death represents theratio between dead cells with PLX4720 versus without PLX4720 (right). �� , P < 0.01. H, the number of macrophages is increased in patient samples treated withvemurafenib and dabrafenib. The number of macrophages was counted in 10 randomly selected microscope fields. See also Supplementary Fig. S8.






marker, proliferating cell nuclear antigen (PCNA; Figs 4F andSupplementary Fig. S8A). Flow cytometric analyses by 7-AAD andAnnexin V staining showed that PLX4720 treatment protectedmacrophages from cell death (Fig. 4G), and cell-cycle analysesshowed that macrophages treated with PLX4720 had a smallersub-G1 population than thosewithout treatment (SupplementaryFig. S8B). Accordingly, the BRAFi dabrafenib similarly increasedmacrophage growth and protected them from cell death (Sup-plementary Fig. S8C and S8D). Consistent with this observation,analyses of melanomas from 10 patients treated with BRAFiindicated a trend toward more macrophages in tumors posttreat-ment than before treatment (Fig. 4H and Supplementary Fig.S8E). Analysis of additional patient samples would be necessaryto determine whether an increase in the numbers of macrophagesafter BRAFi treatment is statistically significant.

BRAF inhibition induces macrophages to produce VEGFProduction of VEGF is induced by activation of the MAPK

pathway in both malignant and normal cells, including macro-phages (50, 51). We determined whether paradoxical activationof the MAPK pathway by BRAFi had similar effects on macro-phages. Flow cytometric analyses by intracellular staining forVEGF indicated that PLX4720 significantly increased the produc-tion of VEGF in macrophages in a biphasic pattern, with increas-ing VEGF production peaking at 3 mmol/L, and declining at 10 to20 mmol/L of PLX4720 treatment (Fig. 5A). This is similar to the

effect seen in increased ERK activation by PLX4720 in macro-phages (see Fig. 4A). ELISA analyses further indicated a 5-foldincrease of VEGF production levels when testing supernatantsfrommacrophages treatedwith 3mmol/LPLX4720 in comparisonwith control (Fig. 5B). Furthermore, PLX4720 treatment resultedin a strong increase in VEGF receptor 1 (VEGFR1) phosphoryla-tion but not total VEGFR1 production, suggesting that BRAFinhibition also exerts an autocrine effect on macrophages trig-gered by VEGF (Fig. 5C and D). Together, our data indicate thatBRAF inhibition elicits potent macrophage responses andincreases the numbers of macrophages, as well as the productionof VEGF, which then represents a potent stimulant for bothmacrophages and melanoma cells.

Targetingmacrophages increases the antitumor effects of BRAFiin mouse models

We investigated the effect of macrophages on melanomasgrown in a murine syngeneic tumor system and treated withBRAFi. After 14 days of treatment with GW2580, a small-mole-cule, ATP-competitive inhibitor of M-CSFR kinase (160 mg/kg),tumor sizes decreased significantly, although the compound wasless efficacious than PLX4720 (25 mg/kg) alone. A combinationof both agents showed greater inhibition of tumor growth andreduced tumor weight (Fig. 6A and Supplementary Fig. S9A)than either alone. It is likely that the inhibitory effect ofGW2580 on tumor growth is due to targetingmacrophages rather

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Figure 5.Paradoxical activation of the MAPK pathway induces VEGF production. A, PLX4720 induces VEGF production. Macrophages were treated with the indicatedconcentration of PLX4720 for 4 hours. Intracellular staining was performed to measure the expression of VEGF. Representative images from one offive independent experiments are shown (left). Fold increase of VEGF-positive macrophages represents the ratio between VEGF-positive cells withPLX4720 versus without PLX4720. �, P < 0.05. B, macrophages were treated with the indicated concentration of PLX4720 for 3 days. Cell culture mediawere harvested for ELISA analysis of VEGF production. C, macrophages were treated with the indicated concentration of PLX4720 for 2 hours. Celllysates were used for immunoblotting with the indicated antibodies. D, macrophages were treated as in C. Expression of VEGFR1 was determined byflow cytometry. Representative images from one of four independent experiments are shown. Relative median fluorescence intensity (MFI, insertednumbers) represents the ratio between VEGFR1-positive macrophages with PLX4720 treatment versus without PLX4720 treatment (right).

Wang et al.





than tumor cells directly, because treatment with GW2580 canreverse macrophage-mediated resistance to BRAFi (Supplemen-tary Fig. S10A), and did not have significant direct effects onmelanoma growth or death when tested on cultured cells in vitro(Supplementary Fig. S10B and S10D), as well as on VEGF pro-

duction in melanoma cells (Supplementary Fig. S10C). GW2580treatment significantly decreased peritoneal F4/80-positivemacrophages, as also shown previously (52). BRAFi treatmentamplified GW2580-inducedmacrophage depletion through a yetto be identifiedmechanism (Fig. 6B and Supplementary Fig. S9B).

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Figure 6.GW2580 increases the antitumor activity of PLX4720 on 1205Lu xenograft tumor growth and the number of macrophages is a poor prognostic markerfor treatment of BRAFi. A, 1205Lu cells were injected subcutaneously into both flanks of nude mice. When the average tumor volume reachedapproximately 100 mm3, the indicated doses of GW2580 and/or PLX4720 or vehicle were administered orally for 14 days. A linear mixed-effect modelwith the random effect at mouse level was used to compare the differences in tumor growth trends between treatment groups over 14 days (n ¼ 10for all groups). �� , P < 0.01; �� , P < 0.001. B, mice were euthanized on day 14 and peritoneal cells were harvested for flow cytometric analysis of thepercentage of F4/80-positive macrophages. Data shown are mean � SD (n ¼ 5). � , P < 0.05; �� , P < 0.01. C, representative images of melanoma specimensfrom patients pretreated with BRAFi. One blue arrow shows one of the representative CD163-positive macrophages. One black arrow shows one of therepresentative Ki67-positive cells. All scale bars, 50 mm. D, univariate Cox regression analysis showed a statistically significant association between thenumber of melanoma-infiltrating macrophages and progression-free survival among 10 patients treated with BRAFi. The number of macrophages wasthe average number counted in 10 randomly selected microscope fields. �, P < 0.05. E, schematic model shows that macrophages switch their roles froma passenger to a driver for melanoma growth and survival under BRAF inhibition. ? marker indicates that there is no direct experimental evidence inthis article. See also Supplementary Figs. S9–S12.






GW2580 treatment abolished F4/80 and CD11b-positive macro-phages in tumors (Supplementary Fig. S11A and S11E). Unlikehuman tumor-infiltrating macrophages, mouse tumor-infiltrat-ing macrophages are mainly located around tumor blood vesselsor necrotic areas, which is consistent with a previous study (22).This may partially explain why PLX4720 treatment results in asignificant decrease in the number of F4/80-positive macro-phages. Likely, the inhibitory effect of BRAFi on angiogenesisresults in a reductionofmacrophagemigration frombloodvesselsto tumor parenchyma (Supplementary Fig. S11B; ref. 53). Therewas decreased signaling of pERK and fewer Ki67-positive cells intumor tissues treated with GW2580 and/or PLX4720 comparedwith control mice (Supplementary Fig. S11C, S11D, and S11F).Toxicity was not detected in the therapy groups and all treatedmice had similar body weight after treatment (SupplementaryFig. S9C). Because themissing immune components in immuno-deficient mice may compromise the infiltration of macrophagesinto tumors, we investigated the combined effects of GW2580with PLX4720 on tumor growth using the syngeneic mouseBRAFV600E, PTEN-null melanoma cell line, Yumm1.7. Similarto human tumor xenografts, GW2580 treatment significantlyincreased the efficacy of PLX4720 (Supplementary Fig. S9D andS9E), as well as reduced the number of peritoneal macrophages(Supplementary Fig. S9F). This does not conclusively demon-strate that the effect of GW2580 is only on macrophages becauseit also targets other types of myeloid cells in addition to macro-phages. Future work on whether CSF-1R inhibitor-inducedtumor growth retardation is solely due to targeting macrophagesis warranted. Together, our data indicate that targeting macro-phages alone can inhibit melanoma growth and increase theefficacy of BRAFi, which provides a rationale for combining BRAFiwith therapies that target macrophages.

Thenumber ofmacrophages correlateswith patients' responsesto BRAF inhibition

To examine the clinical relevance of melanoma-associatedmacrophages on the antitumor responses to BRAFi, we costaineda panel of pretreatment melanoma tissues from 10 stage IVmelanoma patients treated with BRAFi with a proliferation mark-er, Ki67, and a macrophage marker, CD163. Immunohistochem-ical analyses revealed that macrophages were abundant. Ki67-positive melanoma cells were usually surrounded by macro-phages, providing a likely microenvironment for rapid growthof melanoma cells (Fig. 6C). The specificities of anti-Ki67 andanti-CD163 antibodies were confirmed in human lymph nodeand placental tissues (Supplementary Fig. S12). Cox regressionanalysis was used to examine the association between pretreat-mentmacrophage infiltration levels andprogression-free survival.Patients with a higher number of pretreatmentmacrophages weremore likely to have shorter progression-free survival (HR, 1.38;P¼ 0.046; Fig. 6D). Together, our data further support the criticalroles of macrophages in melanoma progression and resistanceto BRAFi. The number of melanoma-associated macrophagesmight be a useful prognostic marker for patients treated withBRAFi, and this could be tested in a future clinical trial.

DiscussionOur studies demonstrate that BRAFi induce paradoxical acti-

vation of theMAPKpathway inmacrophages leading to profoundeffects on bothmacrophages and tumor cells through production

of VEGF. Our data indicate that VEGF plays multifaceted andcentral roles in macrophage-mediated resistance to BRAFi. Theparadoxical activation of the MAPK pathway by BRAFi inducesVEGF production in macrophages, which has an effect on bothmacrophages and tumor cells that express receptors for VEGF.For macrophages, this results in macrophage growth and survival(Fig. 4). Because melanoma cell lines express multiple VEGFreceptors (Supplementary Fig. S5), and primary melanoma cellsalso express high levels of VEGF receptors (42), VEGF can directlyactivate the MAPK pathway in melanomas and promote theirgrowth (41–43, 54). Tumor cells are dependent on their ownVEGF production for autocrine growth stimulation. BRAFi down-regulate the expression of VEGF in many tumors, includingmelanoma cells (44, 55). The production of macrophage-derivedVEGF can replace this growth promoter produced by melanomacells to stimulate melanoma cell growth and survival duringtreatment with BRAFi. VEGF also plays an essential role inangiogenesis. Therefore, macrophage-derived VEGF productioninduced by BRAF inhibition can also exert proangiogenesis effect.Consistent with this observation, the levels of VEGF productionare associatedwith patients' responses to other types of anticancertherapies (56, 57).

Macrophages produce additional growth factors, such asHGF, EGF, or FGF2, which can confer melanoma resistance toBRAFi. Although targeting these factors alone does not reversemacrophage-mediated resistance (Supplementary Fig. S3A), thefactors may synergize with VEGF to stimulate melanomagrowth via activation of the MAPK pathway or other signalingpathways, such as the PI3K/AKT pathway. In support of this,small molecules that target multiple VEGF receptors have bettereffects than anti-VEGF antibodies (Fig. 3 and SupplementaryFigs. S6 and S7). This may be due to VEGFRi that also targetother signaling pathways. Lenvatinib or brivanib alaninate, forexample, inhibit FGF1 receptor signaling at high concentrations(58, 59). A recent study has shown that TNFa produced bymacrophages contributes to melanoma resistance to BRAFi(60), which we did not see in our study (Supplementary Fig.S3C). This may be due to the differences of cell lines and cellculture condition between laboratories.

On the basis of our data, we proposed the following model:macrophages can provide survival signaling for melanoma cells,as targeting macrophages alone can inhibit melanoma growth,albeit the effect is moderate (Fig. 6A and Supplementary Fig.S9D), likely due to the many survival signaling pathways that areactive inmelanomas,whichmayonlypartially dependon stromalcells. Therefore, macrophages generally play a role as passengers(Fig. 6E, left). When melanoma cells are exposed to BRAFi, theirgrowth signaling pathways are interrupted, as evidenced by thelower activity of ERK signaling and the downregulation of growthfactors in tumor cells, and would be more dependent on thesurvival signals from macrophages (Figs. 2 and 3). Importantly,BRAFi induce macrophages to produce growth factors, such asVEGF,whichpromotemelanoma cell growth and survival (Fig. 3).Macrophage produced VEGF also exerts autocrine effect thatactivates VEGFR1 signaling andpotentially, ERK signaling (Fig. 5).In this case, themacrophage transitions from being a passenger toa driver of the malignant process (Fig. 6E, right; ref. 61).

Our study suggests the need to modify the current approach oftargeted therapy that focuses on driver mutations in tumors toalso consider other cells in the tumor environment as targetsfor anticancer therapies. Targeting macrophages, or the tumor


Wang et al.




microenvironment in general, along with therapies that targettumor cells, should be considered an essential part of "cocktails"for melanoma therapy.

Disclosure of Potential Conflicts of InterestM. Herlyn reports receiving commercial research grants from

GlaxoSmithKline, Novartis, and Tetralogic. No potential conflicts of inter-est were disclosed by the other authors.

Authors' ContributionsConception and design: T. Wang, E. Belser, A. Lopez-Coral, G. Zhang, L.M.Schuchter, M. Herlyn, R.E. KaufmanDevelopment of methodology: T. Wang, Y. Ge, E. Belser, A. Lopez-Coral, X. XuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Wang, M. Xiao, C. Krepler, E. Belser, X. Xu,G. Zhang, R. Azuma, R. Liu, L. Li, W. Xu, G. Karakousis, T.C. Gangadhar,M. Lieu, S. Khare, M.B. Halloran, M. HerlynAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Wang, M. Xiao, C. Krepler, E. Belser, A. Lopez-Coral, X. Xu, Q. Liu, R. Liu, R.K. Amaravadi, R.E. KaufmanWriting, review, and/or revision of the manuscript: T. Wang, C. Krepler,E. Belser, A. Lopez-Coral, X. Xu, G. Zhang, Q. Liu, R.K. Amaravadi, W. Xu,G. Karakousis, T.C. Gangadhar, L.M. Schuchter, M. Herlyn, R.E. Kaufman

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T.Wang, A. Lopez-Coral, X. Xu,W. Xu,M.HerlynStudy supervision: T. Wang, R.E. Kaufman

AcknowledgmentsThe authors thank Dr. Dario C. Altieri for discussion and article review; the

Flow Cytometry Core Facility for helping with instrument setup and dataanalysis; the Microscopy Core Facility for helping with images and figures; theAnimal Facility for helpingwithmousework; and theHistologyCore Facility forthe preparation of mouse tissues.

Grant SupportThis work was supported by grants from the National Institutes of Health

(5P30CA 010815-42) and the Commonwealth Universal Research Enhance-ment Program of the Pennsylvania Department of Health (R.E. Kaufman andT. Wang), TheWistar Institute Intramural grants for R.E. Kaufman and T. Wang,andNational Institutes of Health grants forM.Herlyn and T.Wang (CA047159,CA025874, CA114046, CA076674, and CA098101).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 20, 2014; revised January 13, 2015; accepted January 15, 2015;published OnlineFirst January 23, 2015.

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2015;21:1652-1664. Published OnlineFirst January 23, 2015.Clin Cancer Res Tao Wang, Min Xiao, Yingbin Ge, et al. Drive Tumor GrowthBRAF Inhibition Stimulates Melanoma-Associated Macrophages to

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