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. 2020 Sep 23;20(1):913.
doi: 10.1186/s12885-020-07420-0.

Interferon-beta enhances sensitivity to gemcitabine in pancreatic cancer

Amber Blaauboer  1   2 , Stephanie Booy  3   4 , Peter M van Koetsveld  4 , Bas Karels  4 , Fadime Dogan  4 , Suzanne van Zwienen  4 , Casper H J van Eijck  3 , Leo J Hofland  4
Affiliations

Interferon-beta enhances sensitivity to gemcitabine in pancreatic cancer

Amber Blaauboer et al. BMC Cancer. .

Abstract

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-β).

Methods: BxPC-3, CFPAC-1, and Panc-1 cells were pre-treated with IFN-β 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-β 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-β treatment only in BxPC-3 and CFPAC-1 by 12 and 7%, respectively (p < 0.001 and p < 0.05, respectively). Thereby, IFN-β 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-β 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-β in combination with gemcitabine seems promising in patients with pancreatic cancer and needs to be further explored.

Keywords: Chemosensitising effect; Drug transporters; Gemcitabine; Interferon-beta; Pancreatic cancer.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Fig. 1
Fig. 1
In vitro treatment effects of gemcitabine and IFN-β. a Dose response curves of gemcitabine and IFN-β on total DNA amount, as a measure of cell number, in ● BxPC-3, ■ CFPAC-1, and ▲ Panc-1 after 7 days of treatment. b Upper panel represents baseline mRNA expression of IFIT1, OAS1A, and Mx1 in BxPC-3 (left panel), CFPAC-1 (middle panel), and Panc-1 (right panel); Lower panel represent relative mRNA expression in untreated control cells and after 4 (white bar), 12 (light grey bar), 24 (dark grey bar), or 72 h (black bar) pre-treatment with IFN-β. c Experimental design for in vitro experiments. Cell were pre-treated with IFN-β for 4, 12, 24 or 72 h, followed by 72 h gemcitabine monotherapy. d EC50 values of gemcitabine on cell growth in non-IFN-β pre-treated cells vs IFN-β pre-treated cells. EC50 values are presented in nanogram per milliliter (ng/ml, 95% CI). EC50 values depicted in bold represent the strongest decrease. Used concentrations IFN-β: 100 IU/ml for BxPC-3 and CFPAC-1, and 1000 IU/ml for Panc-1. Values represent mean ± SEM of at least two independent experiments in quadruplicate and are shown as the percentage of control. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control
Fig. 2
Fig. 2
Colony-forming assay. a Cytostatic and cytotoxic analysis of colonies in BxPC-3 (left panel), CFPAC-1 (middle panel), and Panc-1 (right panel). Upper panel represents the effect of 72 h IFN-β monotherapy on surviving fraction and colony size. Middle and lower panel represent the effect of 72 h gemcitabine (GEM) in untreated control cells (white bar) versus 72 h IFN-β pre-treated cells (black bar) on surviving fraction and colony size. Used concentrations IFN-β: 100 IU/ml for BxPC-3 and CFPAC-1, and 1000 IU/ml for Panc-1. Used concentrations gemcitabine: 0.5–1 ng/ml for CFPAC-1; and 1–2.5 ng/ml for BxCP-3 and Panc-1. Data are presented as percentage of vehicle treated control. For IFN-β pre-treated cells, effect of IFN-β was set on 100% and used as control. b Photomicrographs of treatment effects on BxPC-3 colonies. Red stained colonies represent the measured colonies. Based on cut-off values for number and size, black stained colonies were excluded. Values represent mean ± SEM of at least two independent experiments and are shown as a percentage of control. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control
Fig. 3
Fig. 3
Cell cycle analysis. Cell cycle distribution in BxPC-3 (left panel), CFPAC-1 (middle panel), and Panc-1 (right panel) after 72 h IFN-β, 72 h gemcitabine, and after 72 h IFN-β pre-treatment followed by 72 h gemcitabine monotherapy. Used concentrations IFN-β: 100 IU/ml for BxPC-3 and CFPAC-1, and 1000 IU/ml for Panc-1. Used concentrations gemcitabine: 1 ng/ml for CFPAC-1 and 2.5 ng/ml for BxPC-3 and Panc-1. Values represent mean ± SEM of at least two independent experiments and are shown as relative percentage of the total cell population. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control
Fig. 4
Fig. 4
mRNA expression of genes involved in transport and metabolism of gemcitabine in BxPC-3 (upper panel), CFPAC-1 (middle panel), and Panc-1 (lower panel). a Schematic overview of the genes encoding for the transporters (SLC29A1, SLC28A1, and SLC28A3), activating enzymes (dCK, CMPK1, and NME1), and inactivating enzymes (CDA, NT5E, and DCTD) of gemcitabine. b Percentage change in mRNA expression between untreated control cells (white bars) and after 72 h IFN-β (coloured bars). Used concentrations IFN-β: 100 IU/ml for BxPC-3 and CFPAC-1, and 1000 IU/ml for Panc-1. Values represent mean ± SEM of at least two independent experiments in quadruplicate and are shown as a percentage of control. **p < 0.01 and ***p < 0.001 versus control
Fig. 5
Fig. 5
Effects of IFN-β and gemcitabine using an ex vivo precision cut tissue slice model. a Experimental design for ex vivo experiments. Slices, derived from xenograft BxPC-3 tumors of untreated mice, were incubated for 72 h without or with IFN-β (100 IU/ml), gemcitabine (1 ng/ml), or the combination of IFN-β plus gemcitabine. b Representative tissue slides of human pancreatic cancer xenograft tissue slices stained for KI-67 (upper panel) or caspase-3 (lower panel) in control and treated slices. c Immunohistochemical analysis of Ki-67 (left) and cleaved caspase-3 (right) expression, representing the proportion of proliferating cells and the proportion of apoptotic cells respectively. Values represent mean ± SEM of at least three different areas within the tumor and are shown as a percentage of control. **p < 0.01 and ***p < 0.001 versus control
Fig. 6
Fig. 6
Treatment effects of IFN-β and gemcitabine in a subcutaneous heterotopic human pancreatic cancer model. a Experimental design for in vivo experiments. BxPC-3 human pancreatic cancer cells (1 × 106 /100 μl PBS) were subcutaneously injected in nude mice. Seven day later, groups of mice received five times a week, on consecutive days, an i.p. injection of IFN-β (1.5 × 105 IU), two times a week (at day 2 and 4) an i.p. injection of gemcitabine (40 mg/kg), or the combination of IFN-β plus gemcitabine. Mice in the control group received five times a week, on consecutive days, an intraperitoneal (i.p.) injection of 100 μl of 0.9% NaCL. b Time course of change in tumor volume (left). After 4 weeks of treatment, mice were sacrificed and tumor volume was measured (right). c Photomicrographs of representative tissue slides of immunohistochemical staining for Ki-67 or cleaved caspase-3, d Immunohistochemical analysis of Ki-67 (left) and cleaved caspase-3 (right) expression, representing the proportion of proliferating cells and the proportion of apoptotic cells respectively. Values represent mean ± SEM of at least three different areas within the tumor and are shown as a percentage of control. **p < 0.01 and ***p < 0.001 versus control
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