Tissue samples and clinicopathologic parameters
ESCC samples (84 paired tumor and peritumor tissues) were obtained from the Affiliated Cancer Hospital of Zhengzhou University. Paired tissue samples were from ESCC patients of the stage IA-IB (n = 26), IIA-IIB (n = 29), IIIA-IIIC (n = 29). None of the patients had previously received chemotherapy, radiotherapy or other therapies, and all patients underwent an informed consent process. Sample collection and the biological experiments in this study were approved by the Ethics Committee of Zhengzhou University (ZZUIRB2021-32). The clinicopathologic parameters were analyzed according to age, gender, stage, tumor differentiation, and lymph node metastasis.
Tissue microarray and immunohistochemistry (IHC) staining assay
Primary tumor and the paired peritumor samples were collected for tissue microarrays. Tissue cores with a diameter of 1.0 mm were taken from the tumor samples by tissue array instrument (Beecher Instruments, Silver Spring, USA). Standard IHC procedures were performed as previously described . The expression of IL-32 and CD206 in the tumor and paired peritumor tissues of the Paraffin-embedded samples were evaluated with anti-human IL-32 (1: 200, BioLegend, 513501, USA) and anti-human CD206 (1: 200, Servicebio, GB11062, China). This assay contained 56 paired samples and all samples of patients without receiving neoadjuvant therapy previously and during the surgery, and all patients underwent an informed consent process. The IHC-score was calculated by multiplying the percentage (P) of positive cells by the intensity (I), according to the formula: H-Score = ∑ (P × I) = (Percentage of cells of weak intensity × 1) + (Percentage of cells of moderate intensity × 2) + (Percentage of cells of strong intensity × 3).
ESCC cell lines and cell culture
Human ESCC cell lines (KYSE150, KYSE450, EC1, EC109 and EC9706), and immortalized normal esophageal cell line Het-1 A were cultured in complete RPMI 1640 medium (Gibco, Grand Island, USA). Complete medium was supplemented with 10% fetal bovine serum (FBS, BI, USA), 100 U/mL penicillin (Solarbio, China) and 100 µg/mL streptomycin (Solarbio, China); and cells were cultured at 37 °C with 5% CO2 under fully humidified conditions. GW4869 (Sigma-Aldrich, D1692, USA) and Y15 (Topscience, T7119, China) were used in cell culture.
Monocyte-derived macrophages (MDMs) were generated from human peripheral blood mononuclear cells (PBMCs) as previously described . Briefly, Human PBMCs were collected from venous blood of healthy volunteers, which were diluted with PBS (pH 7.4) and separated with Ficoll density gradient. The cells were resuspended at 2 × 106 cells/mL in RPMI1640 with 10% FBS. The cells were seeded in culture dishes to adhere and incubated for 4 h at 37 °C. Non-adherent cells were washed with complete media. The adherent monocytes were cultured in the complete medium supplement with 50 ng/mL human M-CSF (Cat: AF-300-25, PeproTech) for 7 days. To obtain EV-IL-32-induced M2 macrophage, MDMs were treated with EV-IL-32 for 72 h.
Identification the isoform of IL-32
Primers (Table S1) were used to amplify all isoforms of IL-32 in ESCC tumor tissues and cell lines. The PCR products were cloned into the T/A cloning vector pMD19-T. Vectors were amplified using Stbl3-competent cells. The monoclonal colonies were cultured and amplified. IL-32 isoforms were confirmed by DNA sequencing. To further certificate the results, different primers were designed and qRT-PCR was performed to distinguish each isoform. Primers were listed in the Table S1 and diagramed in the Fig. S3C.
Plasmid construction and transfection
Lentivirus transfection system was used to establish the stable IL-32β overexpression KYSE150 (KYSE150 IL-32β) cells. The mRNA was extracted from tumor tissues of ESCC patients, and reverse-transcribed into the full-length IL-32β cDNA, which was cloned into the pLVX-puro vector through EcoR I and Xba I restriction sites. Subsequently, the IL-32β overexpression vector was transfected into KYSE150 cells with PowerTrans 293 (Sixiang Biological, SX-TR293-001, China) according to the protocol. Empty vector was transfected into KYSE150 cells as control (KYSE150 vector). Short hairpin RNA (shRNA) was cloned into pSicoR-GFP vector and then was transfected into EC109 (EC109 shIL-32) cell. Empty vector was transfected into EC109 cells as control (EC109 shNC). The target sequence is 5’-AGAGCTCACTCCTCTACTTGA-3’.
Real-time quantitative reverse transcription PCR (qRT-PCR)
Total RNA was extracted from cell lines and tissue samples by the total RNA Isolation Kit (TIANGEN, DP419, China) according to the manufacturer’s instructions. The first-strand cDNA was synthesized from 2 µg of total RNA with the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622, USA). qRT-PCR assay was performed by SYBR Green I Master (Roche, 04887352001, Switzerland) in Roche LightCycler 480 II. The primers for qRT-PCR were listed in the Table S2. GAPDH was used to normalize the data.
The growth of EC109 shNC, EC109 shIL-32, KYSE150 vector and KYSE150 IL-32β cell lines were determined by MTT assay. Briefly, cells were seeded into 96-well culture plates at the density of 4000 cells/well. After 24, 48, and 72 h, cell viability was measured with MTT reagent (Sigma, M2003, USA). Formazan crystals were dissolved in 150 µL DMSO. The absorbance at 490 nm was measured by SpectraMax iD5 (Molecular Devices, USA).
Migration and invasion assays
Cell migration was measured by transwell assays. EC109 shNC, EC109 shIL-32, KYSE150 vector and KYSE150 IL-32β cell lines were resuspended in serum-free RPMI 1640 (200 µL) at the density of 5 × 105 cells/mL, which were seeded into upper chamber (Corning, 353097, USA). The lower chamber was added with complete RPMI 1640 medium. Cells was cultured at 37 °C with 5% CO2 for 48 h. The migration cells were fixed with 4% paraformaldehyde for 30 min. Finally, 0.2% crystal violet was used to stain the cells which migrated to the bottom of the well.
For cell invasion assay, the transwell assay with matrigel (BD, 354234, USA) was performed as previously described . Briefly, 200 µL cell solution (EC109 shILNC, EC109 shIL-32, KYSE150 vector and KYSE150 IL-32β, 1 × 105 cells/well) were seeded into the upper chamber (8 μm pore, Corning, 353097, USA) which was coated by the matrigel. The lower chambers were filed with 600 µL of RPMI 1640 medium including 10% FBS. After 48 h, invasion cells were fixed in 4% paraformaldehyde for 30 min, and stained with 0.2% crystal violet for 30 min.
Wound healing assay
EC109 shNC, EC109 shIL-32, KYSE150 vector and KYSE150 IL-32β cell lines were seeded into 24-well plate in the serum-free RPMI 1640 with the number of 5 × 105 cells/well cultured overnight. Cells were scratched by micropipette tip. After incubation for 12 or 24 h, the wound area was observed by microscope.
In the 24-well Transwell (8.0 μm aperture) co-culture system, EC109 and KYSE150 cell lines were seeded into the upper chamber with 1 × 105/well and EV-IL-32-educated M2 macrophages into the lower chamber with the same density. After 48 h, migrated cells were fixed in 4% paraformaldehyde for 30 min, and stained with 0.2% crystal violet for 30 min.
Enzyme-linked immunosorbent assay
Supernatant was collected from EC109 shNC, EC109 shIL-32, KYSE150 vector and KYSE150 IL-32β cell lines cultured for 48 h. Then, supernatant was centrifuged to eliminate the debris (600 g for 10 min) and large vesicles (10,000 g for 30 min). EV were treated by Native lysis Buffer (Solarbio, R0030, China). Human IL-32 DuoSet ELISA kit (R&D Systems, DY3040-05, USA) was used to measure the concentration of IL-32 according to the manufacturer’s instructions.
For CD206/IL-32/DAPI triple staining, slides were stained with rabbit anti-human CD206 (Servicebio, GB11062, China) and mouse anti-human IL-32 (BioLegend, 513501, USA) according to the manufacturer’s instructions. Sections were scanned by a TissueFAXS imaging system (Tissue Gnostics, Austria).
Isolation and identification of EV
EV were isolated from cultured supernatant by differential centrifugation as previously described . Briefly, supernatant was collected from cells that were cultured for 48 h in EV-free complete medium. Then, the supernatant was centrifuged at 300 g for 10 min, 3,000 g for 10 min to remove cells and debris, 10,000 g for 30 min to remove multivesicular. Subsequently, supernatant was ultra-centrifuged at 100,000 g for 2 h to obtain EV, and the EV were suspended in PBS (pH 7.2). Thereafter, the ultra-centrifugation step was repeated once. Finally, the supernatant was carefully removed and the EV were resuspended in 1 mL PBS (pH 7.2). The concentrations of EV proteins were measured by BCA assay.
Size distribution of the EV were identified by Malvern spray analyzer (Malvern Panalytical, England). The EV makers CD63 and TSG101 were detected by Western blot assay. For Transmission electron microscope (TEM) (HITACHI, Japan), EV were diluted in PBS (pH 7.2) and dropped onto a carbon-coated copper electron microscope grid for staining with uranium acetate.
Labeling and tracking of EV
EV were stained with PKH67 (Umibio, UR52303. China) according to the manufacturer’s protocol. Briefly, 5 µL of PKH67 dye was mixed with 50 µL Diluent C to make the dye solution, which was added into EV for 10 min at room temperature. The stained EV were isolated according to the method mentioned above. To verify whether EV can be phagocytosed by macrophages, MDMs were cultured on 5 mm slides and stained with Hoechst 33342 (Beyotime, C1025, China) and Cellmask orange (Invitrogen, C10045, USA). The EV derived from EC109 were labeled with PKH67 and then cocultured with MDMs. The video was performed by LiT LBS Light-sheet Microscope.
Subsets of M1 and M2 macrophages were defined as described previously . Fluorophore-conjugated monoclonal antibodies specific to CD45, F4/80, CD11c, CD14, CD11b, CD80 and CD206 were listed in Table S3. Then, the phenotype and proportion were analyzed with the BD FACS Celesta flow cytometer (USA). Data were analyzed by FlowJo 7.6.
Gene set enrichment analysis (GSEA)
The GSEA analysis was conducted with the cohort GSE23400 (Probe ID: 203828_s_at for IL-32). We divided the samples into two groups according to the median of IL-32 expression, and h.all.v6.0.synbols.gmt was obtained from the Molecular Signatures Database. RNA-Seq was also performed to analyze the pathway enrichment between IL-32 knockdown EC109 (EC109 shIL-32) and EC109 transfected with empty vector (EC109 shNC) by GSEA.
Total cell lysates were obtained with lysis buffer. Equal amounts of protein were separated by 10% SDS-PAGE gels and transferred to PVDF membrane (Merck Millipore, IPVH00010, USA). Then, PVDF membrane was blocked for 2 h by defatted milk (pH7.2 TBS containing 0.1% Tween 20 and 5% defatted milk) at 4 °C. Subsequently, the PVDF membranes were incubated with antibodies showed in Table S4. Blots were visualized by use of ECL system (Azure C600, USA).
Subcutaneous tumor model was prepared as following. Six-week-old female BALB/c nude mice were subcutaneously injected on the right back with 3 × 106 cells (KYSE150 vector, KYSE150 IL-32β, EC109 shNC and EC109 shIL-32). Tumor size and mouse body weight were measured every two days. After 18 days, all of the mice were sacrificed. Single-cell suspensions were made from tumor tissues and phenotypic analysis of macrophages was performed by flow cytometry.
For mouse lung metastasis models, six-week-old male nude BALB/c mice were intravenously injected with EC109 shNC or EC109 shIL32 (2 × 106 cells/mouse). Seven weeks later, all of the mice were sacrificed. For KYSE150 mouse model, mice were inoculated i.v. with KYSE150 vector or KYSE150 IL-32β (1 × 106 cells/mouse). Six weeks later, all of the mice were sacrificed. Lungs were removed and fixed by 4% formaldehyde. The number of the lung metastatic foci were counted, and tumor lesions within the lung tissues were confirmed by H&E staining.
For macrophages depletion, BALB/c nude mice were intravenously injected with 100 µL clodronate liposome or control liposome (FormuMax Scientific, USA) every 4 days after tail vein injection of 2 × 106 EC109 shNC or EC109 shIL-32 cells until 7 weeks. The efficiency of macrophages depletion was determined by flow cytometry with analysis of the CD45+F4/80+CD11b+cells.
Statistical analysis was performed using GraphPad Prism software version 8.0 (GraphPad Software Inc.). The correlation analysis between IL-32 expression and ESCC clinicopathological characteristics was determined by Chi-square test. Survival analysis was determined by the Kaplan–Meier method and differences between the groups were analyzed by means of log-rank test. Correlation analysis were performed by the Pearson’s rank correlation test. Statistical differences between two groups were evaluated with one-tailed unpaired or paired Student’s t-test. P < 0.05 is considered to be significant difference. The values were expressed as mean ± SEM.