Cell lines

Four CMT cell lines (CMT-U27, CMT-U27e, CF41.Mg, and DMGT), along with two human breast cancer cell lines (MDA-MB-231 and MCF7) were utilized in this study. CMT-U27 and CF41.Mg were purchased from American type culture collection (ATCC; CRL-3456™ and CRL-6232TM, respectively). CMT-U27 originated from a simple carcinoma-subtype CMT obtained from a 14-year-old female Poodle, while CF41.Mg was derived from a mixed-type CMT acquired from a 10-year-old female Beagle. DMGT originated from a complex carcinoma-subtype CMT obtained from a six-year-old female crossbreed dog, originally established by Dr. Shih-Chieh Chang’s team at the Department of Veterinary Medicine, National Chung Hsing University. The identities of CMT cell lines were confirmed by short tandem repeat (STR) profiling analysis.

CMT-U27e, a subline derived from CMT-U27, was obtained after mock selection in parallel with puromycin selection of AGR2-knockout and control CMT-U27 clones (see below). It exhibits elevated levels of AGR2 compared to the original cell line. CMT-U27 and its derivatives were maintained in Roswell Park Memorial Institute 1640 medium (RPMI; Gibco, Thermo Fisher Scientific), while CF41.Mg, DMGT, MDA-MB-231, and MCF7 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Thermo Fisher Scientific) with high glucose. For maintenance, cells were grown in culture media supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) at 37 °C in an incubator supplied with 5% CO2.

Transfection

For ectopic plasmid transfection, cells were seeded in 6-well plates at the following densities: CMT-U27 (4.5 × 105 cells/well), CF41.Mg (2 × 105 cells/well), DMGT (3 × 105 cells/well), MDA-MB-231 (3 × 105 cells/well), or MCF7 (3 × 105 cells/well). The cells were allowed to grow for 18 to 24 h before transfection. CMT-U27, DMGT, MDA-MB-231, or MCF7 was transfected with 1.5 µg of plasmids or the mock control using 4.5 µL of Lipofectamine™ 2000 (Invitrogen, Thermo Fisher Scientific). CF41.Mg was transfected with 2 µg of plasmids using 4 µL of jetPRIME® (Polyplus, Illkirch, France) according to the manufacturer’s instruction.

For siRNA transfection, CMT-U27 or CMT-U27e was transfected with 75 nM siRNA oligonucleotide duplexes using 10 µL of Lipofectamine™ RNAiMAX (Invitrogen, Thermo Fisher Scientific). Following media replacement, transfected cells were cultured for the specified time intervals before further experiments.

AGR2-expressing vectors

The expression vector for canine AGR2 tagged with Myc and His was previously constructed in a pcDNA3.1(+)-A-myc.His backbone [14], denoted pcDNA3.1-myc.His-AGR2 throughout the text. The expression vector for HA-tagged AGR2 was generated by integrating the DNA fragment encoding canine AGR2 into a pcDNA3.1(+)-HA-C vector via the KpnI and XhoI site. To generate the expression vector for human AGR2, the DNA fragment encoding human AGR2 was obtained from human A549 cells using similar methods and then inserted into a pcDNA3.1(+)-A-myc.His vector, referred to as pcDNA3.1-myc.His-hAGR2 in the text.

siRNA oligonucleotide duplexes

Duplex oligonucleotides of siRNA against canine AGR2 (denoted siAGR2) were synthesized by and purchased from GeneDireX Inc. (Taoyuan, Taiwan). Sequences of two siAGR2 duplexes are listed as follows: siAGR2-1: 5ʹ-GGCCAAAGAUAUCACAGUUTT-3ʹ and 5ʹ-AACUGUGAUAUAUCUUUGGCCTT-3ʹ; siAGR2-2: 5ʹ-GACUCAGACCUAUGAAGAATT-3ʹ and 5ʹ-UUCUUCAUAGGUCUGAGUCTT-3ʹ.

siRNA duplexes against canine YWHAE (denoted siYWHAE) or those against canine ACTN4 (denoted siACTN4) were synthesized by and purchased from Eurogentec (Seraing, Liège, Belgium). Sequences of two siYWHAE and siACTN4 duplexes are listed as follows: siYWHAE-1: 5ʹ-GUUGACAGUUGAAGAAAGATT-3ʹ and 5ʹ-UCUUUCUUCAACUGUCAACTT-3ʹ; siYWHAE-2: 5ʹ-CAAGGGAGGAGAAGACAAATT-3ʹ and 5ʹ-UUUGUCUUCUCCUCCCUUG-3ʹ; siACTN4-1: 5ʹ-CCUUCCAAGCCUUCAUUGATT-3’ and 5ʹ-UCAAUGAAGGCUUGGAAGGTT-3ʹ; siACTN4-2: 5ʹ-GUUGACAGUUGAAGAAAGATT-3ʹ and 5ʹ-UCUUUCUUCAACUGUCAACTT-3ʹ.

Generation of AGR2-knockout (KO) CMT cell clones

The CRISPR-Cas9 technique was used to generate AGR2-KO cells in CMT-U27 by transient transfection method. Single guide RNA (sgRNAs) targeting canine AGR2, i.e., canine AGR2 sgRNA-1 or AGR2 sgRNA-2, was constructed into the pU6-gRNA.Ppuro vector by the National RNAi Core Facility, Academia Sinica Taiwan (Nankang, Taipei, Taiwan). AGR2 sgRNA-1 and AGR2 sgRNA-2 sequences are as follows: 5ʹ-GAGTGTAAGAGAGGGCGACG-3ʹ and 5ʹ-GTTGGCCAGAGTGTAAGAGA-3ʹ, respectively. CMT-U27 cells were transfected with the plasmid expressing AGR2 sgRNA-1, AGR2 sgRNA-2, or a mock control using Lipofectamin™ 2000 as described above. The transfectants were selected with 1 μg/mL puromycin at 24 h post-transfection for four days. Live cells were serially diluted and seeded into a 96-well culture plate, then grown until a single cell clone was obtained. Two AGR2-KO clones, KO-S10 and KO-S4, were generated using AGR2 sgRNA-1 and AGR2 sgRNA-2, respectively. The null expression of AGR2 was verified by immunoblotting.

Collection of conditioned media and cell lysates

Conditioned media (CM) were first centrifuged at 300 × g for 10 min to remove detached cells and further centrifuged at 3000 × g for 10 min to remove cell debris. Collected CM was used for following experiments or applied to further processes as indicated elsewhere. Cell lysates were harvested in a homogenization buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, pH 8.0, 1% NP-40) supplemented with the protease inhibitor cocktail (VWR Life Science, USA) and phenyl methyl sulfonyl fluoride (PMSF; Sigma-Aldrich, USA). Protein concentrations of the CM and cell lysates were measured using a BCA protein concentration assay kit (Pierce, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Transwell migration assay

Conditioned media (CM) collected from corresponding cells subjected to various treatments, or fresh media supplemented with recombinant proteins, were added to the bottom well as the attractant (600 µL per well). Cells were suspended in 160 µL of serum-free culture media, stained with 40 µL of Hoechst 33342 Staining Dye Solution (500 nM; Abcam, UK), and then placed in a top hanging insert with a pore size of 8 μm (SPL, Korea) for a transwell migration assay. Cells were seeded into the insert as follows: CMT-U27 (9 × 104 cells), CMT-U27e (9 × 104 cells), CF41.Mg (6 × 104 cells), DMGT (6 × 104 cells), MDA-MB-231 (1 × 105 cells), or MCF7 (1 × 105 cells). Following a 16- to 20-h incubation at 37 °C, cells in the inserts were fixed with 10% formalin at room temperature for 30 min and then washed several times with 1 × PBS. Cells that remained inside the insert (non-migrated cells) were removed with a cotton swap. Images of migrated cells were acquired by using the Leica DMI3000 B Inverted Microscope (Leica, Wetzlar, Germany) equipped with a Zyla 5.5 Megapixel sCMOS camera (Andor Technology, Belfas, Ireland) under a 10 × objective. The number of migrated cells was measured with MetaMorph® NX Software (Molecular Devices, San Jose, CA, USA) and presented as the mean + SD of three independent experiments.

Proteomics analysis of the AGR2-affected CMT secretome by a gel-enhanced liquid chromatography-tandem mass spectrometry (GeLC-MS/MS)

Serum-free CM samples collected from transfected CMT cells were deprived of cells and cell debris and subsequently concentrated and desalted with Vivaspin® 20 (GE Healthcare) following the manufacturer’s instructions. Concentrated CM proteins (10 µg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with 0.5% Coomassie Brilliant Blue G-250 (AppliChem GmbH, Germany). Individual gel lanes were cut into 15 pieces, each of which was dehydrated in acetonitrile (Mallinckrodt Baker) and dried using SpeedVac. The proteins were reduced with 25 mM NH4HCO3 (Sigma-Aldrich) containing 10 mM dithiothreitol (Biosynth AG, Switzerland) at 60 °C for 30 min and alkylated with 55 mM iodoacetamide (Amersham Biosciences, UK) at room temperature for 30 min. The proteins were then digested with trypsin (20 μg/mL; Thermo Fisher Scientific) overnight at 37 °C.

The extracted peptides were analyzed by the LTQ-Orbitrap Discovery (Thermo Fisher Scientific) as described in the previous study [59]. Briefly, peptide extracts were reconstituted in HPLC buffer A (0.1% formic acid; Sigma-Aldrich), loaded across a trap column (Zorbax 300SB-C18, 0.3 × 5 mm; Agilent Technologies, Taiwan) at a flow rate of 0.2 µL/min in HPLC buffer A, and separated on a resolving 100 mm analytical C18 column (inner diameter, 75 µm) using a 15-µm tip (New Objective, USA). The peptides were eluted with a 60-min gradient of HPLC buffer B at a flow rate of 0.25 µL/min across the analytical column. Data-dependent mode was used to detect intact peptides at a resolution of 30,000 and 10 MS/MS scans for the 10 most abundant precursor ions were used to acquire data.

For database searching, the obtained MS/MS spectra were analyzed using the Mascot algorithm (version 2.2.04; Matrix Science, Boston, MA, USA). The search was conducted against the Canis sequence database of Swiss-Prot (released on March 2021; selected for Canis lupus familiaris, 25,491 entries) from the European Bioinformatics Institute. The fragment ion mass tolerance was set to 0.5 Da and the parent ion mass tolerance was set to 10 ppm, with trypsin as the digestion enzyme. Up to one missed cleavage was allowed, and searches included the parameters for variable oxidation on methionine (+ 15.99 Da) and fixed carbamidomethylation on cysteine (+ 57 Da). A random sequence database was used to estimate false-positive rates for peptide matches.

The label-free MS quantification

To identify the AGR2-affected secretome, the label-free MS quantification was used to compare the abundance of CM proteins derived from AGR2-expressing CMT cells with those derived from the vector-expressing control. Each CM sample was meticulously analyzed in triplicate, represented as Rep1, Rep2, and Rep3. The quantification of protein abundance was accomplished by calculating the ratio of the peptide-spectrum match (PSM) count attributed to a specific protein against the PSM counts of all identified proteins within the separate replicates. The cumulative protein abundance, represented as the normalized PSM ratio, within AGR2-expressing CM triplicates was divided by the corresponding cumulative value within vector-expressing CM triplicates. This ratio underwent a logarithmic transformation (Log2), denoted as Log2 (A/V). Proteins that displayed a Log2 (A/V) exceeding the mean + 1.5 SD were classified as AGR2-increased, while those falling below the mean − 1.5 SD were designated as AGR2-decreased.

Immunoprecipitation

Depletion of Myc-tagged AGR2, 14-3-3ε, and α-actinin 4 in the CM was conducted by immunoprecipitation (IP). For depletion of Myc-tagged AGR2, CM of CMT-U27 transfected with an AGR2-expressing vector was incubated with Myc-Trap® (ChromoTek, Germany) according to the manufacturer’s protocol. The slurry of Myc-Trap® beads (25 µL per reaction) was equilibrated with dilution buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA) three times, and the equilibrated beads were incubated with 600 µL of CM and rotated end-over-end at 4 °C for 1 h. For the depletion of 14-3-3ε or α-actinin 4 in the CM, antibodies (3 μg) against 14-3-3ε or α-actinin 4 were first incubated with Dynabeads™ Protein G (40 µL per reaction; Thermo Fisher Scientific) and rotated end-over-end at room temperature for 20 min. Subsequently, Dynabeads™ Protein G-immobilized antibodies were incubated with 600 µL of CM and rotated end-over-end at 4 °C for 16 h. The beads were retained with a magnet, and the remained CM was collected and used for a transwell migration assay. The beads were washed three times with wash buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.05% Triton X-100), and the IP complexes were resolved with 30 µL of 2 × sampling buffer (100 mM Tris–HCl, pH 6.8, 2% β-mercaptoethanol, 4% SDS, 20% glycerol, 0.04% bromophenol blue, 100 mM EDTA) for immunoblotting analysis.

Trichloroacetic acid precipitation of CM proteins

Trichloroacetic acid (TCA; Merck KGaA, Germany) was used to precipitate proteins in the 1–2% FBS-containing CM that was applied to a transwell migration assay. CM was mixed with TCA (20%v/v) and incubated at 4 °C overnight. The mixture was centrifuged at 14,000 rpm at 4 °C for 30 min. Precipitated protein pellets were washed with 200 μL of ice-cold acetone (Merck KGaA, Germany) twice to remove residue of TCA, and then air-dried and resuspended in a sample solution (1% sodium dodecyl sulfate (SDS), 10 mM EDTA, pH 8.0). Resuspended samples were further sonicated at 37 °C for 1 h to thoroughly dissolve the protein pellets.

Immunoblotting

Protein extracts resolved in 1 × sampling buffer (50 mM Tris–HCl, 1% ꞵ-mercaptoethanol, 2% SDS, 10% glycerol, 0.02% bromophenol blue, 50 mM EDTA, pH 6.8) were boiled at 95 °C for 10 min, and then separated by SDS-PAGE with 9% to 15% gradient polyacrylamide gels. Protein samples were transferred to POLYSCREEN® polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Science, Inc., Boston, MA, USA), blocked with BlockPRO™ Blocking Buffer (Visual Protein, Taiwan) at room temperature for 1 h, and subsequently incubated with appropriately diluted primary antibodies (listed in Table 1) at 4 °C overnight.

Table 1 Primary antibodies used for immunoblotting in this study

The membranes were then incubated with secondary horseradish peroxidase (HRP)-conjugated goat-anti-mouse or goat-anti-rabbit IgG (PerkinElmer, at 1:10,000 dilution) at room temperature for 1 h and washed with 1 × Tris-buffered saline containing 0.05% Tween-20 (TBS-T) between steps. Luminescence signals were developed with Western Lightning® ECL-Pro (PerkinElmer), and images were acquired using Hansor Luminescence Image System (Hansor Polymer Technology Corp., Taiwan) with TSGel software (version 3.5). Quantification of protein bands was conducted using ImageJ (version 1.50i).

Reagents and recombinant proteins

Tunicamycin (Cat. No. NC1771734) was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Rapamycin (Cat. No. 51031-RAP-25) and Chloroquine (Cat. No. 581005-CLQ) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). 3-Methyladenine (3-MA; Cat. No. HY-19312) was purchased from Med Chem Express (MCE, Monmouth Junction, NJ, USA). Recombinant canine AGR2 (rcAGR2) was prepared as previously described [14]. Recombinant 14-3-3ε (rc14-3-3ε; Cat. No. PKSH031395) was purchased from Elabscience (Houston, Texas, USA).

Immunofluorescence staining and confocal microscopy

Cells were seeded onto coverslips placed in a 12-well plate and grown to 50–70% confluency, and subsequently cultured in serum-free media supplemented with or without 50 nM rapamycin for 16 h. Cells were fixed with 4% paraformaldehyde containing 2% sucrose in 1 × PBS at room temperature for 20 min and then permeabilized with 0.1% Triton X-100 for 3 min and blocked with BlockPRO™ (Visual protein, Taipei, Taiwan) for 30 min. Cells were incubated with an LC3B antibody (83506S, Cell Signaling Technology, Danvers, MA, USA) at 1:100 dilution, together with a 14-3-3 antibody (Cat. No. 11648-2-AP, Proteintech) at 1:400 dilution or an α-actinin 4 antibody (Cat. No. 19096-1-AP, Proteintech) at 1:300 dilution for 90 min at room temperature, followed by staining with secondary Alexa Fluor 488-conjugated goat-anti-mouse IgG and Alexa Fluor 694-conjugated goat-anti-rabbit IgG (Molecular Probe, Thermo Fisher Scientific), respectively, at 1:200 dilution for 45 min. Nuclei were co-stained with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen) at 0.1 µg/mL in 1 × PBS. Coverslips were washed with 1 × PBS between steps and finally mounted with Fluoro-Gel (Electron Microscopy Science, USA) on slides. Images were acquired using the Zeiss LSM780 confocal laser scanning microscope (Jena, Germany) with a 63 × oil-immersion objective.

Quantification of intracellular puncta numbers and colocalization coefficients

The number of LC3B puncta, α-actinin 4-positive LC3B puncta, or 14-3-3ε-positive LC3B puncta, was quantified using ImageJ. Initially, confocal microscopy-acquired images were filtered with the Difference of Gaussian (DoG) filter to enhance the subcellular puncta structure. To quantify the LC3B puncta per cell, individual cells in each image were selected as the regions of interest (ROIs) and subsequently analyzed using the default “Analyze Particles” plugin to count the number of LC3B puncta within each cell.

For quantifying α-actinin 4-positive LC3B puncta or 14-3-3ε-positive LC3B puncta, the default “Colocalization Threshold” plugin was employed to generate overlay images of LC3B puncta and ACTN puncta, or LC3B puncta and 14-3-3ε puncta, prefiltered with the DoG filter, highlighting the overlapping regions. Subsequently, the same ROIs used for quantifying the LC3B puncta per cell in the previous steps were applied to the overlay images, and the “Analyze Particles” plugin was used to count the number of colocalized puncta within individual cells.

The colocalization coefficient was calculated using ZEN (black edition) software from Carl Zeiss AG in Oberkochen, Baden-Württemberg, Germany. To identify LC3B puncta, α-actinin 4 puncta, or 14-3-3ε puncta, the background subtraction thresholds were established based on the fluorescence intensities of the objects. Subsequently, colocalization coefficients between isolated puncta per cell in the overlay images were determined by measuring the overlapping regions between α-actinin 4 puncta and LC3B puncta or between 14-3-3ε puncta and LC3B puncta.

Isolation of extracellular vesicles

CMT-U27e, Ctrl-S3, KO-S10, and KO-S4 were grown in RPMI supplemented with 2% EVs-depleted FBS for 50 h at 37 °C in a humidified incubator with 5% CO2 supply. For individual cell clones, conditioned media (CM) were collected from cells grown to 80–90% confluency in three 10-cm culture dishes and centrifuged at 300 × g for 10 min and subsequently at 3000 × g for 10 min at room temperature to remove detached cells and cell debris, respectively. The resulting CM was subjected to the isolation of extracellular vesicles using both differential ultracentrifugation (dUC) and size exclusion chromatography (SEC). For dUC, CM was first centrifuged at 16,500 × g at 4 °C for 30 min using the SW 28 Ti Swinging-Bucket Aluminum Rotor (Beckman Life Sciences, Indianapolis, IN, USA). The large EV pellet was washed with 0.22-µm-filtrated DPBS (Gibco, Thermo Fisher Scientific), followed by second centrifugation at 16,500 × g at 4 °C for 1 h to remove non-EV contaminants. The remaining supernatants were further centrifuged at 80,000 × g at 4 °C for 2 h, and the small EV pellet was washed with 0.22-µm-filtrated DPBS, followed by a second centrifugation at 80,000 × g at 4 °C for 2 h to remove non-EV contaminants. The washed large and small EVs were resuspended in 120 µL of 0.22-µm-filtrated DPBS and stored at − 80 °C before further analysis. The remained EV-depleted supernatants were used for a transwell migration assay or immunoblotting.

For SEC, the qEVoriginal/70 nm Gen 2 Column (Izon Science Ltd., New Zealand) was used to isolate EVs. Cell debris-removed CM was first concentrated with Vivaspin® 20 (100 kD cutoff; GE Healthcare) and then added to the qEV column. Once the CM sample was filled in the column, 500 µL of 0.22-µm-filtrated and degassed DPBS was added to the top of the column. Flow-through fractions were immediately collected as follows: void buffer fractions 1 to 6 and sample fractions 1 to 14 (500 µL each), by the manufacturer’s instruction. The collected fractions were stored at − 80 °C until further use. For a transwell migration assay, 500 µL of 2% FBS-containing RPMI supplemented with 100 µL of the EV fraction (Fraction 1) or DPBS was placed in the bottom well as the attractant.

Nanoparticles tracking analysis (NTA)

NTA was employed to determine the absolute size distribution and concentration of extracellular vesicles (EVs). The analysis was conducted using the NanoSight NS300 instrument (NanoSight, Minton Park, UK) in conjunction with NanoSight NTA software (version 3.4; NanoSight) for both data acquisition and analysis. Particles were automatically tracked and sized based on their Brownian motion and diffusion coefficient. Filtered PBS served as the control and blank samples. The NTA measurement conditions were standardized as follows: temperature maintained at 24.0 ± 0.5 °C, viscosity at 0.99 ± 0.01 cP, frames per second set at 25, and a measurement time of 60 s. The detection threshold remained consistent across all samples. Each sample underwent five recordings to ensure accuracy and reliability of the results.

Transmission electron microscopy (TEM)

TEM was utilized to investigate the morphology of EVs. Initially, isolated EVs were resuspended in 4% paraformaldehyde (50–100 μL), and 10 μL aliquots were deposited onto Formvar/carbon-coated EM grids. The grids were then covered, and membrane adsorption was carried out for 20 min in a dry environment. Subsequently, the grids (with the membrane side down) were transferred to drops of PBS (100 μL) using clean forceps for washing, followed by retransfer to a 50 μL drop of 1% glutaraldehyde for 5 min. Afterwards, the grid underwent eight washes with distilled water, each lasting 2 min. Following this, contrast staining was achieved by immersing the grid in a 50 μL drop of uranyl acetate solution for 5 min. Finally, the grid was embedded in 50 μL of methyl cellulose-UA for 10 min on ice. Upon removal of the grid using stainless steel loops, excess fluid was blotted, and the grid was air-dried. The prepared grid was then examined under an electron microscope (JEM 1230, JEOL Ltd., Tokyo, Japan) operating at 80 kV.

Specimen collection

Pre-surgical serum samples were collected from 17 dogs afflicted with CMT who underwent mastectomy at the Veterinary Medical Teaching Hospital (VMTH), National Chung Hsing University (NCHU), between 2017 and 2019. Additionally, serum samples were collected from 15 privately owned, age-matched healthy female dogs for comparison purposes. Blood samples were drawn into serum separating tubes (SSTs) and allowed to clot at room temperature for 30 min before being centrifuged at 2500 × g, 4 °C, for 15 min. The resulting sera were supplemented with a protease inhibitor cocktail (VWP Life Science, Avantor, Radnor Township, PA, USA), divided into 50 μL aliquots, and stored at − 80 °C until utilization.

Paired samples of CMT tissues and non-involved normal mammary gland tissues were collected from 9 out of the 17 CMT patients who underwent simple or bilateral mastectomy at the VMTH, NCHU. These tissues subsequently utilized for collecting the tissue-immersed PBS, mimicking interstitial fluids. All procedures were conducted in compliance with relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee (IACUC) of NCHU (IACUC Number: 109–002). Diagnosis of CMTs was confirmed through radiography and histopathological examination of surgically excised tissues. Classification, histopathological grade, and clinical stage of CMTs were determined on the basis of the modified WHO-TNM system [3].

Preparation of the tissue-immersed PBS

Paired samples of CMT tissues and non-involved normal mammary gland tissues (5 × 5 × 5 mm3) were harvested during surgical procedures. Upon weighting, the tissues were promptly processed as previously described [67]. To minimize blood contamination, the tissues were thoroughly washed with ice-cold PBS and then dissected into 1–3 mm3 fragments using scalpels. The cut tissues were placed into 1.5-mL microcentrifuge tubes and subjected to further washed by ice-cold PBS until the supernatant was clear. Subsequently, the cut tissues were incubated with 600 μL of PBS for 1 h in a humidified incubator at 37 °C containing 5% CO2. Following centrifuging at 8000 × g for 15 min at 4 °C, the resulting supernatants were promptly treated with a protease inhibitor mixture (2 μL/mL; VWP Life Science) and stored at − 80 °C for subsequent analysis.

Statistical analysis

The statistical analysis was conducted by using GraphPad Prism V8.4 software (GraphPad Inc., San Diego, CA, USA). The two-tailed unpaired t-test was applied to evaluate experimental differences between groups, and the Mann–Whitney U test was utilized to assess differences between clinical samples. p < 0.05 was considered statistically significant.