Title: The cholesterol‐binding protein <scp>NPC</scp> 2 restrains recruitment of stromal macrophage‐lineage cells to early‐stage lung tumours
Abstract: Research Article16 July 2015Open Access Source Data The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours Tamihiro Kamata Corresponding Author Tamihiro Kamata Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Hong Jin Hong Jin Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Susan Giblett Susan Giblett Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Bipin Patel Bipin Patel Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Falguni Patel Falguni Patel Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Charles Foster Charles Foster Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Catrin Pritchard Corresponding Author Catrin Pritchard Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Tamihiro Kamata Corresponding Author Tamihiro Kamata Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Hong Jin Hong Jin Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Susan Giblett Susan Giblett Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Bipin Patel Bipin Patel Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Falguni Patel Falguni Patel Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Charles Foster Charles Foster Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Catrin Pritchard Corresponding Author Catrin Pritchard Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Author Information Tamihiro Kamata 1,‡, Hong Jin1,‡, Susan Giblett1, Bipin Patel1, Falguni Patel1, Charles Foster1 and Catrin Pritchard 1 1Department of Biochemistry, University of Leicester, Leicester, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 116 7029; Fax: +44 116 7018; E-mail: [email protected] *Corresponding author. Tel: +44 116 7029; Fax: +44 116 7018; E-mail: [email protected] EMBO Mol Med (2015)7:1119-1137https://doi.org/10.15252/emmm.201404838 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The tumour microenvironment is known to play an integral role in facilitating cancer progression at advanced stages, but its function in some pre-cancerous lesions remains elusive. We have used the V600EBRAF-driven mouse lung model that develop premalignant lesions to understand stroma–tumour interactions during pre-cancerous development. In this model, we have found that immature macrophage-lineage cells (IMCs) producing PDGFA, TGFβ and CC chemokines are recruited to the stroma of premalignant lung adenomas through CC chemokine receptor 1 (CCR1)-dependent mechanisms. Stromal IMCs promote proliferation and transcriptional alterations suggestive of epithelial–mesenchymal transition in isolated premalignant lung tumour cells ex vivo, and are required for the maintenance of early-stage lung tumours in vivo. Furthermore, we have found that IMC recruitment to the microenvironment is restrained by the cholesterol-binding protein, Niemann-Pick type C2 (NPC2). Studies on isolated cells ex vivo confirm that NPC2 is secreted from tumour cells and is taken up by IMCs wherein it suppresses secretion of the CCR1 ligand CC chemokine 6 (CCL6), at least in part by facilitating its lysosomal degradation. Together, these findings show that NPC2 secreted by premalignant lung tumours suppresses IMC recruitment to the microenvironment in a paracrine manner, thus identifying a novel target for the development of chemopreventive strategies in lung cancer. Synopsis M2-polarised immature macrophage-lineage cells (IMCs) found in the early lung adenoma microenvironment and recruited via CCR1 signalling. Adenoma-secreted Niemann-Pick type C2 protein suppresses IMC recruitment to the tumour microenvironment. Characterisation of the V600EBRAF-driven early lung adenoma microenvironment identifies IMCs. IMCs secrete CCR1 chemokines, and CCR1 inhibition prevents IMC recruitment in vivo. IMCs are required for adenoma maintenance since CCR1 inhibition suppresses adenoma burden. Niemann-Pick type C2 (NPC2) protein is secreted by adenoma cells and suppresses IMC recruitment to the microenvironment. Exogenous NPC2 is incorporated by IMCs wherein it suppresses CCR1 chemokine secretion. Introduction Oncogenic mutations are prevalent in human cancers, and some are thought to represent early mutations that initiate and subsequently drive cancer development. RAS and RAF oncogenes are amongst the best-characterised driver oncogenes and are mutated in a significant proportion of human cancers, notably pancreatic (~90%) and lung adenocarcinoma (~30%) in the case of KRAS (Malumbres & Barbacid, 2003), and melanomas (~50%) and thyroid cancers (~30%) in the case of BRAF (Davies et al, 2002). In contrast to KRAS, the incidence of BRAF mutations in human lung adenocarcinoma is relatively low (Naoki et al, 2002), but nearly a half of BRAF mutations in this type of cancer are the most common V600EBRAF mutation (COSMIC: http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), suggesting that V600EBRAF contributes to lung carcinogenesis in some cases. The mechanisms by which KRAS and BRAF oncogenes are involved in early-stage cancer development are beginning to be unravelled by analysis of genetically engineered mouse (GEM) models developing autochthonous tumours, especially for cancer types in which premalignant precursor lesions are difficult to access in humans. Lung adenocarcinoma is one such type of cancer in which atypical adenomatous hyperplasias (AAHs) are the purported precursor, but these early lesions are rarely diagnosed by non-invasive procedures (Gazdar & Brambilla, 2010). Instead, lung-specific expression of G12VKRAS or V600EBRAF in GEM models has provided evidence that activation of these oncogenes initially induces the formation of benign proliferative lesions after which the lesions enter a state of stable cell cycle arrest termed oncogene-induced senescence (OIS) (Collado et al, 2005; Dankort et al, 2007). While V600EBRAF-driven early lung lesions with OIS rarely progress to adenocarcinoma (Dankort et al, 2007), G12VKRAS-driven alveolar hyperplasias progress into malignant adenocarcinomas more frequently (Mainardi et al, 2014), which correlates well with the mutation spectrum of human lung adenocarcinomas. However, malignant progression of early lung lesions in the V600EBRAF model can be facilitated by mutations in key genes including depletion of tumour suppressor TRP53 (Dankort et al, 2007) or constitutive activation of β-catenin (Juan et al, 2014), indicating that these early lesions are indeed precursors for adenocarcinomas. Although the difference between the G12VKRAS and V600EBRAF models with regard to malignant progression could be explained by the nature of intracellular signalling cascades activated by each oncoprotein (Trejo et al, 2012, 2013), cell-extrinsic inflammatory responses have also been shown to contribute to malignant progression of G12VKRAS-driven early lesions, at least in a pancreatic cancer model (Guerra et al, 2011). In contrast, it still remains unclear how inflammatory or other environmental factors could influence the behaviour of V600EBRAF-driven early-stage tumours with OIS. Whereas the microenvironment of advanced cancers, composed of a panoply of different cell types including hematopoietic (immune) cells, vascular components and activated fibroblasts, creates a pro-tumourigenic environment (Whiteside, 2008), there is growing evidence that immune cells in the microenvironment of pre-cancerous lesions could play a tumour-suppressive rather than tumour-promoting role. For example, CD4+ T-cell-mediated adaptive immunity in concert with monocytes/macrophages has been shown to execute the clearance of oncogenic NRAS-induced pre-cancerous lesions in the liver (Kang et al, 2011). In this model, early hepatic lesions exhibited biochemical characteristics of OIS including secretion of inflammatory chemo-cytokines (Kuilman & Peeper, 2009), which likely contributed to the recruitment of the immune cells to the microenvironment. Such a suppressive microenvironment against pre-cancerous OIS lesions could potentially contribute to the less frequent malignant progression of V600EBRAF-driven senescent lung adenomas. To investigate the role of the tumour microenvironment in V600EBRAF-driven premalignant lesions, we have taken advantage of our V600EBRAF-driven autochthonous GEM model in which premalignant papillary adenomas accumulate in the lung. We show here that immature macrophage-lineage cells (IMCs) are recruited to the stroma of senescent lung adenomas through CCR1-dependent mechanisms. Unexpectedly, stroma IMCs are found to play a pro-tumourigenic role in vivo since the suppression of IMC recruitment through CCR1 inhibition profoundly decreases tumour burden. Furthermore, in a screen for proteins secreted from V600EBRAF-expressing premalignant tumour cells, we identified the cholesterol-binding protein Niemann-Pick type C2 (NPC2). Our studies with NPC2 show it is secreted at high levels even at the pre-senescent stage, and is incorporated by IMCs wherein it regulates intracellular cholesterol levels and inhibits secretion of the CCR1 ligand, CC chemokine 6 (CCL6). This results in the suppression of IMC accumulation at the pre-senescent stage. Overall, the data point to a novel role of NPC2 in regulation of the pro-tumourigenic microenvironment. Results V600EBRAF induces the formation of senescent lung adenomas Conditional (Cre-loxP-regulated) knockin mice for oncogenic V600EBRAF and G12V/G12DKRAS have been previously generated by our group and others, and induction of oncogene expression in the lung in both models has been shown to induce premalignant lesions that up-regulate the expression of senescence markers (Collado et al, 2005; Dankort et al, 2007). To obtain an easily manipulated model for biochemical investigations, we utilised Braf+/LSL−V600E;CreER™ (BVE) mice since these developed large numbers of pulmonary papillary adenomas in 100% of mice by spontaneous recombination of the Braf allele in the lung without tamoxifen induction (Fig 1A). Histologically, these tumours were identical to those induced by nasal administration of AdCre (Fig 1A). As previously reported for AdCre-induced tumours (Dankort et al, 2007), pulmonary adenomas in BVE mice exhibited signs of OIS at later stages, including significant loss of Ki67 expression (Fig 1B). These tumours did not stain for senescence-associated β-galactosidase or express p16INK4a or p19ARF at detectable levels but were positive for p21CIP1 and γH2AX (Supplementary Fig S1), suggesting that DNA-damage responses could be the major cause for the cell cycle arrest. Although most tumour cells at 10 weeks or later were negative for Ki67, a small population (2%) of Ki67+ tumour cells remained detectable at this later time point (Fig 1B). Figure 1. Characterisation of lung tissue expressing V600EBRAF (Top) PCR detection of the recombined BrafLox−V600E allele (Lox-V600E) in BVE mouse tissues at 8 weeks of age without tamoxifen induction. Substantial recombination is observed in the lung, while weaker recombination is also detected in the liver. No recombination was detected in hematopoietic tissues (bone marrow and spleen) even after 40 cycles of amplification (right). (Bottom) H&E staining of lung sections from wild-type (WT, 10 weeks of age), BVE (1–10 weeks of age) and Braf+/LSL−V600E mouse 8 weeks after nasal delivery of AdCre is indicated below. V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development. Scale bars, 100 μm. Ki67 immunostaining of lung sections from BVE mice at 3–10 weeks of age. Scale bars, 25 μm. The right bar graph summarises %Ki67+ cells in tumours at different ages post-partum. Four to six mice were analysed for each age group as indicated, and more than 3,500 tumour cells per mouse were evaluated. The data represent mean ± SD. Representative H&E staining and Mac2 immunohistochemistry of serial lung sections from 10-week-old BVE mice at low (upper panels) and high (lower panels) magnifications. Scale bars, 100 μm (upper panels) or 25 μm (lower panels). Source data are available online for this figure. Source Data for Figure 1 [emmm201404838-sup-0005-SDataFig1.zip] Download figure Download PowerPoint For the most part, wherever possible, we utilised the CreER™ strain to induce V600EBRAF expression rather than AdCre because of the known inflammatory phenotypes associated with AdCre delivery to the lung, even in wild-type mice (Mainardi et al, 2014). AdCre was only utilised in situations when the analysis required longer-term survival of mice (see below). Immature macrophage-lineage cells expressing WTBRAF are present in the stroma Interestingly, the development of adenomas in both the BVE and Braf+/LSL−V600E/AdCre models was accompanied by the recruitment of non-tumour cells to the stroma (Fig 1A and C). The majority of these cells displayed an oval-shaped morphology with round nuclei and a relatively low nuclear/cytoplasm ratio reminiscent of myeloid-lineage hematopoietic cells (Fig 1C). Stroma-specific staining with a myeloid marker Mac2 also supported their myeloid origin (Fig 1C). We developed a method to separate the stroma cells from the tumour cells (Supplementary Fig S2) and found that the purified stroma cells were identical in morphology to those in histological sections, but rarely displayed indented nuclei or long cytoplasmic processes that are characteristic of monocytes or dendritic cells (DCs), respectively (Figs 1C and 2A). Cell surface marker analysis confirmed the expression of a myeloid marker CD11b at low levels in these cells, but other hematopoietic lineage markers, including Gr1, were not detected (Fig 2B). The lack of hematopoietic surface marker expression on the isolated stroma cells was not due to enzyme treatment during the isolation procedure since collagenase/DNase treatment of wild-type lung did not perturb the detection of CD11bhighGr1high neutrophils, CD11b+Gr1−/lowF4/80+ monocytes, CD3+ or B220+ lymphocytes or TER119+ red blood cells (Supplementary Fig S3). These findings exclude the possibility that the stroma cells could belong to myeloid cell types with the CD11b+Gr1+ phenotype, including neutrophils, inflammatory monocytes (Geissmann et al, 2003) and myeloid-derived suppressor cells (MDSCs) (Kusmartsev & Gabrilovich, 2006). Figure 2. Characterisation of stroma IMCs Giemsa staining of isolated stroma IMCs. These cells show round or oval-shaped nuclei that lack indentations, and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes, suggestive of immature myeloid-lineage cells that are morphologically distinct from monocytes/DCs. Scale bar, 25 μm. Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b, which is expressed at low levels (overlaid with isotype control histograms shown in grey). Cell surface marker analysis (dot plots) of IMCs demonstrating CD11c+ IMCs do not express macrophage (F4/80), DC (CD24) or M1-polarisation (CD86) markers, but weakly express the M2 marker CD206. Insets show isotype control staining. Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines. The cells grown on culture plates were imaged by phase-contrast microscopy (left). Then, the cells were trypsinised and smeared on glass slides for Giemsa staining (right). These cells are intermediate to large sized, round or amoeboid cells with cytoplasmic vacuolation, consistent with a macrophage morphology. Scale bars, 50 μm. Cell surface marker analysis of cultured IMCs showing differentiation into F4/80+ macrophages with CD11bhighCD11c+CD86+CD206low surface phenotype (overlaid with isotype control histograms shown in light grey). PCR detection of Braf recombination in purified IMCs, AT2 cells and tumour cell aggregates. Source data are available online for this figure. Source Data for Figure 2 [emmm201404838-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint In contrast, the stroma cells strongly expressed CD11c, a common marker for lung macrophages and DCs in mice (Misharin et al, 2013), whereas the macrophage marker F4/80 was not expressed (Fig 2C). Although the CD11blowCD11c+F4/80− phenotype was somewhat consistent with CD11b+ DCs, we think this unlikely as CD24, a marker known to be expressed in circulating DC precursors (O'Keeffe et al, 2003) and lung DCs (Misharin et al, 2013) in mice, was also undetectable (Fig 2C). Instead, these cells weakly expressed CD206, a marker for alternative (M2-type) macrophage activation (Gabrilovich et al, 2012), but not CD86, a marker for DC maturation and classical (M1-type) macrophage activation (Gabrilovich et al, 2012) (Fig 2C). Collectively, the stroma cells did not show typical DC phenotypes but shared some characteristics with M2-polarised macrophages. To further characterise the stroma cells, we cultured the isolated cells for 2 weeks without adding cytokines. The cells were efficiently maintained in these culture conditions, presumably because of support from autocrine chemo-cytokines and growth factors (see below), and developed larger-sized adherent cells (Fig 2D). Morphologically, these cells did not show dendritic cytoplasmic processes, but displayed abundant cytoplasm with numerous vacuoles (Fig 2D), consistent with a macrophage rather than DC morphology. Flow cytometry analysis of the cultured stroma cells demonstrated they maintained the CD11c+Gr1− phenotype with up-regulation of CD11b and acquisition of F4/80 expression (Fig 2E), suggesting macrophage differentiation. Although low-level expression of CD206 was maintained in culture, they also acquired CD86 expression (Fig 2E), indicating that macrophage differentiation of the stroma cells in culture was not skewed towards M1 or M2 polarisation. Although we cannot formally exclude the possibility that the stroma myeloid cells are CD117intCD11b-CX3CR1+ macrophage/DC progenitors (MDPs) (Fogg et al, 2006), CD11b expression on the stroma myeloid cells (Fig 2B) makes this unlikely. In all, we ascribed these stroma cells the name immature macrophage-lineage cells (IMCs). PCR genotyping of the purified IMCs confirmed that they contained the unrecombined LSL-V600E allele but not the recombined Lox-V600E allele (Fig 2F), indicating that they solely express WTBRAF and not V600EBRAF, and thus accumulate as a reactive response to the oncogene-expressing lung tumours. Stroma IMCs secrete growth and EMT-promoting factors To explore the biological functions of IMCs, we co-cultured CMT64 mouse lung adenocarcinoma cells (Franks et al, 1976) with purified IMCs. IMCs enhanced the growth of these cells in a dose-dependent manner, and promoted fibroblast-like morphological changes suggestive of epithelial–mesenchymal transition (EMT) (Fig 3A). Some of the IMCs migrated into CMT64 cell sheets to establish cell-to-cell contacts (Fig 3A, right), and therefore, we assessed whether direct cell contacts were required for the phenotype. CMT64 cells cultured with IMC-conditioned media (IMC-CM) recapitulated the phenotypes (Fig 3B), indicating that secreted factors rather than cell-to-cell contacts are involved in this response. The IMC-CM also induced down-regulation of E-cadherin expression in the CMT64 cells as demonstrated by immunofluorescence (Fig 3B), consistent with an EMT-like response. Figure 3. IMCs promote growth and EMT of lung tumour cells CMT64 cells (1 × 104/well in 12-well plates) were co-cultured with 1.5–3 × 105 IMCs for 4 days. Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate. Harvested cells were subjected to counting (left). Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells. The bar graph (left) shows CMT64 growth (cell counting) data pooled from two independent experiments. Phase-contrast images of CMT64 cells cultured without IMCs (CMT64 only) or co-cultured with IMCs (CMT64/IMC co-culture) are also indicated in the middle and right. A higher magnification image of co-cultured CMT64 cells (right) highlights IMCs migrating into CMT64 cells (arrow heads). Scale bars are 200 μm, except for the high magnification image (right) in which the scale bar is 100 μm. (Left) Growth and EMT-like morphological changes of CMT64 cells cultured with 25% IMC-CM. CMT64 cells cultured for 4 days at low-density in DMEM/10%FCS without IMC-CM (-IMC-CM) or with 25% IMC-CM (+IMC-CM) were counted and normalised to the average of control as 100%. The data in the bar chart represent mean + SD of five cultures utilising IMC-CM obtained from three independent sources. (Middle) Phase-contrast images to indicate representative morphologies of CMT64 cells maintained without IMC-CM (control) or cultured with 25% IMC-CM (+IMC-CM) for 2 weeks. (Right) E-cadherin immunofluorescence of CMT64 cells cultured as above. Scale bars are 200 μm (phase contrast) and 10 μm (immunofluorescence). Co-culture of AT2 cells and autologous IMCs from BVE mice (8- to 10-week-old) for 48 h using the Transwell® culture system. In vitro BrdU incorporation (flow cytometry, left and middle) and EMT marker expression (RT–PCR, right) in co-cultured AT2 cells are indicated. Representative flow cytometry plots are indicated on the left, and the bar chart in the middle indicates % BrdU+ AT2 cell numbers normalised to control cultures without IMCs (n = 4, mean + SD). Gapdh serves as a loading control for the RT–PCR on the right. Morphological alterations and intracellular signalling in AT2 cells cultured with IMC-CM. On the left and middle, primary AT2 cells from 3-week-old BVE mice were cultured for 48 h and then incubated in serum-free IMC-CM (AT2 with IMC-CM) or DMEM (AT2 w/o IMC-CM, serving as a control) for another 48 h, followed by immunostaining for E-cadherin (red) and vimentin (green) and confocal laser scanning microscopy (CLSM) imaging. The 3× zoomed image in the middle right highlights dividing vimentin+ cells that show internalised E-cadherin (arrows). Scale bars, 50 μm. On the right, primary AT2 cells from 10-week-old BVE mice were cultured for 48 h, serum starved for 5 h and treated with or without serum-free IMC-CM for 30 min. Phosphorylation of AKT, SMAD3, MEK and ERK were analysed by immunoblotting. Source data are available online for this figure. Source Data for Figure 3 [emmm201404838-sup-0007-SDataFig3.zip] Download figure Download PowerPoint Alveolar type-2 (AT2) cells, the major epithelial cell type in V600EBRAF-driven lung tumours (Dankort et al, 2007), were also freshly isolated from BVE mice at 10 weeks p.p. using the fractionation method (Supplementary Fig S2). Isolated AT2 cells were validated by the presence of lamellar bodies containing pulmonary surfactants visualised by Papanicolau staining and flow cytometry detection of surfactant protein C (SPC) (Supplementary Fig S2C and D). At this stage, AT2 cells were largely growth-arrested (Fig 1B), but the small proliferating pool (~2%) was detected by in vitro BrdU labelling (Fig 3C). When these cells were co-cultured with autologous IMCs using a Transwell culture system, there was an approximate doubling of BrdU+ proliferating cells, and the expression of EMT markers was also up-regulated (Fig 3C). BrdU incorporation into AT2 cells co-cultured with IMCs was higher than those co-cultured with lung fibroblasts (Supplementary Fig S4), indicating that the increased BrdU incorporation in AT2 cells co-cultured with IMCs is unikely to be due to fibroblast contamination. AT2 cells cultured with IMC-CM displayed a more flattened morphology with down-regulation of membranous E-cadherin, accompanied by vimentin-positive fibroblastic cells surrounding the AT2 cell clusters (Fig 3D, middle). Interestingly, mitotic cells expressing vimentin and internalised E-cadherin were also sometimes observed in the IMC-CM cultures (Fig 3D, arrows in the middle right microphotograph), suggesting a potential relationship between EMT and the proliferation induced by the IMC-CM. Consistent with the growth/EMT-promoting effects, IMC-CM induced phosphorylation of AKT and SMAD3 in the primary AT2 cells in vitro, although no effect on the MEK-ERK pathway was detected (Fig 3D). In order to identify the likely secreted factors involved in this phenotype, we subjected IMC-CM to mass spectrometry analysis. This analysis identified more than 50 secreted proteins including growth factors known to promote cell proliferation and EMT (e.g. TGFβ1, PDGFA, CTGF) (Supplementary Table S1). Secretion of TGFβ and PDGFA was further validated by immunoblotting (Supplementary Fig S5A and B). Secreted proteins previously linked with M2 macrophages and MDSCs (chitinase-3-like 3, arginase-1, S100A9) (Gordon, 2003; Ostrand-Rosenberg & Sinha, 2009) were also identified (Supplementary Table S1). These findings consolidate the M2-polarised nature of the IMCs as well as their potential to promote growth/EMT through paracrine mechanisms. CCR1 signalling is required for IMC recruitment and premalignant tumour development In addition to EMT/growth-promoting factors, CC chemokines CCL6, 7 and 9 were identified in the IMC-CM by mass spectrometry (Supplementary Table S1), and secretion of CCL6 was validated by immunoblotting (Supplementary Fig S5C). CCL6, 7 and 9 are known to be ligands for the chemokine receptor CCR1 (Berahovich et al, 2005). We therefore assessed CCR1 expression using immunofluorescence of separated IMCs/AT2 fractions and immunohistochemistry of BVE lung tissue (Fig 4A and B). Together, these data confirm predominant expression of CCR1 in IMCs, some of which is localised at the plasma membrane along with Mac2, and very little, if any, expression in V600EBRAF-expressing AT2 cells. Figure 4. CCR1 inhibition suppresses IMC recruitment and tumour development in vivo CCR1 immunofluorescence of primary IMCs (left, co-stained with Mac2) and AT2 cells (right, co-stained with E-cadherin) detected by CLSM. Scale bars, 10 μm. CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs, but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 (inset in the left microphotograph). CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs. Scale bar, 50 μm. CT imaging of lungs of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (top) or CCR1 inhibitor (bottom). H: heart; * indicates a tumour region accompanied by atelectasis. H&E staining of lung sections from vehicle (left) or CCR1 inhibitor (right) treated AdCre-infected Braf+/LSL−V600E mice. Scale bars, 250 μm (top) or 50 μm (bottom). Flow cytometry analysis of CD11c+ (top, green) and SPC+ (bottom, red) cells in the lung of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (left) or CCR1 inhibitor (right). Lung CD11c+ (left) and SPC+ (right) cell numbers (per left lobe) were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD). % Lung CD4+ (left) and CD8+ (right) T lymphocytes were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD). Source data are available online for this figure. Source Data for Figure 4 [emmm201404838-sup-0008-SDataFig4.xlsx] Download figure Download PowerPoint To investigate the role of CCR1 signalling in IMC recruitment, we treated AdCre-infected Braf+/LSL−V600E mice with the CCR1 inhibitor J-113863 (Amat et al, 2006), initiated at 5 weeks following AdCre induction before overt IMC accumulation appeared in the lung. AdCre delivery was used to induce V600EBRAF expression in this particular experiment rather than intercrossing with the CreER™ strain since the compromised health conditions of the BVE mice, often as early as immediately after weaning, did not allow us to perform consecutive i.p. drug injections. CT imaging and histological analysis after 4 weeks of treatment showed not only the suppression of IMC recruitment but decreased tumour burden (Fig 4C and D), which was further validated by flow cytometry quantification of CD11c+ IMC and SPC+ AT2 cells (Fig 4E and F) and tumour area quantification of histological sections (Supplementary Fig S6). Of note, the majority of the remaining lung CD11c+ cells in inhibitor-treated mice displayed low side-scatter profiles (Fig 4E right), suggesting that IMCs with increased intracellular granularity were mostly depleted from the lung by the inhibitor. These data demonstrate an essential role for CCR1 signalling in t