Although the contribution of macrophages to metastasis is widely studied in primary tumors, the involvement of macrophages in tumor-draining lymph nodes (LNs) in this process is less clear. We find CD169+ macrophages as the predominant macrophage subtype in naive LNs, which undergo proliferative expansion in response to tumor stimuli. CD169+ LN macrophage depletion, using an anti-CSF-1R antibody or clodronate-loaded liposomes, leads to increased metastatic burden in two mouse breast cancer models. The expansion of CD169+ macrophages is tightly connected to B cell expansion in tumor-draining LNs, and B cell depletion abrogates the effect of CD169+ macrophage absence on metastasis, indicating that the CD169+ macrophage anti-metastatic effects require B cell presence. These results reveal a protective role of CD169+ LN macrophages in breast cancer metastasis and raise caution for the use of drugs aiming at the depletion of tumor-associated macrophages, which might simultaneously deplete macrophages in tumor-draining LNs.

HomeCirculation ResearchVol. 128, No. 2Arterial Delivery of VEGF-C Stabilizes Atherosclerotic Lesions Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBArterial Delivery of VEGF-C Stabilizes Atherosclerotic Lesions Carlos Silvestre-Roig, Patricia Lemnitzer, Julie Gall, Simon Schwager, Albulena Toska, Laurent Yvan-Charvet, Michael Detmar and Oliver Soehnlein Carlos Silvestre-RoigCarlos Silvestre-Roig Institute for Cardiovascular Prevention (IPEK), Klinikum der LMU München (C.S.-R., P.L., O.S.). German Center for Cardiovascular Research (DZHK), Munich (C.S.-R., O.S.). Search for more papers by this author , Patricia LemnitzerPatricia Lemnitzer Institute for Cardiovascular Prevention (IPEK), Klinikum der LMU München (C.S.-R., P.L., O.S.). Search for more papers by this author , Julie GallJulie Gall (INSERM) U1065, Université Côte d’Azur, Centre Méditerranéen de Médecine Moléculaire (C3M), Atip-Avenir, Fédération Hospitalo-Universitaire (FHU) Oncoage, Nice (J.G., L.Y.-C.). Search for more papers by this author , Simon SchwagerSimon Schwager Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich (S.S., M.D.). Search for more papers by this author , Albulena ToskaAlbulena Toska https://orcid.org/0000-0002-4519-9493 Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München (TUM) (A.T.). Search for more papers by this author , Laurent Yvan-CharvetLaurent Yvan-Charvet Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München (TUM) (A.T.). Search for more papers by this author , Michael DetmarMichael Detmar Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich (S.S., M.D.). Search for more papers by this author and Oliver SoehnleinOliver Soehnlein Correspondence to: Oliver Soehnlein, MD, PhD, Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Pettenkoferstr. 9, 80336 Munich. Email E-mail Address: [email protected] https://orcid.org/0000-0002-7854-0694 Institute for Cardiovascular Prevention (IPEK), Klinikum der LMU München (C.S.-R., P.L., O.S.). Physiology and Pharmacology (FyFa), Karolinska Institutet, Stockholm (O.S.). Search for more papers by this author Originally published19 Nov 2020https://doi.org/10.1161/CIRCRESAHA.120.317186Circulation Research. 2021;128:284–286Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: November 19, 2020: Ahead of Print Cell stress elicited by intracellular lipid accumulation causes cell death, a major driver of atherosclerosis. Expansion of the necrotic core and fibrous cap (FC) thinning as consequence of smooth muscle cell (SMC) death are hallmarks of vulnerable plaques. Therefore, therapeutic strategies to limit cell death and SMC rarefication are key to prevent atheroprogression. While recent clinical trials support the overall viability of therapeutically targeting arterial inflammation, they also epitomize the shortcomings of such strategies, as systemic immunomodulation exhibits adverse side effects including impaired host defense.1 Hence, it is a major interest of preventive cardiology to identify means to boycott inflammation locally.Arterial lymphatics represent exit routes for lesional leukocytes and also serve as channels for reverse cholesterol transport, thus limiting atheroprogression.2,3 Here, we hypothesized that targeted delivery of VEGF (vascular endothelial growth factor)-C, the dominant growth factor for lymphatics, to preexisting lesions reverses lipid accumulation, reduces intimal cell death, and confers plaque stability. The F8 antibody specifically targets the extra domain A of fibronectin, an isoform that accumulates in inflamed tissues including atherosclerotic lesions. Extra domain A’s absence in steady-state renders it a suitable target to site-specifically deliver cytokines and hence we tested the ability of F8-VEGF-C conjugates to stabilize vulnerable lesions.Advanced lesions in the carotid artery of Apoe−/− mice were generated as previously described.4 All experiments were approved by the local ethics committee and performed in accordance with institutional guidelines. Only female mice were included in this study as males do not develop advanced lesions after cast insertion. Analyses included in this study were made blindly. Images representing the mean value of a certain readout were chosen as representative images.The F8-antibody showed a preferential binding to atherosclerotic tissue in hypercholesterolemic mice compared to other organs (Figure [A], left). Similarly, the F8-antibody bound to the FC area in advanced human lesions (Figure [A], right). To investigate the effects of VEGF-C on advanced atherosclerosis, Apoe−/− mice with preexisting lesions were treated with F8-VEGF-C (Figure [B], left). This treatment did not significantly affect plasma cholesterol levels or circulating leukocyte counts (not shown) while levels of intimal VEGF-C were strikingly increased (Figure [B], middle/right). In contrast to mice receiving vehicle only, lesion sizes in F8-VEGF-C treated mice did not significantly increase compared to baseline (Figure [C]). In addition, VEGF-C treatment improved signs of plaque instability exemplified by reduced necrotic core sizes (Figure [C]), thicker FCs (Figure [C]) accompanied by expansion of the collagen content within the FC (not shown). Contrasting to our initial hypothesis, however, F8-VEGF-C treatment did not significantly alter arterial lymph vessel area, macrophage burden, and macrophage egress (not shown). Instead, VEGF-C delivery significantly increased intimal SMCs (Figure [C]), and analysis of lipid accumulation in intimal cells showed a significant decrease in the loading of SMCs (Figure [C]) but not of macrophages (not shown) in F8-VEGF-C–treated mice. Overall, these data led us to speculate that VEGF-C lowers lipid content in intimal SMCs, thereby improving their survival.Download figureDownload PowerPointFigure. Arterial delivery of VEGF-C improves plaque stability.A–C, Advanced lesions were generated by insertion of a shear stress modifier around the carotid artery of Apoe−/− mice. A, Representative images of indicated organs (left) or advanced human atherosclerotic lesions (right). B, Schematic representation of arterial-directed delivery of VEGF (vascular endothelial growth factor)-C (left). Representative micrographs showing smooth muscle cells (SMCs) and VEGF-C. Displayed is the quantification of lesional VEGF-C+ area of vehicle-treated and VEGF-C–treated mice (right). C, Representative micrographs of hematoxylin-eosin–stained mouse atherosclerotic lesions. Displayed is the quantification of intima to media ratio, necrotic core area, fibrous cap (FC) thickness, SMC area, and lipid-laden SMC area overall SMCs. Confocal immunofluorescence micrographs showing SMC and lipids. White arrowheads point at lipid droplets in SMA+ area. D, Cholesterol efflux in [3H]-cholesterol-loaded SMCs was performed in medium containing 5% polyethylene glycol (PEG)-HDL (high-density lipoprotein). Cholesterol efflux stimulated by rhVEGF-C (recombinant human VEGF) (500 ng/mL, 24 h) is represented relative to LXR (liver X receptor) agonist (TO901317)–treated SMCs (left). Quantification of the ratio of Abca1 and Gapdh mRNA counts quantified by Digital Drop polymerase chain reaction (right). Both parts show 2 independent experiments each. E, SMCs pretreated with rhVEGF-C (24 h) were incubated with 7-ketocholesterol (200 μg/mL) and indicated amounts of rhVEGF-C for another 24 h. Cell death was measured using propidium iodide (PI) uptake (left). Representative western blot of SMCs treated as indicated probed with antibodies to β-actin or CHOP (middle). Quantification of SMA+TUNEL+ (terminal deoxynucleotidyl transferase dUTP nick end labeling) cells in the FC over total SMA+ cells (right). Data is mean±SEM. Data were analyzed using GraphPad Prism. Normality was assessed by D’Agostino and Pearson test. Significance was assessed by Mann-Whitney test (B) and (D, right); Kruskal-Wallis with Dunn correction for FC thickness and SMC area in (C) and (D, left) and (E, left); 1-way ANOVA with Tukey correction in remaining parts of (C); unpaired 2-sided t test (E, right). F, Scheme summarizing proposed mechanism. G, Table of antibodies used in this study. ABCA1 indicates ATP-binding cassette transporter A1; α-SMA, smooth muscle actin; CHOP, CCAAT-enhancer-binding protein homologous protein; DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; HFD, high fat diet; and L, lumen.To test this idea, we engaged in ex vivo studies. Although oxidized LDL (low-density lipoprotein) uptake by SMCs was not affected by VEGF-C treatment (not shown), VEGF-C favored cholesterol efflux in SMCs to a similar extent as the LXR (liver X receptor) agonist used as positive control (Figure [D], left), an observation that can at least in part be explained by increased expression of the cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in response to VEGF-C (Figure [D], right). Lipid toxicity associates with cell death through increased endoplasmic reticulum stress promoting atherosclerotic plaque destabilization. To evaluate the ability of VEGF-C to reduce lipid-mediated SMC death, we exposed SMCs to 7-ketocholesterol in the presence of VEGF-C. Interestingly, VEGF-C treatment abolished SMC death evoked by 7-ketocholesterol (Figure [E], left). VEGF-C is known to mediate its actions through VEGFR (VEGF receptor) 3, a receptor we could not identify on intimal SMCs (not shown). Neuropilin 1 and neuropilin 2 have been suggested to mediate some of the actions of VEGF-C and indeed we could identify these receptors on some intimal SMC subsets using a recently published scRNAseq (single cell RNA sequencing) database of intimal SMCs5 (not shown). Using siRNA (small interfering RNA)-mediated knockdown of neuropilin 1 and 2 inhibited the VEGF-C-induced increase in ABCA1 expression (not shown). Of note, the VEGF family member VEGF-A was unable to induce cholesterol efflux and to prevent 7-ketocholesterol induced SMC death (not shown). Moreover, the protective effect of VEGF-C was in the range of protection governed by the endoplasmic reticulum stress inhibitor salubrinal (not shown) and was associated with reduced levels of the transcription factor C/EBP (CCAAT-enhancer-binding proteins) Homologous Protein (Figure [E], middle), an important trigger of endoplasmic reticulum stress. In vivo, treatment with F8-VEGF-C reduced SMC death in the fibrous cap (Figure [E], right).Taken together, we here report the beneficial effect of VEGF-C in promoting plaque stability by rescuing atheroprotective SMCs in advanced lesions. The protective effects of VEGF-C treatment unexpectedly stimulated cholesterol efflux in arterial SMCs, diminished endoplasmic reticulum stress, and ultimately prevented cell death (Figure [F]). The use of antibody-based targeted delivery strategies like the one employed here may be therapeutically promising to limit systemic side effects.Nonstandard Abbreviation and AcronymsFCfibrous capSMCsmooth muscle cellVEGFvascular endothelial growth factorSources of FundingDeutsche Forschungsgemeinschaft (SO876/11-1, SFB914 TP B8, SFB1123 TP A6, TP B5), the Vetenskapsrådet (2017-01762), the Else-Kröner-Fresenius Stiftung, and the Leducq foundation.Disclosures The data that support the findings of this study are available from the corresponding author upon reasonable request.FootnotesFor Sources of Funding and Disclosures, see page 285.Correspondence to: Oliver Soehnlein, MD, PhD, Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Pettenkoferstr. 9, 80336 Munich. Email oliver.[email protected]comReferences1. Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, Fonseca F, Nicolau J, Koenig W, Anker SD, et al.; CANTOS Trial Group. Antiinflammatory therapy with canakinumab for atherosclerotic disease.N Engl J Med. 2017; 377:1119–1131. doi: 10.1056/NEJMoa1707914CrossrefMedlineGoogle Scholar2. Martel C, Li W, Fulp B, Platt AM, Gautier EL, Westerterp M, Bittman R, Tall AR, Chen SH, Thomas MJ, et al.. Lymphatic vasculature mediates macrophage reverse cholesterol transport in mice.J Clin Invest. 2013; 123:1571–1579. doi: 10.1172/JCI63685CrossrefMedlineGoogle Scholar3. Milasan A, Smaani A, Martel C. Early rescue of lymphatic function limits atherosclerosis progression in Ldlr-/- mice.Atherosclerosis. 2019; 283:106–119. doi: 10.1016/j.atherosclerosis.2019.01.031CrossrefMedlineGoogle Scholar4. Silvestre-Roig C, Braster Q, Wichapong K, Lee EY, Teulon JM, Berrebeh N, Winter J, Adrover JM, Santos GS, Froese A, et al.. Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death.Nature. 2019; 569:236–240. doi: 10.1038/s41586-019-1167-6CrossrefMedlineGoogle Scholar5. Wirka RC, Wagh D, Paik DT, Pjanic M, Nguyen T, Miller CL, Kundu R, Nagao M, Coller J, Koyano TK, et al.. Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis.Nat Med. 2019; 25:1280–1289. doi: 10.1038/s41591-019-0512-5CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Ambler W, Santambrogio L and Lu T (2021) Advances in understanding and examining lymphatic function: relevance for understanding autoimmunity, Current Opinion in Rheumatology, 10.1097/BOR.0000000000000864, 34:2, (133-138), Online publication date: 1-Mar-2022. Georgakis M, Bernhagen J, Heitman L, Weber C and Dichgans M (2022) Targeting the CCL2–CCR2 axis for atheroprotection, European Heart Journal, 10.1093/eurheartj/ehac094, 43:19, (1799-1808), Online publication date: 14-May-2022. Soehnlein O and Libby P (2021) Targeting inflammation in atherosclerosis — from experimental insights to the clinic, Nature Reviews Drug Discovery, 10.1038/s41573-021-00198-1, 20:8, (589-610), Online publication date: 1-Aug-2021. Andone S, Bajko Z, Motataianu A, Mosora O and Balasa R (2021) The Role of Biomarkers in Atherothrombotic Stroke—A Systematic Review, International Journal of Molecular Sciences, 10.3390/ijms22169032, 22:16, (9032) Miyazaki T and Miyazaki A (2021) Hypercholesterolemia and Lymphatic Defects: The Chicken or the Egg?, Frontiers in Cardiovascular Medicine, 10.3389/fcvm.2021.701229, 8 Dabravolski S, Khotina V, Omelchenko A, Kalmykov V and Orekhov A (2022) The Role of the VEGF Family in Atherosclerosis Development and Its Potential as Treatment Targets, International Journal of Molecular Sciences, 10.3390/ijms23020931, 23:2, (931) January 22, 2021Vol 128, Issue 2Article InformationMetrics © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.120.317186PMID: 33210556 Originally publishedNovember 19, 2020 KeywordscytokineleukocyteinflammationatherosclerosisfibronectinPDF download Advertisement SubjectsAnimal Models of Human DiseaseAtherosclerosisBasic Science ResearchInflammationVascular Biology

Crohn's disease (CD) and ulcerative colitis (UC) are two distinct forms of inflammatory bowel disease (IBD) characterized by an expanded lymphatic network with impaired functionality both in mouse models and in human patients. In this study, we investigated whether targeted delivery of the pro-lymphangiogenic vascular endothelial growth factor C (VEGFC) to the site of inflammation may represent a new, clinically feasible strategy for treating IBD. To achieve targeting of inflamed tissue, we developed a fusion protein consisting of human VEGFC fused to the F8 antibody (F8-VEGFC), which specifically binds to the extradomain A (EDA) of fibronectin, a spliced isoform almost exclusively expressed in inflamed tissues. The therapeutic activity of intravenously administered F8-VEGFC, compared to a targeted construct lacking VEGFC (F8-SIP), was investigated in a mouse model of dextran sodium sulfate (DSS)-induced colitis. The presence of EDA fibronectin was detected in both human and mouse inflamed colon tissue. Biodistribution studies of radiolabeled F8-VEGFC revealed a specific accumulation of the antibody in the colon of DSS-administered mice, as compared to an untargeted VEGFC fusion protein (KSF-VEGFC) (binding the irrelevant hen egg lysozyme antigen). Systemic treatment with F8-VEGFC significantly reduced the clinical and histological signs of inflammation, expanded the lymphatic vascular network, reduced the density of immune cells, and also decreased the expression of inflammatory cytokines in the inflamed colon. Overall, these results reveal that administration of F8-VEGFC represents a novel and promising approach for the treatment of IBD.

The lymphatic vasculature plays a crucial role in regulating the inflammatory response by influencing drainage of extravasated fluid, inflammatory mediators, and leukocytes. Lymphatic vessels undergo pronounced enlargement in inflamed tissue and display increased leakiness, indicating reduced functionality. Interfering with lymphatic expansion by blocking the vascular endothelial growth factor C (VEGF-C)/vascular endothelial growth factor receptor 3 (VEGFR-3) signaling axis exacerbates inflammation in a variety of disease models, including inflammatory bowel disease (IBD), rheumatoid arthritis and skin inflammation. In contrast, stimulation of the lymphatic vasculature, e.g., by transgenic or viral overexpression as well as local injections of VEGF-C, has been shown to reduce inflammation severity in models of rheumatoid arthritis, skin inflammation, and IBD. Strikingly, the induced expansion of the lymphatic vasculature improves lymphatic function as assessed by the drainage of dyes, fluorescent tracers or inflammatory cells and labeled antigens. The drainage performance of lymphatic vessels is influenced by vascular permeability and pumping activity, which are influenced by VEGF-C/VEGFR-3 signaling as well as several inflammatory mediators, including TNF-α, IL-1β, and nitric oxide. Considering the beneficial effects of lymphatic activation in inflammation, administration of pro-lymphangiogenic factors like VEGF-C, preferably in a targeted, inflammation site-specific fashion, represents a promising therapeutic approach in the setting of inflammatory pathologies.

Thrombospondin-2 (TSP2) is an anti-angiogenic matricellular protein that inhibits tumor growth and angiogenesis. Tumor-associated blood vascular endothelial cells (BECs) were isolated from human invasive bladder cancers and from matched normal bladder tissue by immuno-laser capture microdissection. Exon expression profiling analyses revealed a particularly high expression of a short TSP2 transcript containing only the last 9 (3') exons of the full-length TSP2 transcript. Using 5' and 3' RACE (rapid amplification of cDNA ends) and Sanger sequencing, we confirmed the existence of the shorter transcript of TSP2 (sTSP2) and determined its sequence which completely lacked the anti-angiogenic thrombospondin type 1 repeats domain. The largest open reading frame predicted within the transcript comprises 209 amino acids and matches almost completely the C-terminal lectin domain of full-length TSP2. We produced recombinant sTSP2 and found that unlike the full-length TSP2, sTSP2 did not inhibit vascular endothelial growth factor-A-induced proliferation of cultured human BECs, but in contrast when combined with TSP2 blocked the inhibitory effects of TSP2 on BEC proliferation. In vivo studies with stably transfected A431 squamous cell carcinoma cells revealed that full-length TSP2, but not sTSP2, inhibited tumor growth and angiogenesis. This study reveals that the transcriptional program of tumor stromal cells can change to transcribe a new version of an endogenous angiogenesis inhibitor that has lost its anti-angiogenic activity.

The lymphatic vasculature plays an important role in regulating inflammatory responses by influencing drainage of extravasated fluid, inflammatory mediators and leukocytes. We hypothesized that local activation of the lymphatic vessel network in inflamed skin could alleviate disease symptoms. To achieve inflammation-site specific activation of lymphatic vessels, we developed a fusion protein consisting of a human lymphangiogenic factor linked to a diabody specific to a fibronectin splice variant that is only expressed at sites of ongoing angiogenesis such as inflamed skin. The fusion protein was used in two mouse models of chronic, psoriasis-like skin inflammation, namely VEGF-A transgenic mice and imiquimod-induced skin inflammation. In both instances, systemic injections of the fusion protein significantly reduced ear swelling when compared to control protein constructs. Immunofluorescence analyses revealed that treatment with the fusion protein caused a marked increase in lymphatic vasculature in the inflamed skin lesions. Clearance assays employing an exclusively lymphatic-drained near-infrared tracer revealed that the therapy enhanced lymphatic clearance, indicating that the newly formed lymphatic vessels were indeed functional. Flow cytometric analysis of inflamed skin showed a significant reduction of leukocytes, regulatory T cells and γδ T cells upon treatment with the fusion protein. Our results reveal that inflammation site-specific induction of lymphatic vessels by targeted delivery of a lymphangiogenic factor represents a promising new approach for the treatment of chronic inflammatory skin diseases.

VEGF-C is an important mediator of lymphangiogenesis and has been shown to alleviate chronic inflammation in a variety of disease models. In this study, we investigated whether targeted delivery of VEGF-C to sites of inflammation and site-specific activation of lymphatic vessels would represent a clinically feasible strategy for treating chronic skin inflammation. To this end, we generated a fusion protein consisting of human VEGF-C fused to the F8 antibody (F8-VEGF-C), which is specific for the alternatively spliced, angiogenesis-marking extradomain A (EDA) of fibronectin. In two mouse models of psoriasis-like skin inflammation, mediated by transgenic VEGF-A overexpression or repeated application of imiquimod, intravenous treatment with F8-VEGF-C but not with untargeted VEGF-C significantly reduced ear skin edema and was as effective as the clinically used TNF-α receptor-Fc fusion protein (TNFR-Fc). Treatment with F8-VEGF-C led to a marked expansion of lymphatic vessels in the inflamed skin and significantly improved lymphatic drainage function. At the same time, treatment with F8-VEGF-C significantly reduced leukocyte numbers, including CD4+ and γδ T cells. In sum, our results reveal that targeted delivery of VEGF-C and site-specific induction of lymphatic vessels represent a potentially new and promising approach for the treatment of chronic inflammatory diseases.

Chronic itch is a highly debilitating condition affecting about 10% of the general population. The relay of itch signals is under tight control by inhibitory circuits of the spinal dorsal horn, which may offer a hitherto unexploited therapeutic opportunity. Here, we found that specific pharmacological targeting of inhibitory α2 and α3GABA A receptors reduces acute histaminergic and non-histaminergic itch in mice. Systemic treatment with an α2/α3GABA A receptor selective modulator alleviates also chronic itch in a mouse model of atopic dermatitis and in dogs sensitized to house dust mites, without inducing sedation, motor dysfunction, or loss of antipruritic activity after prolonged treatment. Transsynaptic circuit tracing, immunofluorescence, and electrophysiological experiments identify spinal α2 and α3GABA A receptors as likely molecular targets underlying the antipruritic effect. Our results indicate that drugs targeting α2 and α3GABA A receptors are well-suited to alleviate itch, including non-histaminergic chronic itch for which currently no approved treatment exists.

Tumor-associated macrophages (TAMs) have been implicated in the promotion of breast cancer growth and metastasis, and a strong infiltration by TAMs has been associated with estrogen receptor (ER)-negative tumors and poor prognosis. However, the molecular mechanisms behind these observations are unclear. We investigated macrophage activation in response to co-culture with several breast cancer cell lines (T47D, MCF-7, BT-474, SKBR-3, Cal-51 and MDA-MB-231) and found that high granulocyte colony-stimulating factor (G-CSF) secretion by the triple-negative breast cancer (TNBC) cell line MDA-MB-231 gave rise to immunosuppressive HLA-DRlo macrophages that promoted migration of breast cancer cells via secretion of TGF-α. In human breast cancer samples (n = 548), G-CSF was highly expressed in TNBC (p < 0.001) and associated with CD163+ macrophages (p < 0.0001), poorer overall survival (OS) (p = 0.021) and significantly increased numbers of TGF-α+ cells. While G-CSF blockade in the 4T1 mammary tumor model promoted maturation of MHCIIhi blood monocytes and TAMs and significantly reduced lung metastasis, anti-CSF-1R treatment promoted MHCIIloF4/80hiMRhi anti-inflammatory TAMs and enhanced lung metastasis in the presence of high G-CSF levels. Combined anti-G-CSF and anti-CSF-1R therapy significantly increased lymph node metastases, possibly via depletion of the so-called "gate-keeper" subcapsular sinus macrophages. These results indicate that G-CSF promotes the anti-inflammatory phenotype of tumor-induced macrophages when CSF-1R is inhibited and therefore caution against the use of M-CSF/CSF-1R targeting agents in tumors with high G-CSF expression.