TNBC tissue specimen
The study protocol and tissue specimen acquisition were approved by the Ethical Review Committee of Xiangya Hospital (2024121786). From 2024 to 2025, four pairs of samples from patients with histologically confirmed primary triple-negative breast cancer (TNBC) who underwent core needle biopsies at Xiangya Hospital (Hunan, China), received neoadjuvant chemotherapy with carboplatin and paclitaxel for the first time, and subsequently underwent surgery, without prior targeted or immunotherapy, were included in this study. Paraffin-embedded TNBC samples were collected for mass spectrometry analysis, with pre-treatment tissues obtained from core needle biopsies before NAT and post-treatment tissues from surgical resections. Ultimately, Eight TNBC tissue specimens were subjected to 4D-label free proteome analysis (Majorbio, China). To validate our findings, we further utilized paraffin-embedded primary triple-negative breast cancer (TNBC) surgical samples from 44 cases collected between 2023 and 2025. These samples were from patients who received neoadjuvant chemotherapy with carboplatin and paclitaxel for the first time, without prior targeted or immunotherapy, and underwent immunohistochemical analysis to investigate the relationship between THEM6 expression and prognosis. Tumor response to treatment was evaluated using RECIST 1.1, categorizing patients into responder (S) and non-responder (NS) groups. Additionally, human TNBC tissue arrays (WZ-TNBC1201) were purchased from Shanghai Outdo Biotech Co., Ltd.
Mass spectrometry
Paraffin-embedded samples were deparaffinized by incubating them in xylene at 37 °C with constant shaking, followed by centrifugation at 14,000 g to remove residual paraffin. This process was repeated until no wax remained. The samples were then sequentially rehydrated with ethanol and ultrapure water. Following rehydration, lysis was performed using 5% SDT buffer (SDS, 100 mM Tris-HCl, pH 8.5) supplemented with a protease inhibitor cocktail, followed by three rounds of homogenization. Ultrasonic fragmentation was carried out for 1 h, and the lysates were subsequently heated in a boiling water bath at 95 °C for 60 min. After centrifugation, the supernatant was collected for protein quantification using a BCA assay. SDS-PAGE was performed to assess protein integrity. Protein digestion was conducted overnight in TEAB buffer (100 mM) using trypsin, following reduction with TCEP and alkylation with IAM. The resulting peptides were desalted using HLB cartridges, vacuum-dried, and quantified. Finally, data-independent acquisition (DIA) mass spectrometry was performed on a timsTOF Pro2 instrument in DIA-PASEF mode, acquiring MS data across an m/z range of 400–1200 with 64 isolation windows for comprehensive proteomic analysis. Proteins with significant differential expression were identified based on criteria of P < 0.05 and an absolute fold change > 2. The association between THEM6 expression and overall survival (OS) in TNBC patients was analyzed using the KM-Plotter online tool (http://kmplot.com) based on the GSE96058 dataset [13].
Immunohistochemical staining
The tissue sections were dehydrated, subjected to citrate antigen retrieval, and blocked with 5% goat serum for 15 min at room temperature. They were then incubated overnight at 4 °C with a diluted primary anti-THEM6 antibody. Following this, the sections were treated with DAB, counterstained with hematoxylin, and dehydrated using ethanol. Images were captured and analyzed using ImageScope software (Leica Microsystems). The histological score was calculated as Total score = Proportion score × Intensity score, and samples were classified as high or low expression based on a median score of 4.
Cell lines and reagents
The human triple-negative breast cancer cell lines MDA-MB-231 and BT-549 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and RPMI 1640, respectively, each supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cultures were maintained at 37 °C in a 5% CO2 atmosphere. Paclitaxel (HY-B0015) and Carboplatin (HY-17393) were obtained from MedChemExpress (MCE). The ferroptosis inhibitors ferrostatin (Fer-1, HY-100579) and liproxstatin-1 (Lip-1, HY-12726) were also purchased from MCE. Additionally, the pan-caspase inhibitor Z-VAD-FMK (HY-16658B), necrosis inhibitor necrostatin-1 (Nec-1, HY-15760), and autophagy inhibitor 3-methyladenine (3-MA, HY-19312) were sourced from MCE. Antibodies used in the study were acquired from the following suppliers: PGRMC1 (Zen-bio, 122868), THEM6 (Bioss, bs-15296R), FDFT1 (Proteintech, 13128-1-AP), GPX4 (Huabio, ET1706-45), SLC7A11 (Huabio, HA600098), ACSL4 (Huabio, ET7111-43), V5-tag antibody (Proteintech, 14440-1-AP), HA-tag antibody (Proteintech, 51064-2-AP), Flag-tag antibody (Proteintech, 20543-1-AP) and β-actin (Proteintech, 66009-1-Ig). lentiviral vectors including THEM6, PGRMC1 and the Negative Control, were purchased from GENERAL BIOL. Lentiviral particles containing shRNA targeting human FDFT1 (Locus ID 2222) were purchased from Origene.
Lentiviral transduction
Lentiviruses were packaged in HEK293T cells by co-transfection of the expression vector with packaging plasmids psPAX2 and pMD2.G using Lipofectamine 3000 (Invitrogen), following the manufacturer’s protocol. Viral supernatants were collected at 48 h post-transfection, filtered through a 0.45 μm membrane. Target cells (MDA-MB-231 or BT-549) were seeded in six-well plates and infected with lentivirus in the presence of 8 µg/mL polybrene. After 48 h, cells were selected with 2 µg/mL puromycin for one week. Stable expression was confirmed by Western blot analysis.
Cell viability assay
MDA-MB-231 and BT-549 cells were plated in 96-well plates at a density of 5 × 10^4 cells/ml. Following cell adherence, the cultures were treated with specified concentrations of carboplatin or paclitaxel for 48 h. Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay (MCE, HY-K0301), according to the manufacturer’s protocol. Absorbance at 450 nm was quantified using a microplate reader (PerkinElmer). The half-maximal inhibitory concentration (IC50) values were determined from dose-response curves generated using GraphPad Prism software.
Colony formation assay
Breast cancer cells were seeded into six-well plates and allowed to adhere overnight. They were then treated with 0, 0.5, or 1 µM carboplatin for 48 h. Following drug treatment, the medium was removed, and the cells were washed with PBS, trypsinized, and replated at a density of 2000 cells per well in new six-well plates. The cells were incubated for 14 days to allow colony formation. Colonies were subsequently stained with crystal violet, and the number of colonies in each condition was quantified.
Live/dead viability assay
Cells were seeded into 6-well plates at a density of 5 × 10^4 cells per well. After 48 h, cell viability was assessed using the Calcein AM/PI Live-Dead Cell Staining Kit I (APExBIO, K2247). Briefly, cells were incubated with 1 µM propidium iodide (PI) and 1 µM Calcein AM for 30 min at room temperature. Following staining, the cells were washed twice with PBS. Fluorescence microscopy was performed using an Axio Observer 3 fluorescence microscope (Carl Zeiss Microimaging) to visualize and differentiate live (green) and dead (red) cells.
Quantitative real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen). cDNA was synthesized with SuperScript™ II reverse transcriptase (Invitrogen), and quantitative PCR was performed using Power SYBR Green PCR Master Mix (Takara). The primers used for the SYBR Green assays were as follows: THEM6-F:5′-GCAGCACTGGATCTCCTACAACG-3′; THEM-R: 5′- GGTCCTTGGTGACATCACTGAGC-3′; FDFT1-F:5′-GCAACGCAGTGTGCATATTTT-3′; FDFT1-R: 5′-CGCCAGTCTGGTTGGTAAAGG-3′; β-actin-F: 5′-CACCATTGGCAATGAGCGGTTC-3′; and β-actin-R: 5′- AGGTCTTTGCGGATGTCCACGT − 3′. Real-time amplification was carried out using an ABI Prism 7000 SDS (Applied Biosystems). Gene expression levels were quantified using the 2−ΔΔCT method, with normalization to β-actin as the reference gene.
Western blot and ubiquitination assays
Cells were lysed using RIPA lysis buffer (MCE, HY-K1001) supplemented with a protease inhibitor cocktail (MCE, HY-K0010). The protein concentration in the cell lysate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), following the manufacturer’s protocol. Samples were then denatured with SDS-PAGE protein loading buffer (Beyotime, D0071) at 100 °C for 5 min. Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, ISEQ00010). After overnight incubation with the primary antibody, the membranes were probed with the secondary antibody at 37 °C for 1 h. Protein bands were visualized using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, 32106) and captured using Image Lab software version 5.0 (Bio-Rad). For Ubiquitination assays, cells were lysed with 100 µL of NETN buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 20 mM NEM, 1 mM iodoacetamide), boiled for 15 min, and then diluted 10-fold in NETN containing protease inhibitors, NEM, and iodoacetamide. After centrifugation, the supernatant was subjected to immunoprecipitation and analyzed by Western blotting [14].
Cycloheximide (CHX) Chase assay
To evaluate protein stability, MDA-MB-231 and BT-549 cells stably expressing either THEM6 or a negative control were seeded in six-well plates and cultured to approximately 70% confluence. CHX was added to the culture medium at a final concentration of 0.1 mg/mL. Cells were harvested at designated time points (0, 2, 4, 6, and 8 h) following CHX treatment. At each time point, cells were washed with ice-cold PBS and lysed in RIPA buffer supplemented with protease inhibitors. Lysates were clarified by centrifugation at 14,000 g for 15 min at 4 °C, and protein concentrations were quantified using a BCA assay. Equal amounts of protein were subjected to SDS-PAGE, followed by Western blot analysis to assess protein degradation kinetics.
ROS measurement
Intracellular ROS levels were measured using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma, 35845). This probe diffuses into cells and is enzymatically converted to the fluorescent 2′,7′-dichlorofluorescein (DCF). Triple-negative breast cancer (TNBC) cells were plated at a density of 6 × 10^5 cells per well in 6-well plates and incubated for 24 h. The cells were then exposed to 5 µM carboplatin for 48 h. After treatment, the cells were incubated with 10 µM DCFH-DA solution at 37 °C for 30 min, followed by three washes with phosphate-buffered saline (PBS). DCF fluorescence was visualized using an Axio Observer fluorescence microscope (Carl Zeiss Microimaging) with excitation at 485 nm and emission at 535 nm.
Ferrous iron measurement
Intracellular ferrous iron (Fe²⁺) content was quantified using the Iron Assay Kit (Abcam, ab83366). Briefly, samples were incubated at 25 °C for 30 min, followed by an additional 60-minute incubation with the iron probe at 25 °C to form a stable colored complex. Standard curve and reaction solutions were prepared according to the manufacturer’s instructions. The samples were then transferred to a microplate reader (PerkinElmer), and Fe²⁺ levels were determined by measuring absorbance at 539 nm.
Malondialdehyde measurement
Malondialdehyde (MDA) levels were measured in the lysates using the Lipid Peroxidation Assay Kit (Beyotime, S0131) according to the kit’s instructions. Briefly, 0.1 mL sample was mixed with 0.2 mL of the MDA detection working solution and incubated for 15 min at 100 °C. The samples were then allowed to cool to room temperature and centrifuged at 1000 × g for 10 min to collect the supernatant. Next, 200 µL of the supernatant was transferred to a 96-well plate, and absorbance was measured at 532 nm using a microplate reader (PerkinElmer). MDA levels were expressed as the ratio of the absorbance value to that of the control group.
Transmission electron microscope assay
Briefly, cells were seeded in 6-well plates at a density of 5 × 10^4 cells per well and treated with or without 5 µM carboplatin for 48 h. After treatment, cells were collected, washed with PBS, and fixed with 2.5% glutaraldehyde. The samples were then processed according to standard procedures, and images were obtained using a transmission electron microscope (Hitachi). The proportion of mitochondria with increased bilayer membrane formation was quantified using TEM images. To determine the size of mitochondria, TEM images were analyzed using ImageJ software to measure the mitochondrial area, and the relative mitochondrial size was provided.
Xenograft mouse model
The experiments were conducted with approval from the Animal Care and Use Committee of Central South University (China) (XY20240903005). Control or THEM6-overexpressing MDA-MB-231 cells (2 × 10^6) were orthotopically implanted into the mammary fat pads of 20 BALB/c nude mice (4 weeks old). Once the tumors reached approximately 50 mm³ in size, the mice were randomly assigned to four groups, with 5 mice per group: a carboplatin treatment group, in which carboplatin was administered via intraperitoneal (IP) injections at a dose of 50 mg/kg once weekly for three cycles, and a control group. Tumor volumes were measured every 3 days using calipers from the start of treatment and calculated using the formula (length × width2) × 1/2. Mice were sacrificed for tumor dissection on day 35 post-treatment initiation. H₂O₂ levels were measured using an H₂O₂ assay kit (Beyotime). Tumor tissue was homogenized, and absorbance at 560 nm was measured using a microplate reader (PerkinElmer).
Statistical analysis
Overall survival (OS) was analyzed using Kaplan–Meier curves and compared with the log-rank test. Data are presented as mean ± SD (n ≥ 3) and were analyzed using GraphPad Prism 6 software (GraphPad Software). Differences between groups with continuous data were evaluated using the Student’s t-test. p-values less than 0.05 were considered statistically significant.