Abstract
In metazoan organisms, cell competition acts as a quality control mechanism to eliminate unfit cells in favour of their more robust neighbours1,2. This mechanism has the potential to be maladapted, promoting the selection of aggressive cancer cells3,4,5,6. Tumours are metabolically active and are populated by stroma cells7,8, but how environmental factors affect cancer cell competition remains largely unknown. Here we show that tumour-associated macrophages (TAMs) can be dietarily or genetically reprogrammed to outcompete MYC-overexpressing cancer cells. In a mouse model of breast cancer, MYC overexpression resulted in an mTORC1-dependent ‘winner’ cancer cell state. A low-protein diet inhibited mTORC1 signalling in cancer cells and reduced tumour growth, owing unexpectedly to activation of the transcription factors TFEB and TFE3 and mTORC1 in TAMs. Diet-derived cytosolic amino acids are sensed by Rag GTPases through the GTPase-activating proteins GATOR1 and FLCN to control Rag GTPase effectors including TFEB and TFE39,10,11,12,13,14. Depletion of GATOR1 in TAMs suppressed the activation of TFEB, TFE3 and mTORC1 under the low-protein diet condition, causing accelerated tumour growth; conversely, depletion of FLCN or Rag GTPases in TAMs activated TFEB, TFE3 and mTORC1 under the normal protein diet condition, causing decelerated tumour growth. Furthermore, mTORC1 hyperactivation in TAMs and cancer cells and their competitive fitness were dependent on the endolysosomal engulfment regulator PIKfyve. Thus, noncanonical engulfment-mediated Rag GTPase-independent mTORC1 signalling in TAMs controls competition between TAMs and cancer cells, which defines a novel innate immune tumour suppression pathway that could be targeted for cancer therapy.
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Data availability
All data from this study are available in the Article, extended data and supplementary data. All the data from next generation sequencing experiments can be accessed from the Gene Expression Omnibus under accessions GSE230244, GSE230245 and GSE230247. Source data are provided with this paper.
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Acknowledgements
We thank members of the M.O.L. laboratory for helpful discussions. This work was supported by Howard Hughes Medical Institute (Faculty Scholar Award to M.O.L.), MSKCC Geoffrey Beene Cancer Research Center (M.O.L.), MSKCC Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center (M.O.L.), the Mazumdar-Shaw Translational Research Initiative in Kidney Cancer (M.O.L.), Cancer Research Institute (Irvington Fellow awards to X.Z., S.L., C.C. and L.J.), MSKCC Cancer Center Support Grant (P30CA08748), and Federal funds from the National Cancer Institute and National Institutes of Health, under Contract No. HHSN261201500003I (L.S.S.). We also thank Integrated Genomics Operation Core at MSKCC, Cycle for Survival and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology. We acknowledge NYU Langone Health’s Metabolomics Laboratory, partially supported by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center, for acquiring and analysing the amino acid profiling data. We thank M. Aufiero and T. Hohl for transferring key mouse lines. All illustrations were created with BioRender.com.
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X.Z. and M.O.L. were involved in all aspects of this study, including planning and performing experiments, analysis and interpretation of data and writing the manuscript. S.L. and X.Z. generated the Fcgr1-cre mice. I.M. assisted with mouse colony management. L.J. developed the genotyping strategy for Fcgr1-cre mice. L.J. and J.Z. assisted with mice dissection and tissue sample processing. M.H.D., L.J., C.C., W.S., K.J.C., J.Z., T.-W.H., B.G.N., K.X. and X.W. assisted with mouse breeding, tumour measurement and data analysis. L.S.S., W.M.L. and A.B. provided key mouse lines. All authors provided critical scientific feedback on the results and manuscript.
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MSKCC has filed a patent application (number 63/502054) with the US Patent and Trademark Office directed towards targeting the nutrient-sensing pathway in macrophages for cancer immunotherapy. M.O.L. is a scientific advisory board member of and holds equity or stock options in Amberstone Biosciences and META Pharmaceuticals. A.B. is cofounder of Casma Therapeutics and advisory board member of Next Generation Diagnostics, Avilar Therapeutics and Coave Therapeutics. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 mTORC1 signaling supports the MYC-driven ‘winner’ status of cancer cells.
a, Frequency of MYC amplification across human malignancies with breast cancer being the most prevalent cancer type. b, Pam50 molecular subtypes in groups of human breast cancer samples with none, either or both of MYC amplification and PIK3CA driver mutation. c, Immunofluorescence images showing expression of cleaved PARP1 (C-PARP1), MYC, phosphorylated 4E-BP1 (p-4E-BP1), and EpCAM in 6 mm x 6 mm tumor tissue samples from mice of the indicated genotypes. Scale bar: 20 μm. Representative ‘loser’ cells marked by magenta dashed lines and ‘winner’ cells marked by yellow dashed lines. Quantification of percentage of C-PARP1-positive cancer cells (d), percentage of cells with high nuclear MYC expression or high p-4E-BP1 expression among C-PARP1-positive or -negative cancer cells from PyMT (n = 10), S100a8-cre-Rosa26LSL-MYC/+PyMT (n = 10), S100a8-cre-Rptorfl/flPyMT (n = 8) and S100a8-cre-Rosa26LSL-MYC/+Rptorfl/flPyMT (n = 6) tumors (e, f), and cancer cell size for tissue sections in Fig. 1d (g). h, Immunofluorescence images showing expression of cleaved caspase 3 (CC3), MYC, phosphorylated S6 ribosomal protein at serine 240/244 (p-S6), and EpCAM in 6 mm x 6 mm or 12 mm x 12 mm tumor tissue samples from S100a8-cre-Rosa26LSL-MYC/+Rptorfl/flPyMT mice. Scale bar: 20 μm. Representative ‘loser’ cells marked by magenta dashed lines and ‘winner’ cells marked by yellow dashed lines. i, j, Quantification of percentage of cells with high MYC expression or high p-S6 expression among CC3-positive or -negative EpCAM-expressing cancer cells from S100a8-cre-Rosa26LSL-MYC/+Rptorfl/flPyMT mice (n = 5). k–m, RT-qPCR analysis of Rptor, human MYC, and cre mRNA expression in cancer cells isolated from tumors of the indicated genotypes and tumor size (n = 4 mice for each group). All statistical data are shown as mean ± S.D. One-way ANOVA with the Tukey multiple comparison test correction in (d–g, k–m), and Two-sided Student t-test in (i, j).
Extended Data Fig. 2 MYC induces expression of amino acid metabolism genes in PyMT tumors.
A heatmap showing significantly differentially expressed genes (adjusted P < 0.05) from RNA-sequencing experiments between S100a8-cre-Rosa26LSL-MYC/+PyMT (MYC-PyMT) and control-PyMT tumors, categorized in groups that encode for proteins involved in amino acid synthesis, amino acid transporters, and ribonucleoproteins.
Extended Data Fig. 3 A low protein diet triggers TAM reprogramming in MYC-PyMT mice.
a, Immunofluorescence images showing expression of F4/80, phosphorylated 4E-BP1 (p-4E-BP1), and EpCAM in 6 mm x 6 mm tumor tissue samples from mice of the indicated genotypes and dietary treatment conditions. Scale bar: 5 μm. b–c, Quantification of percentage of cells with high p-4E-BP1 expression among EpCAM-expressing cancer cells and F4/80-expressing macrophages (TAMs) in the tumor parenchyma. n = 12 mice for control-PyMT + NP or LP, n = 7 mice for MYC-PyMT + NP, and n = 10 mice for MYC-PyMT + LP. d, Immunofluorescence images showing expression of F4/80, MRC1, and VCAM1 in 6 mm x 6 mm tumor tissue samples from mice of the indicated genotypes and dietary treatment conditions. MRC1 is highly expressed in mammary tissue macrophages (MTMs) in the tumor interstitial region, while VCAM1 is highly expressed in tumor-associated macrophages (TAMs) residing in the tumor parenchyma. Scale bar: 50 μm. T: tumor parenchyma, S: interstitial region or stroma of the tumor tissue. Parenchyma-interstitial region borders marked by yellow dashed lines. e–g, Contour plots and histograms showing the gating strategy and side scatter SSC-A of monocytes, MTMs, and TAMs isolated from mice of the indicated genotypes and dietary treatment conditions. Siglec-F, B220, and Ly6G markers were used to exclude eosinophils, B cells, and neutrophils, respectively. (e). The percentage of monocytes, MTMs, and TAMs among CD45+ cells (f) and their median SSC-A (g) are quantified (n = 8 for each group). NP: control diet with 15% protein in weight (5CC7, TestDiet), LP: low protein diet with 2% protein in weight (5BT9, TestDiet). All statistical data are shown as mean ± S.D. One-way ANOVA with the Tukey multiple comparison test correction in (b,c,f,g).
Extended Data Fig. 4 Transcriptome profiling of TAMs and MTMs.
a, A schematic diagram showing the Rosa26LSL-MYC allele that encodes human MYC (hMYC) and the internal ribosome entry site (IRES)-driven truncated human CD2 (hCD2) protein as a cell surface reporter. b, Reporter assay showing the specificity of S100a8-cre-mediated recombination of the Rosa26LSL-MYC allele and high reconstitution efficiency of wild-type (WT) bone marrow cells. c, Experimental design of bulk RNA-sequencing experiments for tumor-associated macrophages (TAMs) and mammary tissue macrophages (MTMs) isolated from control-PyMT and MYC-PyMT mice reconstituted with WT bone marrow cells and treated with a low protein (LP) diet (2% protein in weight, 5BT9, TestDiet). d, Flow cytometry gating strategy for TAMs and MTMs. Siglec-F, B220, Ly6G, and Ly6C were used to exclude eosinophils, B cells, neutrophils, and monocytes, respectively. e. PCA analysis of TAMs and MTMs isolated from control-PyMT and MYC-PyMT mice reconstituted with WT bone marrow cells and treated with a LP diet, based on the log2 fold change of gene expression compared to the mean. MTMs from MYC3 group are not included due to insufficient cell number.
Extended Data Fig. 5 Generation and validation of Fcgr1-cre mice.
a, A schematic diagram showing the design of Fcgr1-cre knock-in mice with an IRES-tdTomato-T2A-iCre expression cassette targeted to the Fcgr1 gene locus. b, Validation of the Fcgr1-cre knock-in allele by PCR showing germ line transmission of the targeted allele. PCR results were consistently observed across all generations of mice. c, d, Reporter assay showing the specificity of Fcgr1-cre-mediated recombination of the Rosa26LSL-H2B-mCherry allele. Gating strategy of tumor-infiltrating immune cell populations in (c). Top and bottom rows indicate two separate experiments. Histogram overlays in (d) showing H2B-mCherry expression in various tumor-infiltrating immune cell populations isolated from the indicated genotypes. Chromatin-associated H2B-mCherry, but not tdTomato expressed in the cytosol, was retained after fixation and permeabilization in flow cytometric analysis. MTM: mammary tissue macrophage; TAM: tumor-associated macrophage; DC1: type 1 dendritic cell; DC2: type 2 dendritic cell.
Extended Data Fig. 6 TAMs are dietarily reprogramed in MYC-PyMT mice.
a, A schematic diagram showing the design of single-nucleus RNA-sequencing experiments in Control-PyMT and MYC-PyMT mice. Siglec-F, B220, Ly6G, and Ly6C were used to exclude eosinophils, B cells, neutrophils, and monocytes, respectively. VCAM1 is a marker for the tumor parenchyma-localized TAMs. b–d, TSNE plots showing unsupervised clustering of TAM nuclei isolated from the low protein (LP) diet (2% protein in weight, 5BT9, TestDiet)-conditioned control-PyMT and MYC-PyMT tumors, and color-coded as sub-clusters in (b) and genotypes in (c). Percentage of each sub-cluster of TAM nuclei from control-PyMT and MYC-PyMT tumors (d). e, A scatter plot showing enriched gene sets from differentially expressed genes (adjusted P < 0.05) in cluster 3 versus other clusters. f, Immunofluorescence images showing expression of F4/80, EpCAM, and LAMP in 6 mm x 6 mm tumor tissue samples from the LP diet-treated control-PyMT and MYC-PyMT mice. Scale bar: 20 μm. Representative TAMs marked by yellow dashed lines. g, Quantification of AFU (arbitrary fluorescence unit) of LAMP1 expressing in F4/80-expressing TAMs from the LP diet-treated control-PyMT and MYC-PyMT mice (n = 3 mice for control-PyMT + LP, n = 4 mice for MYC-PyMT + LP). Data points are average AFU from each captured images. h, j, RT-qPCR analysis of Tfeb and Rptor mRNA expression in TAMs of the indicated genotypes (n = 4 mice for each group). i, A schematic diagram showing the generation of bone marrow (BM) chimera mice. BM cells from control, Fcgr1-cre-Rptorfl/fl mice were transferred into lethally irradiated MYC-PyMT recipients with a tumor burden around 250–350 mm3 followed by switching to the LP diet two weeks after. All statistical data are shown as mean ± S.D. Over-representation analysis based on hypergeometric test with the Benjamini-Hochberg multiple comparison test correction in (e), two-sided Student t-test in (g,h,j).
Extended Data Fig. 7 Two modes of mTORC1 signaling promoted or suppressed by Rag GTPases.
a. A schematic diagram showing two modes of mTORC1 signaling in TAMs promoted or suppressed by Rag GTPases. Under a normal protein (NP) diet (15% protein in weight, 5CC7, TestDiet) condition, abundant cytosolic amino acids (AAs) suppress the GATOR1 complex, but activate the FLCN complex, and induce Rag GTPases in a RagA/BGTP-RagC/DGDP state that recruits mTORC1 and TFEB/TFE3 to the Rag GTPase complex, supporting or inhibiting their activation, respectively. Under a low protein (LP) diet (2% protein in weight, 5BT9, TestDiet) condition, AA starvation activates GATOR1, but inactivates FLCN, and induces Rag GTPases in a RagA/BGDP-RagC/DGTP state that dissociates mTORC1 and TFEB/TFE3 from the Rag GTPase complex, leading to TFEB/TFE3 nuclear translocation and mTORC1 activation through Rag GTPase-independent mechanisms. Depletion of GATOR1 maintains a RagC/DGDP state under the LP diet condition, while depletion of FLCN maintains a RagC/DGTP state under the NP diet condition, causing inaction or activation of the TFEB/TFE3-mTORC1 signaling pathway, respectively. Depletion of Rag GTPases also activates TFEB/TFE3-mTORC1 signaling under the NP diet condition. b, c, Mass spectrometry quantification of individual AAs in tumor interstitial fluid extracted from control-PyMT and MYC-PyMT tumors under NP or LP diet conditions. AAs shown changes in the LP diet-treated MYC-PyMT tumors were plotted. Due to the absence of spike-in isotope labeled tryptophan, normalized intensity was shown. n = 2 mice each condition for NP treated control-PyMT and MYC-PyMT, and n = 3 mice each condition for LP treated control-PyMT and MYC-PyMT. d, A schematic diagrams showing the generation of bone marrow (BM) chimera mice. BM cells from control and Fcgr1-cre-Depdc5fl/fl mice were transferred into lethally irradiated MYC-PyMT recipients with a tumor burden around 250-350 mm3 followed by switching to the LP diet two weeks after. e, BM cells from control, Fcgr1-cre-Flcnfl/fl, and Fcgr1-cre-Rragafl/flRragbfl/fl mice were transferred into lethally irradiated MYC-PyMT recipients with a tumor burden around 250-350 mm3 and kept under the NP diet condition. f–h, RT-qPCR analysis of Flcn, Rraga, and Rragb mRNA expression in TAMs of the indicated genotypes (n = 4 mice for each group). All statistical data are shown as mean ± S.D. Two-sided Student t-test in (c, f–h).
Extended Data Fig. 8 Dietarily reprogramed TAMs engulf apoptotic cells in MYC-PyMT tumors.
a,b, Experimental design, flow cytometric analysis and quantification of TAM engulfment of apoptotic cells in a complete or low amino acid (AA) RPMI medium (1: 4 mixture of complete RPMI and AA-free RPMI). Apoptotic PyMT cancer cells were labeled by CellTrace Violet (CTV) and induced by H2O2 treatment. TAMs were isolated from tumors of control-PyMT and MYC-PyMT mice (n=5 for each group) treated with a low protein (LP) diet (2% protein in weight, 5BT9, TestDiet). c, Immunofluorescence images showing expression or localization of F4/80, EpCAM, and TUNEL in 6 mm x 6 mm tumors from control-PyMT and MYC-PyMT mice under LP or normal protein (NP) diet (15% protein in weight, 5CC7, TestDiet) conditions. Scale bar: 50 μm. Representative macrophages marked by yellow dashed lines. d, Quantification of percentage of TUNEL-positive cells among all cells. e, Quantification of percentage of TUNEL-positive nuclei inside F4/80-expressing macrophages. Data points are average percentage from random captured images. n = 3 mice for each group in (d,e). f, A schematic diagram showing the generation of bone marrow (BM) chimera mice. BM cells from Fcgr1-cre-Rosa26LSL-H2B-mCherry/+ mice were transferred into lethally irradiated MYC-PyMT recipients with a tumor burden around 250-350 mm3 followed by switching to the LP diet two weeks after. g, Immunofluorescence images showing expression or localization of F4/80, EpCAM, H2B-mCherry, and TUNEL in 6 mm x 6 mm tumor tissue samples from BM chimera mice described in (f). Scale bar: 10 μm. All statistical data are shown as mean ± S.D. One-way ANOVA with the Tukey multiple comparison test correction in (b,d,e).
Extended Data Fig. 9 Apoptotic cancer cells are effectively phagocytosed by reprogramed TAMs.
a, b, Schematic diagrams showing bone marrow (BM) chimera mice to define TAM engulfment of dying cancer cells. BM cells from homozygous H2B-EGFP/H2B-EGFP mice were transferred into lethally irradiated S100a8-cre-Rosa26LSL-MYC/LSL-H2B-mCherryPyMT recipients with a tumor burden around 250-350 mm3 followed by switching to a low protein (LP) diet (2% protein in weight, 5BT9, TestDiet) two weeks after. c, A schematic diagram showing dietary treatment of BM chimera mice. BM cells of control or Fcgr1-cre-Pikfyvefl/fl mice were transferred into lethally irradiated S100a8-cre-Rosa26LSL-MYC/LSL-H2B-mcherryPyMT recipients with a tumor burden around 250–350 mm3, which were either kept under a normal protein (NP) diet (15% protein in weight, 5CC7, TestDiet) condition or switched to a LP diet two weeks after. d, RT-qPCR analysis of PIKfyve mRNA expression in TAMs of the indicated genotypes (n = 4 mice for each group). e, A schematic diagram showing dietary treatment of BM chimera mice. BM cells from control or Fcgr1-cre-Pikfyvefl/fl mice were transferred into lethally irradiated S100a8-cre-Rosa26LSL-MYC/+PyMT or S100a8-cre-Rosa26LSL-MYC/+Pikfyvefl/flPyMT recipients with a tumor burden around 250-350 mm3 followed by switching to a LP diet two weeks after. f, Experimental design of an in vivo engulfment assay with intratumoral injection of CellTrace Violet (CTV) and CellTrace Yellow (CTY)-labeled apoptotic cells and apoptotic bodies. g,h, Experimental design and flow cytometry analysis of an ex vivo engulfment assay with TAMs and cancer cells fed with apoptotic cells and apoptotic bodies in a low amino acid RPMI medium (1: 4 mixtures of RPMI and amino acid-free RPMI). n = 3 independent experiments and cells from independent mice each group. All statistical data are shown as mean ± S.D. Two-sided Student t-test in (d), one-way ANOVA with the Tukey multiple comparison test correction in (h).
Extended Data Fig. 10 Reprogram TAMs to outmatch cancer cell competition.
In S100a8-cre-Rosa26LSL-MYC/+PyMT mice, MYChi cancer cells are fitter than MYClo cancer cells and outcompete them in a process dependent on higher mTORC1 signaling. Cell debris from apoptotic cancer cells are acquired by MYChi cancer cells as a nutrient source to promote mTORC1 activation and the ‘winner’ status. Feeding mice with a low protein diet or depletion of FLCN or Rag GTPases activates TFEB/TFE3 and mTORC1 signaling in tumor-associated macrophages (TAMs) and enhances TAM fitness supported by engulfment of apoptotic cancer cells and apoptotic cell debris. Such a contest in engulfment-dependent nutrient acquisition dictates cell competition between TAMs and MYChi cancer cells, which defines an innate immune tumor suppression pathway that may be targeted for cancer immunotherapy.
Supplementary information
Supplementary Table 1
Differentially expressed genes in human breast cancers with concurrent MYC amplification and PIK3CA gain-of-function driver mutation versus others as well as mouse MYC-overexpressing PyMT tumor versus control-PyMT tumor, and the overlapping significantly regulated gene sets between human and mouse datasets. Student’s t-test with Benjamini–Hochberg multiple comparison test correction for differential gene expression. GSEA based on the Kolmogorov–Smirnov test for identifying enriched gene sets.
Supplementary Table 2
Log fold expression change of highly variable genes among TAMs and MTMs from control-PyMT and MYC-PyMT mice under the LP diet condition.
Supplementary Table 3
Differentially upregulated genes in each cluster of t-SNE plot in single-nucleus RNA-sequencing datasets and overrepresented gene sets of differentially upregulated genes in cluster 3. Wald test with the Benjamini–Hochberg multiple comparison test correction for differential gene expression. Over-representation analysis based on hypergeometric test with the Benjamini–Hochberg multiple comparison test correction for identifying enriched gene sets.
Supplementary Table 4
Individual amino acid levels in tumor interstitial fluids extracted from a LP diet-treated control-PyMT and MYC-PyMT mice.
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Zhang, X., Li, S., Malik, I. et al. Reprogramming tumour-associated macrophages to outcompete cancer cells. Nature 619, 616–623 (2023). https://doi.org/10.1038/s41586-023-06256-5
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DOI: https://doi.org/10.1038/s41586-023-06256-5
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