Abstract
Macrophages have an important role in the maintenance of tissue homeostasis1. To perform this function, macrophages must have the capacity to monitor the functional states of their ‘client cells’: namely, the parenchymal cells in the various tissues in which macrophages reside. Tumours exhibit many features of abnormally developed organs, including tissue architecture and cellular composition2. Similarly to macrophages in normal tissues and organs, macrophages in tumours (tumour-associated macrophages) perform some key homeostatic functions that allow tumour maintenance and growth3,4,5. However, the signals involved in communication between tumours and macrophages are poorly defined. Here we show that lactic acid produced by tumour cells, as a by-product of aerobic or anaerobic glycolysis, has a critical function in signalling, through inducing the expression of vascular endothelial growth factor and the M2-like polarization of tumour-associated macrophages. Furthermore, we demonstrate that this effect of lactic acid is mediated by hypoxia-inducible factor 1α (HIF1α). Finally, we show that the lactate-induced expression of arginase 1 by macrophages has an important role in tumour growth. Collectively, these findings identify a mechanism of communication between macrophages and their client cells, including tumour cells. This communication most probably evolved to promote homeostasis in normal tissues but can also be engaged in tumours to promote their growth.
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Acknowledgements
We thank members of the Medzhitov laboratory for discussions, L. Xu, C. Annicelli, S. Cronin and G. Tokmoulina for animal care and technical help, and N. Palm for critical feedback on the manuscript. O.R.C. is supported by the National Cancer Institute (1K08CA172580-01), the Yale Center for Clinical Investigation (5KL2RR024138), the Yale SPORE in Skin Cancer (1 P50 CA121974), the Damon Runyon Cancer Research Foundation (DRG 108-09) and the Dermatology Foundation. R.M.’s laboratory is supported by The Blavatnik Family Foundation, grants from the National Institutes of Health (AI046688, AI089771 and CA157461) and the Howard Hughes Medical Institute.
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O.R.C. and R.M. conceived the project, designed the experimental approach, interpreted data and wrote the manuscript. N.-Q.C. and A.L.S. designed and performed experiments and wrote the manuscript. T.C., A.M.R., V.J., N.C., C.E.B., G.M.P. and G.W.C. designed and performed experiments and analysed data. S.C.E. and A.J.P. designed experiments, analysed data and provided key expertise.
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Extended data figures and tables
Extended Data Figure 1 Tissue density and histological features of TAMs.
a, LLC and B16 tumours excised at day 19 and stained for F4/80 reveal a similar distribution pattern of macrophages at the periphery and within the centre of the tumours. b, Immunohistochemistry of B16 and LLC cells injected subcutaneously and harvested on day 19. Stains are for F4/80 (B16) and CD11b (LLC). c, d, The density of TAMs within tumours is conserved and depends on the tumour type. LLC (n = 10), B16 (n = 10) and CT26 (n = 5) tumours were harvested 19 days after subcutaneous injection. c, The percentage of macrophages that were F4/80+CD11b+ was determined by FACS analysis. (#, P = 0.0128; *P < 0.0001 using a two-tailed, unpaired t-test). The histograms represent the mean ± s.e.m. Whereas an F test revealed no significant difference in the variance between B16 and CT26, there was a significant difference (P = 0.0128) in the variance between B16 and LLC. Non-parametric analysis using the Mann–Whitney test revealed a significant difference in the percentage of macrophages between LLC and B16 tumours (P = 0.0007) and between CT26 and B16 tumours (P = 0.0007). d, FACS plot of B16 and LLC tumours harvested at day 19 after subcutaneous injection and stained for F4/80 and CD11b. e, Cytology of sorted peritoneal macrophages (PMΦ) and TAMs. The cytology is representative of ten different experiments.
Extended Data Figure 2 Tumour-conditioned medium stabilizes HIF1α and induces expression of Vegf and Arg1.
a, Western blotting of HIF1α and actin on cells grown under conditions of normoxia (N; 20.0% O2) or hypoxia (H; 0.1% O2) or stimulated with control DMEM (C) or LLC-tumour-conditioned medium (LLC). b, c, Luciferase reporter assay of 293T cells transfected with a HIF1α oxygen-dependent domain–luciferase reporter (for protein stabilization) and a Vegf promoter–luciferase reporter (for gene expression). d, e, Expression analysis by qPCR of Hif1a I.1, Hif1a I.2, Pgc1a and Pgc1b mRNA in bone-marrow-derived macrophages stimulated with LLC tumour-conditioned medium. f–h, The active factor in tumour-conditioned medium is <3 kDa and is heat stable. Western blotting for ARG1 in bone-marrow-derived macrophages (f). Expression analysis by qPCR of Vegf mRNA in bone-marrow-derived macrophages (g). Expression analysis by qPCR of Vegf and Arg1 mRNA in bone-marrow-derived macrophages stimulated with boiled (100 °C) or non-boiled LLC tumour-conditioned medium (h). i, j, Adenosine and low pH do not induce the Vegf gene. Expression analysis by qPCR of Vegf mRNA in bone-marrow-derived macrophages stimulated as follows: control medium (DMEM), 50 ng ml−1lipopolysaccharide (LPS) plus the adenosine agonist NECA (10 μM 5′-N-ethylcarboxamidoadenosine), LLC-tumour-conditioned medium, and boiled LLC tumour-conditioned medium, all ±1 μM ZM241385 (an adenosine A2A receptor inhibitor) (i); macrophage growth medium titrated to a range of pHs using sterile HCl (j). LLC-tumour-conditioned medium has a pH of 6.8. Where indicated, the relative expression histograms represent three biological replicates displayed as the mean ± s.e.m.
Extended Data Figure 3 The splice isoform PKM2 is the predominant isoform expressed in tumour cell lines that produce high concentrations of lactic acid in vitro and in vivo.
a, b, Expression analysis by qPCR of Pkm1 and Pkm2 mRNA in tissues from wild-type mice and from four tumour cell lines, normalized to Actb and Rpl13a as housekeeping genes. c, Expression analysis by qPCR of Pkm1 and Pkm2 mRNA in tissues from wild-type mice and from six tumour cell lines. qPCR results were normalized to the housekeeping gene Rpl13a. d, e, The in vivo intratumoral lactate levels in LLC and B16 tumours correspond to the concentrations that have been determined to activate macrophages in vitro. Lactate concentration (mM) in control (DMEM) or LLC-tumour-conditioned medium (unfractionated, <3-kDa fraction and >3-kDa fraction). Most of the lactic acid is present in the <3-kDa fraction (d). The mean in vivo lactic acid concentrations in LLC and B16 tumours were measured using hydrophilic interaction chromatography and mass spectroscopy (e). All relative expression histograms represent three biological replicates displayed as mean ± s.e.m. BAT, brown adipose tissue; WAT, white adipose tissue.
Extended Data Figure 4 Lactic acid induces Vegf at 6 h and Arg1 at 24 h in bone-marrow-derived macrophages.
a, Expression analysis by qPCR of Vegf mRNA in bone-marrow-derived macrophages stimulated with LLC-tumour-conditioned medium at 0 h, 1 h, 6 h and 24 h. b, Expression analysis by qPCR of Arg1 mRNA in bone-marrow-derived macrophages cultured under normoxic conditions (20% O2), hypoxic conditions (0.1% O2) and with 25 mM lactic acid, at 6 h and 24 h. c, Time course of Vegf and Arg1 induction by lactic acid (25 mM), hypoxia (0.1% O2) and lactic acid plus hypoxia in bone-marrow-derived macrophages. Expression analysis by qPCR of Vegf and Arg1 mRNA in wild-type (WT) or Hif1a knockout (KO) bone-marrow-derived macrophages at 0 h, 6 h, 24 h and 48 h. Where indicated, the relative expression histograms represent three biological replicates displayed as mean ± s.e.m.
Extended Data Figure 5 Neither lactic acid nor hypoxia induces foamy cell morphology in peritoneal macrophages.
Wild-type (WT) or Hif1a knockout (KO) peritoneal macrophages were plated in control medium (DMEM) or stimulated with lactic acid (25 mM) or hypoxia (0.1%) for 24 h.
Extended Data Figure 6 Inhibition of monocarboxylate transporters abrogates the activity of lactic acid.
a, Expression analysis by qPCR of bone-marrow-derived macrophages stimulated with unfractionated or fractionated (<3 kDa and >3 kDa) control or LLC-tumour-conditioned medium ±5 mM CHC (α-cyano-4-hydroxycinnamate), a monocarboxylate channel transporter inhibitor. b, Acidic pH is necessary for the effect of lactate on bone-marrow-derived macrophages. Expression analysis by qPCR of bone-marrow-derived macrophages stimulated for 6 h with lactic acid, calcium lactate, HCl plus calcium chloride, or HCl plus calcium lactate. c, Effect of lactic acid on LLC, B16 and CT26 tumour cells. Expression analysis by qPCR of LLC, B16 and CT26 tumour cells stimulated for 6 h (Vegf, left) and 24 h (Arg1, right). Where indicated, the relative expression histograms represent three biological replicates displayed as mean ± s.e.m.
Extended Data Figure 7 TAMs from LLC tumours express a unique profile of TAM-associated and M2-associated markers compared with peritoneal macrophages.
Expression analysis by qPCR of FACS-sorted peritoneal macrophages and LLC TAMs. Results using either Rpl13a or Actb as a housekeeping gene are shown.
Extended Data Figure 8 A subset of TAM markers can be induced by lactic acid and require HIF1α.
Expression analysis by qPCR of MHC II, Mrc1 and Cd11c mRNA from the following cell types: bone-marrow-derived macrophages stimulated with 25 mM lactic acid (a); TAMs from LLC tumours resected from mice with macrophages that are either wild-type (WT; C57BL/6J) or in which Hif1a has been deleted (b). Where indicated, the relative expression histograms represent three biological replicates displayed as mean ± s.e.m.
Extended Data Figure 9 Lactic acid is oxidized by and activates TAMs, characterized by the induction of Arg1, which is important for tumour growth.
a, Lactic acid stimulation of bone-marrow-derived macrophages results in larger tumours when these macrophages are co-injected with LLC cells. The growth rate of LLC tumours in which LLC cells were co-injected 1:1 with bone-marrow-derived macrophages that had been stimulated for 24 h with either control DMEM (n = 15) or 25 mM lactic acid (n = 12) is shown. The tumour volumes were calculated using the formula (width)2 × length × 0.52 (ref. 23); *P = 0.0305 on day 14; #P < 0.0001 on day 16, using a two-tailed, unpaired t-test. Whereas the F test revealed no significant difference in variance between the groups on day 14, there was a significant difference (P = 0.0477) in the variance between the groups on day 16. Non-parametric analysis using the Mann–Whitney test revealed a significant difference between the groups on both days 14 (P = 0.0240) and 16 (P = 0.0467). The data are presented as the mean volume ± s.e.m. b, TAMs oxidize more 14C-lactic acid to 14CO2 than bone-marrow-derived macrophages and cultured LLC cells. TAMs, all other tumour cells (AO), bone-marrow-derived macrophages and LLC cells (1 × 106) were cultured for 2 h in DMEM containing 100 μCi of 14C-lactic acid. c, d, Deletion of Arg1 in macrophages slows the growth of LLC and B16 tumours. c, The images are representative of LLC tumours from wild-type (WT) and Arg1 KO mice. d, The growth rate of B16 tumours in mice with WT (n = 9) versus Arg1 KO (n = 9) macrophages. The tumour volumes were calculated using the formula (width)2 × length × 0.52 (ref. 23). The data are presented as the mean ± s.e.m. P < 0.05 on days 9 and 10 using a two-tailed, unpaired t-test. The F test revealed no significant difference in variance between the compared groups.
Extended Data Figure 10 TAMs express higher levels of urea cycle enzymes than all other tumour cells from LLC tumours.
Expression analysis by qPCR of Cps1, Otc, Ass1, Asl, Arg1, Glul and Gpt mRNA in FACS-sorted TAMs and all other (AO) tumour cells from day 19 LLC tumours.
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Colegio, O., Chu, NQ., Szabo, A. et al. Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature 513, 559–563 (2014). https://doi.org/10.1038/nature13490
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DOI: https://doi.org/10.1038/nature13490
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