Antibodies and reagents
Oleandrin was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Anti- HSP70 (#4876), anti-ATF3 (#33593) and anti-β-actin (#3700) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-PERK (ab79483), anti-eIF2α (ab5369), anti-elF2α (phosphor S52) (ab227593), anti-ATF4 (ab23760), anti-CHOP (ab11419), goat anti-mouse IgG-horseradish peroxidase (HRP) (ab205719), goat anti-Rabbit IgG-HRP (ab6721), anti-PERK (phospho T982) (ab192591), anti-IRE1 (ab37073), anti-IRE1 (phosphor S724) (ab124945), anti-XBP1 (ab37152), anti-Calreticulin (ab2907), Goat anti-rabbit IgG (Alexa Fluor®488) (ab150077) and anti-GADD34 (ab9869) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-HSP90 antibody was purchased from Santa Cruz Biotechnology, INC (Dallas, Texas, USA). PerCP anti-human HLA-DR (307627), APC anti-human CD11c (301614), FITC anti-human CD80 (305406), PE anti-human CD86 (305406) antibodies were obtained from Biolegend (San Diego, CA, USA).
Human breast cancer cell lines MDA-MB-231, MCF7 and T47D were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 cells were cultured in L-15 medium. MCF7 cells were cultured in minimum Eagle’s medium. T47D cells were cultured in RPMI-1640 medium. Cell culture medium was obtained from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). Mouse breast cancer cell lines EMT6 were obtained from ATCC and cultured in Waymouth’s MB 752/1 medium (Biological Industries, Kibbutz Beit-Haemek, Israel). All the medium was supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Inc.,Waltham, MA, USA).
DC culture and CD8+ T cell isolation
This study was approved by the ethics committee of the Cancer Hospital of China Medical University (Ethics Review Approval no. 20170226). Two female volunteers, aged 27 and 35, were recruited and provide the informed consents. Peripheral blood was stained with HLA-A2 and HLA subtype was detected by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from HLA-A2 type volunteer and cultured in 75 cm2 flask for 1 h. Suspended cell were removed, and the adherent cells were cultured with fresh x-vivo 15 medium (Lonza, Alpharetta, GA, USA) containing 5% plasma supplemented with 500 U/ml of IL-4 and 1,000 U/ml of GM-CSF (Promega, Madison, WI, USA). After 5 days of culture, DCs were collected for following experiments. CD8+ T cells were isolated from PBMCs using the CD8+ T Cell Isolation Kit according to manufacturer’s instructions (Miltenyi Biotec, CA, USA).
Immunofluorescence staining for CRT
Breast cancer cells were treated with oleandrin. The concentrations of oleandrin used against MCF7 and MDA-MB-231 cells were 15 nM and 25 nM, respectively. After 24 h of treatment, cells were fixed with 95% ethanol, permeabilized with PBS containing 1% Triton X-100, and blocked using 1% BSA. Cells were incubated with anti-CRT antibody (monoclonal rabbit; 1:75) at 4 °C overnight. Cells were washed 3 times with PBS and incubated with secondary antibody (goat anti-rabbit Alexa Fluor 488, 1:200) for 30 min. Nucleus was stained with 10 μg/ml of Hoechst 33342. Samples were finally observed under a fluorescent microscope (CQ1, Yokogawa, Japan).
In vitro cytotoxicity assay
2×104 of MDA-MB-231 cells were seeded in 6-well plate and divided into the following 5 groups: cells co-cultured with DCs, cells co-cultured with CD8+ T cells, cells pretreated with oleandrin, pretreated cells and co-cultured with DCs, pretreated cells co-cultured with DCs and CD8+ T cells. MDA-MB-231 cells alone were used as control group. After 48 h of co-culture, cells were washed twice with PBS to remove the immune cells and oleandrin. Cells were continued to grow for 14 days. Finally, cells were fixed and stained with crystal violet for 15 min. The numbers of colonies were counted.
Enzyme-linked immunosorbent assays (ELISA)
Breast cancer cells were treated with oleandrin. The concentrations of oleandrin used against MCF7 and MDA-MB-231 cells were 15 nM and 25 nM, respectively. Culture supernatant was collected and secreted ATP (Promega, Madison, WI, USA) and HMGB1 (Signalway Antibody, Maryland, USA) were detected with ELISA kits according to the manufacturer's instructions. MDA-MB-231 cells pretreated with oleandrin or DMSO were co-cultured with DCs for 48 h. IL-2, IL-10 and IFN-γ from the culture supernatant were quantified using Human IL-2 Quantikine ELISA Kit (D2050), Human IL-10 Quantikine ELISA Kit (D1000B) and Human IFN-γ Quantikine ELISA Kit (DIF50C) according to the instructions.
Animal studies were conducted according to the experimental animal guidelines of the China Medical University animal center. 2×105 of EMT6 were inoculated into mammary fat pads of BALB/c mice. After 7 days, both long and short diameters of the tumors reached about 5 mm. The mice were randomly divided into 3 groups with 5 mice in each group: PBS as control group, oleandrin treatment with 0.3 mg/kg and 0.6 mg/kg groups. Oleandrin was administered intraperitoneally every day. Tumor volume was measured every day and quantified as 0.5 × length × width × width. After 7-day administration, mice were sacrificed, and tumors were weighed. Tumor primary cells and splenocytes were harvested for flow cytometric analysis.
Flow cytometric analysis
1x106 of cells were collected and suspended in 100 μl of PBS. Cells were incubated with the following antibodies 5 μl /test of each at room temperature for 30 min, PerCP anti-human HLA-DR, APC anti-human CD11c, FITC anti-human CD80, PE anti-human CD86.
Mouse primary tumor cells were minced and disassociated with the EZ enzyme (Nitta Gelatin Inc., Osaka, Japan). Single cell suspensions from mouse spleen and tumor sites were incubated with TruStain FcX™ (anti-mouse CD16/32) antibody to block non-specific staining and then stained on ice for 30 min with the following combination: CD3-FITC/CD4-PE/CD8-APC, CD45-PerCP/CD11b-FITC/CD11c-APC. Samples were detected by BD AccuriC6 (BD Biosciences, San Jose, CA).
Cells were collected and lysed using radio immunoprecipitation assay (RIPA) buffer. Cell culture supernant was were centrifuged for 30 min with 10,000 xg and protein concentration was measured using the protein concentration kit (2772T, Thermo scientific, Shanghai, China). Protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane. Primary antibodies were diluted and incubated at 4°C overnight. The membranes were then washed and incubated with secondary antibodies at room temperature for 1 h. Chemiluminescence was performed using Supersignal West Pico plus (Thermo Fisher Scientific, Inc.) and detected by BIO-RAD GelDoc XR+ system (Bio‑Rad, Berkeley, CA, USA). Data was analyzed by Image Lab (version 5.2.1).
MCF7, MDA-MB-231 and T47D cells were treated with oleandrin for 10 h. The concentrations of oleandrin used against MCF7, MDA-MB-231 and T47D cells were 15 nM, 25 nM and 12.5 nM, respectively. Total RNA of treated cells and controls were extracted using TRIzol. The mRNA was sequenced using the Illumina Hiseq platform. Differential expression analysis of two groups was performed using the DESeq2 R package (1.16.1). The data were transformed into Venn’s diagrams and heat-map. Gene Ontology (GO) and KEGG enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package.
Quantitative real time polymerase chain reaction (qRT-PCR)
Cells were lysed with 1 mL of TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA), and cDNA was synthesized with the PrimeScript™RT reagent Kit (TaKaRa, Shiga, Japan). qRT-PCR was performed by using SYBR Premix EXtaq (TaKaRa, Shiga, Japan). Primers used are as follow:
CD86: forward, 5’-CTGCTCATCTATACACGGTTACC-3’
CD80: forward, 5’-GGCCCGAGTACAAGAACCG-3’
IL-2: forward, 5’- TCCTGTCTTGCATTGCACTAAG-3’
reverse, 5’- CATCCTGGTGAGTTTGGGATTC-3’
IL-10: forward, 5’-TCAAGGCGCATGTGAACTCC-3’
IFN-γ: forward, 5’-TCGGTAACTGACTTGAATGTCCA-3’
reverse, 5’- TCGCTTCCCTGTTTTAGCTGC-3’
GAPDH: forward, 5’-ACAACTTTGGTATCGTGGAAGG-3’
reverse, 5’- GCCATCACGCCACAGTTTC-3’
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software version 21 (IBM Corp., Armonk, NY, USA). The results were represented as mean ± standard deviation. The comparison between the two groups was conducted by t-test or one-way ANOVA. p<0.05 was considered statistically significant (*, p<0.05, **, p<0.01).