Mid-old cells are a potential target for anti-aging interventions in the elderly

Human tissue experiments

This study complies with the Declaration of Helsinki and was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-BMR-OBS-20-552, Suwon, Korea), and informed consent was obtained from all patients, parents, or legal guardians as appropriate. We obtained serval normal tissues (colon, lung, liver, and skin) from surgically resected specimens adjacent to pathogenic regions by an experienced pathologist. Although histologically normal tissues adjacent to tumors were present within 1 cm from the margins of the tumor, to exclude possibility potential contamination with tumor tissue, we obtained normal tissues from histologically normal (non-pathologic) tissues around the resection margin of surgically resected specimens which were located at 5–12 cm from the pathologic regions. Normal tissues were separately sampled in representative areas by an experienced pathologist, fixed immediately and processed to be formalin-fixed paraffin-embedded (FFPE), according to the tissue specimen regulations of Ajou University Hospital (Supplementary Table 1-5). The standard age was classified into two groups: young (≤40 years) and elderly for colon (≥80 years), lung (≥75 years), liver (≥80 years), serum (≥70 years), and skin (≥70 years).

Animal experiments

Male and female C57BL/6J mice (4-month-old and 12-month-old) were purchased from Korea Basic Science Institute (KBSI, Gwangju, Korea). The 12-month-old mice were raised under clean room system until they became 18 and 22 months old. C57BL/6N aged mice were obtained from ORIENT BIO (Seongnam, Korea) and subsequently raised until they reached 4 months or 23 months old. All animal procedures were approved by the institutional animal research ethics committee at Ajou University Medical Center (approval number: 2020-0051). Blood was immediately collected by intracardial puncture. Blood gas and electrolytes were analyzed by I-Smart 300 Blood Gas & Electrolyte Analyzer (I-SENS Inc., Seoul, Korea). Serum was collected by centrifuging blood samples. Colon, small intestine, stomach, lung, liver, esophagus, and skin were isolated and embedded in paraffin. To determine whether SLIT2 administration caused an anti-aging phenotype, recombinant mouse SLIT2 (5444-SL, R&D Systems, Minneapolis, MN, 2 µg/mouse) was intraperitoneally injected twice weekly for 3 weeks in 22-month-old, 5 weeks in 23-month-old, or 5 weeks in 4-month-old mice. The movement of the mice was recorded on video for a duration of a minute, two times before reaching the experimental endpoint. Movement activity was determined using measurement of the total movement distance for 1 min. Each mouse movement recorded on video was tracked manually. For the four limb-hanging tests, mice were positioned on a metal wired mesh and inverted. Hanging time was normalized to body weight. The results were averaged from 5 trials for each mouse.

Muscle weight and cross-sectional area (CSA) measurements

The skeletal muscles containing gastrocnemius, tibialis anterior and soleus muscle were dissected from the hindlimb and measured a weight and embedded in an OCT compound (Sakura Finetek, Torrance, CA). Sections were cut transversely at 7-μm thickness. For measurement the cross-sectional area (CSA), Laminin antibody (1:500, ab11575, Abcam, Cambridge, UK) was incubated overnight at 4 °C. Sides were washed with PBS and incubated with Alexa-488 conjugated secondary antibody (1:400, A21206, Invitrogen, Carlsbad, CA) for 1 h at room temperature and then stained with DAPI (ab228549, Abcam) for staining nuclei. Images were visualized with a Zeiss LSM 710 confocal laser microscope (Zeiss, Oberkochen, Germany). The CSA was measured as the internal laminin unstained area by the ImageJ software (NIH, Bethesda, MD, freeware imaging software). Two hundred fifty fibers per muscle were measured.

Cell culture

Normal human dermal fibroblasts were isolated from foreskin. Isolated single cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and antibiotics (Invitrogen) at 37 °C in a humidified incubator with 5% CO₂. Young cells were defined as DT < 2 days, mid-old cells as DT = 5–7 days, old cells as DT > 14 days. Primary pulmonary artery smooth muscle cells (PASMCs) were purchased from American Tissue Culture Collections (ATCC, Rockville, MD) and were cultured in vascular cell basal medium (PCS-100-030, ATCC) supplemented with vascular smooth muscle cell growth kit (PCS-100-042, ATCC) at 37 °C in a humidified incubator with 5% CO₂. Human pulmonary alveolar epithelial cells (HPAEpiC) and human primary colonic epithelial cells (HCoEpiC) were purchase from ScienCell Research Laboratories, Inc. (Carlsbad, CA) and Cell Biologics, Inc. (Chicago, IL), respectively, and were cultured in Alveolar Epithelial Cell Medium (ScienCell Research Laboratories, Inc.) and Human Epithelial Cell Basal Medium (Cell Biologics, Inc.), respectively. SW480 cells were purchased from ATCC and were cultured in RPMI1640 medium (WELGENE Inc., Gyeongsan, Korea) supplemented with 10% FBS (Invitrogen) at 37 °C. The IMR90 cells, purchased from ATCC, were cultured in DMEM media with 10% FBS at 37 °C. Heterochromatin foci were assessed by staining the cells with a 1:10,000 diluted DAPI solution (ab228549, Abcam) and examining them using a Zeiss LSM 710 confocal laser microscope.

Real-time PCR

First-strand cDNA was produced from 1 μg total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) and oligo(dT) primer mix. Real-time PCR was conducted with Power SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA) using the following conditions: Initial activation at 95 °C 5 min, by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The primers used for real-time PCR are specified in Supplementary Table 6.

Lentivirus preparation

Lentiviral particles were generated by co-transfection of the lentiviral expression vector (lentivirus plasmid, shRNA lentiviral vector) with lentiviral packaging plasmids (pGagpol, pVSV-G). HEK-293TN cells were transfected with the lentiviral vectors, and the virus-containing supernatant was harvested after 48 h. The SBLC and SLIT2 lentivirus plasmid was constructed by Vector Builder (Chicago, IL) and packaged. Human CXCL12 was cloned from normal human fibroblasts in the laboratory. The coding sequence of CXCL12 was inserted into the pCDH-CMV-MCS-EF1-Puro lentiviral vector (System Biosciences, Mountain View, CA). shRNA sequences are specified in Supplementary Table 7.

Immunocytochemistry

Cell culture plates were placed in an incubator at 37 °C, 5% CO₂ for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.05% Triton X-100 in 1× PBS (0.1% TWEEN-20) for 15 min. Plates were washed twice with 1× PBS-T (0.5% TWEEN-20) and blocked with 1% BSA in 1× PBS for 30 min. Primary antibodies were applied overnight, including Ki67, 1:500 (M7240, Dako, Santa Clara, CA); p-Erk1/2, 1:100 (9101, Cell Signaling Technology, Danvers, MA); p-NFκB, 1:100 (3033, Cell Signaling Technology); p21^Waf1, 1:100 (ab109520, Abcam); p16^INK4A, 1:300 (ab189034, Abcam). Cells were washed two times with 1× PBS-T and incubated with appropriate conjugated secondary antibodies for 1 h at room temperature. Secondary antibodies were as follows: Alexa Fluor 488, 1:600 (A-21206, Thermo Fisher Scientific); Alexa Fluor 555, 1:600 (A-31572, Thermo Fisher Scientific).

EdU proliferation assay

EdU proliferation assay was performed using EdU proliferation kit (ab222421, Abcam) following the protocol. Cells were plated on cover glass and incubated overnight. The cell culture media were then replaced with 10 μM EdU and incubated overnight. Afterward, cells were fixed with 4% formaldehyde and permeabilized using permeabilization buffer. Subsequently, cells were washed with Tris-based saline buffer and treated with the reaction mix containing iFluor 647 azide and EdU additive solution for 30 min. Image acquisition was conducted using a Zeiss LSM 710 confocal laser microscope.

RNA sequencing analysis

Total RNA was extracted from three independent samples using a Macherey-Nagel RNA kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). Briefly, sample quality was checked using a Bioanalyzer RNA Chip (Agilent Technologies, Santa Clara, CA) and RNA-seq was conducted with a Nextseq 500 (Illumina, San Diego, CA). To prepare the data for analysis, Skewer version 0.2.2 was used to trim potentially existing sequencing adapters and raw quality bases from the raw reads. The resulting high-quality reads were then mapped to the reference genome using STAR version 2.5 software. A sequencing library was prepared by LAS Inc. (Gimpo, Korea). Gene models based on the gene annotation of the reference genome hg38 from the UCSC genome in GTF format were used to calculate gene expression values in fragments per kilobase of transcript per million fragments mapped (FPKM) units. Heatmaps drawn in this study used FPKM data to visualize the gene expression. For variance stabilizing transformation (VST) and GSEA, raw sequencing data are trimmed and mapped using AltAnalyze (v.2.1.4.3). Raw count data are loaded in the R (v 4.0.3) DESeq2 package (v.1.38.3). DESeq data set was generated using ‘DESeq’ function. Sample distance was generated by calculating VST and drawn using ‘pheatmap’ function. GSEA was performed using the “fgsea” package (v.1.24.0) from R. Permutations were performed 1000 times, and p values were calculated using statistics from GSEA. The weighted Kolmogorov-Smirnov statistics were calculated to a running sum of a ranked gene list.

Analysis of public scRNA-seq datasets for human tissues

In the analysis of cells from normal tissue, the publicly available scRNA-seq datasets, GSE178341³⁴ (peri-lesional normal colon) was utilized. The gene expression of young (35 and 42 years old) and Old (81, 81, 82, and 90) patients were compared and analyzed. The offered count data and metadata were loaded into ‘Seurat Object’ using the R (version 4.0.3) ‘Seurat’ package (version 4.3.0) Following normalization was performed using the ‘NormalizeData’ function, and principal component analysis (PCA) was performed using the ‘RunPCA’ function. To determine dimensionality, ‘JackStraw’ function was performed with 100 times of replications. Cells with the number of featured RNA >200 & <10,000 and <50% mitochondrial gene percentage were selected. FN1 and ACTA2 were used to annotate fibroblasts and smooth muscle cells, respectively.

IHC and IF staining

Immunohistochemical staining of FFPE tissue sections (4-μm) was performed on Benchmark XT automated IHC stainer (Ventana Medical Systems Inc., Tucson, AZ). The primary antibodies used were as follows: human p16^INK4A, predilution (805-4713, Roche, Tucson, AZ); human Ki67, 1:3000 (M7240, Dako); human H3K9me3, 1:500 (ab176916, Abcam); human p21^Waf1, 1:300 (2990-1, Epitomics, Nanterre, France); human IL1β, 1:100 (ab9722, Abcam); human SAA1, 1:75 (MAB30191, R&D Systems); human SLIT2, 1:80 (HPA023088, Atlas Antibodies, Bromma, Sweden); human type IV Collagen, 1:300 (ab6586, Abcam); human MMP9, 1:100 (GTX100458, Genetex Inc., Irvine, CA); human CXCL12, 1:100 (MAB350, R&D Systems); human CCL5, 1:200 (MAB278, R&D Systems); mouse p16^INK4A, 1:300. (ab189034, Abcam); mouse Ki67, 1:200 (ab16667, Abcam); mouse SAA1/2, 1:1000 (ab199030, Abcam); mouse SLIT2, 1:200 (PA5-31133, Invitrogen); human/mouse NHE3, 1:500 (NBP1-82574, Novus Biologicals, Centennial, CO); human/mouse SCNN1A, 1:100 (ab272878, Abcam); mouse SOX2, 1:100 (ab92494, Abcam). IHC results were presented as the percentage of positive cells in the stromal or epithelial region in the three random areas at high power. The Benchmark XT automated immunohistochemistry stainer was used to perform double immunohistochemistry. The first antibodies were detected using the UltraView universal DAB detection kit (#760-500, Ventana Medical Systems Inc), followed by the detection of the second antibodies using the UltraView Universal Alkaline Phosphatase Red Detection kit (#760-501, Ventana Medical Systems Inc). For IHC analysis of type IV collagen and NHE3, an experienced pathologist evaluated the intensity of immunostaining and graded it as weak, moderate, or strong. Counterstaining of each stained slide was performed using Hematoxylin II (790-2208, Ventana Medical Sytems Inc.). For IF staining of FFPE tissue sections, the following primary antibodies were incubated overnight at 4 °C: SAA1, 1:50 (MAB30191, R&D Systems); p21^Waf1, 1:100 (ab109520, Abcam); IL1β, 1:100 (ab9722, Abcam); SLIT2, 1:200 (PA5-31133, Invitrogen); p16^INK4A, predilution (805-4713, Roche); Vimentin, 1:400 (ab92547, Abcam); Vimentin, 1:100 (AF2105, R&D Systems); Smoothelin, 1:100 (OMA1-06020, Invitrogen); Smoothelin, 1:500 (NBP2-37931, Novus Biologicals); E-cadherin, 1:200 (610181, Becton Dickinson); E-cadherin, 1:100 (ab15148, Abcam); CDX2, 1:100, (235R-15, Cell Margue, Rocklin, CA). Slides were washed twice with PBS and incubated with appropriately conjugated secondary antibodies for 1 h at room temperature. Secondary antibodies for IF staining were as follows: Alexa Fluor 488, 1:300 (ab150113, Abcam); Alexa Flour 488, 1:300 (A-11055, Invitrogen); Alexa Flour 405, 1:300 (A48255, Invitrogen); Alexa Fluor 594, 1:300 (ab150080, Abcam); Alexa Fluor 594, 1:300 (ab150116, Abcam). The stained cells were visualized with a Zeiss LSM 710 confocal laser microscope.

Image processing for Erk1/2 activity reconstruction

Registration and subsequent background subtraction of fluorescence images was performed using ImageJ software. We adopted same strategy as CellProfiler⁵³ for nuclear and cytosol segmentation and quantified the average fluorescence level of Erk-KTR-mClover⁵⁴ for each segment using custom MATLAB codes. Briefly, cell lines were incubated with 500 ng/ml Hoechst (H1399, Invitrogen) for 10 min to label the nuclei. Subsequently, the cytoplasmic area was defined by a 5-pixel (~1.1 μm) annular ring from the nuclear region. Tracking of each cell was conducted using a Munkres assignment algorithm to record the trace of mean fluorescence intensity ratio (cytoplasmic over nuclear, C/N). The extracted C/N ratio was then normalized to frame just prior to stimulation. Normalized C/N ratio curves were further fitted with the following equation to obtain kinetic parameters (trans-localization rate k_on, time delay t_on and C/N ratio at equilibrium R_eq).

If the C/N ratio pattern was recovered (such as old cells in Supplementary Figure 3c), we fitted the C/N curve using the equation below.

Model fitting was conducted using the nlinfit function in MATLAB R2021.

Live cell imaging

Fluorescence (DAPI and FITC filter, Nikon, Tokyo, Japan) and bright field difference interference contrast (BF-DIC, Nikon, TI2-C-DICP-I) microscopy was performed using an inverted microscope (Nikon, ECLIPSE Ti2-E) equipped with a perfect focus system (PFS, Nikon, TI2-N-NDA-P), a motorized stage (Nikon, TI2-S-SE-E), an electron multiplying charge-coupled device (EM-CCD, Andor Technology, Belfast, Northern Ireland, DU-897U-CS0) and a 60 x oil immersion objective lens (Nikon, CFI Apochromat TIRF 60XC Oil, NA 1.49). An epifluorescence illuminator (Nikon, Intensilight C-HFGI, 130 W) was used as a light source for fluorescence. Temperature (37 °C), CO₂ (5%), and humidity (80%) were maintained during the live cell imaging by using stage top incubator (UNO-T-H-PREMIXED, Okolab, Ottaviano, Italy).

In vitro Matrigel degradation assay

SPLInsert™ (SPL Life Sciences Co., Ltd, Pocheon, Korea) with 6.5-mm polycarbonate membrane filters of 0.4 μm pore size were used to form dual compartments. Young or mid-old cells were seeded onto the lower chamber of the SPLInsert™ and the upper chamber of the SPLInsert™ was coated with Matrigel™ (Becton Dickinson) to mimic the conditions of the BM, individually. The upper chamber was transferred into the lower chamber and maintained for 2 days. Two experiments were then individually performed using a Matrigel-coated upper chamber, as follows; for the in vitro Matrigel degradation assay, Matrigel-coated membrane in the upper chamber was cut and boiled with 2× sample buffer (24 mM Tris-HCl,pH 6.8, 10% glycerol, 0.8% SDS, 2% 2-mercaptoethanol and 0.04% bromophenol blue). The supernatant was used for Western blotting to detect type IV collagen (1:1000, ab6586, Abcam) using standard procedures. To analyze the epithelial cell functional marker, HCoEpiC (5 × 10⁴) or SW480 (5 × 10⁴) or HPAEpiC (1.5 × 10⁴) cells were seeded on the Matrigel-coated upper chamber for 2 days and, two experiments were then performed. First, real-time PCR was performed to measure mRNA levels of functional marker genes. Second, immunocytochemistry staining was performed with F-actin staining to compare cell morphology. Cells were fixed in 4% paraformaldehyde for 10 mins and treated with TRITC- phalloidin (1:500, P1951, Sigma, St. Louis, MO) for 1 h and DAPI was counterstained (1:10000, D3571, Invitrogen) for 10 min. Stained cells were visualized with a Lionheart FX automated microscope (BioTek Instruments, Winooski, VT).

Metabolomics analysis

Young (DT1) and mid-old (DT4) fibroblasts (n = 3, each) were cultured in 150 mm culture dishes (353025, Corning Inc., Corning, NY). Both cell types were seeded with the aim of reaching a final cell count of 3 × 10⁶ at the time of harvest, which occurred 2 days later. The conditioned media (CM) was harvested and then centrifuged at 200 × g for 5 min. The supernatant was gently collected, and the subsequent analysis was performed by Basil Biotech (Incheon, Korea). The samples were added with 80% methanol. After vortexing for 1 min and centrifugation at 2000 × g for 10 min, supernatant was transferred to a new 1.5 mL tube and completely dried using a HyperVAC-MAX VC2200 centrifugal vacuum concentrator (Hanil Scientific Inc., Gimpo, Korea). Dried metabolite contents were reconstituted in 100 µL of 0.1% formic acid in water and then subjected to liquid chromatography-mass spectrometry (LC-MS) analysis. LC-MS analysis for metabolomics was performed using a Q Exactive™ Hybrid Quadrupole-Orbitrap MS (Thermo Fisher Scientific) coupled with a 1290 Infinity ultra-high performance liquid chromatography (UHPLC) (Agilent Technology). Metabolite mixtures were loaded using a ZORBAX Eclipse Plus C18 Rapid Resolution High Definition (RRHD) column (2.1 × 50 mm, 1.8 µm particles). The mobile phase solvents consisted of 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile, and the flow rate was fixed at 0.2 mL/min. The gradient of mobile phase was as follows: 2.5% solvent B in 5 min, 2.5–12.5% solvent B in 29 min, 12.5–25% solvent B in 11 min, 25–37.5% solvent B in 11 min, 37.5–80% solvent B in 0.1 min, holding at 80% of solvent B in 13.9 min, 80–2.5% solvent B in 0.1 min, 2.5% solvent B for 19.9 min. The electrospray source was equipped with a Heated Electrospray ionization (HESI‐II) Probe combined with the standard Thermo Scientific™ Ion Max source. Parameters were set as follows: positive mode, spray voltage; 3.8 kV, capillary temperature; and 320 °C. Properties of full MS/dd‐MS2 were set up as follows: full‐MS scans, 150–2000 m/z of scan range, 70,000 of resolution at 400 m/z, 1 × 10⁶ of AGC target, and maximum IT of 60 ms. For MS2 scans, the following parameters were used: 17,500 of resolution at 400 m/z, 2 × 10⁵ of AGC target, maximum IT was 250 ms, ±2 m/z of isolation width, and NCE for dd‐MS2 of 30%. Obtained UHPLC-Orbitrap-MS/MS RAW files were processed using Compound Discoverer 3.1.1.12^TM (Thermo Fisher Scientific). Untargeted metabolomics workflow was used to perform retention time alignment and compound identification. Identification of compounds was performed using mzCloud^TM and ChemSpider.

Lipidomics analysis

The analysis was performed by Basil Biotech. CM was harvested in a same method with the metabolomics analysis (see above). For lipid extraction from the harvest CM samples (young and mid-old, n = 3 each), a two-step method involving neutral and acidic extraction were used. At first, in neutral extraction, lipids from the samples were extracted according to the Folch method using a mixture of chloroform and methanol (2:1, v/v). The samples were vortexed and incubated on ice for 10 min. The samples were centrifuged at 13,800 × g for 2 min at 4 °C, supernatant was transferred to a new 1.5 mL tube. Next, in Acidic extraction, 750 μL of chloroform:methanol:HCl (1 N, 37%) (40:80:1, v/v/v) was added to the remaining samples. After incubating for 15 min at room temperature, 250 μL of cold chloroform and 450 μL of cold 0.1 M HCl were added to the sample. The mixture was vortexed for 1 min and centrifuged at 6500 × g for 2 min at 4 °C. The lower organic phase was collected and combined with the prior extract. The sample was then dried using HyperVAC-MAX VC2200 centrifugal vacuum concentrator (Hanil Scientific Inc.). Dried lipid contents were reconstituted in 50 µL of mobile phase solvent A:solvent B (2:1, v/v) and then subjected to LC-MS/MS analysis. LC-MS analysis for lipidomics was performed using a Q Exactive™ Hybrid Quadrupole-Orbitrap MS (Thermo Fisher Scientific) coupled to a 1290 Infinity UHPLC (Agilent Technology) with heated electrospray ionization (HESI). Lipids mixtures were loaded using a e Hypersil GOLD™ C18 HPLC column (2.1 × 100 mm; 1.9 μm particle size; Thermo Fisher Scientific). The mobile phase solvents consisted of (A) Acetonitrile:Methanol:Water (19:19:2, v/v/v) + 20 mmol/L ammonium formate + 0.1% formic acid (v/v) and (B) 2-propanol + 20 mmol/L ammonium formate + 0.1% formic acid (v/v), and the flow rate was fixed at 0.2 mL/min. The gradient of mobile phase was as follows: 0–5 min, 5% B; 5–15 min, 5–30% B; 15–22 min, 30–90% B; 22–25 min, 90% B; 25–26 min, 90–5% B; 26–30 min, 5% B. Parameters were set as follows: positive mode, spray voltage; 3.8 kV, capillary temperature; and 320 Celsius degree, and S-lens radio frequency(RF); 60%. Properties of full MS/dd‐MS2 were set up as follows: full‐MS scans, 150 to 2000 m/z of scan range, 70,000 of resolution, 1 × 10⁶ of AGC target, and maximum IT of 60 ms. For MS2 scans, the following parameters were used: 35,000 of resolution, 1 × 10⁵ of AGC target, maximum IT was 250 ms, ±1 m/z of isolation width, and NCE for dd‐MS2 of 30%. Obtained UHPLC-Orbitrap-MS/MS RAW files were processed using Lipid Search 4.2TM (Thermo Fisher Scientific). Lipid profiling under the following conditions: parent search; 0.2 Da, product search; 5.0 ppm, precursor ion mass tolerance; 8.0 ppm, M-score threshold; 2.0.

In vitro anti-aging experiment

Mid-old cells were co-cultured with young cells for 30 days. For co-culture, mid-old cells were seeded in a transwell (0.4-µm pore size, 6 well, Corning Inc.) low chamber (1 × 10⁴ cells /well) in a six-well plate. Young or mid-old cells were then seeded in the transwell upper chamber (1 × 10⁴ cells). The upper chamber was replaced weekly. For CM experiments, mid-old cells were treated with CM of young cells for 30 days. Mid-old cells were seeded onto six-well plates (1 × 10⁴ cells/well) and allowed to adhere for 24 h. Culture medium was then replaced with CM, which was harvested from the culture medium of young or mid-old cells (1 × 10⁴ cells) and centrifuged for 3 min at 200 × g. The CM was replaced every 2 days. For SLIT2 experiment, mid-old cells were seeded in a 60-mm dish (5 × 10⁴ cells). Medium was then replaced with serum-free medium with or without recombinant SLIT2 protein (150-11, PeproTech, Rocky Hill, NJ) for 24–48 h.

Immunoblotting

Cells were scraped off the plates, and lysates were collected in 1× RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP40 and 1% sodium deoxycholate) containing protease inhibitor cocktail (K272, Biovision, Milpitas, CA) and phosphatase inhibitor cocktail (K282, Biovision). Samples were resolved by electrophoresis an SDS-PAGE gel and PVDF membranes probed with the antibodies indicated; p16^INK4A, 1:1000 (LS-B1347, LSbio, Seattle, WA); p21^Waf1, 1:1000 (ab109520, Abcam); p53, 1:1000 (MA5-12557, Invitrogen); p-Erk1/2, 1:1000 (9101, Cell Signaling Technology); Total Erk1/2, 1:1000 (9107, Cell Signaling Technology); MDM2, 1:1000 (ab16895, Abcam); Actin, 1:3000 (Abc-2004, Abclon, Seoul, Korea); p-NFκB, 1:1000 (3033, Cell Signaling Technology); Total NFκB, 1:1000 (8242, Cell Signaling Technology); IκBα, 1:1000 (4814, Cell Signaling Technology); p-Pyk2, 1:1000 (3291, Cell Signaling Technology); Total Pyk2, 1:1000 (3292, Cell Signaling Technology); SOX2, 1:500 (ab92494, Abcam); OCT4, 1:1000 (ab19857, Abcam); MMP9, 1:1000 (GTX100458, Genetex Inc.); Tubulin, 1:1000 (sc-32293, Santa Cruz Biotechnology inc., Dallas, TX); GAPDH, 1:1000 (60004-1-lg, Proteintech, Rosemont, IL).

SA-β-Gal staining

Cells were fixed with 4% paraformaldehyde and incubated with SA-β-Gal (1 mg/ml X-gal, 40 mM citric acid/sodium phosphate pH 5.8, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl₂) solution for 12 h at 37 °C. After washing with PBS, SA-β-Gal-positive cells were then analyzed with inverted light microscopy (DMi1, Leica Microsystems, Wetzlar, Germany).

ELISA analysis

Mouse Serum Amyloid A (SAA) Quantikine ELISA Kit (MSAA00, R&D systems) was used for quantitative measurement of circulating SAA levels according to the manufacturer’s instructions. Human SAA1 concentration from plasm obtained from Ajou University Hospital, was measured using a Human Serum Amyloid A1 DuoSet ELISA (DY3019-05, R&D systems). Human SLIT2 (LS-F4840, LSBio), human MMP9 (DMP900, R&D system) and human IL1β (437004, Biolegend, San Diego, CA) concentration were measured from harvested medium of cells.

Long non-coding RNA analysis

To isolate exosomes, conditioned medium was harvested, and differential centrifugation was performed using Ultracentrifugation. Cellular debris was removed by centrifugation at 300 × g for 10 min, followed by centrifugation at 2000 × g for 20 min to remove apoptotic bodies. Macrovesicles were removed by centrifugation at 10,000 × g for 20 min, while exosomes were pelleted by centrifugation at 100,000 × g for 70 min. Pelleted exosomes were resuspended in RNA extraction solution (Macherey-Nagel GmbH & Co. KG) for total RNA extraction. cDNA synthesis was performed using an oligo(dT) primer mix and random hexamer primer mix and PCR was performed using primers (see Supplementary Table 6). Amplified DNA product was analyzed by agarose gel electrophoresis.

Single cell tracking

Cells, labeled with GFP, were tracked over time and through cell divisions using a Lionheart FX automated microscope (BioTek Instruments). Initially, 1 × 10² cells were seeded in a six-well culture plate (30006, SPL Life Sciences Co., Ltd.), and their individual coordinate locations were saved for subsequent tracking.

Microscope image acquisition

IHC staining image acquisition was conducting using a Leica Aperio Scanscope CS and Aperio ImageScope software (Leica Microsystems.). Cell images were acquired using a Leica DMi1 Inverted Microscope and Leica Application Suite (LAS) software (Leica Microsystems). IF images were captured on a Zeiss LSM 710 microscope and analyzed with Zeiss Axio Imager software.

Statistical analysis

Numerical data are presented as mean ± SD of independent determinations. Statistical differences were examined using the Student’s t test and the Mann–Whitney U test. A two-tailed Student’s t test was conducted for samples following a normal distribution. For samples that did not follow a normal distribution, a one-tailed Mann–Whitney U test was performed. The proportional difference between two groups is calculated by Chi-square test.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.