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

Abstract

Cytotoxic chemotherapy remains the standard-of-care treatment for patients with pancreatic ductal adenocarcinoma (PDAC). However, chemotherapy only has modest effects at improving patient survival due to primary or rapidly acquired chemoresistance. The biological underpinnings of PDAC therapy resistance are incompletely defined, but the tumor microenvironment is known to be a major contributor to chemoresistance. We have found chemoresistance is imprinted on PDAC cells by the tumor microenvironment and persists for a period of days after PDAC cells are removed from tumors. However, PDAC chemoresistance is lost upon long term culture in standard laboratory conditions. Interestingly, culture of PDAC cells in Tumor Interstitial Fluid Medium (TIFM), a culture medium we developed to recapitulate the nutrient availability of the tumor microenvironment, maintains PDAC cells in a chemo- and targeted therapy resistant state even after long term culture ex vivo. These findings suggest that microenvironmental metabolic stress keeps PDAC cells in a physiologically relevant, therapy resistant cell state that standard culture models fail to maintain. Using TIFM culture, we sought to understand how PDAC cells in this state resist therapeutic challenge. We found that chemo- and targeted therapies largely retain on-target activity within TIFM medium but fail to activate cell death, enabling a "chemotolerant" cell state, which is also observed in PDAC tumors. This chemotolerant state is driven by suppression of apoptotic priming and can be overcome by targeting the anti-apoptotic regulator BCL-XL. Taken together, these findings suggest that reprogramming of cell death mechanisms by the PDAC nutrient microenvironment is a key contributor to therapy resistance in this disease.

Figure 1:. The tumor microenvironment imprints chemotherapy resistance in PDAC cells.

(A) Diagram of workflow for the comparison of chemotherapeutic killing of mPDAC cell lines derived from genetically engineered mouse models of PDAC into standard culture conditions and orthotopic murine PDAC tumors derived from the same cell lines. (B) Viability of mPDAC3-RPMI cells measured by IncuCyte cell viability assay after treatment with indicated concentrations of gemcitabine for 72 hours (n = 3). (C) mPDAC3-RPMI orthotopic tumor weights from animals treated with vehicle (n = 6) or gemcitabine (n = 8). (D) Experimental schematic for the comparison of chemotherapeutic sensitivity of cultured mPDAC3-RPMI cells that were kept in culture or were grown as orthotopic allografts in mice and then sorted from tumors and returned to culture for indicated periods of time (tumor-adapted). (E-F) Viability of mPDAC3-RPMI cells measured by propidium iodide exclusion. mPDAC3-RPMI cells were kept in culture or tumor-adapted and then returned to culture as shown in (D) and treated with the indicated concentrations of (E) gemcitabine (n = 3) or (F) SN-38 for 72 hours (n = 3). Statistical significance in C was assessed using the Mann-Whitney U test. For E and F, the area under the curve (AUC) was calculated and unpaired t-test was performed to determine significance in the difference of the AUC for the indicated cultures. *** p≤0.001 and **** p≤0.0001.

Figure 2:. Tumor nutrient conditions are sufficient to imprint therapy resistance in PDAC.

(A) Diagram of the generation of paired PDAC cell lines from murine PDAC tumors in TIFM or in RPMI. (B) Representative images of mPDAC1-TIFM and mPDAC1-RPMI cells expressing Nuclight incubated with Cytotox Green Dye and treated with 19 nM gemcitabine or vehicle for 72 hours. (C-F) Viability of indicated mPDAC-TIFM and mPDAC-RPMI cultures measured by IncuCyte cell viability assay treated with the indicated concentration of (C) gemcitabine (n = 3), (D) 5-fluorouracil (n = 3), (E) SN-38 (n = 3) or (F) RMC-7977 (n = 3) for 72 hours. (G) Diagram of experiment moving mPDAC1-TIFM cultures to RPMI medium (TIFM → RPMI) for various period of time and assessing chemotherapeutic response of these cultures. (H) Viability of mPDAC1-TIFM, mPDAC1-RPMI and mPDAC1-TIFM→RPMI cells measured by IncuCyte cell viability assays after the indicated time point in culture and treated with the indicated concentration of gemcitabine (n = 3) for 72 hours. (I) Diagram of the protocol for transitioning mPDAC-RPMI and human PDAC cell lines and organoids to grow in TIFM. We term these mPDAC-RPMI→TIFM cultures. (J) Viability of mPDAC1-RPMI and mPDAC1-RPMI→TIFM cultures measured by IncuCyte cell viability assay when treated with the indicated concentration of gemcitabine (n = 3) for 72 hours. (K) Viability of patient derived organoids measured by CellTiter-Glo assay in standard organoid culture media or TIFM following treatment with the indicated concentration of gemcitabine (n = 3) for 72 hours. Viability of indicated human PDAC cell lines measured by IncuCyte cell viability assay cultured in RPMI or TIFM when treated with the indicated concentration of (L) gemcitabine (n = 3), (M) 5-fluorouracil (n = 3) or (N) SN-38 (n = 3) for 72 hours. For C-F and J-N, the area under the curve (AUC) was calculated and unpaired t-test was performed to determine significance in the difference of the AUC for the indicated cultures. *** p≤0.001 and **** p≤0.0001.

Figure 3:. TME nutrient stress induces chemotolerance by suppressing apoptotic priming

(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.

Figure 4:. The TME imprints a chemotolerant phenotype on PDAC cells.

(A) Diagram of the generation of orthotopic allograft murine PDAC tumors from mPDAC3-RPMI cells and assessing chemotherapy tolerance by immunofluorescence analysis of cell death and DNA damage markers after gemcitabine treatment. (B) Representative immunofluorescence images of γH2AX and cleaved caspase-3 in vehicle and gemcitabine treated mPDAC3-RPMI orthotopic allograft tumors. Scalebar indicates 250 μm. (C,D) Quantification of PDAC cells staining positive for (C) γH2AX and (D) Cleaved Caspase-3 in vehicle (n = 6) and gemcitabine (n = 8) treated mPDAC3-RPMI orthotopic allograft tumors. (E) Diagram of workflow for the comparison of apoptotic priming of mPDAC cell lines adapted to growth in orthotopic allograft tumor conditions and returned to standard culture versus maintained in standard culture conditions. (F,G) Relative mitochondrial depolarization following treatment with the indicated concentration of BIM peptide for mPDAC3-RPMI cells, which were tumor-adapted and returned to standard culture conditions for (F) 4 days or 4 weeks (G) or maintained continuously in standard culture (n = 3). For F,G, the area under the curve (AUC) was calculated and unpaired t-tests were performed to determine significance in the difference of the AUC for the indicated cultures. Statistical significance in C,D was assessed using the Mann-Whitney U test. * p≤0.05 and **** p≤0.0001.

Figure 2 - Figure supplement 1:. Tumor nutrient conditions renders PDAC cells insensitive to a variety of chemotherapeutic and target therapies.

(A-C) Viability of mPDAC1-TIFM and mPDAC1-RPMI cultures measured by IncuCyte cell viability assay treated with the indicated concentration of (A) paclitaxel (n = 3), (B) oxaliplatin (n = 3) or (C) trametinib (n = 3) for 72 hours. (D) Measurement relative organoid size of TIFM- and standard media-cultured patient-derived organoids over time following treatment with vehicle (TIFM n = 9, DMEM/F12 n = 6) and gemcitabine (100 nM) (TIFM n = 9, DMEM/F12 n = 6) for 72hrs. For A-D, the area under the curve (AUC) was calculated and unpaired t-test was performed to determine significance in the difference of the AUC for the indicated cultures. *** p≤0.001 and **** p≤0.0001.

Figure 2 - Figure supplement 2:. Differences in growth rate do not account for differential sensitivity of mPDAC-RPMI and mPDAC-TIFM cultures to chemotherapy treatment.

(A) Percentages of mPDAC1-TIFM and mPDAC1-RPMI cultures in G1, S and G2/M phases of the cell cycle (n = 3). (B-E) GR_toxic estimates for the indicated mPDAC-TIFM and mPDAC-RPMI cell line following treatment with the indicated concentration of (B) gemcitabine (n = 3), (C) 5-fluorouracil (n = 3), (D) SN-38 (n = 3), and (E) RMC-7977 (n = 3) for 72 hours. (F) GR_max estimates for the indicated mPDAC cell lines, culture condition and drug treatment (n = 3 cell lines). (G) Percentages of mPDAC1-TIFM cells treated with 100 nM gemcitabine for the indicated time periods in G1, S and G2/M phases of the cell cycle (n = 3). (H) Growth rates of vehicle treated mPDAC1-TIFM and mPDAC1-RPMI cultures and mPDAC1-RPMI cultures treated with 2.5 μM palbociclib (n = 3). (I) Viability of mPDAC1-TIFM and mPDAC1-RPMI cultures and mPDAC1-RPMI cells treated with 2.5 μM palbociclib cultures measured by IncuCyte cell viability assay following treatment with the indicated concentration of gemcitabine (n = 3) for 72 hours. For A, unpaired t-tests were performed to assess significance in differences in cell cycle phase between culture conditions. For F, paired t-tests were performed to assess significance in differences in GR_max estimates. For G, unpaired t-tests were performed to assess significance in differences in cell cycle phase between the indicated timepoint of gemcitabine treatment and the initial population. For H, unpaired t-tests were performed to assess significance in differences in proliferation rate. For I, the area under the curve (AUC) was calculated and unpaired t-tests were performed to determine significance in the difference of the AUC for the indicated cultures. * p≤0.05, *** p≤0.001 and **** p≤0.0001.

Figure 2 - Figure supplement 3:. TIFM-cultured PDAC cells are quiescent during chemotherapy treatment but can resume proliferation upon drug removal.

(A) Growth rates of mPDAC1-RPMI and mPDAC1-TIFM cultures measured every 6 hours over 2 days following exposure to 100 nM gemcitabine for 24 hours (n = 3). For A, the area under the curve (AUC) was calculated and unpaired t-test was performed to determine significance in the difference of the AUC for the indicated cultures. **** p≤0.0001.

Figure 2 - Figure supplement 4:. Transitioning mPDAC-RPMI to grow in TIFM does not select for rare clonal populations.

(A) Diagram of the lentiviral barcoding of mPDAC3-RPMI cells prior to transitioning these cells into TIFM as shown in Fig. 2I or maintenance of the culture in RPMI. (B) Barcode recovery of mPDAC3-RPMI cells either maintained in RPMI or transitioned to grow in TIFM. (C) Gini index for barcode distribution of mPDAC3-RPMI cells either maintained in RPMI or transitioned to grow in TIFM. (D) Correlation of log2 fold change in barcode abundance between mPDAC-RPMI cells transitioned into TIFM or maintained in RPMI. For D, Spearman’s rank correlation test was used to determine significant correlation in barcode abundance change between culture conditions.

Figure 3 – Figure supplement 1:. TIFM cultures have intact on-target activity and cellular responses to SN-38 treatment but do not undergo apoptosis.

(A) Immunoblots for γH2AX and H2AX in the indicated mPDAC cell line and culture condition following treatment with 10 μM SN-38 for 18 hours. (B) Alkaline comet assay of mPDAC1-RPMI and mPDAC1-TIFM cells treated with 10 μM SN-38 (RPMI n = 45, TIFM n = 70) or vehicle (RPMI n = 55, TIFM n = 70) for 18 hours. (C) Immunoblots for γH2AX, BIM, and Cleaved Caspase-3 in mPDAC1-RPMI and mPDAC1-TIFM cells treated with 10 μM SN-38 Gemcitabine over time at the indicated timepoints. For B, unpaired t-tests were performed to determine significance of differences between sample conditions. **** p≤0.0001.

Figure 3 – Figure supplement 2:. Exogenous thymidine in TIFM suppresses on-target activity of 5-fluorouracil.

(A) Immunoblots for γH2AX and H2AX in the indicated mPDAC cell line and culture condition following treatment with 5 μM 5-FU for 18 hours. (B) Alkaline comet assay of mPDAC1-RPMI and mPDAC1-TIFM cells treated with 5 μM 5-FU (RPMI n = 38, TIFM n = 34, RPMI + thymidine n = 46) or vehicle (RPMI n = 38, TIFM n = 50) for 18 hours. (C) Diagram of metabolic routes for thymidylate nucleotide synthesis. Cells possess a de novo synthesis route dependent on the activity of TYMS, the target of 5-fluorouracil. With loss of TYMS function, thymidylate nucleotide pools may still be maintained through a salvage pathway, dependent on the presence of exogenous thymidine in the environment and TK1 activity. (D) Cell viability of mPDAC1-RPMI cells measured by IncuCyte cell viability assay following treatment with indicated concentrations of 5-FU in the presence or absence of the indicated concentrations of thymidine after 72 hours (n = 3). For B, unpaired t-test was performed to determine significance of differences between sample conditions. For D, the area under the curve (AUC) was calculated and unpaired t-test was performed to determine significance of differences of AUC for the indicated cultures. *** p≤0.001, and **** p≤0.0001.

Figure 3 – Figure supplement 3:. RMC-7977 retains on-target activity in TIFM-cultured PDAC cells.

(A) Immunoblots for phospho-ERK and ERK in mPDAC1-RPMI and mPDAC1-TIFM cells treated with 2.5 μM RMC-7977 or vehicle for 24 hours.

Figure 3 – Figure supplement 4:. Upregulation of pro-apoptotic BCL2 family members in mPDAC1-TIFM and mPDAC1-RPMI cells in response to gemcitabine.

(A) Heatmap of normalized count values for known pro-apoptotic BH3-only genes following RNA-seq analysis of mPDAC1-RPMI and mPDAC1-TIFM cells treated with 125 nM gemcitabine or vehicle for 24 hours.

Figure 3 – Figure supplement 5:. Cells under chronic nutrient stress acquire impaired apoptotic potential.

(A) Diagram of experimental workflow for generating RPMI → TIFM cultures. Parental mPDAC1-RPMI adapted cultures are placed in TIFM containing 1% RPMI-1640 (V/V) for 2 weeks. Following this period, cells are transferred into 100% TIFM medium for 4 weeks prior to experimentation. (B) Immunoblots for γH2AX, BIM, and Cleaved Caspase-3 in mPDAC1-RPMI or mPDAC1-RPMI → TIFM cells treated with 150 nM gemcitabine over time at the indicated timepoints. (C) Relative mitochondrial depolarization of mPDAC1-RPMI and mPDAC1-RPMI → TIFM cells in response to treatment with recombinant BIM peptide at the indicated concentrations (n = 3). (D) Diagram of experimental workflow for generating chronically low arginine stressed PDAC cultures. Parental mPDAC1-RPMI adapted cultures are placed in RPMI containing 10 μM Arginine for 6 weeks prior to experimentation. (E) Immunoblots for γH2AX, BIM, and Cleaved Caspase-3 in mPDAC1-RPMI or mPDAC1 low arginine cultured cells treated with 150 nM gemcitabine over time at the indicated timepoints. (F) Relative mitochondrial depolarization of mPDAC1-RPMI and mPDAC1 low arginine cultured cells in response to treatment with recombinant BIM peptide at the indicated concentrations (n = 3). For C,F, the area under the curve (AUC) was calculated and unpaired t-tests were performed to determine significance of differences in AUC for the indicated sample conditions. * p≤0.05 and *** p≤0.001.

Figure 3 – Figure supplement 6:. Mcl-1 overexpression in mPDAC TIFM cultures rescues synergistic cell death between BCL-XL targeting mimetic compounds and chemotherapy.

(A) Cell viability of mPDAC1-TIFM cells measured by IncuCyte cell viability assay following transduction with Mcl-1 overexpression or empty vector constructs and treatment with the indicated drug conditions (Gemcitabine, 150 nM), (A1331852, 1 μM), (Navitoclax, 1 μM) for 72hrs. Unpaired t-tests were performed to determine significance in cell viability measurements. *** p≤0.001, and **** p≤0.0001.