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The cellular and molecular origin of tumor-associated macrophages

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Science  23 May 2014:
Vol. 344, Issue 6186, pp. 921-925
DOI: 10.1126/science.1252510

Abstract

Long recognized as an evolutionarily ancient cell type involved in tissue homeostasis and immune defense against pathogens, macrophages are being rediscovered as regulators of several diseases, including cancer. Here we show that in mice, mammary tumor growth induces the accumulation of tumor-associated macrophages (TAMs) that are phenotypically and functionally distinct from mammary tissue macrophages (MTMs). TAMs express the adhesion molecule Vcam1 and proliferate upon their differentiation from inflammatory monocytes, but do not exhibit an “alternatively activated” phenotype. TAM terminal differentiation depends on the transcriptional regulator of Notch signaling, RBPJ; and TAM, but not MTM, depletion restores tumor-infiltrating cytotoxic T cell responses and suppresses tumor growth. These findings reveal the ontogeny of TAMs and a discrete tumor-elicited inflammatory response, which may provide new opportunities for cancer immunotherapy.

Origins of tumor macrophages

To help the immune system fight cancer, it is important to understand the origins and functions of immune cells in tumors and the surrounding tissues. One type of immune cells, macrophages, is present both in tumors and in nearby noncancerous tissue, but the relationship between these two cell populations is unclear. Franklin et al. found that tumor-associated macrophages in mouse mammaries differed in form, function, and origin from macrophages found in nearby noncancerous mammary tissue. Moreover, when they removed macrophages from the tumors but not the other mammary tissue, tumors shrank and cytotoxic T cells—another kind of immune cell that kills tumor cells—infiltrated the tumors. Tumor-associated macrophages may thus be an important therapeutic target.

Science, this issue p. 921.

Macrophages are tissue-resident innate immune cells important in homeostasis and host defense against pathogens (1). These functionally diverse phagocytes differentiate from yolk sac–derived embryonic precursors and locally self-renew both during steady state (24) and helminth infection (5). Additionally, bone marrow–derived monocytes give rise to macrophages in the intestine and the dermis (6, 7), as well as during acute infection and inflammation (8). However, the precise ontogeny and function of macrophages in chronic disorders, such as cancer, are incompletely understood (9).

To investigate myeloid cells during cancer progression, we utilized the MMTV-PyMT (PyMT) mammary tumor model (10). Myeloid cells made up more than 50% of CD45+ tumor-infiltrating leukocytes and consisted of three major populations (I, II, and III), distinguishable by morphology and cell surface expression of major histocompatibility complex class II (MHCII), CD11b, Ly6C, Ly6G, CD11c, CD115, and F4/80 (fig. S1). Populations II and III phenotypically resembled Ly6C+ inflammatory monocytes and neutrophils, respectively, while population I expressed classical dendritic cell (DC) markers MHCII and CD11c and the macrophage marker F4/80. Owing to the ambiguity of characterizing cell populations with surface markers (11, 12), we sought to define these cells on the basis of transcriptional phenotype (13). Using principal component analysis of DC and macrophage populations from the ImmGen Project (14, 15), we defined “population I” cells as tumor-associated macrophages (TAMs) because they clustered with macrophage subsets (Fig. 1A). A support vector machine learning algorithm corroborated this classification (fig. S2). Moreover, cells of population I did not express the DC lineage–specific transcription factor Zbtb46 or DC markers c-Kit, CD26, BTLA, and Flt3, but expressed the macrophage transcription factor Mafb and macrophage markers CD64 and MerTK (14,15) (Fig. 1, B and C). Furthermore, Flt3L-deficient PyMT mice, which lack cells of the classical DC lineage (16), showed no defect in population I, confirming a pre-DC–independent origin of TAMs (fig. S3).

Fig. 1 Macrophages constitute the dominant myeloid cell population in mammary tumors.

(A) Principal component analysis of population I: TAM (mammary) and CD11b+ splenic DC (sp) gene expression compared to populations collected by the Immunological Genome Project (GSE15907). Expression data are pooled from two replicate microarray experiments. (B) Expression of Zbtb46 and Mafb mRNA in sorted TAMs and CD11b+ splenic DCs (spDCs) relative to Actb as determined by quantitative polymerase chain reaction (n = 3). (C) Flow cytometric analysis of DC and macrophage (mΦ) signature surface markers expressed on TAMs and CD11b+ spDCs. Data are representative of two independent experiments. (D) Flow cytometric analysis of myeloid cell populations found in wild-type (WT) mammary glands and pooled tumors from 8-, 16-, and 20-week-old PyMT mice. Data are representative of three independent experiments. (E) Flow cytometry of TAM and MTM populations in individual tumors from the same mouse. (F) Pooled data of individual tumors as in (E) from multiple mice (n = 3). Data are shown as mean ± SEM. Dot plots are gated on CD45+ leukocytes.

Macrophages populate mammary tissues during steady state and are required for mammary gland development (17). Upon tumor growth, we observed a decrease in the proportion of MHCIIhiCD11bhi cells found in untransformed wild-type (WT) mammary glands and an increase in TAMs (Fig. 1D). We defined MHCIIhiCD11bhi cells as “mammary tissue macrophages” or “MTMs” because they also phenotypically resembled macrophages (fig. S4). TAM expansion was associated with the growth of individual tumors (Fig. 1, E and F), demonstrating that CD11blo TAMs, but not CD11bhi MTMs, are bona fide tumor-associated macrophages that accumulate with increased tumor burden.

Tissue-resident macrophage expansion or differentiation of macrophages from blood-borne precursors could account for TAM accumulation. To distinguish between these mechanisms, we connected congenically marked PyMT mice using parabiosis (fig. S5A). We observed Ly6C+ inflammatory monocytes, MTMs, and TAMs from both parabionts in developing tumors (fig. S5, B and C), demonstrating that TAMs and MTMs required input from the circulation. The chimerism of inflammatory monocytes and T cells (fig. S4, C and D) was in accordance with published studies (2, 18, 19). This was in contrast to red pulp macrophages, which are maintained independently from monocytes (2) and consequently exhibited minimal chimerism (fig. S5C).

Circulating monocytes are critical progenitors for macrophages (20). To determine whether Ly6C+CCR2+ inflammatory monocytes contributed to TAMs and MTMs, we crossed PyMT mice to Ccr2−/− mice, which exhibit reduced numbers of circulating inflammatory monocytes due to impaired bone marrow egress (21). At 16 weeks, MTMs were significantly reduced in Ccr2−/− PyMT mice (Fig. 2A and fig. S6), implying that MTMs are constitutively repopulated by inflammatory monocytes. Owing to the loss of both the monocyte and MTM populations (Fig. 2A and fig. S6), a concomitant increase in TAMs would be expected if their maintenance was independent of CCR2+ monocytes. However, the TAM percentage in Ccr2−/− PyMT mice was not significantly different compared to controls (Fig. 2A and fig. S6). Similar trends were observed in 20-week-old mice (Fig. 2A and fig. S6), suggesting that inflammatory monocytes contribute to MTMs, and to a lesser extent, TAMs.

Fig. 2 TAMs differentiate from CCR2+ inflammatory monocytes.

(A) Flow cytometric analysis of tumor monocytes (mono), TAMs, and MTMs from 16- and 20- week-old Ccr2+/+ PyMT and Ccr2−/− PyMT mice (n = 5 to 8). Data are pooled from five independent experiments. (B) Flow cytometric analysis of tumor monocytes, TAMs, and MTMs from WT PyMT and CCR2DTR PyMT mice after DT treatment (mice were treated intraperitoneally every 3 days, seven treatments total) (n = 6). Data are pooled from four independent experiments. (C) Ki67 staining in TAMs and MTMs from 16-week-old PyMT mice. Data are representative of four independent experiments. (D) EdU incorporation in MTMs and TAMs from 16-week-old PyMT mice 20 hours after intraperitoneal EdU injection. MTM and TAM populations are first gated on CD45+ cells and then gated as shown in (C). Data are representative of two independent experiments. (E) Surface expression of CD11c and CD11b on transferred CCR2+ bone marrow cells from CCR2GFP mice 5 and 7 days after transfer, gated on total transferred cells. (F) Percentage of total CD45+ leukocytes that are of CCR2GFP donor origin as identified by congenic marker 5, 7, and 11 days after transfer (n = 3 per time point). Data are pooled from three independent experiments. (G) Cell proliferation of transferred cells 11 days after transfer. All comparisons were made using student’s t test, and data are shown as mean ± SEM. Statistical significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001; ns, not statistically significant.

To determine whether inflammatory monocytes were required for TAM maintenance, we generated CCR2DTR PyMT mice expressing diphtheria toxin receptor (DTR) under control of the Ccr2 locus (22). DT treatment resulted in 96% depletion of tumor-associated monocytes (Fig. 2B and fig. S7), compared to 80% depletion in Ccr2−/− mice (Fig. 2A and fig. S6). With this more potent depletion strategy, both MTM and TAM numbers were significantly reduced (Fig. 2B and fig. S7), suggesting that TAMs are derived from CCR2+ monocytic precursors, but require less input from the blood compared to MTMs. We considered that a higher proliferative capacity of TAMs compared to MTMs might account for their differing precursor requirement. Indeed, TAMs expressed higher levels of Ki67 staining and EdU incorporation relative to MTMs (Fig. 2, C and D).

To investigate whether monocytes could differentiate into TAMs in vivo, we transferred CCR2+ bone marrow cells isolated from CCR2GFP reporter mice (23) into congenically marked CCR2DTR PyMT mice depleted of endogenous monocytes. At days 5, 7, and 11 after transfer, we observed transferred cells in developing tumors (fig. S8A). Five and 7 days after transfer, we detected up-regulation of F4/80, CD11c, and MHCII and down-regulation of Ly6C and CD11b on the transferred cells (Fig. 2E and fig. S8B). Additionally, transferred cells expanded and were Ki67+ 11 days after transfer (Fig. 2, F and G). Transfer of hematopoietic stem cell–depleted CCR2+ bone marrow monocytes (CCR2+Flt3c-Kit-) provided similar results (fig. S9). Collectively, these observations demonstrate that tumor growth induces the differentiation of CCR2+ monocytes into TAMs.

We performed gene-expression profiling to further distinguish TAMs from MTMs. As expected, the integrin CD11b (Itgam) was expressed at lower levels in TAMs than in MTMs (Fig. 3A). However, several other integrins and the integrin receptor Vcam1 were up-regulated in TAMs (Fig. 3A). “M2” or alternatively activated macrophages (AAMs) have been proposed to be associated with tumor progression (24). Surprisingly, we found that the TAM population did not express AAM markers such as Ym1, Fizz1, and Mrc1; instead, MTMs more closely resembled AAMs (Fig. 3A and fig. S10). In line with the expression data, Vcam1 and Mrc1 (CD206) proteins were detected in TAMs and MTMs, respectively (Fig. 3B). Combining these markers with our monocyte transfer system, we addressed whether TAMs differentiated from MTMs. The lack of Mrc1 expression on transferred monocytes at all examined time points suggested that TAM differentiation from monocytes was a distinct pathway, rather than MTM conversion (Fig. 3C). Additionally, we detected Vcam1 up-regulation on TAMs as a late differentiation event (Fig. 3C). Altogether, we identified sequential phenotypic changes in monocytes during TAM differentiation (fig. S11).

Fig. 3 TAMs are not phenotypically AAMs and can be identified by Vcam1 expression.

(A) Gene expression of sorted TAMs and MTMs from 16-week-old PyMT mice. Data are pooled from three replicate microarray experiments (two MTM samples and three TAM samples). Differentially expressed genes were determined with a P-value threshold of 0.05. Fold change is depicted with a log2 scale. (B) Flow cytometry of Vcam1 and Mrc1 expression on TAMs and MTMs. Data are representative of more than five independent experiments. (C) Mrc1 and Vcam1 expression on transferred CCR2+Flt3c-Kit bone marrow monocytes and their progenies at 5, 7, and 11 days after transfer into DT-treated CCR2DTR PyMT recipients (n = 2 mice per time point). Mrc1 and Vcam1 expression on MTMs or TAMs, respectively, are shown for comparison.

The finding that TAMs did not resemble AAMs was unexpected, because the type 2 cytokine interleukin-4 (IL-4) produced by T cells and/or tumor cells has been implicated in TAM polarization (25, 26). Additionally, in other M2-polarizing environments, IL-4 is crucial for the expansion of tissue-resident macrophage populations (5). However, we found that Il4−/− PyMT mice had normal proportions of CD11bloVcam1+ TAMs (fig. S12, A to C). Furthermore, TAM differentiation was intact in the absence of lymphocytes (fig. S12, D to F). These observations suggest that TAMs are not AAMs, and their differentiation is not secondary to tumor-elicited adaptive immune responses.

While investigating the mechanisms of TAM differentiation, we observed that TAMs display a gene expression signature (27) associated with the Notch signaling pathway (fig. S13). Notch signaling is a conserved developmental pathway instrumental in hematopoietic cell fate specification (28). In DCs, canonical Notch signaling mediated by the key transcriptional regulator RBPJ controls lineage commitment and terminal differentiation (27, 29, 30). To explore whether Notch signaling played a role in TAM differentiation, we used CD11ccre mice that efficiently deleted floxed DNA sequences to a greater extent in TAMs than MTMs, but not in monocytes or neutrophils (fig. S14). CD11ccreRbpjfl/fl PyMT mice exhibited a selective loss of MHCIIhiCD11blo TAMs (Fig. 4A). However, a MHCIIhiCD11bhi population still remained (Fig. 4A and fig. S15, A and B). Transcriptional profiling comparing this population to WT TAMs confirmed a loss of the Notch-dependent program in RBPJ-deficient cells (fig. S15C). MHCIIhiCD11bhi macrophages in CD11ccreRbpjfl/fl PyMT mice did not express the TAM marker Vcam1 or the MTM marker Mrc1 in most cells (Fig. 4B), suggesting that the majority are TAM intermediates. Moreover, we observed increased Ly6CMHCII–/lo TAM precursors (fig. S11) in CD11ccreRbpjfl/fl PyMT mice compared to WT PyMT mice (fig. S15, D and E). These data reveal that in the absence of RBPJ, inflammatory monocytes are unable to terminally differentiate into TAMs.

Fig. 4 RBPJ-dependent TAMs modulate the adaptive immune response.

(A) Flow cytometric data of myeloid populations in 20-week-old CD11ccre Rbpjfl/fl PyMT and WT PyMT mice. Plots are gated on CD45+B220 cells. Data are representative of more than five independent experiments. (B) Flow cytometry of Vcam1 and Mrc1 expression on TAMs and MTMs from WT PyMT mice and MHCII+ cells from CD11ccre Rbpjfl/fl PyMT mice. Data are representative of three independent experiments. (C) Total tumor burden of WT PyMT and CD11ccreRbpjfl/fl PyMT mice at 16 and 20 weeks of age (n = 13 to 14). (D) Flow cytometric analysis of GzmB and PD-1 expression in CD8+ T cells infiltrating PyMT tumors at 8, 16, and 20 weeks. Data are representative of three independent experiments. (E) Quantification of TAMs (gated on CD45+ cells) and PD-1+CD8+ T cells (gated on CD45+TCRβ+CD8+ cells) from 8-, 16-, and 20-week-old PyMT mice (n = 3 per time point). (F) PD-1 and GzmB expression in CD8+ T cells from WT PyMT or CD11ccreRbpjfl/fl PyMT mice. Data are representative of more that five independent experiments. (G) Quantification of PD-1+ or GzmB+ CD8+ T cells as in (F) (n = 8 to 12). Results represent pooled data and are shown as mean ± SEM. Student’s t test was performed and statistical significance is indicated by **P < 0.01.

The Mrc1+ cells found within the CD11bhi population in CD11ccreRbpjfl/fl PyMT mice (Fig. 4B) indicated that MTM differentiation was not compromised. To address the specificity of this RBPJ-dependent pathway during tumorigenesis, we analyzed non-PyMT mice. As expected, MHCIIhiCD11bhi MTMs from WT and CD11ccreRbpjfl/fl mammary glands expressed Mrc1 (fig. S16). MHCIIhiCD11blo myeloid cells were present in mammary glands from WT mice (Fig. 1D). However, these cells did not express Vcam1 (fig. S16A), and their differentiation was not affected in CD11ccreRbpjfl/fl mice (fig. S16B), suggesting that they are distinct from MHCIIhiCD11blo TAMs.

Associated with the TAM differentiation defect, CD11ccreRbpjfl/fl PyMT mice had reduced tumor burden (Fig. 4C). In Ccr2−/− PyMT mice, which have reduced MTMs (Fig. 2A and fig. S6), tumor development was unaffected (fig. S17A), implying a nonredundant function for RBPJ-dependent TAMs in promoting tumor growth. In the PyMT model, CD11c+ myeloid cells act as antigen-presenting cells, forming stable, yet unproductive, interactions with tumor-infiltrating T cells (12). We hypothesized that one tumor-promoting function of TAMs may be their control of the adaptive immune response. Granzyme B (GzmB) is a cytolytic molecule important for tumor immunosurveillance. Conversely, Programmed Death-1 (PD-1) is an inhibitory co-receptor denoting “exhausted” T cells. As PyMT tumors progressed, an increase in PD-1+GzmBCD8+ T cells was observed (Fig. 4D), paralleling TAM expansion, with PD-1+ cells making up ~50% of late-stage tumor-infiltrating CD8+ T cells (Fig. 4E). In CD11ccreRbpjfl/fl PyMT mice, the PD-1+ population decreased, whereas the GzmB+ population increased (Fig. 4, F and G). In contrast, the T cell phenotype in Ccr2−/− mice was unchanged (fig. S17, B to D). Classical DCs also delete RBPJ in the CD11ccreRbpjfl/fl system, so we examined Flt3L-deficient PyMT mice that lack cells of the DC lineage, but have a comparable TAM population (fig. S3). Flt3l−/− PyMT mice showed no difference in tumor growth or CD8+ T cell phenotype (fig. S17, E to H). These data suggest a specific function for RBPJ-dependent TAMs in promoting tumor immune tolerance in part by modulating the CD8+ T cell response (fig. S18).

Other RBPJ-dependent TAM functions, including nonimmune regulatory roles, require further investigation. In addition, TAM differentiation is not completely abolished in the absence of RBPJ, raising the possibility that the remaining TAM intermediates may have tumor-promoting activities. Furthermore, to what extent TAM differentiation from monocytes occurs in other murine and in human tumors remains to be determined. Nonetheless, our findings suggest that a better understanding of this distinct tumor-elicited inflammatory response may create new opportunities for cancer treatment.

Supplementary Materials

www.sciencemag.org/content/344/6186/921/suppl/DC1

Materials and Methods

Figs. S1 to S18

References (3133)

References and Notes

  1. Acknowledgments: We thank T. Honjo for the Rbpjfl/fl mouse strain and B. Reizis for the CD11ccre mouse strain. We also thank J. Joyce and the M. Li lab for their insightful discussions. The data presented in this paper are tabulated in the manuscript and in the supplementary materials (Gene Expression Omnibus accession no. GSE56755). MSKCC has filed a provisional patent application with the U.S. Patent and Trademark Office (application no. 61/935,318).  The application is directed toward methods and compositions for targeted cancer therapy and to identify potential responders to targeted therapy based on the presence of specific tumor-associated macrophages. R.A.F and M.O.L are listed as inventors on this patent application. This work was supported by the Cancer Research Institute Tumor Immunology Predoctoral Fellowship Training Grant (R.A.F), NIH grant AI101251 (K.L.), Cancer Research Institute Clinic and Laboratory Integration Program Grant (M.O.L.), and the American Cancer Society Research Scholar Award (M.O.L.).
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