Patients and specimens
Three distinct cohorts of GBC patients were recruited for this study. For the first cohort, a tissue microarray containing 62 cases of GBC tumor tissues, accompanied by comprehensive clinicopathological and follow-up data, was retrospectively obtained from GBC patients who underwent radical cholecystectomy before receiving gemcitabine-based chemotherapy at Renji Hospital affiliated with Shanghai Jiao Tong University School of Medicine between January 2008 and April 2015, with the patients’ consent. For the second cohort, 35 cases of GBC tumor tissues, accompanied by comprehensive clinicopathological and follow-up data, was retrospectively obtained from GBC patients who underwent radical cholecystectomy before receiving gemcitabine-based chemotherapy at Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine between May 2020 and July 2024, with the patients’ consent. The inclusion criteria for patients were: (1) definitive GBC diagnosis by pathology. (2) radical cholecystectomy, including complete resection of primary tumorous tissues with confirmation of negative margins by histological examination. (3) absence of radiotherapy or chemotherapy prior to surgery. (4) receipt of gemcitabine-based chemotherapy post-surgery. The sample size for the Cohort 1 and 2 was determined by the total number of consecutive, eligible patients available during the study period, which is a standard approach for retrospective biomarker studies.
In the third cohort, 18 cases of primary GBC tissues were utilized to assess gemcitabine sensitivity in mini-PDX models, alongside the acquisition of relevant clinical information from patients. These primary tissues were obtained via radical cholecystectomy or tissue biopsy before the initiation of guided chemotherapy, guided by mini-PDX results, at Shanghai Sixth People’s Hospital Affiliated with Shanghai Jiao Tong University School of Medicine and Renji Hospital affiliated with Shanghai Jiao Tong University School of Medicine between July 2016 and August 2023, with the patients’ consent.
This study received approval from the Ethics Committee of Shanghai Sixth People’s Hospital Affiliated with Shanghai Jiao Tong University School of Medicine (No. 2020-243), and written informed consent was obtained from all subjects involved in the study.
Cell culture and reagents
NOZ cells (CTCC-003-0099) and GBC-SD cells (CTCC-400-0165) were procured from Zhejiang Meisen Cell Technology Co., Ltd (Jinhua, China), while human embryonic kidney 293 T (HEK293T) cells were acquired from the American Type Culture Collection. The wild-type and gemcitabine-resistant PANC-1 cells were purchased from Yaji Biotechnology Co., Ltd. (Shanghai, China). All cell lines were recently authenticated by short tandem repeat (STR) profiling and tested negative for mycoplasma contamination. NOZ cells were cultured in William’s E medium (Gibco), whereas GBC-SD, PANC-1 and 293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco). Wild-type NOZ cells were subjected to escalating concentrations of gemcitabine (HY-17026, MedChem Express) ranging from 500 nM to a final concentration of 16 μM over a 24-week period and were subsequently cultured in 16 μM gemcitabine to sustain their chemoresistance, leading to the development of GEM-R cells. All cell lines were supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin and were incubated in a humidified chamber with 5% CO2 at 37 °C. H2O2 (323381, Sigma-Aldrich) and NAC (A7250, Sigma-Aldrich) were employed to modulate intracellular ROS levels, while MitoSOX Red (HY-D1055, MedChem Express) was utilized for intracellular ROS detection via flow cytometry. Mdivi-1 (HY-15886, MedChem Express) was utilized for mitophagy inhibition, CCCP (HY-100941, MedChem Express) was utilized for mitophagy activation, and CB-6644 (HY-114429, MedChem Express) was used to hinder the ATPase activity of Reptin.
Plasmids, shRNA, and sgRNA
Flag-tagged SENP3 was inserted into the pCDH or pcDNA3.1 vector, while HA-tagged WT or mutant (K115R, K132R, K269R, K288R, K368R, K456R) Reptin was incorporated into the pcDNA3.1 vector. SENP3-targeting shRNAs and a non-specific control shRNA (shNC) were integrated into the pLKO.1 vector. Reptin-targeting sgRNA and a non-specific control sgRNA (sgNC) were inserted into the lentiCRISPR-V2 vector. The 2 kb region upstream of the transcription start site of PINK1 was designated as its promoter region, cloned into the pGL3-basic vector, and labeled as PINK1-Luc.
Virus production and infection
293 T cells cultured in 10 cm dishes reached optimal transfection readiness at 70% to 80% confluency. Co-transfection was performed using 12 µg of the necessary plasmids (overexpression constructs, shRNAs, or sgRNAs), along with 9 µg of psPAX2 and 3 µg of pMD2.G, employing 50 µl of polyethyleneimine. Following transfection, the 293 T cells were maintained at 37 °C, with the transfection medium being refreshed after 4 h. Virus-containing medium was harvested between 48 to 72 h post-transfection and augmented with 5 µg/ml polybrene for infecting target cells in dishes or microplates, a process lasting 12 to 24 h. Subsequently, the infected cells underwent positive selection with 2.5 µg/ml puromycin to eliminate uninfected cells and establish stable cell lines.
Generation of single-cell Reptin knockout clones
The modified lentiCRISPR-V2 vector, housing Cas9 nuclease and sgRNA targeting the sixth exon in the Reptin genomic locus, was employed to establish stable Reptin knockout (RKO) cell lines in both NOZ and GBC-SD cells. Subsequently, single-cell knockout clones were generated, and the knockout efficiency was validated through genomic PCR sequencing and immunoblotting.
Cell viability assays
Cells in single-cell suspension were seeded at a density of 4000 cells per well for chemical reagent treatment or 2000 cells per well for growth rate assays in 96-well plates containing 100 µl of culture medium. Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay at specified time points: 72 h post-chemical reagent treatment or every 24 h after plating. Briefly, 10 µl of CCK-8 solution was directly added to the cells, followed by a 3 h incubation at 37 °C, and absorbance was measured at 450 nm.
Colony formation assays
For colony formation, GBC cells in single-cell suspension were seeded and cultured in 6-well plates at a density of 500 cells per well or in 12-well plates at a density of 250 cells per well for 10 days. Subsequently, cells were treated with gemcitabine at the concentration of IC50 or vehicle for 96 h. Colonies were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.
Cell apoptosis assays
GBC cells were plated in dishes or microplates overnight and treated with gemcitabine or vehicle for the indicated time and dose. Subsequently, all cells were harvested by trypsinization without EDTA and doubly stained with Annexin V-FITC/PI (C1062, Beyotime Biotechnology) and analyzed using flow cytometry.
Cell migration assays
GBC cells in single-cell suspension were seeded at a density of 10000 cells per well into the upper compartment of a Transwell chamber (Corning). The lower chamber was filled with 500 µl of DMEM supplemented with 20% FBS. After incubating the cells for 24 h, those in the upper chamber were removed with a cotton swab. Migrated cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.
Western blot assays
Standard procedures were followed for immunoblotting. Cell lysates were prepared using radioimmunoprecipitation lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.1% SDS, 2 µM EDTA) supplemented with proteinase inhibitor and quantified using the BCA Protein Assay Kit (P0012, Beyotime Biotechnology). Twenty micrograms of protein aliquots were electrophoresed through 10% or 12% SDS polyacrylamide gels and transferred to polyvinyl difluoride membranes (Millipore). Membranes were then blocked in 5% skim milk at room temperature for 1 h, followed by overnight incubation with primary antibodies at 4 °C. Horseradish peroxidase-conjugated secondary antibodies were used, and signals were detected using the ECL Kit (Millipore). Antibodies against SENP3 (5591, 1:1000), SUMO2/3 (4971, 1:1000), PINK1 (6946, 1:500), LC3B (3868, 1:1000), α-actinin (6487, 1:1000) were obtained from Cell Signaling Technology; Antibodies against GAPDH (60004-1-Ig, 1:1000), β-Tubulin (10094-1-AP, 1:1000), SQSTM1 (66184-1-Ig, 1:1000) were obtained from Proteintech; Antibodies against Reptin (A1905, 1:1000), HA (AE008, 1:1000), Lamin A/C (A17319, 1:1000) were obtained from ABclonal. Full, uncropped scans of all Western blots are provided in the Supplementary Material.
Immunoprecipitation (IP) assays
For exogenous co-IP assays, 293 T cells were transfected with plasmids for 72 h. For endogenous co-IP assays, NOZ cells were used without plasmid transfection. Cells were lysed with IP lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and protease inhibitor cocktail), followed by incubation with antibodies at 4 °C overnight. Protein A/G Magnetic Beads (88803, Thermo Fisher Scientific) were employed to capture antibodies. The immunocomplexes were then washed with lysis buffer and subjected to immunoblotting. For SUMOylation assays, cells were transfected with plasmids for 72 h, lysed with denaturing buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 40 mM DTT, 5% glycerol, 20 mM NEM, and protease inhibitor cocktail) for 30 minutes, and subjected to immunoblotting following IP with SUMO-IP buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl). Antibody against SENP3 (5591, 1:100) was obtained from Cell Signaling Technology; Antibodies against Reptin (A1905, 1:100), HA (AE008, 1:100), Flag (AE005, 1:100) were obtained from ABclonal.
Mass spectrometry
Beads were resuspended in 100 µl of 50 mM NH4HCO3, reduced with 10 mM DTT at 56 °C for 1 h, alkylated with 50 mM iodoacetamide at room temperature in the dark for 40 min, and subjected to enzymatic digestion at 37 °C overnight. Salt was removed from the sample using a C18 tip, and extracted peptides were lyophilized to near dryness. Experiments were conducted using a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer coupled with Easy-nLC1200 (Thermo Fisher Scientific). Data were acquired using a data-dependent top20 method, and raw MS files were analyzed and searched against a protein database using MaxQuant (1.6.2.10).
Data-Independent Acquisition (DIA) Proteomics
There are 3 biological replicates for the samples. Protein extraction was performed using SDT lysis buffer (4% SDS, 100 mM Tris-HCl [pH 7.6]), with protein concentration determined via BCA assay. A total of 15 μg of each protein sample was subjected to SDS-PAGE on a 4–20% gradient gel, stained with Coomassie Blue R-250, and a pooled QC sample was prepared. Filter-aided proteome preparation (FASP) was used for trypsin digestion, followed by C18 desalting. Peptides were reconstituted in 0.1% formic acid with iRT standards and analyzed via DIA-MS using an Astral mass spectrometer coupled to a Vanquish Neo UHPLC system. Parameters included a 380–980 m/z MS1 scan range (240,000 resolution), 299 DIA windows (2 m/z isolation), and HCD collision energy of 25 eV. Data were processed using DIA-NN with trypsin specificity, allowing 1 missed cleavage, carbamidomethylation of cysteine as a fixed modification, and oxidation of methionine and N-terminal acetylation as variable modifications. Protein identifications were filtered at 1% FDR.
Chromatin immunoprecipitation Sequencing (ChIP-seq)
There are 2 biological replicates for the samples. NOZ cells were cross-linked with formaldehyde, and chromatin was extracted and sheared into fragments of 200–1000 bp. Immunoprecipitation was performed using antibodies against Reptin (ab91462, 1:50, Abcam), and antibody-protein-DNA complexes were captured using Protein A/G beads, washed, and eluted. DNA was purified and subjected to library preparation, followed by high-throughput sequencing. Data analysis included aligning reads to the reference genome, identifying enriched regions, and annotating peaks.
Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR)
Following the ChIP procedure described above, the immunoprecipitated DNA was analyzed by quantitative PCR. Specific primers were designed to amplify the promoter region of the PINK1 gene. The relative enrichment of target DNA fragments was calculated by normalizing the amount of immunoprecipitated DNA to the corresponding input DNA, with technical replicates performed for each sample.
Real-time quantitative PCR (RT-qPCR) assays
Total RNA was extracted from cells using TRIzol Reagent (Invitrogen), and 500 ng of total RNA was reverse transcribed into cDNA using the ABScript Neo RT Master Mix (ABclonal). RT-qPCR was performed in triplicate using the QuantStudio 5 System (Applied Biosystems). Ct values were compared using the 2−ΔΔCt method, with 18S serving as an internal reference gene. Primers used for RT-qPCR are listed in Supplementary Materials.
Immunofluorescence (IF) assays
GBC cells were seeded into confocal dishes, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Primary antibody against Reptin (A1905, 1:50, ABclonal) was applied overnight at 4 °C, followed by incubation with fluorophore-conjugated secondary antibodies (8889, 1:500, Cell Signaling Technology) and nuclear staining with DAPI dye. Images were captured using a confocal laser scanning microscope.
Luciferase assays
For luciferase assays, 293 T cells were seeded in 12-well plates at a density of 20000 cells per well and incubated overnight. Transfection of PINK1-Luc and pRL-TK plasmids was performed using polyethyleneimine. Cells were harvested 48 h post-transfection, lysed, and Rluc and Fluc activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity was calculated as the ratio of Fluc to Rluc.
Mitophagy staining
Mitophagy in live cells was monitored using the Mitophagy detection kit MD01 (Dojindo) according to the manufacturer’s instructions. Gemcitabine treatment induced mitophagy, and the level of mitophagy was evaluated based on the Mtphagy dye area of each cell. Colocalization of Mtphagy and lysosomal dyes was also analyzed.
Mt-Keima mitophagy assay
Cells were transiently transfected with the pHAGE-mt-mKeima plasmid using an appropriate transfection reagent. After transfection, cells were treated with gemcitabine or vehicle for the indicated time and dose. Subsequently, cells were harvested and subjected to dual-excitation flow cytometry (excitation: 488 nm for neutral pH, 561 nm for acidic pH) to quantify the red (lysosomal) and green (mitochondrial) fluorescence signals, reflecting mitochondrial delivery to lysosomes.
Transmission electron microscopy (TEM) analysis
For TEM, fresh 1 mm3 tissues were fixed in Servicebio fixative (G1102) within 1–3 min, then post-fixed with 1% osmium tetroxide. Dehydrated in ethanol/acetone, infiltrated with 812 embedding medium (SPI), polymerized at 60 °C for 48 h. 60–80 nm sections (Leica UC7) on 150-mesh grids were stained with uranyl acetate and lead nitrate, observed via HT7800 TEM (Hitachi).
Xenograft model
For SENP3-related experiments, 4-week-old male BALB/c nude mice were housed under specific pathogen-free conditions and injected subcutaneously with 2 × 106 NOZ or GBC-SD cells in 100 µl of PBS. Gemcitabine (20 mg/kg) or vehicle (saline) injections were administered intraperitoneally every 3 days, with six mice per group.
For CB-6644-related experiments, 4-week-old male NOD-Scid mice were housed under specific pathogen-free conditions and injected subcutaneously with 2 × 106 GBC-SD cells in 100 µl of PBS. Vehicle (saline), gemcitabine (20 mg/kg), CB-6644 (20 mg/kg), or combination (20 mg/kg gemcitabine and 20 mg/kg CB-6644) injections were administered intraperitoneally every 3 days, with five mice per group.
After tumor formation, mice were randomly allocated into groups. Tumor measurements were taken every 6 days before each injection, and tumor volume was calculated using the formula (length × width2)/2. Mice were euthanized at the end of the study, and tumors were collected and weighed. Animal procedures were conducted in accordance with the National Institutes of Health Guidelines and approved by the Institutional Animal Care and Use Committee of Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (No. 2024-0273). The sample size (n ≥ 5 mice per group) was chosen based on common practice in similar xenograft studies in our field and was constrained by ethical principles of the 3Rs (Replacement, Reduction, Refinement) to use the minimum number of animals necessary to achieve reliable results.
Mini-patient derived xenograft (mini-PDX) model
Drug sensitivity was evaluated using the OncoVee mini-PDX assay (LIDE Biotech Inc., China) [27]. GBC tissues were obtained after surgical resection, processed, and implanted subcutaneously in BALB/c nude mice. Gemcitabine (60 mg/kg) or placebo (saline) treatments were administered for 7 days, followed by evaluation of anti-tumor activity based on relative fluorescence units (RFU) using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Proliferation rates were calculated based on RFU measurements. All procedures were performed under sterile conditions and approved by the Institutional Animal Care and Use Committee.
Immunohistochemistry (IHC) analysis
Tissue slides were deparaffinized, treated with 3% H2O2, autoclaved, and incubated with primary antibodies overnight at 4 °C. Biotinylated secondary antibodies were applied, followed by signal amplification and detection using the DAB system. Stained sections were photographed under a light microscope, and protein expression was evaluated based on staining intensity and proportion scores. Antibodies against SENP3 (ab247139, 1:200), Reptin (ab91462, 1:200), and Ki-67 (ab15580, 1:200) were obtained from Abcam. Antibody against 4-HNE (bs-6313R, 1:200) was obtained from Bioss. TUNEL Assay Kit (ab66108) was obtained from Abcam.
Hematoxylin and eosin (H&E) staining
Tissue sections of liver and kidney were deparaffinized in xylene and rehydrated through a graded series of ethanol to distilled water. The sections were then stained with hematoxylin for nucleus visualization, followed by differentiation in acid alcohol and bluing in ammonia water. Subsequently, the cytoplasm was counterstained with eosin. After staining, the sections were dehydrated through an ascending ethanol series, cleared in xylene, and mounted with a neutral resinous medium. Stained sections were examined and photographed under a light microscope to assess tissue morphology and pathological changes.
Plasma concentration measurement of CB-6644
Mice received a single intraperitoneal injection of CB-6644. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-injection. Plasma was separated by centrifugation, and the concentration of CB-6644 was quantified using mass spectrometry. The plasma concentration-time profile was analyzed to evaluate the systemic exposure of CB-6644.
Measurement of serum ALT and AST levels
To assess liver function, the enzymatic activities of ALT and AST in mouse serum were quantified using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China. Catalog No. Z002-1-1) following the manufacturer’s protocols.
Statistical analysis
Data were presented as means ± SEM. Statistical analysis was performed using R (4.5.1) software. Kolmogorov-Smirnov test and Shapiro-Wilk test were used for normality tests. For normally distributed quantitative data, Student’s t-tests and analysis of variance (ANOVA) were used; for skewed distributed quantitative data, Mann-Whitney U test and Kruskal-Wallis test were used. Survival analysis was conducted using the Kaplan-Meier method and log-rank test. Cox regression analysis was used for multivariate analysis, and Pearson correlation coefficient was calculated for IHC staining. A two-sided p-value < 0.05 was considered statistically significant.
