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. Author manuscript; available in PMC: 2018 Jan 4.
Published in final edited form as: Cell Rep. 2017 Dec 26;21(13):3662–3671. doi: 10.1016/j.celrep.2017.12.011

Tumor-associated macrophages derived from circulating inflammatory monocytes degrade collagen through cellular uptake

Daniel Hargbøl Madsen 1,3,4,*, Henrik Jessen Jürgensen 1,4, Majken Storm Siersbæk 5, Dorota Ewa Kuczek 3, Loretta Grey Cloud 1, Shihui Liu 1, Niels Behrendt 4, Lars Grøntved 5, Roberto Weigert 2,6, Thomas Henrik Bugge 1,7,*
PMCID: PMC5753792  NIHMSID: NIHMS926597  PMID: 29281816

Summary

Physiologic turnover of interstitial collagen is mediated by a sequential pathway, in which collagen is fragmented by pericellular collagenases, endocytosed by collagen receptors, and routed to lysosomes for degradation by cathepsins. Here, we used intravital microscopy to investigate if malignant tumors, which are characterized by high rates of extracellular matrix turnover, utilize a similar collagen degradation pathway. Tumors of epithelial, mesenchymal, or neural crest origin all displayed vigorous endocytic collagen degradation. The cells engaged in this process were identified as tumor-associated macrophage (TAM)-like cells that degraded collagen in a mannose receptor-dependent manner. Accordingly, mannose receptor-deficient mice displayed increased intra-tumoral collagen. Whole transcriptome profiling uncovered a distinct extracellular matrix-catabolic signature of these collagen-degrading TAMs. Lineage-ablation studies revealed that collagen-degrading TAMs originated from circulating CCR2+ monocytes.

The study identifies a function of TAMs in altering the tumor microenvironment through endocytic collagen turnover and establishes macrophages as centrally engaged in tumor-associated collagen degradation.

Keywords: Tumor-associated macrophages, Extracellular matrix remodeling, Cathepsins, Collagen endocytosis, Tumor microenvironment, Collagenases, CCR2-derived TAMs, Cancer invasion, M2-polarized macrophages, Endocytic matrix turnover

eTOC blurb

Madsen et al. identify a population of tumor-associated macrophages with a distinct matrix catabolic signature as key effectors of collagen turnover during invasive tumor growth. These matrix-degrading macrophages largely are derived from CCR2+ monocytes reprogrammed by the tumor microenvironment and degrade collagen through mannose receptor-dependent cellular uptake.

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Introduction

Invasive tumor growth causes loss of tissue architecture and organ failure (Danø et al., 1985; Liotta et al., 1983; Sloane and Honn, 1984). It involves extensive degradation of interstitial collagen, which is the most abundant extracellular matrix (ECM) component. ECM degradation allows for physical expansion, and facilitates tumor growth by growth factor release from the ECM (Hynes, 2009). ECM degradation is often accompanied by deposition of a tumor-supporting ECM with altered stiffness, composition and organization (Kaukonen et al., 2016; Lu et al., 2012; Naba et al., 2014; Schedin and Keely, 2011). Interstitial collagen is a dense network of collagen fibers stabilized by multiple inter- and intramolecular crosslinks (Linsenmayer, 1991; Ricard-Blum and Ville, 1989), forming a supramolecular structure refractory to all but a handful of proteolytic enzymes of the matrix metalloproteinase (MMP) family. These collagenases cleave collagen at a single site (Aimes and Quigley, 1995; Birkedal-Hansen, 1987; Freije et al., 1994; Hasty et al., 1987; Hotary et al., 2000, 2006; Knäuper et al., 1996; Netzel-Arnett et al., 2002; Stricklin et al., 1977) to release large collagen fragments that are internalized through the engagement of endocytic receptors of the mannose receptor (MR) family and collagen-binding β1-integrins. The internalized collagen fragments are routed to lysosomes and degraded by lysosomal cathepsins (Engelholm et al., 2003; Everts et al., 1985; Everts and Beertsen, 1992; Kjøller et al., 2004; Lee et al., 2006, Lee et al., 1996; Madsen et al., 2007, 2011, 2012, 2013; Martinez-Pomares et al., 2006).

Tumor-associated macrophages (TAMs) are the most abundant leukocytes in tumor stroma. TAMs derive from tissue-resident macrophages or from recruited inflammatory monocytes conditioned by the tumor microenvironment (Franklin et al., 2014; Lahmar et al., 2016; Noy and Pollard, 2014), and the density of TAMs correlates with prognosis (Biswas et al., 2013; Zhang et al., 2012).

We recently established an imaging assay that enables the visualization of cellular collagen degradation in situ (Madsen et al., 2013). Here we used this assay to study endocytic turnover of interstitial collagen in the tumor microenvironment of syngrafted and genetically-induced mouse cancers. Irrespective of tumor type, endocytic interstitial collagen turnover was vigorous and mediated by cells phenotypically resembling TAMs. The cells expressed high levels of M2-markers, and were dependent on the MR for endocytic collagen degradation. Transcriptomic profiling of FACS-isolated collagen-endocytosing TAMs revealed a distinct ECM-catabolic signature, characterized by high expression of ECM-degrading enzymes and low expression of ECM proteins. Collagen-endocytosing TAMs mostly were derived from CCR2-positive inflammatory monocytes recruited to the tumor microenvironment. The study establishes TAMs as mediators of tumor-associated endocytic collagen degradation.

Results

The endocytic pathway for collagen turnover is highly active in solid tumors

Acid-extracted fluorescence-labeled rat tail collagen fibrils spontaneously form collagen fiber networks when placed in connective tissue. The fate of this collagen can be followed by two-photon or confocal microscopy (Madsen et al., 2013). We injected fluorescent collagen and dextran, a lysosomal marker, into subcutaneous Lewis lung carcinoma (LLC) growing in syngeneic mice, and, separately, into adjacent dermis. The mice carried a Col1a1-GFP transgene allowing for identification of cancer-associated and dermal fibroblasts. Multiple cells endocytosing collagen were seen in the tumors 24 h after collagen injection, showing that the endocytic collagen degradation pathway is operative within the tumor microenvironment (Figure 1A). These collagen-endocytosing cells also endocytosed large amounts of dextran and were not fibroblasts, as determined by their lack of GFP expression (Figure 1A).

Figure 1. Tumors are infiltrated by high-level collagen-endocytosing M2-polarized macrophages.

Figure 1

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(A-B) Projections of subcutaneous LLC (A) or adjacent dermal tissue (B) 24 h after injection of fluorescent collagen and dextran into Col1a1-GFP reporter mice expressing GFP in fibroblasts. (C) Quantification of high-level collagen-endocytosing cells in tumor (red bar, examples in A) or in adjacent dermis (green bar, example in B). N=3 mice (3 z-stacks per mouse). Error bars, standard deviations (S.D.). *, P < 0.01. (D and E) Tumor tissue 24 h after injection of fluorescent collagen into dTomato-expressing LLC (D) and dTomato-expressing T241 fibrosarcoma (E). Examples of high-level collagen endocytosing cells are indicated with arrows in A, B, D, and E. (F and G) LLC in situ stained for CD11b (F) or MR (G) 24 h after collagen and dextran injection. Arrows show high-level collagen-endocytosing cells positive for CD11b (F) or MR (G). (H-J) Flow cytometry analysis of single cell suspensions of a LLC for CD11c expression by collagen- and dextran- endocytosing cells (top right gate) or collagen- and dextran-non-endocytosing cells (bottom right gate). (G) Percent dextran- and collagen-endocytosing cells (green bars) or dextran- and collagen-non-endocytosing cells (red bars) from tumor single cell suspensions positive for the indicated surface-markers based on flow cytometry analysis exemplified in H-J. N=4 mice, error bars, S.D.

Serial z-stack analysis showed that collagen-endocytosing cells were far more abundant in tumor tissue than in adjacent dermis (Figure 1A and B, quantified in Figure 1C). This is consistent with the high rate of collagen turnover in neoplastic tissue, as compared to normal tissue (Jones and De Clerck, 1982; Lu et al., 2012). High level collagen- and dextran-endocytosing cells were also abundant in syngeneic B16BL6 melanoma of neural crest origin (Figure S1A) and T241 fibrosarcoma of mesenchymal origin (Figure S1B), as well as in MMTV-PymT transgene-induced mammary adenocarcinomas (Figure S1C). In all tumors, collagen-endocytosing cells had identical morphology, and were not tumor-associated fibroblasts, as shown by the lack of GFP expression (Figure 1A and S1A-C). Taken together, this shows that the endocytic collagen degradation pathway is highly active in solid tumors, irrespective of tumor cell origin, and that it is executed by a tumor microenvironment-abundant population of cells different from tumor-associated fibroblasts.

Macrophages mediate endocytic collagen degradation in tumors

To identify cells mediating endocytic collagen degradation, we made LLC cells stably expressing dTomato fluorescent protein, and formed tumors in syngeneic Col1a1-GFP mice, to simultaneously visualize tumor cells and tumor-associated fibroblasts. Surprisingly, when tumors were imaged 24 h after collagen injection, high-level collagen endocytosing cells were neither tumor cells nor tumor-associated fibroblasts (Figure 1D). An absence of collagen endocytosis by tumor cells and by tumor-associated fibroblasts was also observed when the experiment was performed using dTomato-expressing T241 fibrosarcoma cells (Figure 1E). Aligned with this, collagen-endocytosing cells expressed the macrophage marker CD11b, and the M2-macrophage marker, MR, as shown by in situ immunostaining of LLC tumors (Figure 1F-G), indicating that collagen-endocytosing cells are TAMs.

To extend these findings, we analyzed single cell suspensions from subcutaneous LLC injected with fluorescent collagen and dextran by flow cytometry (Figure 1H-K). Consistent with the in situ immunostaining, most collagen- and dextran-internalizing cells expressed the pan-macrophage marker F4/80. Interestingly, these cells also frequently expressed the dendritic cell markers CD11c and MHCII, which were expressed infrequently on collagen- and dextran non-endocytosing cells (Figure 1K). This resemblance of TAMs to dendritic cells is aligned with recent findings (Lohela et al., 2014).

Collagen-endocytosing cells are M2-like TAMs with a distinct matrix catabolic signature

We further characterized cells mediating endocytic collagen degradation in tumors using markers of macrophage polarization. For this, we generated subcutaneous LLC, injected fluorescent collagen, prepared single cell suspensions 24 h later, and isolated CD45-positive and CD11b-positive cells endocytosing high levels of collagen and dextran by FACS (Figure 2A). For comparison, we FACS-isolated CD45-positive and CD11b-positive cells not endocytosing dextran and collagen (Figure 2B). qPCR analysis of collagen- and dextran-endocytosing cells showed high expression of the M2-macrophage markers, Mrc1 (encoding MR), Retnla (encoding Fizz1), and Arg1 (encoding arginase), and, correspondingly, a low expression of the M1-macrophage marker, Nos2 (encoding iNOS) (Figure 2C).

Figure 2. A distinct ECM-catabolic transcriptomic signature of collagen-endocytosing TAMs.

Figure 2

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(A-B) Post-sorting flow cytometry analysis of single cell suspensions of LLC FACS-sorted into CD45+;CD11b+ cells endocytosing dextran and collagen (A) or not endocytosing dextran and collagen (B). (C) qRT-PCR analysis for M2-polarization markers (green bars) and an M1-polarization marker (blue bar). Data were generated by normalizing the expression level of dextran- and collagen-endocytosing CD45+;CD11b+ cells to dextran- and collagen-non-endocytosing CD45+;CD11b+ cells. N=3, error bars, S.D. (D-E) Flow cytometry dot-plots of single cell suspensions of LLC. Cells were FACS-sorted into dTomato-expressing LLC cells, GFP-expressing fibroblasts (D) and F4/80-positive cells endocytosing fluorescently labeled collagen (E). (F) Principal component analysis of each biological RNseq replicate from total tumor RNA, isolated LLC cells, fibroblasts, or collagen-endocytosing TAMs as shown in (D-E). (G-H) Heat maps of normalized (z-score) RNAseq read counts of genes encoding collagens, proteoglycans, and glycoproteins (G), cathepsins, MMPs, and fibrinolytic factors (Plasminogen activation system) (F).

TAMs may contribute to both ECM turnover and to the construction of a tumor-specific ECM by secreting ECM components (Afik et al., 2016). To further define collagen-endocytosing TAMs, we generated subcutaneous LLCs using dTomato fluorescent protein-expressing LLC cells in Col1a1-GFP mice, followed by injection of fluorescent collagen to enable the FACS-isolation of cancer cells, tumor-associated fibroblasts, and collagen-endocytosing TAMs (Fig 2D-E). Each of these cell populations, as well as tumor cell suspensions prior to FACS-isolation, was then profiled by RNAseq. The full data set is in Table S1. RNAseq dataset, Related to Figure 2. Principal component analysis showed clear separation of the three cell types, with the total tumor sample placed closest to the LLC cells (Figure 2F). RNAseq data confirmed the expression of M2-macrophage markers identified by imaging, flow cytometry, and qRT-PCR, and also verified that the sorted dTomato- and GFP-expressing cells were indeed cancer cells and fibroblasts, respectively (Figure S2).

Interestingly, collagen-endocytosing TAMs differed from TAMs described by Afik et al. by low expression of collagen genes, including fibril-forming collagens, basement membrane collagens, and fibril-associated collagens with interrupted triple helices, proteoglycans, and other ECM glycoproteins, which were expressed at much higher levels in tumor-associated fibroblasts (Figure 2G). Collagen-endocytosing TAMs expressed high levels of cathepsins, which is consistent with high lysosomal degradation capacity (Figure 2H). Collagen-endocytosing TAMs also expressed the highest levels of the genes encoding the collagenases MMP-8, and MMP-13, as well as high levels of the genes encoding the gelatinase, MMP-9, and the elastase, MMP-12. Genes encoding MMP-3, and the collagenases, MMP-2 and MMP-14, were expressed at higher levels in tumor-associated fibroblasts (Figure 2H). Finally, genes encoding fibrinolytic proteins were mostly expressed by either tumor-associated fibroblasts or the collagen-endocytosing TAMs (Figure 2H).

Collagen-endocytosing TAMs originate from CCR2+ monocytes

To determine the origin of collagen-degrading cells in tumors, we used a mouse strain that expresses the human diphtheria toxin (DT) receptor under control of the Ccr2 promoter (CCR2-depleter+/0 mice). This strain was crossed to Ccr2+/RFP mice, which carry a RFP reporter gene inserted into the endogenous Ccr2 gene, to generate Ccr2+/RFP;CCR2-depleter+/0 bi-transgenic mice and Ccr2+/RFP littermates. This allowed for selective depletion of CCR2+ cells from tumors by the administration of DT and a simultaneous monitoring of depletion efficacy using flow cytometry. Accordingly, these mice were inoculated with LLC and treated with DT. Flow cytometry analysis of single cell suspensions of tumors showed that CCR2+ cells constituted 21 % of all cells in the tumors of DT-treated Ccr2+/RFP mice (Figure 3A), and that DT-treatment of Ccr2+/RFP;CCR2-depleter+/0 littermates resulted in near complete ablation of CCR2+ cells (Figure 3B). Analysis of single cell suspensions from collagen-injected LLC tumors growing in DT-treated Ccr2+/RFP mice also revealed that about half of the collagen-endocytosing cells in tumors were CCR2+ and about half were CCR2− (Figure 3C). As expected, the CCR2+ collagen-endocytosing cells were essentially absent in DT-treated Ccr2+/RFP;CCR2-depleter+/0 bi-transgenic mice (Figure 3D). Interestingly, CCR2− collagen-endocytosing cells were also highly reduced after the CCR2 expression-dependent depletion (compare Figure 3C and D, quantified in Figure 3E), suggesting that most collagen-endocytosing CCR2− cells in tumors are derived from CCR2+ tumor-recruited monocytes.

Figure 3. Collagen-endocytosing M2-TAMs originate from CCR2+ monocytes.

Figure 3

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LLC growing in Ccr2+/RFP (A and C) or Ccr2+/RFP;CCR2-depleter+/0 mice (B and D) treated with DT during tumor growth. Ccr2+/RFP mice are DT-insensitive, whereas DT depletes CCR2+ cells in Ccr2+/RFP;CCR2-depleter+/0 mice. (A and C) Flow cytometry analysis of single cell suspensions of LLC from Ccr2+/RFP mice shows that about 20% of cells in LLC are CCR2+ (A) and the majority of collagen-endocytosing TAMs (CD45+;CD11b+ cells) are CCR2+ (C). (B and D) Flow cytometry analysis of LLC from DT-treated Ccr2+/RFP;CCR2-depleter+/0 bi-transgenic mice demonstrates efficient depletion of CCR2+ cells (B) and removal of the vast majority of all collagen-endocytosing M2-TAMs (D). (E) Collagen-endocytosing M2-TAMs (%) in LLC from DT-treated Ccr2+/RFP mice (red bar, n=3) or Ccr2+/RFP;CCR2-depleter+/0 mice (grey bar, n=3). Error bars, S.D. *, P < 0.01.

MR mediates endocytic collagen degradation by TAMs

In situ immunostaining (Figure 1G) and transcriptomic profiling of collagen-endocytosing cells (Figure 2C) revealed a high expression of MR, a key collagen endocytosis receptor (Madsen et al., 2013; Martinez-Pomares et al., 2006). To determine the role of MR in endocytic collagen degradation in tumors, we established subcutaneous LLC in syngeneic wildtype (Mrc1+/+) mice and MR-deficient (Mrc1−/−) littermates, and injected fluorescent collagen into the tumors. Analysis of tumors growing in Mrc1+/+ mice showed numerous cells endocytosing collagen when analyzed 24 h later (Figure 4A). In contrast, we found very few collagen-endocytosing cells in tumors growing in Mrc1−/− littermates (Figure 4B). To further analyze the role of MR in tumor-associated collagen degradation, we affinity purified CD45-positive cells from collagen-injected LLC growing in Mrc1+/+ and Mrc1−/− mice and analyzed the macrophage populations by flow cytometry (Figure 4C and D). Consistent with the image analysis, the fraction of CD45+, CD11b+, F4/80+ cells endocytosing high levels of collagen was dramatically reduced by loss of MR (Figure 4E). In further support of a pivotal role of MR in endocytic collagen turnover, when CD45+,CD11b+ cells from LLC were sorted into MR-positive and MR-negative cells and cultured with fluorescently labeled collagen, MR-positive cells endocytosed 11-fold more collagen than MR-negative cells (Figure 4F-G). Next, we followed the growth of subcutaneous LLC in MR-deficient mice and wildtype littermates. The tumors grew slightly slower in MR-deficient mice (Figure 4H), although the difference did not reach statistical significance. When analyzing the collagen content of the tumors, we observed a striking 2.7-fold increase in collagen in tumors of MR-deficient mice (Figure 4I-J).

Figure 4. MR mediates collagen endocytosis by M2-TAMs.

Figure 4

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(A-B) Images of collagen-injected LLC from Mrc1+/+ mice (A) or Mrc1−/− littermates (B). (C and D) Flow cytometry analysis of collagen endocytosis by CD45+; F4/80+ cells from LLC grown in Mrc1+/+ mice (C) or Mrc1−/− littermates (D). (E) Percentage of CD45+;CD11b+;F4/80+ cells from Mrc1+/+ mice (black bar, n=3) and Mrc1−/− littermates (grey bar, n=3) with high collagen endocytosis based on flow cytometry analyses as exemplified in C and D. Error bars, S.D. *, P < 0.01. (F-G) Single cell suspension of LLC were enriched for CD45-positive cells and sorted into CD11b+;MR− (red) and CD11b+;MR+ (green) cells. Plastic-adhered cells were incubated with Alexa 647-labeled collagen, and analyzed by flow cytometry (F). (G) Flow cytometry quantification. Error bars, S.D. **, P < 0.001. (H) LLC cells were inoculated subcutaneously into Mrc1+/+ mice (blue line) and Mrc1−/− littermates (red line) and tumor sizes were measured from the appearance of the first palpable tumor until the largest tumor had reached the maximal permitted size. Error bars, standard error of the mean. (I) Images from picrosirius red-stained LLC sections from Mrc1+/+ mice (upper panel) or Mrc1−/− littermates (lower panel) showing collagen fibers. Bars: 50 μm. (J) Quantification of picrosirius red (PSR) -positive areas of LLC from Mrc1+/+ mice (blue dots) or from Mrc1−/− littermates (red dots). Data are individual values for each tumor, as well as mean values and standard error of the means. *, P < 0.05. **, P < 0.001.

Discussion

Tumors are rapidly expanding pseudo-organs that efficiently dissolve existing ECM structures. Accordingly, we found here that the endocytic pathway for interstitial collagen degradation is highly active in tumors of multiple different origins, and in all tumors, was executed by cells with characteristics of TAMs. These collagen-degrading TAMs largely originated from circulating CCR2+ monocytes recruited to the tumor microenvironment, and MR was identified as the principal collagen endocytosis receptor on these cells.

TAMs are central orchestrators of a diverse array of processes critical to tumor initiation, propagation, and dissemination. These include suppression of anti-tumor cytotoxic T cells, promotion of tumor cell growth, motility, invasion, intravasation, angiogenesis, and ECM deposition (Afik et al., 2016; Gocheva et al., 2010; Hughes et al., 2015; Lahmar et al., 2016; Lewis et al., 2016; Noy and Pollard, 2014; De Palma et al., 2005). Many of these functions are ascribed to the M2-polarization of TAMs, and therapeutic approaches aimed at driving TAMs toward M1-polarization have shown promise in mouse models of cancer (Georgoudaki et al., 2016; Pyonteck et al., 2013).

Recently, it was reported that CCR2+ monocyte-derived TAMs were involved not only in ECM turnover, but also in the deposition of a tumor-specific ECM that stimulates tumor progression (Afik et al., 2016). This included the synthesis of ECM components, including collagens. In our transcriptomic comparison of carcinoma cells, fibroblasts, and collagen-endocytosing TAMs from subcutaneous LLC, we, however, observed very little contribution from TAMs to the generation of a tumor-specific ECM. Tumor-associated fibroblasts were the dominant producers of collagen, proteoglycans, and other ECM glycoproteins, whereas tumor cells expressed high levels of some proteoglycans and a few glycoproteins. Consequently, TAMs may encompass distinct subpopulations involved in ECM degradation and synthesis, respectively.

IL-4-induced M2 polarization of macrophages within the tumor microenvironment upregulates cysteine cathepsins, which may function both in the lysosome and as secreted enzymes to promote ECM turnover (Olson and Joyce, 2015). Our study shows that collagen-endocytosing TAMs, in addition to high levels of cathepsins, express a range of ECM-degrading enzymes, including collagenases and gelatinases. This implies that endocytic collagen turnover by TAMs may be a cell-autonomous process, in which each step of the collagen catabolic sequence leading to lysosomal collagen degradation is executed by the TAMs. However, our assay, as currently configured, cannot identify the cells executing the initial collagen fragmentation. It follows that other cellular constituents of the tumor microenvironment may actively contribute to TAM-mediated endocytic collagen degradation, for example by aiding the initial collagen fragmentation. In support of this, we found high expression of the collagenase, MMP-2, and its activator (and collagenase) MMP-14, in tumor-associated fibroblasts.

The high expression of the elastase MMP-12 and components of the fibrinolytic system by the collagen-endocytosing TAMs suggests that other prominent constituents of the tumor ECM, such as elastin and fibrin, may also be degraded by TAMs. The degradation of such non-collagenous ECM proteins may also involve endocytosis (Motley et al., 2016).

In conclusion, we show that tumor-associated collagen degradation involves receptor-mediated endocytic uptake and lysosomal degradation of collagen by TAMs displaying a distinct matrix-catabolic signature.

Experimental Procedures

Animal experiments

Work was done in an AAALAC-certified facility with protocols approved by the NIDCR Institutional Animal Care and Use Committee (NIH) or according to institutional guidelines approved by the Animal Experiments Inspectorate in Denmark (Finsen Laboratory/BRIC). See Supplemental experimental procedures for details on animal husbandry and genotyping.

Subcutaneous tumors

Subcutaneous tumors were generated by injection of syngeneic C57BL/6-derived tumor cell lines as described (Bugge et al., 1997). See Supplemental experimental procedures for details.

Histology

Tumors were fixed in zinc-buffered formalin for 24 h, paraffin embedded, sectioned, and stained with picrosirius red. Two central areas of each tumor were selected by light microscopy, and images were acquired using polarization filters. Picrosirius red-positive areas were determined by ImageJ-based threshold-analysis. Acquisition and image analysis was blinded.

Monocyte ablation

Mice were inoculated subcutaneously with 5×105 LLC cells, and received bi-daily intraperitoneal injections of 50 ng/g body weight DT, starting on the day of inoculation and continuing until experimental endpoint 11 days later.

Collagen labelling

Labeling of acid-extracted rat tail tendon type I collagen with fluorescent dyes was performed by modification of a previously described procedure (Birkedal-Hansen et al., 2003). See Supplemental experimental procedures for details.

In vivo collagen endocytosis

Mice were injected intratumorally with 80 μl of 0.4-mg/ml fluorescently labeled collagen and 0.3 mg/ml TMR- or FITC-conjugated 10 kD dextran (Invitrogen) neutralized immediately before injection using Hepes-buffered PBS. 18–24 h later, mice were injected intravenously with 150 μl of 4-mg/ml Hoechst 33342 trihydrochloride trihydrate dye (Invitrogen). Mice were euthanized 4–6 h later, and tumors immediately imaged by confocal microscopy.

Confocal microscopy

Confocal imaging was performed using an (IX81; Olympus) confocal microscope equipped with a scanning head (FluoView 1000; Olympus). All images were acquired using a U Plan S Apochromat 60× numerical aperture, 1.2 water immersion objective. See Supplemental experimental procedures for details.

Quantitation of cellular collagen and dextran uptake

Four z stacks of 27-μm sections were collected for each mouse, each stack at the same depth within the tissue. Total number of cells in each z stack was determined by counting all nuclei stained with Hoechst dye. Final values for cell numbers per mouse were the mean of 4 z stacks.

In situ staining

Tumors were fixed for 4 h in zinc-buffered formalin 24 h after collagen and dextran injection. For MR staining, tumors were permeabilized with 1% Triton X-100 in PBS for 45 min, washed in PBS, 0.5% BSA, 0.2% Triton X-100 (PBS-BT), blocked in PBS-BT, 2% goat serum for ≥1 h, and incubated overnight at 4°C with primary antibody in PBS-BT, 2% goat serum at these dilutions: anti-CD11b (Biolegend) 1:200, and rat anti-MR (AbD Serotec) 1:200. Tumors were washed in PBS-BT and incubated overnight at 4°C with Alexa Fluor 488–conjugated secondary antibodies diluted 1:500 in PBS-BT. After washing in PBS-BT, the tissue was incubated for 1 h with Hoechst diluted 1:10,000 in PBS-BT, washed, and imaged as described in the “Confocal microscopy” section.

FACS and RNA isolation

Alexa 647-collagen and TMR-dextran were injected into LLC as described above. Tumors were harvested 24 h later, and single-cell suspensions prepared. Cells were blocked with FC receptor-blocking reagent and enriched for CD45+ cells using magnetic anti-CD45 microbeads (Miltenyi). Cells were stained with Zombie aqua and anti-CD11b antibody (cl. M1/70, Biolegend) for 1 h on ice, gated for the Zombie aqua-negative and CD11b-positive cell population, and sorted into dextran- and collagen-endocytosing cells and dextran- and collagen-non-endocytosing cells on a FACS Aria II (BD Biosciences). 1–2 × 105 cells of each of the two cell populations were isolated from each sample. In other experiments, Alexa 647-collagen was injected into dTomato-expressing LLC growing in Col1a1-GFP mice. 24 h later, single-cell suspensions were prepared, FC receptor-blocked, and stained with Zombie aqua and anti-F4/80 antibody. Cells were sorted into dTomato+ cells (cancer cells), GFP+ cells (fibroblasts), and dTomato−;GFP−;F4/80+;collagen+ cells (M2-TAMs). RNA was isolated by Trizol (Invitrogen) lysis, phase separation with chloroform, and purification using a Qiagen RNA micro purification kit.

qPCR

100 ng total RNA was used for oligo(dT)-mediated first-strand cDNA synthesis (Superscript III First-Strand Synthesis SuperMix; Invitrogen). Gene expression analysis was performed in cell populations from 3 individual tumors using the real-time PCR detection system (iCycler iQ; Bio-Rad Laboratories) and iQ SYBR Green Supermix (Bio-Rad Laboratories). Primers are in Table S2. Details on PCR and data analysis are in Supplemental experimental procedures.

RNAseq

RNA quality was assessed by Agilent 2100 Bioanalyzer. Total RNA (200 ng) was prepared using polydT-mediated cDNA synthesis according to the manufacturer’s (Illumina) instructions. Libraries were made with a NEBNext RNA library preparation kit for Illumina. Library quality was assessed using Fragment Analyzer (AATI), followed by library quantification (Illumina library quantification kit). Sequencing was done on a HiSeq1500 platform (Illumina) with a read length of 50 bp. Sequencing depth and alignment information is in Table S3. Details on data analysis are in Supplemental experimental procedures.

Ex vivo collagen endocytosis

Single cell suspensions of LLC were enriched for CD45-positive cells and stained with Zombie aqua, anti-CD11b (Biolegend), and anti-MR (Biolegend) antibodies as described above. Cells were sorted into CD11b+;MR+ and CD11b+;MR− cells using a FACS Aria I (BD Biosciences), and plated in 24-well plates (5–9 × 104 cells/well). Alexa 647-labeled collagen was added overnight at a final concentration of 15 μg/ml. Cells were trypsinized, stained with Zombie aqua and analyzed by flow cytometry with a LSRII flow cytometer (BD Biosciences).

Statistics

Significance was tested by unpaired 2-tailed Student’s t-test, assuming independent variables, normal distribution and equal variance of samples. Calculations were made with GraphPad Prism software.

Supplementary Material

1

Table S1. RNAseq dataset, Related to Figure 2.

2

Highlights.

  • -

    CCR2+ monocyte-derived tumor-associated macrophages degrade collagen in tumors

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    Collagen-degrading macrophages display a matrix catabolic transcriptomic signature

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    Tumor-mediated collagen degradation entails cellular uptake and lysosomal routing

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    The mannose receptor mediates cellular collagen uptake by matrix-degrading cells

Acknowledgments

We thank Mary Jo Danton for reviewing this manuscript. Supported by the NIDCR Intramural Research Program (T.H.B.), European Commission/Marie Curie program (D.H.M), Danish Cancer Society (D.H.M., H.J.J.), Region Hovedstadens Forskningsfond (D.H.M.), The Novo Nordisk Foundation (D.H.M. and L.G.), The Danish Council for Independent Research (L.G., H.J.J). We thank Ronni Nielsen, University of Southern Denmark, for assistance with Illumina sequencing. Research was supported by the NIDCR Veterinary Research Core and Combined Technical Research Core (Z numbers DE000729-08, DE000740-05).

Footnotes

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Supplemental information

Supplemental information includes Supplemental Experimental Procedures, 2 figures and 3 tables.

Author Contributions

Conceptualization, D.H.M. and T.H.B; Methodology, D.H.M., L.G., R.W., and T.H.B.; Investigation, D.H.M., H.J.J., M.S.S., D.E.K., L.G.C., S.L., and L.G.; Writing – Original Draft, D.H.M. and T.H.B., Writing – Review & Editing, D.H.M., H.J.J., M.S.S., D.E.K., L.G.C., S.L., L.G., N.B., R.W., and T.H.B.

Accession Numbers

RNAseq data: GSE107053

Declaration of Interests

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Table S1. RNAseq dataset, Related to Figure 2.

NIHMS926597-supplement-1.pdf (1.5MB, pdf)
2
NIHMS926597-supplement-2.xlsx (3.1MB, xlsx)

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