Cell lines and culture
TNBC cell lines MDA-MB-231 and HCC 38 and luminal cell line MCF-7 were obtained from American type culture collection (ATCC; Manassas, VA, USA). All of them were cultured in RPMI-1640 medium(Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Cosmo bio, USA) and 100 µg/ml penicillin/ streptomycin (Gibco BRL, Grand Island, NY, USA). The cells were incubated at 37◦C in 5% CO2.
Cell Proliferation Assay
MDA-MB-231, HCC38 and MDA-MB-231 cells were evenly planted in 96-well plates at a concentration of 4000 cells/well. According to a concentration gradient for every 5 wells, the medium was replaced with the medium with various doxorubicin concentrations (from 0.001µM to 80µM) for 6H and 24H after the cells’ adherence. The cell viability was tested by using the WST-8 colorimetric assay (Cell Counting Kit-8; Dojindo Laboratories, Kumamoto, Japan). After adding WST-8 reagent solution to each well and incubating the microplates for 2 h at 37 ◦C, we used the cell counter (Sysmex CDA-500, Sysmex Corporation, Hyogo, Japan) to measure absorbance at 450 nm(23).
Confocal Microscope
The ΔφM, mitochondrial quantity and ROS distribution were detected immune-fluorescence assay under a laser scanning confocal microscopy (Nikon Instruments A1 Confocal Laser Microscope Series With NIS-Elements C Software). Cells planted in glass bottom 35mm petri dishes were stained with 100µl of both tetramethyl rhodamine, methyl ester(TMRM) (400nM, Marker Gene Technologies INC, USA) and Mito Marker Green(MTG) (120nM, Marker Gene Technologies, Inc, USA ) for 30minutes, Cellular Ros Assay kit (1:1000, ab186029, Abcam) for 45minutes. After dyes removed and washing by PBS, added 100µl of Hoechst 33342(1;10000, H3570, Thermo Scientific, USA) for 10 minutes at room temperature in the dark. Washed samples with PBS. For TMRM, MTG, ROS and Hoechst 33342, the excitation wavelength is at 548, 490, 650 and 361nm, and the emission wavelength is at 574, 516, 675 and 486nm, respectively.
Flow Cytometry
We measured the ΔφM, the total amount of mitochondria, and the ROS content in cells by semi-quantitative experimental method flow cytometry (BD FACS Aria™ III Cell Sorter). After incubation with DMSO and 100nM doxorubicin for 6h, cells were washed with PBS, and then added TMRM and MTG (400nM and 120nM, 37 ◦C 30mins in the dark). After washing with PBS and harvested by trypsinization and centrifugation(1000rpm,4 ◦C,5mins), cells were counted and adjusted to 1 million cells /ml by using cell staining buffer. Added 7-AAD (Bio Legend Way, San Diego) (5ul/0.5ml cell staining buffer) to control the cell survival rate above 85% and incubate it in the dark for 10min, then immediately measured by flow cytometry.
Western Blot
Western blot
Western blotting was used for evaluating the mitophagy proteins. Cell lines were planted at 6-well microplate, after washing twice with PBS, added 300µl M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, USA) and scraped for 5minutes. The turbid sample liquid was added in an Amicon ultra 0.5ml tube, centrifuged at 1500 rpm at 18°C for 3 minutes, and then centrifuged again at 14000g for 30 minutes. Recover the filter in a new tube and centrifuge again at 1000g for 2min to obtain the sample concentrate and quantify the protein. The proteins were separated by SDS-PAGE (12% acrylamide gel) (Bio-Rad,#1610175) 20µg per lane and transferred to 0.2µm PVDF membranes (Bio-Rad, USA). Then the membranes were blocked with 4% skimmed milk for 1 hour and incubated with primary antibodies with anti β-Actin (Millipore Sigma, mouse, #a5441, 1:5000), anti DRP1( Abcam, rabbit, ab184247,1:1000), anti p62(Abcam, mouse, #ab207305, 1:1000) and anti- light chain 3 (LC3B) (Abcam, rabbit, ab18709, 1:1000) at 4°C overnight, followed by incubation with the corresponding HRP-conjugated secondary antibody, and the bands were visualized by ECL™ Prime western blotting Detection Reagent(Millipore Sigma) and LAS-4000 mini image analyzer.
Seahorse Xf Mitochondrial Stress Assay
The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Seahorse XFe96 Analyzer(Agilent, Santa Clara, USA). Cell plates with 3 ×104 cells per well (10wells per sample) were seeded into Cell-Tak coated seahorse 96 well plates and preincubated at 37°C for 30 min without CO2. OCR and ECAR were measured in XF medium (non-buffered RPMI 1640 containing 10 mM glucose, 2mM L-glutamine, and 1 mM pyruvate) under basal conditions and in response to 2µM oligomycin, 2µM fluoro-carbonyl cyanide phenylhydrazone (FCCP), and 1µM rotenone + 1µM antimycin A.
Patient Samples
We used biopsy specimens taken before chemotherapy and surgical tissues from 108 breast cancer patients who underwent NAC and surgery at the Tohoku University Hospital and Tohoku Kosai Hospital in Sendai, Japan, between 2015 and 2020. All the specimens had been fixed in 10% neutral buffered formalin and embedded in paraffin wax. The patient's prognosis information of disease-free survival(DFS) was updated in January 2023.
Immunohistochemistry
We performed immunohistochemistry on 3-µm-thick 10% formalin-fixed paraffin-embedded tissues. Sections were deparaffinized with xylene and rehydrated with a graduated series of ethanol. After antigen-retrieval using a microwave in PH6 citrate buffer for DRP1, or using an autoclave (Tomy SX-500 High-Pressure Steam Sterilizer, Tomy Seiko Co., Ltd., Tokyo, Japan) in PH9 Tris EDTA buffer for p62, and in PH8 EDTA buffer for Parkin, the sections were blocked with a blocking solution of 10% rabbit serum (Nichirei Biosciences, Tokyo, Japan) and then incubated with primary antibodies against DRP1 (Abcam, mouse, ab56788, 1:1000), p62(Abcam, rabbit, ab207305, 1:2000) and Parkin(Santa Cruz Bio, mouse, sc-32282,1:50) at 4°C overnight. Endogenous peroxidase activity was blocked using absolute methanol containing 0.3% H₂O₂ for 30 min at room temperature. Sections were incubated with a biotinylated secondary anti-mouse/ anti-rabbit antibody (Nichirei Bioscience, Tokyo, Japan) at a dilution of 1:100 for 30 min at room temperature, and peroxidase-conjugated avidin (Nichirei Bioscience, Tokyo, Japan) was used. Human kidney, Human hepatocellular cancer and human gall bladder were used as the positive control for DRP1,p62 and Parkin respectively.
Assessment Of Immunoreactivity
HALO was used to detect cells and calculate the H-score. The digital analysis software "HALO TM Area Quantification ver. 2.2 (Indica Labs, Corrales, NM)" was used to quantitatively evaluate the specific morphology of tumor cells and the immunoreactivity of target proteins in the cytoplasm(24). This application first detects the IHC image into hematoxylin and DAB channels, then detects individual cells and their subcellular compartments, nucleus, and cell membrane, then scores the cells as high, medium and low based on the average DAB signal associated with the cell membrane. The thresholds for high, medium, and low were determined separately for each biomarker. HALO outputs the number and ratio of negative, high medium, and low cells, which is then used to calculate the total score. Immunohistochemical staining was evaluated using an intensity score (0:negative, 1: low intensity, 2: medium intensity, 3 high intensity) multiplied by the positive ratio per tumor area from 0 to 10(0–100%), resulting in a score from 0 to 30.
Data Analysis
All experiments were performed at least three times. ImageJ was used to analyze the intensity of immunofluorescence, and unpaired t-test was used to ensure the consistency of repeated experimental results. To analyze the change trend of immunohistochemical results before and after chemotherapy, a paired t test was carried out. The analysis software utilized was GraphPad Prism version 9.0. The relationship between clinicopathological results and protein expression were analyzed using two-tailed Student’s t-test in SPSS Statistics. The statistical significance of all the P values were defined as P < 0.05.