DOI: 10.6084/m9.figshare.30153916
title: PreTA-mediated metabolism of 5-Fluorouracil by intratumoral <i>Citrobacter freundii</i> drives chemoresistance in pancreatic cancer

description: Quantitative analysis of 5-FU and its bacterial-derived metabolites (5-FU co-incubated with either the preT-knockout or wild-type clinical isolate of <i>C. freundii</i>): To assess the ability of <i>preT</i>-knockout and wild-type <i>C. freundii</i> strains to metabolize 5-FU, 40 μM 5-FU was incubated with 4 × 10⁸ CFU/mL of either<i> </i><i>preT</i>-knockout or wild-type clinical isolates in 600 μL of NB medium. The incubation time points were set at 0 h, 6 h, and 24 h (n = 3) and a blank control group was included. At the end of each incubation, 600 μL of ice-cold acetonitrile was added to stop the reaction. After vortexing, 200 μL of each sample was taken, and an equal volume of acetonitrile along with 10 μL of the internal standard 5-Chlorouracil was added. After vortexing, samples were centrifuged at 12,500 rpm for 10 min at 4°C. The supernatant (100 μL) was collected, diluted with four volumes of pure water, vortexed, and centrifuged again. The resulting supernatant was collected for subsequent analysis. The samples were analyzed using liquid chromatography with tandem quadrupole mass spectrometry (LC-MS/MS QQQ) using the AB SCIEX Triple Quad™4500MD. Chromatographic separation was performed using a Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). The mobile phase consisted of solution A (26.5 mM HCOOH and 1.0 mM NH4OAC in H2O) and solution B (methanol). A flow rate of 0.3 mL/min was used with the following gradient elution profile: 0–0.50 min, 60% A 40% B; 0.5–2 min, 40–85% B; 2–3 min, 85% B; 3–3.5 min, 85–40% B; 3.5–4 min, 40% B. The injection volume injected was consistently set at 10 μL. This assay used multiple reaction monitoring in electrospray negative ionization, and the ions measured for quantitation were as follows: 5-FU, 129.1-&gt;42.1; 5-chlorouracil, 145.1-&gt;42.1; and FBAL, 106.0-&gt;85.9.<b> </b>The retention time for 5-FU and FBAL in the column and gradient was 0.82 and 0.76 min, respectively. The quantitative analysis of 5-FU was performed using an internal standard method with a seven-point calibration curve. Calibration curves for the analytes were obtained using seven calibration standards (100, 200, 500, 1250, 2500, 5000, and 10000 ng/mL for 5-FU and FBAL). Calibration standards were analyzed in duplicate, and curves were constructed using 1/x-weighted linear regression (R² ≥ 0.995). Limit of detection (LOD): 5-FU: 0.1 ng/mL, FBAL: 0.4 ng/mL, both defined at a signal-to-noise ratio (S/N) ≥ 3. Quantitative analysis of 5-FU and its bacterial-derived metabolites (<i>preTA</i><i> </i>inhibitors testing): For inhibitor testing, C. freundii strains (4 × 10⁸ CFU/mL) were incubated with 40 μM 5-FU in 600 μL of the NB medium. Each incubation was performed in the presence of one of the previously reported DPD inhibitors—Gim, Eniluracil, Thymidine, Uridine, and Uracil (each at 40 μM). The incubation time points were set at 0 h, 6 h, and 24 h (n = 3) and a blank control group was included. At the end of each incubation, 600 μL of ice-cold acetonitrile was added to stop the reaction. After vortexing, 200 μL of each sample was taken, and an equal volume of acetonitrile along with 10 μL of the internal standard 5-Chlorouracil was added. After vortexing, samples were centrifuged at 12,500 rpm for 10 min at 4°C. The supernatant (100 μL) was collected, diluted with four volumes of pure water, vortexed, and centrifuged again. The resulting supernatant was collected for subsequent analysis. The samples were analyzed using liquid chromatography with tandem quadrupole mass spectrometry (LC-MS/MS QQQ) using the AB SCIEX Triple Quad™4500MD. Chromatographic separation was performed using a Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). The mobile phase consisted of solution A (26.5 mM HCOOH and 1.0 mM NH4OAC in H2O) and solution B (methanol). A flow rate of 0.3 mL/min was used with the following gradient elution profile: 0–0.50 min, 60% A 40% B; 0.5–2 min, 40–85% B; 2–3 min, 85% B; 3–3.5 min, 85–40% B; 3.5–4 min, 40% B. The injection volume injected was consistently set at 10 μL. This assay used multiple reaction monitoring in electrospray negative ionization, and the ions measured for quantitation were as follows: 5-FU, 129.1-&gt;42.1; 5-chlorouracil, 145.1-&gt;42.1; and FBAL, 106.0-&gt;85.9.<b> </b>The retention time for 5-FU and FBAL in the column and gradient was 0.82 and 0.76 min, respectively. The quantitative analysis of 5-FU was performed using an internal standard method with a seven-point calibration curve. Calibration curves for the analytes were obtained using seven calibration standards (100, 200, 500, 1250, 2500, 5000, and 10000 ng/mL for 5-FU and FBAL). Calibration standards were analyzed in duplicate, and curves were constructed using 1/x-weighted linear regression (R² ≥ 0.995). Limit of detection (LOD): 5-FU: 0.1 ng/mL, FBAL: 0.4 ng/mL, both defined at a signal-to-noise ratio (S/N) ≥ 3.Quantitative analysis of 5-FU and its bacterial-derived metabolites by targeted metabolomics (during co-incubation with bacterial strains and Gimeracil): To evaluate the metabolism of 5-FU by multiple bacterial strains and the inhibitory effect of Gim, 40 μM 5-FU and 40 μM Gim were incubated with 4 × 10⁸ CFU/mL of each bacterial strain (<i>C. freundii</i>, <i>P. putida</i>, <i>P. aeruginosa</i>, <i>P. fluorescens</i>, <i>L. reuteri</i>, <i>E. coli</i>, <i>K. pneumoniae</i>, and <i>E. asburiae</i>) in 600 μL of the corresponding medium. The incubation time points were set at 0 h, 6 h, and 24 h (n = 3) and a blank control group was included. At the end of each incubation, 600 μL of ice-cold acetonitrile was added to stop the reaction. After vortexing, 200 μL of each sample was taken, and an equal volume of acetonitrile along with 10 μL of the internal standard 5-Chlorouracil was added. After vortexing, samples were centrifuged at 12,500 rpm for 10 min at 4°C. The supernatant (100 μL) was collected, diluted with four volumes of pure water, vortexed, and centrifuged again. The resulting supernatant was collected for subsequent analysis. The samples were analyzed using liquid chromatography with tandem quadrupole mass spectrometry (LC-MS/MS QQQ) using the AB SCIEX Triple Quad™4500MD. Chromatographic separation was performed using a Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). The mobile phase consisted of solution A (26.5 mM HCOOH and 1.0 mM NH4OAC in H2O) and solution B (methanol). A flow rate of 0.3 mL/min was used with the following gradient elution profile: 0–0.50 min, 60% A 40% B; 0.5–2 min, 40–85% B; 2–3 min, 85% B; 3–3.5 min, 85–40% B; 3.5–4 min, 40% B. The injection volume injected was consistently set at 10 μL. This assay used multiple reaction monitoring in electrospray negative ionization, and the ions measured for quantitation were as follows: 5-FU, 129.1-&gt;42.1; 5-chlorouracil, 145.1-&gt;42.1; and FBAL, 106.0-&gt;85.9.<b> </b>The retention time for 5-FU and FBAL in the column and gradient was 0.82 and 0.76 min, respectively. The quantitative analysis of 5-FU was performed using an internal standard method with a seven-point calibration curve. Calibration curves for the analytes were obtained using seven calibration standards (100, 200, 500, 1250, 2500, 5000, and 10000 ng/mL for 5-FU and FBAL). Calibration standards were analyzed in duplicate, and curves were constructed using 1/x-weighted linear regression (R² ≥ 0.995). Limit of detection (LOD): 5-FU: 0.1 ng/mL, FBAL: 0.4 ng/mL, both defined at a signal-to-noise ratio (S/N) ≥ 3.Quantitative analysis of 5-FU and its bacterial-derived metabolites by untargeted metabolomics: We incubated 5-FU (40 μM) with <i>C. freundii</i> strain (4×10<sup>8</sup> CFU/mL) in 600 μL of NB medium for 24 h (n=3). A blank control group was included. Post-incubation, reactions were stopped with 600 µL ice-cold acetonitrile. After vortexing, 200 µL aliquots were mixed with an equal volume of acetonitrile and precipitated at -20°C for 1 h. Samples were then revortexed, centrifuged (12,500 rpm, 15 min, 4°C), and 100 µL of supernatant was collected for quantitative analysis using an ultraperformance liquid chromatography (UPLC)-Orbitrap-Exploris-120-MS/MS apparatus with a Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). The mobile phase consisted of NH<sub>4</sub>HCO<sub>3</sub> (6.5 mM) (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The gradient elution program was established: 0–1 min, 2% B; 1–3 min, 2–60% B; 3–5 min, 60% B; 5–7 min, 60–100% B; 7–8 min, 100% B; 8–8.1 min, 100–2% B; and 10 min, 2% B. Samples (10 μL of injection volume) were separated and analyzed with the column temperature at 40°C. Data acquisition was performed using Xcalibur software. Mass spectrometry analysis employed full-scan (60,000 resolution, 60–900 <i>m/z</i><i> </i>range) and data-dependent MS<sup>2</sup> (dd-MS<sup>2</sup>; 15,000 resolution) modes. Ion source parameters included: 2500 V spray voltage (negative mode), sheath/aux gases at 50/10 arb, and 300°C transfer tube temperature. Full-MS used 100 ms maximum injection time with standard AGC target; dd-MS<sup>2</sup> used auto injection time and stepped HCD collision energy (20, 40, 60%). Retention times for 5-FU and FBAL were 0.666 and 0.687 min, respectively. Data processing utilized MS-DIAL (ppm calculation), Sirius (validation), and MZmine (MS<sup>2</sup> export).the supernatant metabolomics data of <i>C. freundii</i> strain: After thawing glycerol-preserved C. freundii strain at -80°C, the bacteria were inoculated separately in DMEM containing 10% FBS, creating group 1 and group 2 (n=3). In both groups, DMEM with 10% FBS was used as the blank control. Both groups were cultured, one to the logarithmic growth phase and the other to the stationary phase, then centrifuged at 4200 rpm and 4°C for 15 min. The supernatant was collected and transferred to prechilled cryogenic vials. A 1 mL sample was loaded onto an SPE column, and 3 mL of methanol eluate was collected. The evaluation was dried using nitrogen gas and reconstituted with 300 μL of pre-chilled methanol-water (4:1, v/v) containing an internal standard mix (4 μg/mL). The mixture was vortexed for 1 min, sonicated on an ice-water bath for 10 min, allowed to stand at -40°C for 2 h, and centrifuged for 10 min (13000 rpm, 4°C). A 150 μL aliquot of the supernatant was filtered through a 0.22 μm organic phase syringe filter into an LC vial and stored at -80°C until LC-MS analysis. QC samples were prepared by pooling equal volumes of extract from each sample. Note: All extraction reagents were pre-chilled at -20°C before use. LC-MS was performed using a Waters ACQUITY UPLC I-Class Plus/Thermo QE HF system. Chromatographic conditions were as follows: column, ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); column temperature, 45°C; mobile phase: A, water with 0.1% formic acid, B, acetonitrile; flow rate, 0.35 mL/min; injection volume, 2 μL. The gradient elution was programmed as follows: 0–2 min, 95% A and 5% B; 2.1–4 min, 70% A and 30% B; 4.1–8 min, 50% A and 50% B; 8.1–10 min, 20% A and 80% B; 10.1–15 min, 100% B; 15.1–16 min, return to 95% A and 5% B. Spectral data was collected on a Waters Thermo QE HF spectrometer, in ESI positive and negative ion modes, with spray voltage of 3800 V and -3000 V, respectively. The capillary temperature was maintained at 320°C, and the auxiliary gas heater temperature was set to 350°C. The sheath gas flow rate was 35 Arb, and the auxiliary gas flow rate was 8 Arb. The S-lens RF level was configured at 50. The mass range was set to 70-1050 m/z, with a full MS resolution of 60,000 and an MS/MS resolution of 15,000. Collision energy was applied in stepped NCE mode, with values of 10, 20, and 40. The LC-MS raw data were processed using Progenesis QI v3.0 (Nonlinear Dynamics, Newcastle, UK) for feature alignment, signal integration, and normalization, generating a 3D data matrix of retention time-m/z pairs, peak intensities, and sample information. Metabolite identification and annotation were performed using Progenesis QI (Waters Corporation, Milford, USA) based on MS/MS fragmentation data from public databases, including HMDB and LipidMaps. Data quality control was ensured using internal standards, and preprocessing was evaluated through multivariate and univariate statistical approaches. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was applied to identify altered metabolites between groups, with model validation conducted via 7-fold cross-validation. Differential metabolites were selected based on VIP &gt; 1 in OPLS-DA and p &lt; 0.05.<br>