Hematopoietic aging promotes cancer by fueling IL-1⍺-driven emergency myelopoiesis

Abstract

Age is a major risk factor for cancer, but how aging impacts tumor control remains unclear. In this study, we establish that aging of the immune system, regardless of the age of the stroma and tumor, drives lung cancer progression. Hematopoietic aging enhances emergency myelopoiesis, resulting in the local accumulation of myeloid progenitor-like cells in lung tumors. These cells are a major source of interleukin (IL)-1⍺, which drives the enhanced myeloid response. The age-associated decline of DNA methyltransferase 3A enhances IL-1⍺ production, and disrupting IL-1 receptor 1 signaling early during tumor development normalized myelopoiesis and slowed the growth of lung, colonic, and pancreatic tumors. In human tumors, we identified an enrichment for IL-1⍺-expressing monocyte-derived macrophages linked to age, poorer survival, and recurrence, unraveling how aging promotes cancer and offering actionable therapeutic strategies.

Fig. 1. Age is associated with an attrition of tissue-resident alveolar macrophages, despite increased myelopoietic potential.

(A) Hematoxylin and eosin staining of lung sections derived from lungs of naïve young (7-week-old, 7.wks) and old (72-week-old, 72.wks) mice (left) and quantification of cell density (right). (B) Vessel leakage assessed by retro-orbital administration of either Evans Blue (left) or fluorophore-conjugated Dextran (right). (C) Schematic of fate-mapped GMP-derived cells produced during adult hematopoiesis using the Ms4a3^CRE-tdTomato (TdT) mouse. (D) Absolute number of alveolar macrophages (AM) in the lungs of young and old Ms4a3^CRE-TdT mice at steady-state. Quantification of (E) TdT^NEG and self-renewing (Ki67^POS) TdT^NEG AM and of (F) TdT^POS AM and self-renewing TdT^POS AM in the lungs of young and old Ms4a3^CRE-TdT mice at steady-state. (G) Frequency distribution of major myeloid cell types identified from scRNAseq of TdT^NEG and TdT^POS myeloid cells in naïve lungs of young and old Ms4a3^CRE-TdT mice. (H) Absolute number of Ly6C^HI and Ly6C^LO monocytes and neutrophils in the blood of naïve young and old mice. (I) Absolute number of CMP, MDP, GMP, GP, and cMoP in the bone marrow of naïve young and old mice. Across all experiments, n=4-5 mice were used per group. Data shown in panels (A), (B), and (G) are each representative of one independent experiment; panels (D)-(F), (H), and (I) are each representative of two independent experiments. Across all panels, data represent mean ± SEM. P-values were computed by unpaired t-test. (AM, alveolar macrophage; cDC1, type 1 conventional dendritic cell; cDC2, type 2 conventional dendritic cell; mregDC, mature DCs enriched in immunoregulatory molecules; moDC, monocyte-derived DC; IM, interstitial macrophage; CMP, common myeloid progenitor; MDP, monocyte-dendritic cell progenitor; GMP, granulocyte-monocyte progenitor; GP, granulocyte progenitor; cMoP, common monocyte progenitor)

Fig. 2. Aging of the hematopoietic compartment promotes lung cancer progression.

An orthotopic model for primary lung adenocarcinoma involving the Intravenous injection of Kras^G12D/+ Tp53^-/- Rosa26^A3Bi- Rag1^-/- (KPAR) cells was used to assess tumor growth in young (7-week-old, 7.KPAR) and old (72-week-old, 72.KPAR) mice. (A) (Left) Longitudinal kinetic analysis of tumor burden in the lungs of young and old mice at 5-, 10- and 20-days post-tumor cell inoculation, and (Right) tumor burden in the lungs of young and old mice at 17 days post-inoculation (n=3-5 mice per group) with representative H&E cross-sections shown at left. Scale bar = 1 mm. (B) Survival curve of tumor-bearing young (n=10) and old (n=14) mice. P-value was computed using the Log-rank Mantel-Cox test. (C) Experimental design of heterochronic bone marrow transplantation, involving the transfer of (1) donor bone marrow from young CD45.2 mice into young CD45.1 recipient mice, (2) donor bone marrow from old CD45.2 mice into young CD45.1 recipient mice, and (3) donor bone marrow from young CD45.1 mice into old CD45.2 recipient mice. Chimeric mice were inoculated with tumor cells after an eight-week engraftment period. (D) Tumor burden in the lungs of chimeric mice at 10 days post-tumor cell inoculation. Scale bar = 1mm. (E) Tumor burden in the lungs of chimeric mice at 20 days post-inoculation. Scale bar = 1mm. (F) Absolute number of lung MP in the lungs of chimeric mice at 10 (left) and 20 (right) days post-inoculation. (G) Tumor burden in the lungs of (black) old recipients of old donor bone marrow and of (yellow) old recipients of young donor bone marrow at 17 days post-inoculation. (H) Absolute number of lung MP, mo-macs, and neutrophils in the tumor-bearing lungs of chimeric mice shown in (G). Across all experiments, except in panel (B), n=3-5 mice were used per group. Data shown in panels (A), (D), (G), and (H) are from one experiment. Data shown in panels (B), (E), and (F) are each representative of two independent experiments. Across all panels, data represent mean ± SEM. P-values were computed by either unpaired t-test or a one-way ANOVA. (Lung MP, lung myeloid progenitor-like cell; mo-mac, monocyte-derived macrophage)

Fig. 3. An IL-1α signature defines myeloid progenitor-like cells born from age-enhanced emergency monopoiesis.

CD45^POS cells were sorted from young (n=3) and old (n=3) tumor-bearing mice and sequenced at the single-cell level. (A) Sub-clustering of myeloid progenitors (MPs) using a K-nn graph partitioning approach and annotation of MP cell states (i.e., MonoP, GranuloP, NeuP) based on defining markers and shown through a heatmap plotting UMI counts per cell. (B) Transcriptional similitude amongst myeloid cells was determined by hierarchical clustering based on mRNA expression profile. Transcriptomes of bone marrow progenitors (BM-GMP, BM-GP, BM-cMoP) in the tumor-bearing setting were used. (C) Frequency of total lung MPs in tumor-bearing lungs of young and old mice. (D) Differentially expressed genes (DEGs) between (top) NeuP, (middle) GranuloP, and (bottom) MonoP from lung tumors of old vs. young mice. (E) DEGs between (left) Ly6C^HI monocytes, (middle) Ly6C^LO monocytes, and (right) TREM2 monocyte-derived macrophages (mo-macs) from lung tumors of old vs. young mice (F) Fold change in the frequency of NeuP, GranuloP, and MonoP, relative to the mean frequency of each respective cell state in lung tumors from young mice. (G) DEGs that define MonoP, based on Wilcoxon Rank Sum testing of MonoP vs. all other MPs. Hallmark genes plotted in red. Significant gene networks identified by gene ontology analysis. (H) Mean UMI of Il1a, Il1b, and Il1r1 across immune cells. (I) Concentration of IL-1α (left) and IL-1β (right) in the lung homogenate of digested naïve and tumor-bearing lungs of young and old mice. In panel (I), n=5 mice were used per group. Data shown in (A)-(G) are from one independent experiment. Raw data from (B) are taken from Caiado et al., 2023. Data represent mean ± SEM. P-values were computed by unpaired t-test. (MonoP, monocytic progenitor; GranuloP, granulocytic progenitor; NeuP, neutrophilic progenitor; GMP, granulocyte-monocyte progenitor; GP, granulocyte progenitor; cMoP, common monocyte progenitor)

Fig. 4. IL-1α signaling fuels aging-driven tumor growth.

Brefeldin A was administered to young (n=5) and old (n=5) tumor-bearing mice to quantify in vivo production of IL-1α and IL-1β. (A), (B) IL-1α and (C), (D) IL-1β production levels in MPs, macrophages (macs), monocytes (mono), neutrophils (neu) from lung tumors and in GMP, macrophages, monocytes, and neutrophils from bone marrow of the same mice. Expression of (E) IL-1α and (F) IL-1β by lung MPs in young and old tumor-bearing mice. (G) Tumor burden in the lungs of old mice that received either isotype control, anti-IL-1α neutralizing antibody, anti-IL-1β neutralizing antibody, or anakinra after 16 days post-tumor cell inoculation. Scale bar = 1mm. (H) Survival curve of control (n=20) and anakinra-treated (n=19) tumor-bearing old mice. Solid line: control; Dotted line: anakinra-treated. Difference in median survival of 8 days. P-value was computed using the Log-rank Mantel-Cox test. (I) Frequency of activated NK cells in lung tumors of control (PBS) old mice and old mice that received anakinra immediately after tumor cell inoculation. (J) Tumor burden in the lungs of young and old mice that received either control (PBS) or anakinra after 16 days post-tumor cell inoculation. Scale bar = 1mm. (K) Tumor burden in chimeric mice: wild-type or Il1a^-/- recipient mice were transplanted with either young or old donor bone marrow and were either treated with control (PBS) or anakinra. Scale bar = 1mm. (L) Levels of phosphorylated p38 (phospho-p38) in bone marrow HSCs of old mice that either received control (PBS) or anakinra. (M) Frequency of bulk GMPs in the bone marrow of young and old mice that either received control (PBS) or anakinra immediately after tumor cell inoculation. (N) Absolute number of lung MP in lung tumors of control (PBS) old mice and old mice that received anakinra immediately after tumor cell inoculation. (O) Absolute number of mo-mac in lung tumors of mice shown in (N). Data shown in panels (A)-(J) and (M)-(O) are representative of at least two independent experiments; data in panels (K) and (L) are each from one independent experiment. Data are represented as mean ± SEM. P-values were computed by either one-way ANOVA or by unpaired t-test. (Lung MP, lung myeloid progenitor-like cell; GMP, granulocyte-monocyte progenitor; NK cell, natural killer cell; HSC, hematopoietic stem cell; mo-mac, monocyte-derived macrophage)

Fig. 5. Aging of myeloid cells results in DNMT3A deficiency and promotes expression of the IL-1α program.

IL-1α production by (A) bone marrow monocytes and (B) GMPs, GPs, cMoPs from young and old mice in the presence of control media, LPS, or apoptotic cell debris. (C) DEGs between young and old HSCs sorted from bone marrow of young and old mice. Relative expression of (D) Dnmt3a and (E) Tet2 and Asxl1 mRNA by sorted bone marrow HSCs from young and old mice. (F) Protein expression of DNMT3A by HSCs from bone marrow of naïve, young and old mice. (G) Relative expression of DNMT3A mRNA by HSCs from younger (n=3; ages 30, 31, 41) and older (n=3; ages 60, 60, 84) healthy donors. (H) Protein expression of DNMT3A by circulating HSPCs from the peripheral blood of younger (age<65) and older (age≥65) healthy donors. (I) Frequency of circulating HSPCs from the peripheral blood of NSCLC patients. (J) Protein expression of DNMT3A by circulating HSPCs from younger (age<65) and older (age≥65) NSCLC patients. (K) DEGs between Dnmt3a^-/- and Dnmt3a^+/+ (WT) murine HSCs (left) and GMPs (right). (L) DEGs between DNMT3A-proficient and –deficient (left) murine and (right) human bone marrow monocyte-derived macrophages and blood monocyte-derived macrophages, respectively. (M) IL-1α, (N) IL-1β and (O) TNF-α production by LPS-stimulated young and old bone marrow monocytes that were either untreated or treated with the DNMT3A inhibitor across independent experiments. (P) IL-1α production by LPS-stimulated blood monocytes from younger (age<65) and older (age≥65) healthy donors that were either untreated or treated with the DNMT3A inhibitor. Data shown in (A), (B), (M)-(P) are representative of at least two independent experiments. Data shown in (H)-(J) were collected across at least three independent experiments. Raw data for panels (C) and (D) were obtained from Itokawa et al., 2022, Kovtonyuk et al., 2022, and Sun et al., 2014; for panel (G) were obtained from Oetjen et al., 2018 and Ainciburu et al., 2023; for panels (K) and (L) were obtained from Zhang et al., 2022 and Guryanova et al., 2016 and Rausch et al., 2023 and Cobo et al., 2022. Data are represented as mean ± SEM. P-values were computed by either unpaired t-test or one-way ANOVA.

Fig. 6. The IL-1α mRNA program is a marker for aging and outcome in cancer patients.

(A) Tumor burden in lungs of old mice either treated with a control isotype antibody, PD-1 blocking antibody, or the combination of PD-1 blocking antibody and anakinra. (B) Frequency of cytotoxic CD8 T cells (top) and NK cells (bottom) in the tumor-bearing lungs of mice shown in (B). (C) (Left) mRNA expression of cell type-defining genes, IL1A, IL1B, and a composite score for the IL-1α-associated program, defined in Fig. 2, according to scRNA-seq of immune cells in lung tissues from NSCLC patients, and (right) heatmap of UMI counts of individual genes per cell (each individual row is a single cell) belonging to the mo-mac cluster. (D) Frequency mo-macs expressing the IL1A program (IL1A^POS) in paired tumor and adjacent, normal lung (nLung) tissue specimens from NSCLC patients. (E) Frequency of IL1A^POS mo-macs in resected tumor lesions from (left) all patients and (right) just the top quartile of patients of each age group, based on frequency values after outlier exclusion. (F) (left) Distribution of patients that experienced a recurrence of cancer depending on age, and (right) frequency of tumoral IL1A^POS mo-macs per patient depending on status of recurrence. (G) Kaplan-Meier curve showing overall survival difference between high and low scorers of the IL1A mRNA program among NSCLC patients in The Cancer Genome Atlas (TCGA) and other studies belonging to the Genomic Data Commons Data Portal and independent datasets. (H) Associations between risk of lung cancer incidence and cytokines and chemokines measured in the blood of lung cancer patients, collected prior to diagnosis, and age-matched and smoking controls without cancer. Statistical values shown as (p-value, odds ratio). (I) Frequency of IL1A^POS mo-macs (left) and monocytes (right) in the CRC tumor and adjacent, non-involved colon tissues from 31 CRC patients and separated by age. (J) Summary diagram of mechanism. Raw data from panels (C)-(F) were obtained from Leader et al., 2021. Processed data for panel (H) were obtained from the Lung Cancer Cohort Consortium (LC3). Data are represented as mean ± SEM. P-value was computed by either paired two-tailed t-test or unpaired t-test.