Cell culture
Murine breast cancer cells Py8119 was purchased from ATCC (Manassas VA, USA). To establish obesity-associated breast cancer cell line, C57BL/6 mice were fed with a low-fat diet (LFD; 10 kcal% fa/no sucrose) or a high-fat diet (HFD; 60 kcal% fat) for 3 months to generate lean or obese mice, following orthotopically injected Py8119 cells into the no. 4 mammary of mice, following observation for 6 weeks. The mice were sacrificed and fresh tumor tissues were digested using a gentleMACS tumor dissociation kit (Miltenyi Biotec). The mixture was incubated with an RBC lysis buffer. 3×106 isolated cells were seeded in 10-cm plates. All cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 7% fetal bovine serum, 1% glutamine, and 1% antibiotic-antimycotic (Corning, New York, USA).
Transwell assay
To assess the migratory ability of tumor cells, 5×104 cells were seeded in serum-free medium in the upper transwell insert. For cell invasion, 105 cells were seeded in serum-free medium in the upper transwell insert coated with 70 μl Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The complete medium was filled in lower wells to serve as a chemoattractant. After 24 (migration) and 48 (invasion) h incubation, tumor cells on transwell insert were fixed with ice-cold methanol for 15 min, following stained with 1% crystal violet. Cells on upper surface of transwell insert were removed with cotton swabs before being observed under a microscope.
Colony-formation and cell-proliferation assays
To assess the ability of single cells to grow into colonies, 103 cells were seeded in six-well plates and cultured in complete medium for 2 weeks, with medium being refreshed twice a week. Cells were washed with PBS twice, and then stained with 1% crystal violet. For the cell-proliferation assay, 104 cells were seeded in 24-well plates with complete medium. On the indicated days, cells were trypsinized and the cell number was counted.
RNA isolation and real-time quantitative polymerase chain reaction (qPCR)
RNA was extracted with a GENzolTM TriRNA Pure kit (Geneaid, Taipei, Taiwan). Complementary (c)DNA was synthesize using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Amplification of cDNA was conducted on a StepOne Plus Real-Time PCR system (Applied Biosystems, Darmstadt, Germany) using GoTaq qPCR Master Mix (Promega) with the specific primers as follows: SOX2, 5’-GCGGAGTGGAAACTTTTGTCC-3’ (forward) and 5’-GGGAAGCGTGTACTTATCCTTCT-3’ (reverse); Nanog, 5’-CACAGTTTGCCTAGTTCTGAGG-3’ (forward) and 5’-GCAAGAATAGTTCTCGGGATGAA-3’ (reverse); Oct4, 5’-CGGAAGAGAAAGCGAACTAGC-3’ (forward) and 5’-ATTGGCGATGTGAGTGATCTG-3’ (reverse); IL33, 5’-ATTTCCCCGGCAAAGTTCAG-3’ (forward) and 5’-AACGGAGTCTCATGCAGTAGA-3’ (reverse); ST2, 5’-CTGGCACTGGCATTTGGT-3’ (forward) and 5’-GCCTACGAGCAGGAGATT-3’ (reverse); Casp1, 5’-TGGTCTTGTGACTTGGAGGA-3’ (forward) and 5’-TGGCTTCTTATTGGCACGAT-3’ (reverse); and 18S ribosomal (r)RNA, 5’-GTAACCCGTTGAACCCCATT-3’ (forward) and 5’-CCATCCAATCGGTAGTAGCG-3’ (reverse). Relative mRNA levels were calculated using the ΔΔCT equation(Lee et al. 2020).
Chromatin immunoprecipitation (ChIP)
Cells were incubated with 1% formaldehyde with glycine to crosslink the protein-DNA complexes. Chromatin complexes were extracted with a Chromatin Extraction Kit (ab117152) and processed with a ChIP Kit Magnetic - One Step (ab156907), following incubated with anti-YAP (Cell Signaling #14074) or mouse IgG overnight. After DNA was reverse-crosslinked and purified, a qPCR analysis was performed. The qPCR primers are listed as follow: IL33 (-1179~73), 5’-AATGGCCTCAATCTGTCTC-3’ (forward) and 5’-ACTGATGTGGAGTTGAAAC-3’ (reverse); and IL33 (-555~550), 5’-AGCTCTGAGAGTTTCTATC-3’ (forward) and 5’-AGACTCATAGCAGAATTAGC-3’ (reverse).
Western blotting
Cells were lysed using ice-cold RIPA buffer containing a protease and phosphatase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Total protein (30 μg) were resolved by SDS-PAGE and then transferred onto PVDF membranes (Millipore, Temecula, CA, USA). The membranes were incubated overnight at 4 °C with the primary antibody against IL33 (AF3626, R&D Systems, Minneapolis, MN, USA), NFκB (#8242, Cell Signaling, Danvers, MA, USA), phosphorylated (p)-NFκB (#3033, Cell Signaling), α-tubulin (GTX112141, GeneTex, San Antonio, TX, USA), and β-actin (GTX11003, GeneTex). The membranes were then washed three times with TBST for 10 min each and further probed with an HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 1 h. Proteins were visualized on the enhanced chemiluminescence (ECL) system (Millipore)(Lai et al. 2021).
Short hairpin (sh)RNA and lentiviral infection
Mouse IL33 shRNAs (TRCN0000173324 and TRCN0000173352) and mouse YAP shRNAs (TRCN0000238432 and TRCN0000238436) in pLKO.puro vectors were purchased from the National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). HEK293T cells were transfected following the manufacturer’s instructions, and virus-containing supernatants were collected after 2 d. Cells were incubated with supernatants for 2 d, and then stably selected using puromycin(Tsai et al. 2021).
Immunohistochemistry (IHC) and immunofluorescence (IF)
For IHC staining, breast cancer tissue microarrays were purchased from US Biomax (Rockville, MD, USA), and tumor tissues from mice were formalin-fixed and paraffin-embedded on slides. Paraffin-embedded tissues were deparaffinized and rehydrated. Antigen was retrieved in a heated citrate buffer (Vector Laboratories, Burlingame, CA, USA) for 1 h, and then the slides were incubated with anti-YAP (1:200, Cell Signaling), anti-IL33 (1:200, R&D system), anti-FoxP3 (1:200, Cell Signaling), and anti-PD-L1 (1:200, Cell Signaling) primary antibodies overnight at 4 °C. The slides were further incubated with the SignalStain Boost IHC detection reagent (Cell Signaling), following visualized using DAB peroxidase substrate and counterstained with hematoxylin (Vector Laboratories). For IF staining, cells were seeded in an eight-well chamber slides (Millipore) coated with poly-L lysine. Cells were treated with recombinant IL33 for 1 h, fixed with 2% paraformaldehyde at room temperature for 10 min, and permeabilized with 0.3% TritonX-100/PBS for 30 min. Blocking was done using 1% BSA/PBS for 1 h. anti-NFκB antibody (#8242, Cell Signaling) was applies overnight at 4 °C, and Alexa Flour 488-conjugated secondary antibody (Thermo Fisher Scientific) was applied at room temperature for 1 h. Nuclei were s with UltraCruz mounting medium (Santa Cruz Biotechnology) for 5 min. The slide were mounted with a coverslip before imaging using microscope.
Tumor-infiltrating lymphocyte (TIL) analysis
Fresh tumor tissues were dissected into small fragments and digested using the gentleMACS tumor dissociation kit (Miltenyi Biotec). After red blood cell lysis, isolated cell suspensions were incubated with antibodies conjugated to fluorophores including CD45-APC, CD11b-PE-Cy7, CD3-FITC, CD8-APC-Cy7, Ly6G-APC, CD4-PE-Cy7, F4/80-FITC, PD-L1-PE (Miltenyi Biotec) at 4 °C for 1 h. Cells were washed with PBS twice before analysis on the BD FACSVia flow cytometer (BD Biosciences). The gating strategy is shown in Supplemental Fig. S1.
Bioinformatic analyses
Gene expression patterns of IL33 pathway-, YAP signaling pathway-, and obesity-associated genes were downloaded from UCSC Xena browser (https://xenabrowser.net/). An ‘obesity score’ was defined as the sum of normalized expressions of obesity-related genes to present the degree of obesity of breast cancer patients. A ‘YAP signaling score’ was defined as the sum of normalized expressions of YAP signaling-related genes to present the degree of activation of YAP signaling. An ‘IL33 pathway’ score was defined as the sum of normalized values of the gene expressions of IL33, ST2 and Casp1. IL33 gene expression and the infiltration levels of Tregs and macrophages were downloaded from the Tumor IMmune Estimation Resource (TIMER, http://timer.cistrome.org/).
Statistical analyses
GraphPad Prism 6.0 software were used for all statistical analyses. Data are presented as the mean±standard deviation (SD), and all data points represent independent experiments. Unpaired, two-tailed Student's t-test was performed to compare the differences between two groups. Pearson test analysis was performed to evaluated a correlation coefficient.