Ethics statement<\/p>\n
All animal experiments were performed in accordance with and approved by the Institutional Animal Care and Use Committee of the University of Southern California. The maximal tumor size permitted by the approved animal protocol was 15\u2009mm in diameter. Mice were euthanized when tumors reached this limit or when they became moribund and showed signs of cachexia, lateral recumbency, observable weight loss, or lack of response to noxious stimuli. The maximal tumor burden permitted by the institutional guidelines was not exceeded in any experiment.<\/p>\n
Mice<\/p>\n
Female and male CD45.1 or CD45.2 C57BL\/6 mice, as well as BALB\/c mice, were obtained from Charles River Laboratories or The Jackson Laboratory. Rag2 knockout (Rag2\u2013\/\u2013; B6.Cg-Rag2tm1.1Cgn\/J) mice were obtained from the Jackson Laboratory and were bred in-house. Mice were maintained under specific pathogen-free conditions according to institutional guidelines. Based on established lifespan comparisons, mice at 8\u201312 weeks of age are generally considered young adults, corresponding to early adulthood in humans (approximately 20\u201330 years). In contrast, mice at 65\u201375 weeks are commonly used to represent a late-life or aging stage, broadly analogous to late middle to early elderly stages in humans (approximately 50\u201370 years). However, such cross-species comparisons are inherently non-linear and depend on the physiological parameter assessed52<\/a>. In this study, C57BL\/6 mice aged 8\u201312 weeks and 65\u201375 weeks were used as the young and aged cohorts, respectively. For BALB\/c mice used in this study, animals aged \u226565 weeks were defined as aged based on experimental availability.<\/p>\n Cell lines<\/p>\n The E0771 cell line was purchased from the CH3 BioSystems. B16-F10 (B16), RAW 264.7, and EMT6 cell lines were purchased from the American Type Culture Collection (ATCC). The AT-3 cell line was a gift from Dr. Scott Abrams (Roswell Park Comprehensive Cancer Center, Buffalo, NY). E0771, B16, and RAW 264.7 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Sigma-Aldrich), 1% nonessential amino acid (NEAA; Gibco), 2 mmol\/L L-glutamine (Gibco), 0.5% penicillin\/streptomycin (Gibco), and 50 \u00b5mol\/L 2-mercaptoethanol (Gibco). AT-3 and EMT6 cells were cultured in DMEM (Gibco) supplemented with 10% FBS, 1% NEAA, 2 mmol\/L L-glutamine, 0.5% penicillin\/streptomycin, and 50 \u00b5mol\/L 2-mercaptoethanol. These cell lines were authenticated by morphology, phenotype, and growth, and routinely screened for Mycoplasma and maintained at 37\u2009\u00b0C in a humidified 5% (E0771, B16, RAW 264.7, EMT6) or 7% (AT-3) CO2 atmosphere.<\/p>\n Plasmids<\/p>\n PmIL-6 FL and pmIL-6 mut NF-\u03baB were gifts from Dr. Gail Bishop (Addgene plasmid # 61286; http:\/\/n2t.net\/addgene:61286<\/a>; RRID:Addgene_61286 and Addgene plasmid # 61293; http:\/\/n2t.net\/addgene:61293<\/a>; RRID:Addgene_61293, respectively)53<\/a>.<\/p>\n Tumor model<\/p>\n E0771 (5 \u00d7 105), AT-3 (5 \u00d7 105), or EMT6 (2 \u00d7 105) tumors were surgically implanted under anesthesia with isoflurane into the fourth mammary gland of female mice as described54<\/a>,55<\/a>. B16 (5 \u00d7 105) tumor cells were injected subcutaneously on the left flank of male mice. Tumor growth was measured 3\u20135 times a week, and the volumes were calculated by determining the length of short (l) and long (L) diameters (volume = l2 \u00d7 L\/2) as previously described54<\/a>,55<\/a>.<\/p>\n In vivo treatment<\/p>\n For in vivo thymulin-conditioning, thymulin solution (1.5\u2009mg\/kg of body weight) was prepared from thymulin acetate (MedChemExpress, Monmouth Junction, NJ, USA) with an equimolar concentration of ZnCl2 (39059, Sigma-Aldrich, St. Louis, MO, USA) in PBS (Gibco), and was injected intraperitoneally (i.p.) starting on day 1 after tumor cell injection and continued daily for 4 weeks. Control mice received ZnCl2 in PBS (vehicle). For PD-L1 blockade, anti-PD-L1 Ab (clone 10\u2009F.9G2, BioXcell) was given i.p. on day 7, 10, 13, 16, 19, 22 after the tumor cell inoculation at a dose of 200\u2009\u03bcg\/mouse54<\/a>,55<\/a>.<\/p>\n Flow cytometry<\/p>\n Single-cell suspensions of mouse peripheral blood and tumors were prepared for flow cytometric analysis. Red blood cells in blood were lysed using ACK Lysis Buffer (Life Technologies). Tumor tissues were weighed, minced, filtered through 70-\u03bcm filters, and stored at \u221280\u2009\u00b0C in FBS (Hyclone, Waltham, MA) with 10% DMSO (Sigma) before analysis. Cells were blocked with anti-mouse CD16\/32 (553142, BD Biosciences, San Diego, CA, USA) and surface-stained with indicated markers. Live\/dead cell discrimination was performed using LIVE\/DEAD Fixable Near-IR Dead Cell Stain Kit (Life Technologies). To evaluate changes in the myeloid and lymphoid compartments of peripheral blood and the tumor microenvironment, we used the 23-color flow cytometry gating strategy (Supplementary Fig.\u00a01<\/a>) as described previously54<\/a>. For analysis of cytokine expression, intracellular IL-1\u03b1, IL-1\u03b2, IL-6, TNF-\u03b1, and IFN-\u03b3 assays were performed using Fixation\/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer\u2019s recommendations. Prior to intracellular staining, cells were stimulated ex vivo for 4\u2009h at 37\u2009\u00b0C in stimulation media composed as follows: RPMI 1640 medium with 10% FBS (Hyclone), 50\u2009\u03bcg\/ml of gentamicin (Gibco), 25\u2009mM HEPES (Gibco), 2 mM L-glutamine (Gibco), 0.1\u2009mM sodium pyruvate (Gibco), 0.1\u2009mM NEAA (Gibco), LPS (100\u2009ng\/ml; LPS-SM ultrapure, InvivoGen), and 1\u00d7 Cell Stimulation Cocktail with protein transport inhibitors (containing phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin; eBioscience, Thermo Fisher Scientific, Waltham, MA). Antibodies used in this study are listed in Supplementary Table\u00a0S1<\/a> and were diluted using Brilliant Stain Buffer (566349, BD Biosciences). Samples acquired on Aurora (Cytek, San Diego, CA) cytometers were analyzed with FlowJo software (Treestar). Visualization of the multidimensional data was performed using the dimensionality reduction algorithm Uniform Manifold Approximation and Projection (UMAP), provided in FlowJo Plugin Exchange (v4.0.4, Treestar). Annotated pie charts were generated by SPICE software developed by the National Institutes of Health (v6.1).<\/p>\n Parabiosis<\/p>\n Parabiosis surgery was performed as previously described56<\/a>. In brief, mirror-image skin incisions were made from the elbow to the knee in each mouse anesthetized with isoflurane. The peritoneal openings of the adjacent parabionts were sutured together. Forelimbs and hindlimbs were sutured together, and the skin of each incision was closed using continuous suture. Each mouse was injected subcutaneously with buprenorphine, as directed, for pain and monitored during recovery. Parabiosed pairs were analyzed more than 2 weeks after surgery.<\/p>\n Generation of bone marrow chimeras<\/p>\n Bone marrow and mixed bone marrow chimeras were generated as previously described54<\/a>,57<\/a>. First, recipient C57BL\/6 mice were irradiated with 500\u2009cGy followed by a second dose of 550\u2009cGy 3\u2009h apart. To obtain donor bone marrow, femurs and tibiae were harvested, and the bone marrow was flushed out. For bone marrow chimeras, 1 \u00d7 107 bone marrow cells from aged or young mice were injected into irradiated young or aged mice. For mixed bone marrow chimeras, 1 \u00d7 107 bone marrow cells from young and aged mice of a 1:1 mixture were injected into irradiated young or aged mice. After 8\u201312 weeks, the recipients were used for the experiments.<\/p>\n Generation of bone marrow-derived macrophages<\/p>\n Bone marrow-derived macrophages were generated as described previously58<\/a>. Briefly, bone marrow cells from flushed marrow cavities of femurs and tibiae of >65 weeks old female C57BL\/6 mice were cultured in RPMI 1640 with 10% FBS containing 10\u2009ng\/ml macrophage colony-stimulating factor (M-CSF, PeproTech, Rocky Hill, NJ, USA) with\/without thymulin (1\u2009\u00b5g\/ml) and an equimolar concentration of ZnCl2. A control group was cultured in the same media with M-CSF and ZnCl2. Medium was changed daily. One week later, bone marrow-derived macrophages were harvested. Cells were stimulated with LPS (100\u2009ng\/ml; LPS-SM ultrapure, InvivoGen, San Diego, CA, USA) for 24\u2009h before ELISA analysis.<\/p>\n ELISA<\/p>\n Levels of IL-1\u03b1, IL-1\u03b2, IL-6, and TNF-\u03b1 secreted by aged bone marrow-derived macrophages were measured using ELISA MAX Deluxe Set Mouse IL-1\u03b1 (BioLegend), Mouse IL-1\u03b2 ELISA Kit (Sigma-Aldrich), Quantikine Mouse IL-6 ELISA Kit (R&D Systems), and BD OptEIA Mouse TNF ELISA Set II (BD Biosciences), respectively. The absorbance of each ELISA plate was measured using a microplate reader CLARIOstar Plus (BMG LABTECH, Weston Parkway, NC).<\/p>\n NF-\u03baB DNA-binding assay<\/p>\n The NF-\u03baB p65 DNA-binding activity was measured with the TransAM NF-\u03baB p65 according to the manufacturer\u2019s recommendations (Active Motif, Carlsbad, CA, USA). Nuclear extracts were prepared from 1 \u00d7 106 bone marrow-derived macrophages generated from aged mice. After cells are cultured with thymulin (1\u2009\u03bcg\/ml) and an equimolar concentration of ZnCl2 or ZnCl2 only (control) for 1 week, the cells were activated with LPS (100\u2009ng\/ml) for 1\u2009h, and cell lysates were prepared in 200\u2009\u03bcl lysis buffer [10\u2009mM Hepes-KOH, pH 7.8, 10\u2009mM KCl, 0.1\u2009mM EDTA, pH 8.0, protease inhibitor mixture (Roche), and 0.1% Nonidet P-40], and nuclear extracts were obtained in 30\u2009\u03bcl buffer [50\u2009mM Hepes-KOH, pH 7.8, 420\u2009mM KCl, 0.1\u2009mM EDTA, pH 8.0, 5\u2009mM MgCl2, protease inhibitor mixture (Roche), and 20% glycerol]58<\/a>. Nuclear extracts (5\u2009\u03bcl) in microwells were used to evaluate NF-\u03baB DNA-binding activity.<\/p>\n Immunoblot analysis<\/p>\n Cells were lysed with 200\u2009\u03bcl lysis buffer [10\u2009mM Hepes-KOH, pH 7.8, 10\u2009mM KCl, 0.1\u2009mM EDTA, pH 8.0, protease inhibitor mixture (Roche), and 0.1% Nonidet P-40]. The cell lysates were separated by standard SDS-PAGE (Bioland Scientific, Paramount, CA) and analyzed by immunoblotting using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The following antibodies were used: anti-phospho-I\u03baB\u03b1 (Ser32\/36; 5A5, Cell Signaling Technology), goat anti-mouse IgG-HRP (Poly4053, BioLegend), and anti-\u03b2-actin-HRP (2F1-1, BioLegend). The dilution ratios for anti-phospho-I\u03baB\u03b1, goat anti-mouse IgG-HRP, and anti-\u03b2-actin-HRP were 1:1000, 1:2000, and 1:1000, respectively. The Western HRP Substrate (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific) was used for the development of positive signals, and chemiluminescence was detected using an iBright 1500 (Thermo Fisher Scientific).<\/p>\n Luciferase reporter assay<\/p>\n Luciferase reporter assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). For transfections, 1 \u00d7 105 RAW 264.7 cells were transfected with 0.8\u2009\u03bcg of pmIL-6 FL (WT NF-\u03baB) or pmIL-6 mut NF-\u03baB plasmid (mut NF-\u03baB), and 0.1\u2009\u03bcg of pRL-TK using Lipofectamine 2000 (Invitrogen) and then plated in a 24-well plate at 1 \u00d7 105 cells\/well. After 24\u2009h, cells were activated by LPS (100\u2009ng\/ml, InvivoGen) and thymulin (1\u2009\u03bcg\/ml) with an equimolar concentration of ZnCl2 or ZnCl2 only (control) for 24\u2009h. Following activation, cell extracts were prepared using Passive Lysis Buffer (Promega). Luciferase activity was determined from a 20-\u03bcl cell extract and measured on the CLARIOstar Plus (BMG LABTECH) microplate reader.<\/p>\n CTL killing assay<\/p>\n CTL killing assays were performed as previously described59<\/a>. Cytotoxicity was assessed using a 7-AAD\/CFSE cell-mediated cytotoxicity assay kit (Cayman Chemical, MI, USA) according to the manufacturer\u2019s instructions. Briefly, whole splenocytes were isolated from AT-3 or E0771 tumor-bearing aged mice 2 weeks after tumor implantation, following in vivo thymulin or vehicle (control) conditioning as described above. A total of 4 \u00d7 10\u2077 splenocytes were co-cultured with 2 \u00d7 10\u2076 IFN-\u03b3-treated (100\u2009U\/ml; PeproTech) AT-3 or E0771 tumor cells in the presence of thymulin (1\u2009\u03bcg\/ml) and an equimolar concentration of ZnCl\u2082 or ZnCl\u2082 alone (control). After 5 days, cells were harvested and used as effector cells (E). For target cell (T) preparation, AT-3 or E0771 tumor cells were treated with IFN-\u03b3 (100\u2009U\/ml) for 48\u2009h prior to use. Target cells were labeled with CFSE (5\u2009mM stock solution in PBS) for 10\u2009min at 37\u2009\u00b0C, washed twice with PBS, and immediately used in the assay. CFSE-labeled target cells were co-incubated with effector cells at the indicated effector-to-target (E:T) ratios (12.5:1, 25:1, and 50:1) for 4\u2009h. Flow cytometric analysis was performed as described above. The percentage of specific lysis was calculated as follows: % specific lysis = 100 \u00d7 (% sample lysis \u2212 % basal lysis) \/ (100 \u2212 % basal lysis), where % sample lysis and % basal lysis represent the percentage of CFSE\u207a 7-AAD\u207a target cells in the presence or absence of effector cells, respectively.<\/p>\n Human PBMC analysis<\/p>\n Written informed consent was obtained from 93 healthy donors aged between 21 and 87 with no personal history of cancer for the collection, storage, and analysis of blood samples under the Institutional Review Board of the University of Southern California (approval number: HS-22-00354) in accordance with the Declaration of Helsinki. Peripheral blood was obtained, and PBMCs were isolated using Lymphocyte Separation Medium (Corning) density gradient centrifugation and stored as previously described60<\/a>,61<\/a>,62<\/a>,63<\/a>. For the evaluation of the effect of thymulin, cells were cultured with thymulin (1\u2009\u03bcg\/ml) and an equimolar concentration of ZnCl2 or ZnCl2 only (control) for 24\u2009h and were activated by LPS (100\u2009ng\/ml, InvivoGen) and 1\u00d7 Cell Stimulation Cocktail with protein transport inhibitors (Thermo Fisher Scientific) for 4\u2009h and were analyzed by flow cytometry.<\/p>\n IPA database exploration<\/p>\n To identify candidate factors associated with aging and inflammation, we performed a systematic exploration of the QIAGEN Ingenuity Pathway Analysis (IPA) database64<\/a>. Within the \u201cDiseases and Functions\u201d category, the term \u201cAging\u201d was used as a query. This search initially yielded 163 molecules, all of which were extracted for further analysis (Supplementary Dataset\u00a03<\/a>). These molecules were then filtered based on the \u201cCausal or Correlated\u201d annotation provided in IPA, and 35 molecules classified as \u201cCausal\u201d were selected. Among these, we further narrowed down the candidates by applying the criteria \u201cDisease or Function = Longevity\u201d and \u201cEffect on Disease or Function = decreases\/affects,\u201d resulting in 9 molecules. Finally, from these 9 candidates, we identified molecules for which knockout mouse models have been reported to exhibit increased levels of pro-inflammatory cytokines, including IL-1, IL-6, and\/or TNF-\u03b1. Based on this criterion, three factors\u2014FOXO3, LMNA, and SIRT family\u2014were selected for subsequent analyses.<\/p>\n Single-cell RNA sequencing<\/p>\n Count matrices, barcodes, feature data, and patient metadata for the eight oldest and eight youngest untreated primary breast tumors were downloaded from the Gene Expression Omnibus (GEO) (GSE176078). Cells with more than 200 detected genes (nFeature_RNA\u2009>\u2009200), more than 250 total detected transcripts (nCount_RNA\u2009>\u2009250), and less than 20% mitochondrial content were retained for downstream analyses. Doublets were removed using the DoubletFinder R package (v2.0.6)65<\/a>. Briefly, each patient sample is preprocessed using Seurat (v5.4.0), and doublet detection was performed using the top 50 principal components. Artificial doublets were generated and integrated into each patient dataset at a defined proportion (pN = 0.25). The optimal neighborhood parameter (pK) was determined empirically using the paramSweep, summarizeSweep, and find.pK functions. Optimal pK was selected based on the maximum BCmetric score. The expected number of doublets (nExp) was estimated as 7.5% of the total cell number and adjusted for homotypic doublets based on cluster identities. Cells were then classified using DoubletFinder, and predicted doublets were removed from downstream analyses. Filtered datasets were merged, normalized using SCTransform, and subjected to principal component analysis (PCA) for dimensionality reduction. The ElbowPlot function was used to determine the optimal dimensionality of the dataset (PCs = 30). Data integration was performed using Seurat\u2019s IntegrateLayers function with canonical correlation analysis (CCA) to minimize batch effects. A shared nearest neighbor graph was constructed using the FindNeighbors function, and cell clustering was performed using the FindClusters function at a resolution of 0.8. Differentially expressed genes (>25% cells, >0.25-fold change) were identified using the FindAllMarkers function and used for cluster annotation. Differential expression between annotated clusters was determined using the MAST method via the FindMarkers function, using a minimum expression proportion of 25% and a minimum fold change of 0.1. Single-cell gene set enrichment analysis (GSEA) was performed using VISION (v3.0.2) to calculate enrichment scores for curated gene signatures and biological pathways.<\/p>\n Statistical analysis<\/p>\n Statistical analysis was performed using a two-tailed Student\u2019s t-test for comparisons between 2 groups, a one-way ANOVA with Tukey\u2019s multiple comparisons test for comparisons among more than two groups, a two-way ANOVA with Bonferroni\u2019s multiple comparisons test, the Mantel\u2013Cox method (log-rank test) for survival analysis, or Pearson correlation analysis to assess relationships among variables, using GraphPad Prism 10.5.0 (GraphPad Software). p\u2009<\u20090.05 was considered statistically significant. Data are presented as mean\u2009\u00b1\u2009SEM.<\/p>\n Reporting summary<\/p>\n