Immunomodulatory antitumor effect of interferon­beta combined with gemcitabine in pancreatic cancer.

Resistance to gemcitabine is common and critically limits its therapeutic efficacy in patients with pancreatic cancer. Interferon­beta (IFN­ß) induces numerous antitumor effects and synergizes with gemcitabine treatment. The immunomodulatory effects of this treatment regimen have not yet been described. In the present study, the antitumor effect of IFN­ß combined with gemcitabine was investigated in immune competent mice. Mouse KPC3 cells were used in all experiments. Treatment effects were determined with cell proliferation assay. Reverse transcription­quantitative PCR was used to measure gene expression. For in vivo experiments, cells were subcutaneously injected in immune competent mice. For immune profiling, NanoString analysis was performed on tumor samples of treated and untreated mice. Baseline expression of Ifnar­1 and Ifnar­2c in KPC3 cells was 1.42±0.16 and 1.50±0.17, respectively. IC50 value of IFN­ß on cell growth was high (>1,000 IU/ml). IFN­ß pre­treatment increased the in vitro response to gemcitabine (1.3­fold decrease in EC50; P<0.001). In vivo, tumor size was not statistically significant smaller in mice treated with IFN­ß plus gemcitabine (707±92 mm3 vs. 1,239±338 mm3 in vehicle­treated mice; P=0.16). IFN­ß alone upregulated expression of numerous immune­related genes. This effect was less pronounced when combined with gemcitabine. For the first time, to the best of our knowledge, the immunomodulatory effects of IFN­ß, alone and combined with gemcitabine, in pancreatic cancer were reported. Prognostic markers for predicting effective responses to IFN­ß therapy are urgently needed.

The Class I HDAC Inhibitor Valproic Acid Strongly Potentiates Gemcitabine Efficacy in Pancreatic Cancer by Immune System Activation.

Background: Gemcitabine efficacy in pancreatic cancer is often impaired due to limited intracellular uptake and metabolic activation. Epi-drugs target gene expression patterns and represent a promising approach to reverse chemoresistance. In this study, we investigate the chemosensitizing effect of different epi-drugs when combined with gemcitabine in pancreatic cancer. Methods: Mouse KPC3 cells were used for all experiments. Five different epi-drugs were selected for combination therapy: 5-aza-2'-deoxycytidine, hydralazine, mocetinostat, panobinostat, and valproic acid (VPA). Treatment effects were determined by cell proliferation and colony forming assays. Expression of genes were assessed by real-time quantitative PCR. The most promising epi-drug for combination therapy was studied in immune competent mice. Intratumor changes were defined using NanoString PanCancer panel IO360. Results: All epi-drugs, except hydralazine, potentiated the gemcitabine response in KPC3 cells (range decrease IC50 value 1.7−2-fold; p < 0.001). On colony formation, the cytotoxic effect of 0.5 ng/mL gemcitabine was 1.4 to 6.3 times stronger (p < 0.01). Two out of three drug-transporter genes were strongly upregulated following epi-drug treatment (a range fold increase of 17−124 and 9−60 for Slc28a1 and Slc28a3, respectively; all p < 0.001). VPA combined with gemcitabine significantly reduced tumor size with 74% compared to vehicle-treated mice and upregulated expression of immune-related pathways (range pathway score 0.86−1.3). Conclusions: These results provide a strong rationale for combining gemcitabine with VPA treatment. For the first time, we present intratumor changes and show activation of the immune system. Clinical trials are warranted to assess efficacy and safety of this novel combination in pancreatic cancer patients.

Interferon-beta enhances sensitivity to gemcitabine in pancreatic cancer.

BACKGROUND: Adjuvant gemcitabine for pancreatic cancer has limited efficacy in the clinical setting. Impaired drug metabolism is associated with treatment resistance. We aimed to evaluate the chemosensitising effect of interferon-beta (IFN-ß). METHODS: BxPC-3, CFPAC-1, and Panc-1 cells were pre-treated with IFN-ß followed by gemcitabine monotherapy. The effect on cell growth, colony formation, and cell cycle was determined. RT-qPCR was used to measure gene expression. BxPC-3 cells were used in a heterotopic subcutaneous mouse model. RESULTS: IFN-ß increased sensitivity to gemcitabine (4-, 7.7-, and 1.7-fold EC50 decrease in BxPC-3, CFPAC-1, and Panc-1, respectively; all P < 0.001). Findings were confirmed when assessing colony formation. The percentage of cells in the S-phase was significantly increased after IFN-ß treatment only in BxPC-3 and CFPAC-1 by 12 and 7%, respectively (p < 0.001 and p < 0.05, respectively). Thereby, IFN-ß upregulated expression of the drug transporters SLC28A1 in BxPC-3 (252%) and SLC28A3 in BxPC-3 (127%) and CFPAC-1 (223%) (all p < 0.001). In vivo, combination therapy reduced tumor volume with 45% (P = 0.01). Both ex vivo and in vivo data demonstrate a significant reduction in the number of proliferating cells, whereas apoptosis was increased. CONCLUSIONS: For the first time, we validated the chemosensitising effects of IFN-ß when combined with gemcitabine in vitro, ex vivo, and in vivo. This was driven by cell cycle modulation and associated with an upregulation of genes involving intracellular uptake of gemcitabine. The use of IFN-ß in combination with gemcitabine seems promising in patients with pancreatic cancer and needs to be further explored.