Cell culture and hypoxia mode
HepG2, Hep3B, MCF7, MDA-MB-231, H1299, SiHa, C33A and 4T1 cells were cultured in high glucose Dulbecco ’s Modified Eagle ’s medium (HyClone, Logan UT, USA) and 10% fetal bovine serum (HyClone, Logan UT, USA). A549 and B16 cells were cultured in RPMI-1640 medium (HyClone, Logan UT, USA) and 10% fetal bovine serum. All cells were maintained at 37 ℃ and 5% CO2 in incubator. All cell lines were obtained from China Infrastructure of Cell Line Resource (Beijing, China)
Cells were cultured in a 1% O2, 5% CO2 incubator for 24h to establish a physical anoxia state.
Colony formation
Cells were treated with SVA and/or MET for 24 hours and then reseeded in new dishes for 14 days. The cells were fixed using 4% paraformaldehyde (PFA) and then stained with 0.5% crystal violet.
Carboxyfluorescein diacetate succinimidyl ester (CFSE) assay
Cells were stained with 2.5μM CFSE for 30min and then treated with relevant concentration SVA and/or MET, analyzed by flow cytometry (BD Biosciences, USA). The protocol was described as previously [19].
Apoptosis assays
After treatment with SVA and/or MET, cells were collected and followed the Annexin V-PE apoptosis detection kit (BD Biosciences, USA) to analysis the percentage of apoptosis cells.
RNA sequencing assay
HepG2 cells were untreated or treated with 2.5μM SVA, 5mM MET and combination of 2.5μM SVA and 5mM MET for 24h, quickly extracted and cryopreserved with liquid nitrogen. Then RNA sequencing were performed by Guangzhou RiboBio (China, Guangzhou). Every group were contain 3 independent samples for gene expression analysis.
Mouse models
All animal procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee at Xi ’an Jiaotong University. The 5×105 4T1 cells were injected into the fat pad of the 4th left mammary of four-week-old female BALB/c mice, and 1×106 B16 cells were subcutaneously injected into four-week-old female C57BL/6J mice. And the mice were treated with 15mg/kg/day simvastatin and/or 100 mg/kg/day metformin by intragastric administration.
Immunoblotting
After treatment with SVA and/or MET, cells were harvested by RIPA buffer, and the lysates were underwent SDS-PAGE and transferred onto PVDF membrane. Then the membrane were incubated with primary antibody and HRP-conjugated sencondary antibody, and the chemiluminescent signals were detected by ChemiDocTM XRS+ (Bio-rad, USA). The antibodies anti-p-RB, anti-RB, anti-cleaved caspase3, anti-caspase3, anti-Hif1α were obtained from Cell Signaling Technology (USA), anti-MCM7, anti-ET-1, anti-PHD2 were purchased from Santa Cruz (USA), anti-ETAR and anti-ETBR were purchased from Abcam (UK), and surviving were purchased from Proteintech (China).
Immunofluorescence and immunohistochemistry
Cells or mouse tissues were fixed by 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100 in PBS, followed blocking with 5% BSA, supplemented with 10% goat serum. Subsequently the slides were stained with CD31 (Abcam, UK), NG2 (BD Biosciences, USA), VE-cadherin (Biolegend, USA), Hif1α (Cell Signaling Technology, USA) and ETBR (Abcam, UK). Nuclei were stained with 5 μg/mL 4, 6-diamidino-2-phenylindone (DAPI). The images were taken by Leica TCS SP5 confocal laser scanning microscopy (Leica, Germany). The detailed procedures were similarly performed as described previously [19].
The perfusion status of vessels were labeled by Rhodamine-labeled lectin (Vector Labs, USA) for 15 min. 20 mg/kg Lectin were intravenous injected into mice, then intracardiac perfusion of 40 ml 4% PFA with a flux of 10 ml/min. Tumors were extracted and fixed with 4% PFA, embedded in OCT (Sakura Finetek, USA), and produced frozen sections. Finally, co-staining with CD31 and imaging with confocal laser scanning microscopy.
Tissues were fixed in 10% neutralized formaldehyde and embedded in paraffin, and then stained with PCNA (Santa Cruz, USA), cleaved caspase3 and Hif1α. Images were taken by Leica SCN400 slide scanner (Leica, Germany). The detailed procedures were similarly performed as described previously [19, 20].
Patient-derived organoid model
Breast cancer tissues were obtained from First Affiliated Hospital of Xi ’an Jiaotong University with informed consent and approved by the Ethics Review Committee of the First Affiliated Hospital of Xi ’an Jiaotong University. The breast cancer tissues were chopped up and digested by Collagenase at 37 ℃ for 1h followed by filtering on a 150 mesh filter and centrifuging 10min at 400g. The precipitate was resuspended in organoid medium, and centrifuged one more time, then resuspended with 40μL BME type 2 (Trevigen, USA). After BME was solidified, 400μL organoid medium was added for culture. The detailed procedures and reagents were performed as described previously [21]
The viability were assayed as followed: the PDOs were dispensed into 384-well microplates and treated with SVA and/or MET for 0, 24h, 48 or 72h, then analyzed by CellTiter-Glo (Promega, USA) accordance with its’ technical manual.
Statistical analysis
The quantitative analysis performed by Prism 7.0 software, and the quantitative data were represented by mean ± SEM. Statistical significance was calculated using two-tailed student ’ s t test or one-way ANOVA t-test. Each experiment data was represented a minimum of three biological replicates. *P < 0.05, ** P < 0.01, *** P < 0.001.