Mice and cell lines
Male C57BL/6 mice (6 weeks old) and BALB/c nude mice (immunodeficient mice, 6 weeks old) were obtained from Dae-Han Experimental Animal Center (Eumsung, Republic of Korea). Mice were housed in a controlled environment with a 12:12-h light-dark cycle. All experimental procedures were conducted in accordance with current laws and guidelines for the care and use of laboratory animals approved by the Animal Ethics Committee of Kyung Hee University [KHUASP (SE)-17-157].
Mouse melanoma B16F10 cell and human B cell line U266 cells were purchased from ATCC. B16F10 cells were grown in Dulbecco’s Modified Eagle’s medium (Gibco BRL, Grand Island, NY, USA) and U266 cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma, St. Louis, MO, USA). Cells were routinely tested for Mycoplasma contamination using a polymerase chain reaction (PCR)-based Mycoplasma Detection Kit (iNtRON Biotech Inc., Seongnam, Republic of Korea).
Tumor models
C57BL/6 and BALB/c nude mice were injected subcutaneous (s.c.) with 3 × 105 cells into the right or flank of mice. The time of mouse death was recorded and used to calculate the survival rate.
Caspase assay
To estimate the apoptosis, activities of caspases were measured using colorimetric caspase activity assay kits (R & D Systems), according to the protocol of the manufacturer. In brief, the melanoma tissues and cells were lysed in the lysis buffer provided in the kit. The protein extracts (150 µg) assayed by bicinchoninic acid (Sigma) were incubated with the supplied reaction buffer containing 1 mM DTT with or without substrates (Asp-Glu-Val-Asp for caspase-3; Ile-Glu-Thr-Asp for caspase-8; Leu-Glu-His-Asp for caspase-9) labeled with p-nitroaniline at 37°C for 4 h. The optical density was measured at 405 nm wavelength using ELISA reader.
Immunofluorescence staining
Melanoma tissue samples were fixed with 10% formaldehyde in PBS, embedded in optimal cutting temperature compound, and the sections were incubated with anti-melan A (Cell Signaling Technology, MA, USA), anti-LC3 (Santa Cruz Biotechnology), anti-TSLP neutralizing (Anti-TSLP, R & D Systems), anti-tryptase (Santa Cruz Biotechnology), rat IgG-FITC (Abcam, Cambridge, MA, USA), rabbit IgG-TRITC (Abcam, Cambridge, MA, USA), or mouse IgG-FITC antibodies (Santa Cruz Biotechnology). The mounting medium containing 4', 6-diamidino-2-phenylindole (Sigma-Aldrich Co.) was used to counterstain DNA. Confocal microscopic images were visualized under a Fluoview FV10i confocal microscope (Olympus Co., Tokyo, Japan).
Transmission electron microscopy (TEM)
For TEM, melanoma tissue of mice was fixed with 2% paraformaldehyde, 0.05 M sodium cacodylate buffer, 2% glutaraldehyde, and pH 7.2 PBS at 4°C. The samples were then postfixed in 1% osmium tetroxide, dehydrated through a graded series of ethanol, and placed in Embed 812. Ultrathin sections were cut in an ultramicrotome (MT-X, RMC, Tucson, AZ, USA), stained with uranyl acetate and Reynolds’ lead citrate, and examined using a Libra 120 TEM (Carl Zeiss Inc., Weimar, Germany).
Histamine assay
Histamine levels were measured using a histamine assay kit (Oxford Biomedical Research, Oxford, MI, USA). The fluorescent intensity was measured at 460 nm (excitation at 355 nm) using a spectrofluorometer.
Enzyme-linked immunosorbent assay (ELISA)
Cytokine levels were analyzed following the manufacturer’s instruction (R&D Systems, Minneapolis, Minnesota, USA; BD Biosciences, San Diego, CA, USA).
Mast cell staining
Melanoma tissue samples were immediately fixed with 10% formaldehyde and embedded in paraffin. Next, samples were cut into 4 µm thick sections that were stained with alcian blue and safranin O after dewaxing and dehydration. Staining procedure was performed according to previous study [17].
Blood biochemical parameters
After the experiment, blood samples were collected from the animals in all groups and the serum was prepared by centrifugation at 3,000 × g at 4°C for 10 min. A DRI CHEM NX500 analyzer (Fujifilm, Tokyo, Japan) determined the contents of lactate dehydrogenase (LDH), blood urea nitrogen (BUN), alanine transaminase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), glucose, and C-reactive protein (CRP).
Single-cell RNA sequencing analysis
Single-cell transcriptomic amplification and library preparation were performed by ROKIT GENOMICS Inc. (Seoul, Republic of Korea) using BD Rhapsody System (BD Biosciences) according to manufacturer’s instructions. For further details regarding the materials and methods, please refer to the supplementary information.
Forced swimming test (FST)
A preliminary FST was conducted to evaluate differences between individual mice on day 0. On the basis of the immobility time, the mice were split into three groups. The transparent cylinder used to perform the FST has a diameter of 20 cm and a height of 40 cm. The FST lasted for 6 min in total. After a 2 min delay, the total time of immobility was timed for 4 min. On day 26, the immobility time of the last FST was assessed. After performing FST, animals were euthanized with CO2 gas and blood was taken from the heart, and the serum was then prepared by centrifugation at 3000 × g for 10 min at 4°C. Tumor weights were also measured. For extracting proteins, tumor samples were cut into pieces promptly frozen in liquid nitrogen. For histological investigation, the rest of the tumor fragments had been fixed in formalin at a concentration of 10%.
Patients and samples
Human study was approved by the Bioethics Committee of Kyung Hee University [KHSIRB-19-326(EA)]. The biospecimens for this study were provided by the Chonnam National University Hwasun Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare and Family Affairs. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. The biospecimens were taken from healthy volunteers (n = 10; 5 males, 5 females) and patients with melanoma (n = 10; 6 males, 4 females, stage 1 or 2 melanoma) (Table S1).
Differentiation of mouse bone marrow-derived mast cells (BMMCs)
BMMCs were generated from the femoral bone marrow cells of female mice (BALB/c) and these cells were incubated in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µM 2-mercaptoethanol, 10 mM sodium pyruvate, 10 µM MEM nonessential amino acid solution (Invitrogen, Carlsbad, CA, USA), 100 U/ml murine IL-3 (R & D system), and 0.5 U/ml murine stem cell factor (R & D system) at 37°C in a humidified atmosphere in the presence of 5% CO2. After 4 to 5 weeks of culturing, more than 96% of the cells were identified as mast cells as determined by toluidine blue staining and the fluorescence-activated cell sorting analysis of the cell surface expression of c-kit and FcεR I. BMMCs were stimulated with phorbol 12-myristate 13-acetate plus A23187 (PMACI), IgE, or compound 48/80.
Cytotoxicity assay
For the cytotoxicity assay, cell death was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma). For this assay, B16F10 cells were stimulated with TSLP (20 ng/ml), Anti-TSLP (20 ng/ml), or IgG. To inhibit the autophagy, 3-MA was treated before TSLP treatment. MTT reagent (5 mg/ml) was added to each well and incubated at 37°C for 4 h. The insoluble formazan product was dissolved in DMSO. OD was analyzed at 562 nm wavelength using ELISA reader.
Bromodeoxyuridine (BrdU) incorporation assay
For the proliferation assay, BrdU incorporation into B16F10 cells was assessed using BrdU colorimetric immunoassay (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer’s protocol. Briefly, BrdU was added, and cells were allowed to incubate for an additional 4 h. Fixed cells were incubated with anti-BrdU antibodies complex in PBS (1:100) for 90 min. OD was analyzed at 405 nm wavelength using ELISA reader.
Flow cytometry analysis
Apoptosis was analyzed using an Annexin V-FITC apoptosis detection kit (Sigma-Aldrich, St. Louis, MO, USA) and analysis was performed according to the manufacturer’s protocol. To stain the cells, 5 µl of Annexin V-FITC and 10 µl of propidium iodide (PI) solution was added to each suspension and incubated for 10 min at room temperature in the dark. Induction of cell death was measured using Sysmex CyFlow Ploidy Analyzer (Sysmex Partec GmbH, Gorlitz, Germany). For TSLPR analysis, cells were stained with anti-TSLPR and mouse IgG-FITC antibodies (Santa Cruz Biotechnology).
Statistical analysis of data
SPSS 12.0 (IBM Corp., Armonk, NY, USA) software was used for all statistical analysis. Group comparisons were conducted by independent t-test, one-way ANOVA followed by Tukey’s post-hoc test, or Mann-Whitney test, with the mean values and the standard error of the mean (SEM) calculated for each group. Kaplan–Meier survival curves were calculated using overall survival. In vitro studies were conducted independently at least-three experiments with duplicate. Throughout the figures, figure legends, and text the following terminology is used to denote statistical significance: #, *, ##, p < 0.05. GraphPad Prism software was used to create the graphs (version 8.0.1, GraphPad Software Inc., CA, USA).