Animal modelsXenograft mouse models of PDAC

We used the two PDAC patient-derived xenograft (PDX) xenograft models of relapse that we described in Reyes-Castellanos et al. [32], namely PDAC032T and PDAC084T, which always relapse after cancer remission induced by the chemotherapy, i.e., gemcitabine alone or combined with perhexiline, respectively. Recipient mice were 5–6-week-old athymic female, Swiss nude mice, SOPF (Specific and Opportunistic Pathogen Free) health status strain Crl:Nu(lco)-Foxn1nu (Charles River, France). To obtain the xenografted mice, the subcutaneous tumor from an initial mouse donor was removed and finely minced with a scalpel. Then, 150 mg of tumor’s pieces were mixed with 50 μl of Matrigel and implanted with a trocar (10 Gauge) in the subcutaneous space of recipient isoflurane-anesthetized mice. A cohort of 15 recipient mice, on average, was generated with the same donor mouse tumor, thus limiting the variability of results within one experiment. Tumor volume was measured twice per week using a digital caliper, and tumor volume was calculated using the formula V = Length × (width)2/2.

When xenografts reached ~200 mm3 volume, mice were randomly assigned in a treatment group in which the average of all tumors was 200 mm3. Treatments were administered by intraperitoneal (IP) injection during 1 month as follows: gemcitabine 120 mg/kg IP twice a week for the PDX PDAC032T, or gemcitabine plus perhexiline (120 mg/kg IP twice a week and 5 mg/kg IP every other day, respectively) for the PDAC084T PDX. Gemcitabine (Gemzar) was provided by Eli Lilly & Co., and perhexiline maleate salt by Sigma-Aldrich. Vehicle-injected mice (controls) were injected with PBS in the case of gemcitabine controls or 3% DMSO in PBS for combination treatment controls. Mice whose tumor volume reached 1.5 cm3 were ethically sacrificed and tumors removed. Treatment to induce tumor regression is limited to a duration of 1 month, which is sufficient to lead to remission. Then, treatment is stopped, and the mice are left alive to monitor their relapse.

The in vivo relapse-preventing effect of arsenic trioxide (ATO, AS2O3, Sigma-Aldrich) and L-buthionine sulfoximine (BSO, Sigma-Aldrich) was evaluated in the xenograft mouse model. Mice were divided into different groups (at least n = 4 per condition): PBS (vehicle control), AS2O3 (0.2 mg/kg), and AS2O3 (0.2 mg/kg) + BSO (0.3 mg/kg). AS2O3 was administrated IP daily (5 days a week), starting right after the end of the 1-month chemotherapy treatment, inducing complete tumor regression, and during one, two, or three months. BSO was administrated IP 3 times per week for 2 months, starting 9 and 16 days (for PDAC032T and PDAC084T xenografts, respectively) after the end of the 1-month chemotherapy treatment and ATO start of treatment, for the sake of animal welfare. The use of low doses, even though both molecules are non-toxic in vivo, is based on the literature [39,40,41]. Treatment with BSO alone was not tested, as it has never shown antitumor action as a single treatment [41, 42] and also because of the limitation in the number of mice xenografted with the same donor mouse tumor.

Syngeneic allograft mouse model of PDAC

Orthotopic syngeneic allografts were generated as previously described [30]. The murine KPCluc2 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 600 μg/ml Hygromycine B (ThermoFisher Scientific) for selection of cells containing the vector encoding luciferase and GFP proteins, at 37 °C with 5% CO2 in a humidified atmosphere. One million KPCluc2 cells were IP injected into 5- to 6-week-old C57BL/6 female mice (immunocompetent strain, SOPF health status, Charles River, France). Tumoral growth was followed by bioluminescence upon injection of 3 mg luciferin-EF (Promega) using a Photon Imager device (Biospace Lab). Twelve days post-grafting, tumor-bearing mice were randomly assigned to two treatment cohorts (at least n = 5 per condition): vehicle control (treated with PBS), and gemcitabine 120 mg/kg IP twice a week during one month. Mice were sacrificed when they reached the ethical limit point.

All mice were kept under specific pathogen-free conditions and according to the current European regulation; the experimental protocol was approved by the Institutional Animal Care and Use Committee (#16711).

Tumor dissociation

Xenograft and allograft tumors were dissociated using the gentle MACS™ Octo Dissociator with Heaters and the Tumor Dissociation Kit, mouse (130-096-730), as per manufacturer’s instructions (Miltenyi Biotec). Briefly, tumors were cut into 2–4 mm3 pieces and resuspended in RPMI 1640 medium supplemented with 2% FBS (Thermo Scientific, Waltham, MA, USA). These pieces then underwent mechanical and enzymatic digestion for 1 h. Immediately following dissociation, all single-cell suspensions were filtered using a MACS SmartStrainer (70 µm). The suspensions were centrifuged for 7 min at 300 × g at room temperature, the supernatant was aspirated, and the cells were resuspended in RPMI 2% FBS medium. Cells were counted by flow cytometry using the MACSQuant-VYB cell cytometer (Miltenyi Biotec).

Flow cytometry ex vivo

All metabolic fluorescent probes were purchased from InvitrogenTM.

Mitochondrial mass and mitochondrial membrane potential measurements

Mitochondrial mass and mitochondrial membrane potential measurements were performed using MitoTracker Deep Red (M22426) and the MitoProbe™ TMRM Kit (M20036), respectively. Briefly, 200,000 cells were collected after tumor dissociation, centrifuged, and labeled with MitoTracker or TMRM to a final concentration of 200 nM and 20 nM in PBS at 37 °C for 10 min and 30 min, respectively. Then, 10,000 events per sample were acquired in a MACSQuant-VYB cytometer (Miltenyi Biotec), and data analysis was performed using the FlowJo software.

Total ROS and mitochondrial superoxide anions detection

Total ROS and mitochondrial superoxide anions measurements were performed using CellROX Orange (C-10443) and MitoSOX Red (M36008), respectively. Briefly, after tumor dissociation, 200,000 cells were collected, centrifuged, and labeled with CellROX or MitoSOX to a final concentration of 5 µM and 10 µM (in PBS for MitoSOX, and culture medium for CellROX) for 30 min and 20 min at 37 °C, respectively. After incubation, cells were centrifuged and resuspended in PBS 1x for flow cytometry analysis. 10,000 events per sample were acquired in a MACSQuant-VYB cytometer (Miltenyi Biotec), and data analysis was performed using the FlowJo software.

ATP level measurement ex vivo

Total ATP level was measured using the cell viability assay (Cell-Titer Glo Kit; Promega) according to the manufacturer’s instructions. Immediately after tumor dissociation, 50,000 cells from each single-cell suspension were resuspended in 100 µl of RPMI 2% FBS medium and distributed in four replicates in a 96-well flat-bottom culture plate. Cells were then treated with PBS (Control), 1 µM oligomycin (inhibitor of mitochondrial respiration), or 100 mM 2-DG (inhibitor of glycolysis). Following 1 hour of incubation at 37 °C, 100 µl of Cell Titer Glo reaction mix solution was added to each well for a final volume of 200 µl. Plates were then analyzed by luminescence using Tristar LB 941 apparatus (Berthold Technologies). The background relative light unit (RLU) was subtracted from each RLU value. By comparing the different conditions, total ATP (PBS condition) and percentages of both mitochondrial and glycolytic ATP were determined: mitochondrial ATP = Total ATP - ATP (oligomycin); mitochondrial ATP (%) = mitochondrial ATP/Total ATP *100; glycolytic ATP = Total ATP - ATP (2DG); glycolytic ATP (%) = glycolytic ATP/Total ATP *100.

Quantification of small antioxidant moleculesGSH/GSSG measurement

GSH/GSSG-Glo Assay kit (Promega, V6611) was used following manufacturer’s protocol with some modifications. Briefly, 10 mg of tumor tissue was crushed using a Precellys® Evolution device, resuspended in 50 µl of PBS/EDTA, and distributed in a 96-well plate. After homogenization, 50 μl of Total Glutathione Lysis Reagent (for Total glutathione measurement) or Oxidized Glutathione Lysis Reagent (for GSSG measurement) was added. Luciferin Generation Reagent and Detection Reagent were added to all wells, and luminescence was recorded using Tristar LB 941 apparatus (Berthold Technologies). By comparing the different conditions, GSH/GSSG ratio was calculated using the following equation: GSH/GSSG = (total glutathione RLU - GSSG RLU)/(GSSG RLU/2).

NADPH measurement

Ten micrograms of tumor tissue were crushed using a Precellys® Evolution, resuspended in 50 µl of PBS/EDTA, and distributed in a 96-well plate. Measurement was performed according to the manufacturer’s protocol. Briefly, 50 µl of NADP/NADPH-Glo™ Detection Reagent (Promega, G9081) was added to each well and incubated for 30 min at RT. Luminescence was recorded using Tristar LB 941 apparatus (Berthold Technologies).

Gene expression analysis by RT-qPCR

Total RNA was isolated from 20 to 25 mg of tumor piece using both TRIzol (Invitrogen) and the Qiagen total RNA isolation kit (Qiagen, ref 74004) to avoid protein and extracellular matrix accumulation in the columns. The tumor piece was lysed in 600 µl RLT Buffer (from Qiagen kit) with 1% β-Mercaptoethanol using beads tube from Precellys lysing kit for hard tissue (ref P000917-LYSK0-A, 3x Cycle 1500 rpm, 15 s of mix, 10 s of rest) and directly centrifuged 3 min at 10,000 × g at RT. Supernatant was transferred in 400 µl of TRIzol and incubated for 5 min at RT followed by addition of 150 µl of chloroforme. Tubes were carefully mixed and incubated 3 min at RT before centrifugation (12,000 × g, 5 min, 4 °C). The transparent upper phase containing RNAs was transferred in a tube containing 500 µl of 70% ethanol, mixed, and transferred inside Qiagen RNA isolation kit columns according to manufacturer’s instructions, which were followed until the end of extraction. RNA samples were subjected to reverse-transcription (RT) using the Go Script reagent (Promega) following manufacturer’s instructions. Next, Real-Time quantitative PCR was performed in triplicate using Takara reagents and the Stratagene cycler Mx3005P QPCR System. Raw values were normalized with the housekeeping gene TBP1 for the same cDNA sample. We used the human primers for Nrf2, HO-1, SLC7A11, and GPX4, involved in the antioxidant defense, and PGC-1α and TFAM, involved in mitochondrial homeostasis. Primer sequences can be found in [43]. For each RNA sample, RT reaction was done twice to generate 2 batches of cDNA, which were amplified by qPCR in three independent experiments for each gene.

Transcriptomic analysis by bulk RNA sequencingTotal RNA isolation from tumors

RNA was extracted from PDAC xenografts using the RNeasy Mini kit (Qiagen) as described above. RNA integrity and concentration were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The average RIN (RNA integrity number) values for all samples were comprised between 9.3 and 10, ensuring a high quality of isolated RNAs.

Bulk RNA sequencing

The preparation of mRNA libraries was realized following manufacturer’s recommendations (kapa mRNA HyperPrep from ROCHE). Final samples pooled library prep were sequenced on ILLUMINA Novaseq 6000 with S1-200cycles cartridge (2 × 1600Millions of 100-base reads), corresponding to 2 × 30Millions of reads per sample after demultiplexing.

Bulk RNA sequencing data analysis

Quality control has been performed on the fastq files using FastQC (v0.11.9) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). To map the sequenced reads to the human reference genome, we made use of STAR (v2.7.3a). From these mapped reads, gene counts were then quantified using featureCounts (v2.0.1). Starting from the raw gene counts, normalization and differential expression analysis have then been performed using DESeq2 (v 1.22.2).

Differential gene expression analysis

Differential expression analysis was performed using the limma package (R/Bioconductor). Genes with a log₂ fold change (logFC) > 1.25 and an adjusted p value < 0.05 were considered significantly differentially expressed. A total of 688 upregulated genes were identified in the PDAC032T relapse group compared to untreated.

Functional enrichment analysis

Functional enrichment analysis of the upregulated gene set was conducted in R using the following tools: enrichGO (from the clusterProfiler package) for Gene Ontology (GO) biological processes, enrichKEGG for KEGG pathway analysis, enrichR for Reactome. Default parameters were used for all enrichment tools. Significantly enriched terms and pathways were defined as those with an adjusted p value < 0.05.

Gene set enrichment analysis (GSEA)

Gene set enrichment analysis (GSEA) was performed using GSEA v4.1 tool (Broad Institute). Following parameters were used: Number of permutations = 1000, permutation type = gene set, Chip = Human_Ensembl_Gene_ID_MSigDB.v2022.1.Hs. chip. Other parameters were left at default values. The normalized enrichment scores (NES) were computed from PDAC032T relapse group compared to PDAC032T control group. Genes with FDR q value < 0.05 and fold change ≥ 1.5 were considered of interest.

In vitro assaysCell viability

The human PDAC032T and PDAC084T primary PDAC cells were cultured in serum-free ductal media (SFDM) at 37 °C with 5% CO2 in a humidified atmosphere as reported previously [30, 32]. SFDM is a complex medium supporting the PDAC primary cell growth and containing DMEM-F12, nicotinamide, glucose, hormones, growth factors, and Nu-serum, providing a low-protein alternative to FBS. Cells were seeded in 96-well plates in triplicate (5000 cells per well) and the corresponding treatment was administered the day after. Cells were treated with perhexiline (7 µM), gemcitabine (1 µM), or the combination for 24 h. These treatments were done in the presence and absence of the antioxidant N-acetylcysteine (NAC, Sigma-Aldrich) at 2.5 mM. Next, cell viability was determined by the Crystal violet viability assay, which is independent of cell metabolism. For this, cells were fixed in glutaraldehyde (1%), washed twice with PBS, stained with Crystal violet (0.1%) for 10 min, and then washed three times with PBS. Crystals were solubilized in SDS (1%), and absorbance was measured at 600 nm using an Epoch-Biotek spectrophotometer.

Total ROS measurement by flow cytometry

Cells were seeded in 12-well plates in duplicates (200,000 cells/1 ml medium/well) and the day after, treatments were administered. Cells were treated with perhexiline (7 µM), gemcitabine (1 µM), or the combination for 24 h. After treatments, the medium was supplemented with CellROX Orange at a final concentration of 5 μM. Cells were incubated for 30 min at 37 °C, then harvested with pre-warmed Accutase (Gibco) and resuspended in PBS for flow cytometry analysis. 10,000 events per sample were acquired in a MACSQuant-VYB cytometer (Miltenyi Biotec), and data analysis was done with the FlowJo software.

Immunoblotting

Tumor pieces were lyzed using Precellys Lysing Kit (MK28R Hard Tissue 2 mL, Bertin Technologies). Each tumor sample was homogenized on ice in 400 µl of RIPA Buffer (supplemented with Protease and Phosphatase Inhibitor) using the Precellys Evolution with the Hard Tissue Programme. Protein concentrations were determined with the Pierce BCA protein assay kit (23225, Thermo Fisher Scientific). Equal protein amounts (35 μg) diluted in a 4× Laemmli buffer were denatured by heating at 95 °C for 5 min and separated by electrophoresis on 4–20% Mini-PROTEAN TGX Precast Protein Gel, then transferred onto a 0.45μm nitrocellulose membrane. All membranes were systematically stained with Ponceau red to confirm equal protein loading and transfer. Membranes were blocked with 5% non-fat dry milk in TBS-T (TBS with 0.1% Tween-20) for 1 h at room temperature and then incubated in 5% BSA in TBS-T with appropriate primary antibodies at 4 °C overnight: HO-1 (1:2000, Abnova - A303-662A), FTH (1:1000, abcam 75973), and GPX4 (1:1000, Abcam 125066). Membranes were then washed three times with TBS-T, and incubated with the appropriate HRP-coupled secondary antibody at 1:2000 for 1h30min at RT, before being revealed with ECL (Immobilion Western ECL, Millipore). Signal detection was performed using Fusion FX7 imagine system. Protein band intensities (Raw Integrated Density) were quantified using ImageJ Software after background subtraction (rolling ball radius: 50 pixels). Signal of proteins of interest was normalized to Ponceau Red to account for loading variation.

Statistical analyses

Results are expressed as the mean ± SD of duplicates or triplicates, except for in vitro cell viability data shown in mean ± SEM, and at least two or three independent experiments were done for each analysis. Statistical analysis of data was performed with GraphPad Prism 8 (GraphPad Software) using two-tailed unpaired Student’s t-test. P values < 0.05 were considered to be statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).