Research Article

Circulating Tumor Microparticles Promote LungMetastasis by Reprogramming Inflammatoryand Mechanical Niches via a Macrophage-Dependent PathwayHuafeng Zhang1, Yuandong Yu1, Li Zhou1, Jingwei Ma1, Ke Tang1, Pingwei Xu1, Tiantian Ji1,Xiaoyu Liang2, Jiadi Lv2,Wenqian Dong2, Tianzhen Zhang2, Degao Chen2, Jing Xie2,Yuying Liu2,3, and Bo Huang1,2,3

Abstract

Despite the frequency of lung metastasis and its associatedmortality, the mechanisms behind metastatic tumor cell sur-vival and colonization in the lungs remain elusive. Here, weshow that tumor cell–released microparticles (T-MPs) fromthe primary tumor site play a critical role in the metastaticprocess. The T-MPs remodeled the lung parenchyma via amacrophage-dependent pathway to create an altered inflam-matory and mechanical response to tumor cell invasion.Mechanistically, we show that circulating T-MPs readily enterthe lung parenchyma where they are taken up by local macro-

phages and induce CCL2 production. CCL2 recruitsCD11bþLy6Chigh inflammatory monocytes to the lungswhere they mature into F4/80þCD11bþLy6C� macrophagesthat not only produce IL6 but also trigger fibrin deposition.IL6 and the deposited fibrin facilitate the survival andgrowth of tumor-repopulating cells in the lungs by provid-ing chemical and mechanical signals, respectively, thussetting the stage for lung metastasis. These data illustratethat T-MPs reprogram the lung microenvironment promot-ing metastasis. Cancer Immunol Res; 6(9); 1046–56. �2018 AACR.

IntroductionDespite metastasis being the leading cause of cancer-related

mortality, the underlying mechanism of how primary tumor cellssurvive and colonize distant organs remains an enigma. Althoughgenetic differences between primary tumors and metastases havebeen documented (1, 2), studies have also shown that themajority of genomic alterations present in the metastases weresimilarly present in the corresponding primary tumors (2–5),suggesting that nongenomic elements contribute to themetastaticprocess. Emerging evidence indicates that cancermetastasis can befacilitated by tumor-induced systemic environmental changes(6, 7). In line with the "seeds and soil" theory (8), it has beenproposed that the primary tumor can influence the micro-environment of distant organs prior to metastasis, a processtermed premetastatic niche formation (8, 9). Bone marrow–

derived cells have been shown to be involved in this process(10). However, the detailed molecular and cellular mechanismsof premetastatic niche formation remain unclear.

In addition to releasing soluble factors into circulation, tumorscan also communicate with distant organs, through the release ofvarious microvesicles that mediate intercellular communications(11, 12). Exosomes are small, endosome-derived extracellularmicrovesicles (30–100 nm), delivering contents such as proteins,messenger RNAs and microRNAs to recipient cells (13). Tumorcell–derived exosomes contribute to premetastatic niche forma-tion by means of their message delivery (14, 15). Tumor cells canalso rearrange their cytoskeleton, leading to encapsulation ofcytosolic contents within the cellular membrane to form large-sized vesicles, which are subsequently released into extracellularspaces (16). Such specialized subcellular vesicles of 0.1 to 1 mmsizes are termed microparticles (MPs; ref. 17). Studies fromcolorectal and pancreatic cancer patients have demonstrated thattumor cells release theirMPs into the circulation (18, 19).Withoutdisregarding current research on tumor exosomes in premetastaticniche formation, we propose that tumor cell–derived MPs(T-MPs) also contribute to this process. Capillary vessels are phys-iologically permeable to particles up to 5 to 12 nm in diameter(20), undoubtedly preventing circulating T-MPs (0.1–1 mm insizes) from entering parenchymal tissues. However, one notableexception is the lungs, where the preexisting apertures in the basallamina of alveolar capillaries and epithelium range from 0.3 to3 mm in width (21), allowing MPs to freely cross membranebarriers. Therefore, circulating T-MPs may enter and deliver mes-sages from primary tumors to the lung parenchyma, implying apossibility that primary tumors use T-MPs as a means to remodelthe local lung microenvironment and pave the way for futurelung metastasis.

1Department of Biochemistry and Molecular Biology, Tongji Medical College,Huazhong University of Science and Technology,Wuhan, China. 2Department ofImmunology, Institute of Basic Medical Sciences and State Key Laboratory ofMedical Molecular Biology, Chinese Academy of Medical Sciences and PekingUnion Medical College, Beijing, China. 3Clinical Immunology Center, ChineseAcademy of Medical Sciences, Beijing, China.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

H. Zhang and Y. Yu contributed equally to this article.

Corresponding Author: Bo Huang, Chinese Academy of Medical Sciences, 5Dong Dan San Tiao, Beijing 100005, China. Phone: 86-10-69156447; Fax: 86-10-65229258; E-mail: tjhuangbo@hotmail.com

doi: 10.1158/2326-6066.CIR-17-0574

�2018 American Association for Cancer Research.

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Macrophages are the principal immune cells within the tumormicroenvironment and are obligate partners for tumor cell migra-tion and metastasis (22). Secondary lung tumors are one of themost frequent human metastatic lesions. Normal lung tissuecontains an abundance of alveolar and interstitial macrophages(23). Whether and how lung macrophages are involved in theprocess of lung metastasis remains unclear. Our previous studieshave shown that T-MPs polarized macrophages toward an M2phenotype, leading to tumor growth and metastasis (24). In thepresent study, we provide evidence that lung macrophages effec-tively take up circulating tumor MPs and respond by creating aninflammatory andmechanical niche that promotes the survival ofand colonization by immigrant tumorigenic cells, thus setting thestage for the formation of subsequent lung metastasis.

Materials and MethodsAnimals and cell lines

Female BALB/c and C57BL/6 mice (6–8 weeks old) werepurchased from the Centre of Medical Experimental Animals ofHubei Province (Wuhan, China). CCR2�/� mice were a gift fromDr. LuGao (HuazhongUniversity of Science and Technology). Allanimal experiments were approved by the Animal Care and UseCommittee of Tongji Medical College. B16-F10melanoma, Lewislung carcinoma (LLC), and 4T1 breast cancer cell lines werepurchased from the China Centre for Type Culture Collection(CCTCC) and cultured according to the manufacturer's guide-lines. (B16-F10 and LLC were purchased in 2014, and 4T1 werepurchased in 2012). B16-F10 and LLCweremaintained inDMEMsupplemented with 10% FBS, and 4T1 cells were maintained inRPMI-1640 supplementedwith 10%FBS. Cells were thawed fromliquid nitrogen stocks and passaged 3 times before being used forin vivo or in vitro experiments. All cell lines were tested anddetermined to be free of mycoplasma and other rodent pathogensby the CCTCC.

Isolation of tumor microparticles and splenocytemicroparticles

Tumor cells or splenocyteswere cultured in serum-freemediumand hypoxic conditions for 24 hours after which themediumwascollected for microparticle isolation as described previously (25).Briefly, supernatants were centrifuged at 1,000� g for 10minutesto remove whole cells and then centrifuged for 2 minutes at14,000 � g to remove debris. The supernatant was further cen-trifuged for 60minutes at 14,000� g to pellet microparticles. Thepellets were washed three times and resuspended in PBS for thesubsequent experiments. The protein concentration of T-MPs wasmeasured using a BCA kit, according to manufacturer's protocol(Pierce, Thermo Fisher Scientific). T-MPs size and particle numberwere analyzed using a NanoSight NS300 system.

T-MPs labeling and treatmentTo assess the T-MPs distribution in the lung, liver, and spleen,

purified T-MPs were fluorescently labeled using PKH26 mem-brane dye (Sigma-Aldrich) according to the manufacturer's pro-tocol. Labeled T-MPs (50 mg) were intravenously injected inmice.The lungs, liver, and spleen were collected and embedded inTissue-Tek OCT (Sakura) for cryosections 2 hours later. Tissuecryosections (10 mm) were stained with DAPI and analyzed bytwo-photon fluorescent microscopy (Confocal Laser ScanningMicroscope Leica TCS SP8; Leica).

Lung leakage experimentsFifty micrograms of B16-MPs was injected by tail vein. Control

mice were injected with 50 mg splenocyte-derived MPs (S-MPs)or PBS. Twenty-four hours after B16-MPs treatment, micewere injected with 2 mg of FITC-labeled dextran 70,000 MW(Sigma-Aldrich) by tail vein injection. Two hours after dextraninjection, mice were euthanized and perfused with PBS. Lungswere fixed and inflated with a 50% vol/vol mixture of Tissue-TekOCT for cryosections.

Spontaneous metastasis and lung colonization studiesFor the generation of spontaneous lung metastatic models, 6-

to 8-week-old C57BL/6J female mice were injected in the flankwith 5 � 105 B16-F10 cells. After 5 days, 50 mg of B16-MPs wasinjected intravenously every other day for a total of 10 treatmentswithin 3 weeks. Lungs were collected at days 21 and 35. Forparaffin sections, before removal, lungs were perfused and fixedwith 10% buffered formalin. Lungs were excised and fixed informalin overnight. Paraffin-embedded lungs were systematicallysectioned and stained with hematoxylin and eosin (H&E) stain-ing, and images were taken. Metastasis index (the percentage ofmetastasis tumor areas to total lung areas based on calculationfrom 10 slides) and the number of metastatic foci (averagednumber of metastasis sites per square millimeter of lung area)were used to describe the metastatic lesions. A subset of micewas treated with 60 mL clodronate liposome or CSF1R inhibitorGW2580 (Med Chem Express) intranasally to deplete lungmacrophages. Clodronate liposomes were purchased fromwww.clodronateliposomes.org (Vrije University, Amsterdam,the Netherlands).

Tumor volumewasmeasuredby calipers and calculated accord-ing to the formula: length � width2/2. To assess the number ofcirculating tumor cells (CTC), whole blood was collected andcentrifuged at 2000 � g � 5 minutes; buffy coat was isolated andcultured in DMEM (10% heat inactivated FBS, 1% penicillin/streptomycin and 2 mmol/L L-glutamine). Tumor colonies werecounted on day 10 of culture and the number of CTCs wascalculated as the colonies present in the dish. To analyze theCTCs by flow cytometry, buffy coat was collected and stainedwithAPC-conjugated anti-CD45 antibody (clone OX1, eBioscience).CD45-negative cells were measured.

For the 4T1 breast cancermodel, 5� 105 4T1 breast cancer cellswere injected into the second mammary pad of female BALB/cmice. Five days following this, 50 mg of 4T1-MPs was injectedintravenously for a total of 10 treatments within 3 weeks.

To analyze the role of T-MPs education in tumor metastasis,6- to 8-week-old C57Bl/6 female mice preeducated with 50 mgB16-MPs every other day for 20 days, followed by intravenousinjection of 1 � 105 B16-F10 cells. Mice were sacrificed 21 dayslater, and the black melanoma nodules on the lungs were assay-ed. A subset ofmice was treatedwith clodronate liposome, CSF1Rinhibitor GW2580, anti-IL6 (clone MP5-20F3, BioLegend), orplasmin (Sigma-Aldrich), where indicated.

CCR2�/� bone marrow transplantationBone marrow cells were harvested by flushing the femurs and

tibias of CCR2�/� mice or wild-type (WT) littermates. RecipientC57BL/6 mice were irradiated with a single dose of 9 Gy, and allmice received 5 � 106 bone marrow cells from either CCR2�/�

mice or WT littermates within 4 hours. After 4 weeks, the recon-stituted C57BL/6 mice were used in subsequent experiments.

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Flow-cytometric analysis and lung macrophage isolationLungs tissueswere collected, cut into small pieces (�1–2mm2),

and digested at 37�C for 45 to 60 minutes with an enzymecocktail (collagenase A and DNase I, Sigma-Aldrich). Single-cellsuspensions were filtered through a 40-mm cell strainer. Cellsuspensions were washed in PBS and 1% FBS and incubat-ed with the following fluorochrome-conjugated antibodies:CD11b (M1/70), CD11c (clone N418), F4/80 (clone BM8),Ly-6C (clone HK1.4), CD3 (clone 17A2), CD19 (clone 6D5),NK1.1 (clone PK136), CD206 (clone MR6F3), CD45 (cloneOX1). For intracellular cytokine staining, lung single cells weretreated with Fix/Perm (eBioscience) solution following stainingwith a surface marker, and restained with CCL2 (clone 2H5),IL6 (clone MP5-20F3), IL10 (clone JES5-16E3), TNFa (cloneMP6-XT22), or Ki-67 (clone 16A8) antibody.

For isolation of macrophages from lung tissues, single-cellsuspensions from lungs were stained with CD45 (clone OX1)and F4/80 (clone BM8) antibodies and then were sorted using aBD FACS Aria III Cell Sorter (BD Biosciences) with purities of>95%.

T-cell proliferations assayF4/80þCD11bþLy6C� macrophages were isolated from lung

tissues of mice treated or not with B16-MPs (10 injections) usingBD FACS Aria III Cell Sorter. After additional 24 hours culture invitro, supernatants of lung macrophages were collected for fol-lowing experiments.

For the analysis of antibodies induced T-cell proliferation,splenic CD8þ T cells were isolated by Magnetic-activated cellsorting using a CD8þ T-cell isolation kit (Miltenyi). T cells werelabeled with CFSE (Sigma) and activated by anti-CD3/28 beads(Thermo Fisher Scientific) in the presence of supernatants frommacrophages. T-cell proliferations were examined by CFSE dilu-tion assay 72 hours later using flow cytometry.

For the analysis of antigen-induced T-cell proliferation in vitro,CD8þ T cells purified from the spleen of OT-I mice were labeledwith CFSE and then cultured with dendritic cells (DCs) whichpulsedwithOVA257-264 at a T-cell/DC ratio of 10:1 in the presenceof supernatants from macrophages. T-cell proliferations wereexamined by CFSE dilution assay 72 hours later using flowcytometry.

For the analysis of antigen-induced T-cell proliferationin vivo, 1 � 106 CD8þ T cells purified from the spleen of OT-Imice were labeled with CFSE and adoptive transferred intoB16-MP or PBS treated mice. Next day, mice were infected with5 � 105 CFU of listeria monocytogenes expressing ovalbumin(OVA; LmOVA). T-cell proliferations in lung were examined72 hours later.

Tissue processing and immunofluorescenceFor immunofluorescence, before removal, lungs were per-

fused with PBS and inflated with a 50% vol/vol mixture ofTissue-Tek OCT. The perfused lungs were then removed andembedded in Tissue-Tek OCT, following by freezing on dry ice.OCT tissue cryosections (10 mm) were stained with antibodiesagainst F4/80 (1:200 dilution, Abcam) and fibrin (1:200 dilu-tion, Abcam). Secondary antibodies conjugated to Alexa Fluor488 or 594 were used. All experiments were performed accord-ing to the manufacturer's protocol and visualized by two-photon fluorescent microscopy fibrin expression was quanti-fied using ImageJ software by determining the ratio between the

areas of fibrin and DAPI staining and described as arbitraryunits (a.u.).

3D fibrin gel cell culture of tumor cellsThree-dimensional (3D) fibrin gel cell culture of tumor cells

was conducted according to our previously described method(26). In brief, tumor cells were detached froma conventional rigidplate by 0.25% trypsin digestion and suspended in DMEM (10%FBS). Cell density was adjusted to 1 � 104 cells/mL. Fibrinogen(Searun Holdings Company) was diluted into 2 mg/mL with T7buffer (pH 7.4, 50 mmol/L Tris, 150 mmol/L NaCl). Fibrinogenand cell solution mixture (1:1) was made, resulting in 1 mg/mLfibrinogen and 5,000 cells/mL in the mixture. Cell/fibrinogenmixtures (250mL)were seeded into eachwell of 24-well plate andmixed well with preadded 5 mL thrombin (0.1 U/mL, SearunHoldings Company) for culture under 37�C condition.

Real-time PCRTotal RNA was extracted from cells with TRIzol reagent

(Invitrogen). Real-time PCR was performed with 2 mg of cDNAas a template, using Fast SYBR Green PCRmaster mix (TOYOBO)on a CFX96 Touch real-time PCR detection system (Bio-Rad).mRNA levels were normalized to b-actin. The primer sequencesare shown as follows: S100A8, 50-AAATCACCATGCCCTCTA-CAAG-30 (sense) and 50-CCCACTTTTATCACCATCGCAA-30

(antisense); S100A9, 50-ATACTCTAGGAAGGAAGGACACC-30

(sense) and 50-TCCATGATGTCATTTATGAGGGC-30 (antisense);SAA3, TGCCATCATTCTTTGCATCTTGA-30 (sense) and 50-CCGT-GAACTTCTGAACAGCCT-30 (antisense); CCL2, TTAAAAACCTG-GATCGGAACCAA-30 (sense) and 50-GCATTAGCTTCAGATTTA-CGGGT-30 (antisense); IL6, CCAAGAGGTGAGTGCTTCCC-30

(sense) and 50-CTGTTGTTCAGACTCTCTCCCT-30 (antisense);b-actin, GGCTGTATTCCCCTCCATCG-30 (sense) and 50-CCAGT-TGGTAACAATGCCATGT-30 (antisense).

Cytokine detection assaysCCL2, IL6, and VEGF levels were assessed using the murine

ELISA kit (Dakewei) for mouse cells and lung homogenateaccording to the manufacturer's protocol.

Western blot analysisB16-MPs or macrophages protein were extracted and analyzed

by Western blot using anti-mouse HIF1-a (1:1,000 dilution, CellSignaling Technology), anti-mouse VEGF-A (1:500 dilution,Abcam), anti-phospho-TBK (Ser172; 1:1,000 dilution, Cell Sig-naling Technology), anti-TBK (1:1,000 dilution, Cell SignalingTechnology), anti-phospho-STAT6 (Tyr641; 1:500 dilution,Abcam), anti-STAT6 (1:1,000 dilution, Abcam), or anti-mouseb-actin (1:1,000 dilution, Cell Signaling Technology), followedby secondary horseradish peroxidase–coupled antibodies incu-bation and visualized by enhanced chemiluminescence accordingto the manufacturer's protocol (ECL kit; Pierce).

Statistical analysisAll experiments were performed at least three times. Results are

expressed as mean � SEM and analyzed by unpaired two-tailedStudent t test. The Kaplan–Meier method was used to estimateoverall survival, and the differences in survival were analyzedusing the log-rank test. Differences were considered to be statis-tically significant when the P value was <0.05. The analysis was

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conducted using the GraphPad Prism version 6 (GraphPadSoftware).

ResultsCirculating T-MPs promote primary tumor cell lung metastasis

Although lung metastasis is a common phenomenon for var-ious types of solid tumors,whether primary solid tumor cells use acommon pathway for their lung metastasis remains elusive.Tumor cells are capable of releasing MPs into the extracellularspace after exposure to environmental cues. Hypoxia is ubiqui-tously present in solid tumor microenvironments. We found thathypoxia (1% O2) caused more than a 10-fold release of T-MPs intumor cell lines (B16-F10 melanoma, 4T1 breast or LLC lungtumor cells), compared with normoxia, as evidenced by theNanoSight system (Fig. 1A). The quantification of MP-containedproteins also showed a 10-fold increase in proteins (Fig. 1B).However, these hypoxia-induced T-MPs did not display a differ-ence in the mean size or size distribution between hypoxia-induced and normoxia-formed T-MPs (Fig. 1C and D; Supple-mentary Fig. S1A—S1C). We analyzed hypoxia-inducible pro-teins, such as HIF-1a and VEGF-A byWestern blot. We found thatthe level of HIF-1a, but not VEGF-A, exhibited a 5-fold increase inthe hypoxia-MPs, comparedwith normoxia-MPs (SupplementaryFig. S1D). In addition to hypoxia, various exogenous and endog-enous signals are also able to induce tumor cells releasing MPs,which are capable of entering the circulation (27–29). Given thelarge permeability of alveolar capillaries (0.3–3 mm), we hypoth-esized that circulating T-MPs might enter the lung parenchymaleading tometastasis of theprimary tumor to the lungs. To test thishypothesis, C57BL/6 mice were subcutaneously inoculated with1 � 105 B16-F10 melanoma cells. Five days later, we injectedhypoxia-induced, B16-F10-derived MPs (B16-MPs) to the mice

every other day for a total of 10 injections. Normal splenocyte-derived MPs (S-MPs) were used as control MPs. We found thatcompared with the control, mice treated with B16-MPs showeda greater lung metastasis burden (metastasis index and numberof metastatic foci) as measured by H&E staining on day 21 (Fig.1E). On day 35, macroscopic melanoma nodules were visible inthe B16-MPs group (Fig. 1E). Consistently, the intravenousinjection of 4T1 breast cancer cell–derived MPs (4T1-MPs) alsopromoted breast cancer lung metastasis in mice (Fig. 1F). Toverify whether the repeated injection of T-MPs is necessary fortumor cell metastasis, we conducted 3, 7, or 10 injections ofB16-MPs to B16-F10 tumor-bearing mice. We found that 3injections of B16-MPs did not result in the increase of B16-F10metastasis. However, 7 and 10 injections both increasedB16-F10 metastasis (Supplementary Fig. S1E). Collectively,these data suggest that circulating T-MPs are capable of pro-moting primary tumor cell lung metastasis.

Local macrophages are essential for T-MP–promoted lungmetastasis

Next, we investigated the underlying mechanism throughwhich circulating T-MPs promote lung metastasis. Exosomesare smaller extracellular microvesicles (30–100 nm), whichhave been known to stimulate tumor growth (14). However,we found that the above described intravenous injection ofT-MPs did not promote the growth of the primary melanoma orbreast tumors (Fig. 2A), nor did it increase the number of CTCs(Fig. 2B; Supplementary Fig. S2A), suggesting that the maineffect of T-MPs is not directly on the primary tumor but ratheron the lungs where they promote lung metastasis. In line withthis notion, pretreatment of animals with B16-MPs increasedlung metastases following intravenous injection of circulatingB16-F10 tumor cells (Fig. 2C), leading to significantly

Figure 1.

Circulating T-MPs promote tumor cell lung metastasis. A–D, Tumor cells were cultured in serum-free medium under either 20% or 1% O2 for 24 hours. Thenumber (A) and total protein (B) of the isolated T-MPs were analyzed. C, T-MP size was measured. D, B16-MPs were analyzed by scanning electronic microscopy.E, mice (n ¼ 6) were injected with B16-F10 cells, followed by 3 weeks of B16-MPs or S-MPs treatment. Lung micrometastasis was determined by H&Estaining on day 21 (scale bar, 1 mm). Macroscopic lung metastasis was observed on day 35. F, BALB/c mice (n¼ 6) were injected with 4T1 cells, followed by 3 weeksof treatment with 4T1-MPs or S-MPs. Measurement of lung metastasis burden is shown in E. Data in bar graphs represent mean � SEM, three independentexperiments were performed. Data were analyzed using Student t test, � , P < 0.05; ��� , P < 0.001.

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shortened survival of the mice (Fig. 2D). A similar result wasalso obtained from preinjection of 4T1-MPs (SupplementaryFig. S2B and C), suggesting that different T-MPs have a similarability to influence the lung microenvironment. As furtherreinforcement, fluorescence-labeled, primary tumor cell–released MPs were found to access to the lungs via the circu-lation (Fig. 2E; Supplementary Fig. S3A). Those T-MPs in thelungs could be taken up by both F4/80þCD11cþ alveolarmacrophages and F4/80þCD11c� interstitial macrophages(30, 31), as evidenced by flow cytometry and immunostaining(Fig. 2F; Supplementary Fig. S3B). To dissect whether thosemacrophages are involved in T-MP–promoted lung metastasis,we used clodronate liposomes, a widely used macrophage-depleting agent to deplete lung macrophages (32). We foundthat macrophage depletion significantly blunted the metastasis-promoting effect of B16-MPs (Fig. 2G). In addition to theclodronate liposome, we also used a CSF1R inhibitor,GW2580, to disrupt the macrophages' effect in B16-MP–treatedtumor-bearing mice. Consistently, the metastasis-promotingeffect of B16-MPs was impaired (Supplementary Fig. S3C).These data suggest that lung macrophages mediate T-MP–promoted primary tumor cell lung metastasis.

Proinflammatory microenvironments are known as a "pro-moting force" that drives metastasis (33). In parallel with beingtaken up by macrophages, injection of B16-MPs resulted in the

enhanced endothelial permeability in the lungs, as evaluatedby the extravasation of FITC dextran (Fig. 2H), a typical featureof inflammation and inflammatory microenvironment (34).Consistently, the expression of S100A8, S100A9, and serumamyloid A3 (SAA3), three inflammatory mediators that havebeen known to remodel the lung microenvironment for pri-mary tumor cell metastasis (35, 36), was upregulated in thelungs after the injection of B16-MPs (Fig. 2I). However, if wedepleted macrophages in advance, neither vascular permeabil-ity nor the expression of S100A8, S100A9, and SAA3 wasinfluenced by the injection of T-MPs (Supplementary Fig.S4A and S4B). Together, these results suggest that, uponuptake, T-MPs confer to macrophages the ability to build aninflammatory microenvironment in the lungs, leading to pri-mary tumor cell metastasis.

T-MP–reprogrammed macrophages produce CCL2 formonocyte recruitment

Next, we investigated how the inflammatory microenviron-mentwas transformedby lungmacrophages after taking upT-MPs.Recruitment of myeloid cells is an essential step during theprocess of inflammation. To examine this, we analyzed immunecells in the lung 48 hours following an intravenous injection ofT-MPs. We found that although proportions of T cells, B cells, andNK cells were not changed in the lungs (Supplementary Fig. S5A

Figure 2.

Macrophages are essential for T-MP–promoted lung metastasis. A and B, Analysis of primary B16-F10 or 4T1 tumor growth after 3 weeks of treatment with B16-MPsor 4T1-MPs (A). The number of CTCs per mL blood was analyzed (B). C and D, Mice pretreated with B16-MPs or S-MPs were challenged with B16-F10 cellsvia tail vein. Twenty-one days later, a subset of mice (n ¼ 6) were sacrificed, and tumor nodules in the lungs were assessed (C). The long-term survivalof mice (n ¼ 8) was assessed (D). E, Two hours after PKH26-labeled B16-MPs injection, the distribution of B16-MPs in the lungs, liver, and spleen in mice wasanalyzed by confocal microscopy (scale bar, 20 mm). F, Mice were injected with PKH26-labeled B16-MPs. The expression of CD11c and F4/80 in PKH26-positivecells in the lungs was analyzed by flow cytometry. G, B16-F10 tumor-bearing mice were intranasally administrated with clodronate liposomes twice a week,followed byB16-MPs treatment for 3weeks. The lungmetastasiswas analyzed byH&E staining (scale bar, 1mm).H,Micewere treatedwith B16- or S-MPs for 24 hoursand then received FITC-labeled dextran injection. Two hours later, the lung tissue was analyzed under a microscope (scale bar, 50 mm). I, Real-time PCRanalysis of indicated gene expression of the lungs in mice 24 hours after intravenous injection of B16- or S-MPs. Data in bar graphs represent mean � SEM,n ¼ 3 independent experiments. Data were analyzed using Student t test or log-rank test, �, P < 0.05; �� , P < 0.01.

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and S5B), the proportion of CD11bþLy6Chigh inflammatorymonocytes (37) was increased �4-fold in the lungs (Fig. 3A).These cells can express myeloid chemokine receptor CCR2 that isattracted by CCL2 (34, 37). Injection of B16-MPs resulted in theupregulation of CCL2 in the lungs (Fig. 3B). Further investigationrevealed that CCL2 was mainly produced by lung macrophagesthat had taken up T-MPs, evidenced by (i)CCL2mRNAwhich wasupregulated in macrophages following injection of T-MPs (Sup-plementary Fig. S6A); and (ii) macrophage depletion (chlodro-nate) or inhibition (GW2580)which blocked the increase ofCCL2levels and the recruitment of the inflammatory monocytes (Sup-plementary Fig. S6B and S6C). To directly confirm the CCL2induction in lung resident macrophages by B16-MPs, we isolatedthe lung macrophages (CD11bþF4/80þ Ly6C�) for CCL2 detec-tion. We found that CCL2 expression was increased in lungmacrophages treated with B16-MPs (Supplementary Fig. S6Dand S6E). Therefore, circulating T-MPs can enter the lung paren-chyma where they are taken up by lung macrophages to inducethe production of CCL2 in order to attract inflammatory mono-cytes. To provide additional support for this hypothesis, weinjected B16-MPs into CCR2�/� mice, and found the recruitmentof CD11bþLy6Chigh inflammatorymonocytes to the lungs by B16-MPs was abrogated (Fig. 3C). In parallel, B16-MPs neither pro-moted primary tumor cells lung metastasis in CCR2�/� mice (Fig.3D), nor shortened the overall survival of themice (Fig. 3E). Then,we investigated the pathway throughwhich T-MPs activate macro-phages to produce CCL2. Previous studies showed that MPs fromapoptotic tumor cells contain genomic and mitochondrial DNAfragments that can activate the cGAS-STING-TBK1 pathway, lead-ing to type I IFN production in dendritic cells (27) and STAT6activation inmacrophages (24).Here,we also found that B16-MP–induced CCL2 expression in macrophages is dependent on the

activation of TBK1/STAT6 (Supplementary Fig. S7A–S7C).Moreover, the use of STINGor cGAS siRNA consistently abrogatedthe expression of CCL2 in macrophages (Supplementary Fig. S7Dand S7E), indicating that DNA fragments within hypoxic tumorcell-MPs contributed to CCL2 upregulation in macrophagesvia the cGAS/STING/STAT6 pathway. Together, these data sug-gest that CCL2 production by T-MP–affected lung macrophagesis critical for the formation of a prometastatic inflammatorymicroenvironment.

CCL2-attracted monocytes transform to dual-phenotypemacrophages

The above data showed that entry of T-MPs into the lungsresulted in the attraction of CD11bþLy6Chigh monocytes via theCCL2/CCR2 signaling pathway. However, this population ofCD11bþLy6Chighmonocyteswas not persistentlymaintained andeven showed adecreasing trend (Supplementary Fig. S8A), regard-less of continuous injection of B16-MPs and high levels ofCCL2 in the lungs (Supplementary Fig. S8B). In contrast, theproportion of F4/80þCD11bþLy6C� macrophages significantlyincreased after 10 injections with B16-MPs, as evaluated by flowcytometry (Fig. 4A) and further confirmed by immunostaining ofF4/80 (Fig. 4B). Macrophages are normally considered nonpro-liferating cells. Using the proliferation marker Ki-67, we didnot find that B16-MPs treatment caused the proliferation ofF4/80þCD11bþLy6C� cells in the lungs (Supplementary Fig.S8C–S8E), suggesting that the recruited CD11bþLy6Chigh mono-cytes differentiated into F4/80þCD11bþLy6C� macrophages. Toconfirm this, CD45.2þmicewere injectedwithB16-MPs for 6hours,followed by an adoptive transfer of CD45.1þCD11bþLy6Chigh

monocytes. We found that the transferred CD45.1þ monocytesmigrated to the lungs and maintained the monocyte phenotype of

Figure 3.

T-MP–affected macrophages produce CCL2 for monocyte recruitment. A and B, Mice were treated with B16- or S-MPs for 48 hours. CD11bþLy6Chigh inflammatorymonocytes in the lungs were analyzed by flow cytometry (A). CCL2 in the lungs was analyzed by real-time-PCR and ELISA (B). C, WT or CCR2�/� micewere treated with B16-MPs for 48 hours. CD11bþLy6Chigh cells in lungs were analyzed. D and E, CCR2�/� mice pretreated with B16-MPs were injected with B16-F10cells. After 21 days, a subset of mice (n ¼ 6) were sacrificed, and tumor nodules in the lungs were counted. The bottom is a macroscopic analysis of lungmetastases on day 30 (D). The long-term survival of tumor-bearingmice (n¼ 8) was assessed (E). Data shown are representative of three independent experimentsand error bars represent mean � SEM. Data were analyzed using Student t test or log-rank test. NS, not significant (P > 0.05); � , P < 0.05; �� , P < 0.01.

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CD11bþLy6Chigh during the first 24 hours, but most of theseswitched to a F4/80þCD11bþLy6C� macrophage phenotype 96hours later (Supplementary Fig. S8F). Thesemonocyte-transformedmacrophages were seemingly capable of taking up 5-fold moreB16-MPs than monocytes (Supplementary Fig. S8G). In addition,inhibition of the recruitment of CD11bþLy6Chigh monocytes by aCCL2-neutralizing antibody abrogated the number of T-MP–increased macrophages in the lungs (Fig. 4C). Such transformedmacrophages appeared to have an inflammatory phenotypebecause high levels of proinflammatory cytokines, TNFa and IL6,were found in those cells, as evidenced by the intracellular flow-cytometric analysis (Fig. 4D). We also found that M2-relatedmarkers including CD206 and IL10 were upregulated inF4/80þCD11bþLy6C�macrophages in the lungs after 10 injectionswith B16-MPs (Fig. 4E). Such macrophages also highly expressedarginase 1, a typical marker of the M2 phenotype (SupplementaryFig. S8H). In line with the immunosuppressive trait of M2macrophages, we found that these F4/80þCD11bþLy6C�

macrophages effectively inhibited CD3/CD28 antibodies-stim-ulated T-cell proliferation as well as OVA257-264 peptide-stimulated OT-1 T-cell proliferation in vitro (Fig. 4F; Supple-mentary Fig. S9A). Pretreatment with B16-MPs inhibited theproliferation of OT-1 T cells in vivo, which, however, could berescued by the administration of a CSF-1R inhibitor GW2580(Supplementary Fig. S9B). Therefore, CCL2-attracted mono-cytes are transformed to a new class of macrophages withboth inflammatory and immunosuppressive traits. To clarifythe role of these newly differentiated macrophages in lungmetastasis, mice, pretreated with 10 injections of B16-MPs,were subjected to a combination of intravenous and intranasaladministration of clodronate liposomes to deplete macro-phages, followed by an intravenous injection of B16-F10 tumorcells. As expected, depletion of F4/80þCD11bþLy6C� macro-

phages by clodronate liposomes or inhibition of macrophagesby GW2580 blocked the prometastatic effect of B16-MPs(Fig. 4G; Supplementary Fig. S9C). Together, these data suggestthat T-MP–triggered production of CCL2 attracts monocyteswhich can mature into F4/80þCD11bþLy6C� metastasis-promoting macrophages with both inflammatory and immuno-suppressive phenotypes.

Macrophages promote growth of tumor-repopulating cells viaIL6 signaling

Given the important role of inflammation in metastasis andthat the above F4/80þCD11bþLy6C� macrophages highlyexpress the key inflammatory cytokine IL6,we further investigatedIL6.We found that the concentration of IL6 in the lungs graduallyincreased in line with the multiple intravenous injections ofB16-MPs (Fig. 5A). However, this increase of IL6 was blocked byeither macrophage depletion or CCL2 neutralization in B16-MP–treated mice (Fig. 5B). To clarify the role of IL6 in the above lungmetastasis, we then used an IL6 antibody to neutralize IL6. As aresult, B16-MP–promoted lung metastasis was blocked (Fig. 5C)and the mice had a prolonged survival (Fig. 5D), suggesting thatT-MP–promoted lung metastasis is mediated through IL6 signal-ing. Next, we investigated how IL6 promoted lung metastasis.

Stem cell–like tumorigenic cells are thought to be the key cellpopulation that repopulates ametastatic tumor in distant organs.We have previously established a highly effective soft 3D fibrin gelculture system to generate tumorigenic cells, termed tumor-repo-pulating cells. These soft fibrin gels correspond to 90 Pa in elasticstiffness and the cells were individually trapped inside allowingfor colony formation (26). Such tumor-repopulating cells notonly showed spheroid-like morphologic changes resemblingstem-like cells, but also as few as 10 gel-selected B16 tumor-repopulating cellswere able to repopulate a lungmetastatic tumor

Figure 4.

CCL2-attracted monocytes transform to macrophages. A, F4/80þCD11bþLy6C� macrophages in the lungs of mice were analyzed by flow cytometry after 10injections of B16- or S-MPs. B, Immunostaining of F4/80þ cells in the lungs (scale bar, 50 mm). C,Macrophage numbers in the lungs of mice were analyzed after 10-time B16-MPs treatment combined with anti-CCL2. D and E, mice were treated with 10 injections of B16- or S-MPs. Macrophage IL6 and TNFa (D) as well asCD206 and IL10 (E) was assessed by flow cytometry. F, Anti-CD3/28 bead-activated splenic T-cell proliferation was examined by a CFSE dilution assayin the presence or absence of macrophage supernatants. G, B16-MPs pretreated mice were administered with intravenous and intranasal clodronate liposomes,followed by B16-F10 cell injection. After 21 days, mice (n¼ 6) were sacrificed, and tumor nodules in the lungs were counted. Data shown are representative of threeindependent experiments and error bars represent mean � SEM. Data were analyzed using Student t test, � , P < 0.05; �� , P < 0.01.

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in immunocompetent mice. Here, we found that B16-F10 tumor-repopulating cells had an increased ability to survive andcolonize in the lungs, compared with differentiated B16-F10cells (Fig. 6A). The survival and colonization of B16-F10 tumor-repopulating cells in the lungs could be further enhancedby the injection of B16-MPs (Fig. 6B), leading to a greaterlung metastatic burden (Fig. 6C) and shorter overall survival(Fig. 6D), suggesting that stem-like tumor-repopulating cellsare a major contributor to lung metastasis. IL6 has been asso-ciated with induction of tumor cell stemness (38), whichprompted us to hypothesize that IL6 contributes to lung meta-stasis by regulating tumor-repopulating cells. To test this, weused B16-MP–conditioned macrophage supernatants to treatB16-F10 tumor-repopulating cells, and we found an increaseof the size (�86%) and number (�57%) of B16-F10 tumor-

repopulating cells colonies in soft 3D fibrin gels, which, however,was abrogated by anti-IL6 or anti-IL6R treatment (Fig. 6E and F).Although the injection of T-MPs promoted tumor-repopulatingcells survival and colonization in the lungs of mice, IL6 neutral-ization impaired this process (Fig. 6G). Together, these resultssuggested that macrophages promote the growth of tumor-repopulating cells via IL6 signaling in order to enhance lungmetastasis.

Fibrin(ogen) deposition essential for tumor growth and lungmetastasis

The above data suggest that T-MP–triggered lung inflamma-tion results in the production of IL6, leading to tumor-repo-pulating cell colonization and growth. However, in addition tochemical signaling, stem cells also require an extracellular

Figure 6.

IL6 facilitates tumor-repopulating cell growth. A, PKH26-labeled B16-F10 cells cultured in 3D soft fibrin gels or two-dimensional (2D) rigid plastic were injected intomice. Confocal analysis of B16-F10 cells in the lungs was performed 24 hours after injection (left; scale bar, 50 mm). The number of B16-F10 cells per field isshown (right). B, PKH26-labeled 3D B16-F10 cells were injected to mice pretreated with B16- or S-MPs. B16-F10 cells in the lungs were analyzed (scale bar, 20 mm).C and D, Mice pretreated with B16- or S-MPs were challenged with 3D B16 cells. After 21 days, a subset of mice (n ¼ 6) were sacrificed, and tumor nodulesin the lungs were counted (C). The long-term survival of tumor-bearing mice (n ¼ 8) is shown in D. E and F, Macrophages were treated with B16-MPs for 24 hoursand the supernatants were used to culture B16-F10 cells in 3D fibrin gels in the presence or absence of anti-IL6 or anti-IL6R. On day 5, the colony size (E)and number (F) of B16-F10 tumor-repopulating cells were analyzed (scale bar, 50 mm). G, PKH26-labeled 3D B16-F10 cells were injected into mice pretreatedwith B16-MPs, B16-MPs/anti-IL6, or PBS. The colonization of B16-F10 cells in the lungs was analyzed. Data shown are representative of three independentexperiments and error bars represent mean� SEM. Data were analyzed using Student t test, or log-rank test, NS, not significant (P > 0.05); �, P < 0.05; �� , P < 0.01;��� , P < 0.001.

Figure 5.

Macrophages promote B16 cell lung metastasis via IL6 signaling. A, IL6 in the lungs was measured by ELISA after 1, 3, 7, or 10 B16-MPs treatments. B, IL6 levelsin the lungs after 10 B16-MPs treatments combined with anti-CCL2 or clodronate liposomes. C and D, Mice pretreated with B16-MPs, B16-MPs/anti-IL6, or PBSwere challenged with B16-F10 cells. After 21 days, a subset of mice (n ¼ 6) were sacrificed, and tumor nodules in the lungs were counted (C). D, Thelong-term survival of mice (n ¼ 8) was assessed. Data in bar graphs represent mean � SEM, three independent experiments performed. Data were analyzedusing Student t test or log-rank test, � , P < 0.05; �� , P < 0.01.

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matrix-generated mechanical niche for their growth and sur-vival (39). Collagen is a main stromal component in the lungs;however, tumor-repopulating cells only grow well in fibrin gelsbut not in collagen matrices (26), implying that lung-infiltrat-ing T-MPs may build a fibrin(ogen) physical microenviromentfor tumor-repopulating cells growth and survival. In line withthis possibility, we found that the deposition of fibrin in thelungs was increased after an injection of B16-MPs (Fig. 7A).Such fibrin deposition could be the result of the enhanced lungendothelial permeability. When we injected fluorescence-labeled fibrinogen to mice following an injection of B16-MPs,after 24 hours, abundant fibrinogen was observed in the lungs(Fig. 7B). However, B16-MP–caused fibrin deposition could bedegraded by an intranasal administration of plasmin (Supple-mentary Fig. S10A). Under this condition, we found that thesurvival of tumor-repopulating cells in the lungs of B16-MP–pretreated mice was impaired, evidenced by the decreasedtumor-repopulating cell numbers (Fig. 7C). Consistently, plas-min-mediated fibrin degradation resulted in blunting the pro-metastatic effect of B16-MPs in the lungs (Fig. 7D). We isolatedtumor cells from the lungs of the above treated mice and seededthem into soft 3D fibrin gels. The result showed that there werefewer colonies in the plasmin group as compared with thecontrol (Fig. 7E), suggesting that fibrin deposition is essentialfor the survival and colonization of tumor-repopulating cells in

the lungs. In addition, we found that either depleting (chlo-dronate) or inhibiting (GW2580) macrophages or neutralizingCCL2 resulted in decreases in fibrin deposition in T-MP–treatedmice (Fig. 7F; Supplementary Fig. S10B), suggesting that fibrindeposition in the lungs is caused by T-MP–triggered macro-phages. Fibrin deposition occurs during inflammation due tothe increased permeability of blood vessels. VEGF plays acritical role in vascular permeability, which might lead to fibrindeposition into the lungs. Here, we found that B16-MPs treat-ment resulted in the upregulation of VEGF expression in thelungs, which was abrogated by GW2580 administration (Sup-plementary Fig. S10C). In addition, when we used B16-MPs totreat bone marrow–derived macrophages or macrophages iso-lated from the lungs, the expression of VEGF was also upre-gulated (Supplementary Fig. S10D and S10E). Furthermore,VEGF neutralization inhibited the leakage of fibrinogen in thelungs (Supplementary Fig. S10F), suggesting that T-MP–induced VEGF by macrophages contributes to fibrin depositionin the lungs. Unlike VEGF, IL6 neutralization did not affect T-MP–triggered fibrin deposition in the lungs (Fig. 7F). Thus, IL6signaling and fibrin deposition are two parallel biologicalevents that promote lung metastasis. Together, these datasuggest that circulating T-MPs can cause fibrin(ogen) deposi-tion in the lungs which supports tumor-repopulating cellgrowth and consequently promotes lung metastasis.

Figure 7.

Fibrin(ogen) deposition is essential for tumor-repopulating cell growth and lung metastasis. A, Fibrin deposition in the lungs of mice treated with B16- or S-MPs.B, Mice pretreated with B16- or S-MPs for 24 hours were injected with fluorescence-conjugated fibrinogen. Two hours later, fibrinogen in the lungs wasanalyzed. C, PKH26-labeled 3D B16-F10 cells were injected into mice pretreated with B16-MPs or B16-MPs/plasmin. The colonization of B16-F10 cells in thelung was analyzed. D, Mice treated with B16-MPs or B16-MPs/plasmin was challenged with B16-F10 cells. After 21 days, mice (n ¼ 6) were sacrificed, and tumornodules in the lungs were counted. E, CFSE-labeled B16-F10 cells were injected into mice pretreated with B16-MPs or B16-MPs/plasmin. CFSEþ tumor cellsisolated from the lungs were seeded into soft 3D fibrin gels for 5 days and the colony numbers were analyzed. F, Fibrin deposition was analyzed in the lungstreated with B16-MPs, B16-MPs/clodronate liposomes, B16-MPs/anti-CCL2, or B16-MPs/anti-IL6. G,Workingmodel of circulating tumor microparticles that promotelung metastasis via a macrophage-mediated reprograming of lung inflammatory and mechanical niches. Data in bar graphs represent mean � SEM, threeindependent experiments performed. Data were analyzed using Student t test, � , P < 0.05; �� , P < 0.01.

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DiscussionDespite the abundant literature on tumor cell metastasis, the

manner by which primary tumor-derived signals help the tumorcell to survive and colonize distant organs is a poorly definedprocess. In the present study, we provide evidence that primarytumor cells form lung metastasis via a series of biological eventstriggered by circulating T-MPs and illustrated in Fig. 7G: (i) T-MPsenter the lungs via circulation; (ii) lung macrophages take upT-MPs and release CCL2; (iii) CCL2 recruits monocytes to thelungs where they are transformed to macrophages; (iv) macro-phages release IL6 which facilitates the growth and survival ofhighly tumorigenic tumor-repopulating cells in the lungs; (v) inparallel, T-MP–conditioned macrophages induce lung inflamma-tion which increases endothelial permeability, leading to fibrindeposition in the lungs; (vi) fibrin deposition provides mechan-ical signals for tumor-repopulating cell growth and survival; and(vii) tumor-repopulating cell growth results in the formation ofmetastatic lesions in the lungs.

The presence of T-MPs in tumor microenvironments is ageneral pathophysiological phenomenon (40). Such continu-ously produced T-MPs in the interstitial fluid of extracellularspace may enter lymph or papillary vessels. After entering thedraining lymph node through lymphatic circulation, T-MPs canbe cleared by local macrophages. However, T-MPs may circulateto the lungs if they enter blood vessels due to their abnormallylarge apertures in the basal lamina of alveolar capillaries andepithelium (21).

This study shows that T-MP–reprogrammed lung macro-phages to both M1 and M2 phenotypes. Although immuno-suppression conferred by macrophages undoubtedly favorstumor cell immune evasion and survival in the lungs; theproinflammatory phenotype provides inflammatory signalsthat facilitate the growth and survival of tumor-repopulatingcells in the lungs. IL6, a pleiotropic inflammatory cytokine, notonly mediates the activation of the MAPK and PI3K/Akt sig-naling pathways for tumor cell growth, but also classicallyactivates STAT3 which can maintain the stemness of tumorcells (41). Previously, we developed a mechanics-based methodto select and amplify a subpopulation of cancer cells that areparticularly tumorigenic can grow very round spheroids in 3Dsoft matrices and are called tumor-repopulating cells (26, 42).Although 3D fibrin matrices are an artificial culture system, wefound, using immunohistochemical staining, that fibrin(ogen)was present in tumor tissues. CD45�ALDHþ tumor cells arereported to be stem cell–like tumor cells (43); however, wehave previously shown that only ALDHþ rather than CD45�

breast cancer cells isolated from MMTV-PyMT mice growinto colonies in soft 3D fibrin gels (44). In addition, we havedemonstrated that only CD133þ tumorigenic but not CD133�

B16 melanoma cells effectively grow into colonies in soft3D fibrin gels (45). Findings presented in the current studysuggest that tumor-repopulating cells are tumorigenic in vivoand fibrin is likely required for tumorigenic cell development.In the present study, we demonstrated that stem-like tumor-repopulating cells are important players for lung metastasis. Wefurther confirmed that IL6 contributes to lung metastasis bypromoting tumor-repopulating cell growth and survival. Thus,T-MP–reprogrammed macrophages can facilitate lung metas-tasis via releasing the inflammatory signal molecule IL6.

Cells apply contractile forces to sense the physical microenvi-ronment and respond accordingly by binding of extracellularmatrix (ECM) proteins such as collagen and fibrin to integrins,leading tomechanotransduction along clustered integrins to focaladhesions (46). In this study, we found that inflammatory signalsby T-MP–reprogrammed macrophages caused an increased per-meability of lung capillaries, leading to fibrin(ogen) deposition.Consequently, deposited fibrin provided mechanical signals fortumor-repopulating cell survival and growth in the lungs. Wepropose that T-MPs act as a pathway that primes the lungs formetastasis by creating an immunosuppressive, inflammatory, andphysical mechanical microenvironment that is permissive to thegrowth of metastatic tumor cells. Our findings also evoke con-cerns regarding current EGFR-targeted therapy and high-dosechemotherapies. Although such treatments cause widespreadtumor cell death in a short time, apoptotic tumor cells may betriggered to produce large amounts of T-MPs, whichmay promotelung metastasis or worsen primary tumors by facilitating tumor-repopulating cell growth and survival. This possibility has impli-cations for current therapies.

In summary, the data presented in this study demonstratethat T-MPs can initiate a pathway for the generation of pre-metastatic niche-associated macrophages in the lungs, whichcan reprogram the lung immune, inflammatory, and mechan-ical microenvironments and thus promote tumor-repopulatingcell growth and lung metastasis. Our findings provide mech-anistic insights regarding premetastatic niche formation andorgan site–specific tropism of metastasis. Further research onthe metastatic axis of T-MPs may reveal effective targets toprevent and treat lung metastasis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: B. HuangDevelopment of methodology: H. Zhang, Y. Yu, J. Ma, D. Chen, B. HuangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Zhang, Y. Yu, L. Zhou, K. Tang, P. Xu, T. Ji,X. Liang, T. Zhang, J. XieAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Zhang, J. Ma, T. ZhangWriting, review, and/or revision of themanuscript:H. Zhang, Y. Liu, B. HuangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Ma, P. Xu, T. Ji, J. Lv, Y. LiuStudy supervision: Y. Liu, B. HuangOther (assisted in conducting experiments): W. Dong

AcknowledgmentsThis work was supported by the National Natural Science Foundation of

China (81788101, 81661128007, 81530080, 81701544), the Chinese Acade-my of Medical Sciences Initiative for Innovative Medicine (2016-I2M-1-007),and China Postdoctoral Science Foundation funded project (2017M610478).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 10, 2017; revised February 27, 2018; accepted June 28,2018; published first July 12, 2018.

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References1. Schmidt-Kittler O, Ragg T, Daskalakis A, Granzow M, Ahr A, Blankenstein

TJ, et al. From latent disseminated cells to overt metastasis: genetic analysisof systemic breast cancer progression. Proc Natl Acad Sci USA 2003;100:7737–42.

2. Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signature ofmetastasis in primary solid tumors. Nat Genet 2003;33:49–54.

3. Weigelt B, Glas AM, Wessels LF, Witteveen AT, Peterse JL, van't Veer LJ.Gene expression profiles of primary breast tumors maintained in distantmetastases. Proc Natl Acad Sci USA 2003;100:15901–5.

4. Ding L, Ellis MJ, Li S, Larson DE, Chen K, Wallis JW, et al. Genomeremodelling in a basal-like breast cancer metastasis and xenograft. Nature2010;464:999–1005.

5. Yachida S, Jones S, Bozic I, Antal T, Leary R, Fu B, et al. Distant metastasisoccurs late during the genetic evolution of pancreatic cancer. Nature2010;467:1114–7.

6. Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. NatRev Cancer 2009;9:239–52.

7. McAllister SS,Weinberg RA. The tumor-induced systemic environment as acritical regulator of cancer progression and metastasis. Nat Cell Biol2014;16:717–27.

8. Psaila B, Lyden D. The metastatic niche: adapting the foreign soil. Nat RevCancer 2009;9:285–93.

9. PeinadoH, Lavotshkin S, LydenD. The secreted factors responsible for pre-metastatic niche formation: old sayings and new thoughts. Semin CancerBiol 2011;21:139–46.

10. Kaplan RN, Riba RD, Zacharoulis S, Bramley AH, Vincent L, Costa C, et al.VEGFR1-positive haematopoietic bone marrow progenitors initiate thepre-metastatic niche. Nature 2005;438:820–7.

11. Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors ofimmune responses. Nat Rev Immunol 2009;9:581–93.

12. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, RatajczakMZ. Membrane-derived microvesicles: important and underappreciatedmediators of cell-to-cell communication. Leukemia 2006;20:1487–95.

13. Simons M, Raposo G. Exosomes–vesicular carriers for intercellular com-munication. Curr Opin Cell Biol 2009;21:575–81.

14. Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, et al. Melanoma exosomes educate bone marrow progenitorcells toward a pro-metastatic phenotype through MET. Nat Med 2012;18:883–91.

15. Costa-Silva B, Aiello NM, Ocean AJ, Singh S, Zhang H, Thakur BK, et al.Pancreatic cancer exosomes initiate pre-metastatic niche formation inthe liver. Nat Cell Biol 2015;17:816–26.

16. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: artefacts nomore. Trends Cell Biol 2009;19:43–51.

17. Mause SF, Weber C. Microparticles: protagonists of a novel communi-cation network for intercellular information exchange. Circ Res 2010;107:1047–57.

18. Mege D, Panicot-Dubois L, Ouaissi M, Robert S, Sielezneff I, Sastre B, et al.The origin and concentration of circulating microparticles differ accordingto cancer type and evolution: a prospective single-center study. Int J Cancer2016;138:939–48.

19. Geddings JE, Mackman N. Tumor-derived tissue factor-positive micro-particles and venous thrombosis in cancer patients. Blood 2013;122:1873–80.

20. Sarin H. Physiologic upper limits of pore size of different blood capillarytypes and another perspective on the dual pore theory of microvascularpermeability. J Angiogenes Res 2010;2:14.

21. Sirianni FE, Chu FS, Walker DC. Human alveolar wall fibroblasts directlylink epithelial type 2 cells to capillary endothelium. Am J Respir Crit CareMed 2003;168:1532–7.

22. Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cellmigration, invasion, and metastasis. Cell 2006;124:263–6.

23. Tan SY, KrasnowMA. Developmental origin of lungmacrophage diversity.Development 2016;143:1318–27.

24. Ma R, Ji T, Chen D, Dong W, Zhang H, Yin X, et al. Tumor cell-derivedmicroparticles polarize M2 tumor-associated macrophages for tumorprogression. Oncoimmunology 2016;5:e1118599.

25. Tang K, Zhang Y, Zhang H, Xu P, Liu J, Ma J, et al. Delivery of chemo-therapeutic drugs in tumor cell-derived microparticles. Nat Commun2012;3:1282.

26. Liu J, Tan Y, Zhang H, Zhang Y, Xu P, Chen J, et al. Soft fibrin gels promoteselection and growth of tumorigenic cells. Nat Mater 2012;11:734–41.

27. Zhang H, Tang K, Zhang Y, Ma R, Ma J, Li Y, et al. Cell-free tumormicroparticle vaccines stimulate dendritic cells via cGAS/STING signaling.Cancer Immunol Res 2015;3:196–205.

28. Constantinescu P, Wang B, Kovacevic K, Jalilian I, Bosman GJ, Wiley JS,et al. P2X7 receptor activation induces cell death and microparticlerelease in murine erythroleukemia cells. Biochim Biophys Acta 2010;1798:1797–1804.

29. Baran J, Baj-Krzyworzeka M, Weglarczyk K, Szatanek R, Zembala M,Barbasz J, et al. Circulating tumor-derived microvesicles in plasmaof gastric cancer patients. Cancer Immunol Immunother 2010;59:841–50.

30. Chang YJ, Kim HY, Albacker LA, Baumgarth N, McKenzie AN, Smith DE,et al. Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity. Nat Immunol 2011;12:631–8.

31. Song C, Luo L, Lei Z, Li B, Liang Z, Liu G, et al. IL-17-producing alveolarmacrophages mediate allergic lung inflammation related to asthma.J Immunol 2008;181:6117–24.

32. van Rooijen N, Hendrikx E. Liposomes for specific depletion ofmacrophages from organs and tissues. Methods Mol Biol 2010;605:189–203.

33. Bald T, Quast T, Landsberg J, Rogava M, Glodde N, Lopez-Ramos D, et al.Ultraviolet-radiation-induced inflammation promotes angiotropism andmetastasis in melanoma. Nature 2014;507:109–13.

34. Hiratsuka S, Ishibashi S, Tomita T, Watanabe A, Akashi-Takamura S,Murakami M, et al. Primary tumors modulate innate immune signallingto create pre-metastatic vascular hyperpermeability foci. Nat Commun2013;4:1853.

35. Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumor-mediated upregu-lation of chemoattractants and recruitment of myeloid cells predetermineslung metastasis. Nat Cell Biol 2006;8:1369–75.

36. Hiratsuka S, Watanabe A, Sakurai Y, Akashi-Takamura S, Ishibashi S,Miyake K, et al. The S100A8-serum amyloid A3-TLR4 paracrinecascade establishes a pre-metastatic phase. Nat Cell Biol 2008;10:1349–55.

37. Meierovics AI, Cowley SC. MAIT cells promote inflammatory monocytedifferentiation into dendritic cells during pulmonary intracellular infec-tion. J Exp Med 2016;213:2793–809.

38. Korkaya H, Kim GI, Davis A, Malik F, Henry NL, Ithimakin S, et al.Activation of an IL6 inflammatory loop mediates trastuzumab resistancein HER2þ breast cancer by expanding the cancer stem cell population.Mol Cell 2012;47:570–84.

39. Malanchi I, Santamaria-Martinez A, Susanto E, Peng H, Lehr HA, DelaloyeJF, et al. Interactions between cancer stem cells and their niche governmetastatic colonization. Nature 2011;481:85–9.

40. Yang F, Liu S, Liu X, Liu L, Luo M, Qi S, et al. In vivo visualization of tumorantigen-containing microparticles generated in fluorescent-protein-elicited immunity. Theranostics 2016;6:1453–66.

41. Iliopoulos D, Hirsch HA, Wang G, Struhl K. Inducible formation ofbreast cancer stem cells and their dynamic equilibrium with non-stemcancer cells via IL6 secretion. Proc Natl Acad Sci USA 2011;108:1397–1402.

42. Tan Y, Tajik A, Chen J, Jia Q, Chowdhury F, Wang L, et al. Matrix softnessregulates plasticity of tumour-repopulating cells via H3K9 demethylationand Sox2 expression. Nat Commun 2014;5:4619.

43. Wan L, Lu X, Yuan S, Wei Y, Guo F, ShenM, et al. MTDH-SND1 interactionis crucial for expansion and activity of tumor-initiating cells in diverseoncogene- and carcinogen-induced mammary tumors. Cancer Cell 2014;26:92–105.

44. Liu Y, Liang X, Dong W, Fang Y, Lv J, Zhang T, et al. Tumor-repopulatingcells induce PD-1 expression in CD8þ T cells by transferring kynurenineand AhR activation. Cancer Cell 2018;33:480–94.

45. Liu Y, Lv J, Liu J, Liang X, Jin X, Xie J, et al. STAT3/p53 pathway activationdisrupts IFN-b-induced dormancy in tumor-repopulating cells. J ClinInvest 2018;128:1057–73.

46. Leight JL, Drain AP, Weaver VM. Extracellular matrix remodeling andstiffening modulate tumor phenotype and treatment response. Annu RevCancer Biol 2017;1:313–34.

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