Journal: bioRxiv

Article Title: Tumor nutrient stress gives rise to a drug tolerant cell state in pancreatic cancer

doi: 10.1101/2025.09.04.673818

Figure Lengend Snippet: (A) (Left) Immunoblot confirmation of Bax and Bak1 double knockout ( Bax/Bak1 DKO) in mPDAC1-RPMI cells. (Right) Cell viability measured by IncuCyte cell viability assay of mPDAC1-RPMI NTC and Bax/Bak1 DKO cultures treated with the indicated concentration of gemcitabine (n = 3) for 72 hours. (B) Diagram of cell death by chemotherapy. Initial chemotherapeutic response requires exposure mediated by cellular transporters and damage from on-target activity such as DNA double stranded breaks, indicated by the presence of γH2AX. Following cellular damage, a pro-death cellular response is generated, which may feature the upregulated expression of pro-apoptotic BH3-only proteins. A sufficiently strong death signal, in the case of apoptotic cell death, results in mitochondrial outer membrane permeabilization (MOMP). This event results in a commitment to cell death leading to cellular disassembly mediated by the activation of executioner caspases, which finally leads to loss of plasma membrane integrity. (C) (Right) Alkaline comet assay of mPDAC1-RPMI and mPDAC1-TIFM cultures treated for 18hrs with 150nM Gemcitabine (RPMI n = 58, TIFM n = 64) or DMSO vehicle (RPMI n = 61, TIFM n = 52). (Left) Representative images of comets following treatment with gemcitabine or vehicle. (D) Heatmap of C2 MSigDB gene sets with differential enrichment from GSEA analysis of mPDAC1 RPMI cultures treated for 24 hours with 125nM Gemcitabine versus DMSO vehicle control. All gene sets displayed a statistically significant enrichment of less than 0.05 after p-value adjustment by Benjamini-Hochberg correction. Right column displaying enrichment scores for the corresponding gene set following GSEA analysis of mPDAC1 TIFM cultures treated with gemcitabine under identical conditions. Values in heatmap displayed reflect normalized enrichment score (NES). (E) ULM enrichment scores predicting enhanced transcription factor activity for the top 5 differentially enriched TFs in mPDAC1-RPMI cells in response to gemcitabine treatment, with the enrichment scores for mPDAC1-TIFM cells displayed in parallel. (F) Immunoblots for γH2AX, BIM, and Cleaved Caspase-3 in mPDAC1-RPMI and mPDAC1-TIFM cells treated with 150nM Gemcitabine over time at the indicated timepoints. (G) Red fluorescence intensity of Omi-mCherry reporter expressing mPDAC1-RPMI and mPDAC1-TIFM cells treated with either gemcitabine or vehicle. (H) Percent of Omi-mCherry low cells (post-MOMP) in mPDAC cells cultured under the indicated medium and drug treatment conditions (n = 4). (I) Immunoblots for BAX in mitochondrial and cytosolic isolates from mPDAC1-RPMI and mPDAC1-TIFM cells treated with 250 nM gemcitabine over time at the indicated timepoints. (J) Relative mitochondrial depolarization of mPDAC1-RPMI and mPDAC1-TIFM cells in response to treatment with recombinant BIM peptide at the indicated concentrations (n = 3). (K) Immunoblots for BCL2 family members in the indicated mPDAC cell line and culture condition. (L) Immunoblots for BCL-XL, MCL-1, BIM and BAX following immunoprecipitation of BCL-XL or MCL-1 in mPDAC1-RPMI cell lysates. “Iso” sample reflects pulldown with isotype control rabbit IgG antibody. (M) Immunoblots for BCL-XL and BAX following immunoprecipitation for BCL-XL in mPDAC1-TIFM or mPDAC1-RPMI cell lysates following treatment with gemcitabine (125 nM) and A1331852 (100nM) as indicated for 24 hours. (N) Cell viability of mPDAC1-TIFM cells measured by IncuCyte cell viability assay following treatment with the indicated compounds for 72 hours (Gemcitabine, 150 nM), (A1331852 1 µM), (S63845 1 µM), (Venetoclax 1 µM), (Navitoclax 1 µM). Individual datapoints reflect the mean viability of 3 replicates for a single cell line (mPDAC1-4), as indicated in the figure legend (n = 4 cell lines). (O) (Left) Immunoblot confirmation of Bcl-xL knockout in mPDAC1-TIFM cells. (Right) Cell viability of Bcl-xL knockout or NTC mPDAC1-TIFM cells measured by IncuCyte cell viability assay, following treatment with the indicated concentrations of gemcitabine for 72 hours (n = 3). (P) Immunoblots for γH2AX, BIM, and Cleaved Caspase-3 in Bcl-xL knockout or NTC mPDAC1-TIFM cells following treatment with 150 nM Gemcitabine at the indicated timepoints. For A,J,O, the area under the curve (AUC) was calculated and unpaired t-test was performed to determine significance of differences in AUC for the indicated cultures. For C, H, N, unpaired t-tests were performed to determine significance of differences between sample conditions. For E, all gene sets displayed for the mPDAC1-RPMI sample had significant enrichment with a p-value of less than p=0.05 after multiple tests correction. For J, all transcription factors displayed had significant enrichment with a p-value of less than p=0.0001 after multiple tests correction. * p≤0.05, ** p≤0.01, and **** p≤0.0001.

Article Snippet: In vitro cell viability assays were performed using the following compounds: Gemcitabine hydrochloride (Sigma, G6523), SN-38 (Cayman, 15632), 5-Fluorouracil (Cayman, 14416), Palbociclib (Cayman, 16273), Paclitaxel (Cayman, 10461), Oxaliplatin (Cayman, 13106), Trametinib (Cayman, 16292), RMC-7977 (MedChem Express, HY-156498), S63845 (Selleck Chem, S8383), Venetoclax (Selleck Chem, S8048), Navitoclax (Selleck Chem, S1001), A-1331852 (Selleck Chem, S7801).

Techniques: Western Blot, Double Knockout, Viability Assay, Concentration Assay, Activity Assay, Generated, Expressing, Membrane, Activation Assay, Clinical Proteomics, Alkaline Single Cell Gel Electrophoresis, Control, Fluorescence, Cell Culture, Recombinant, Immunoprecipitation, Knock-Out