Chronic stress accelerates pancreatic cancer growth and invasion: a critical role for beta-adrenergic signaling in the pancreatic microenvironment

Abstract

Pancreatic cancer cells intimately interact with a complex microenvironment that influences pancreatic cancer progression. The pancreas is innervated by fibers of the sympathetic nervous system (SNS) and pancreatic cancer cells have receptors for SNS neurotransmitters which suggests that pancreatic cancer may be sensitive to neural signaling. In vitro and non-orthotopic in vivo studies showed that neural signaling modulates tumour cell behavior. However the effect of SNS signaling on tumor progression within the pancreatic microenvironment has not previously been investigated. To address this, we used in vivo optical imaging to non-invasively track growth and dissemination of primary pancreatic cancer using an orthotopic mouse model that replicates the complex interaction between pancreatic tumor cells and their microenvironment. Stress-induced neural activation increased primary tumor growth and tumor cell dissemination to normal adjacent pancreas. These effects were associated with increased expression of invasion genes by tumor cells and pancreatic stromal cells. Pharmacological activation of β-adrenergic signaling induced similar effects to chronic stress, and pharmacological β-blockade reversed the effects of chronic stress on pancreatic cancer progression. These findings indicate that neural β-adrenergic signaling regulates pancreatic cancer progression and suggest β-blockade as a novel strategy to complement existing therapies for pancreatic cancer.

Keywords: Beta-adrenergic; Beta-blocker; Chronic stress; Invasion; Pancreatic cancer; Tumor microenvironment.

Figure 1. Chronic stress increased pancreatic tumor progression

A. Mice were exposed to chronic stress (daily restraint) vs home cage control conditions for 2 hours per day for 28 days commencing 7 days prior to tumor cell injection. Where described, mice were treated with β-blockers for the duration of the experiment. B. Primary tumor size was measured over time by non-invasive bioluminescence imaging. Luciferase activity: ×10¹⁰ p/sec. Inset shows increased resolution over days 0–21, luciferase activity: ×10⁸ p/sec. C. Representative images of tumor-specific bioluminescence in mice exposed to control vs stress conditions. Luminescence scale: p/sec/cm²/sr. D. Primary tumor mass at 42 days after tumor cell injection. Data shown are representative of three replicate experiments.

Figure 2. Stress increased metastatic dissemination from primary pancreatic tumors

A. Representative images of intact (left panel), resected primary tumor and adjacent normal pancreas with bioluminescent tumor signal (middle panel, luminescence scale: p/sec/cm²/sr). Right panel: Hematoxylin and eosin staining of pancreatic tumor (T) grown orthotopically in normal pancreas (P). Asterisk shows region of local invasion. Scale bar: 150 µm. B. Tumor cell dissemination into adjacent normal pancreas was quantified by bioluminescence imaging after resection of the primary tumor. Luciferase activity: ×10⁶ p/sec. C. Metastasis was quantified in control vs stressed mice at 42 days after tumor cell injection. D. Pancreatic tumor metastasis in liver (left panel, arrow: metastasis, scale: 2 mm). Middle panel: Metastasis was confirmed by bioluminescence imaging (luminescence scale: ×10⁵ p/sec/cm²/sr). Right panel: Hematoxylin and eosin staining of liver metastasis (T: tumor, L: liver, scale: 100 µm).

Figure 3. Beta-blockade reversed stress-enhanced pancreatic cancer progression

A. Tumor progression was tracked using non-invasive bioluminescence imaging in mice that were exposed to control (black) vs. stress conditions (red) and treated with propranolol (dotted line) or placebo (solid line). Luciferase activity: ×10⁸ p/sec. B. Representative images of mice taken on day 42 after tumor cell injection. Mice were exposed to control vs. stress conditions and treated with propranolol vs. placebo. Black tape in each panel covered autoluminescent osmotic minipumps used for drug delivery. Luminescence scale: p/sec/cm²/sr. C. Primary tumor mass was determined on day 42 after tumor cell injection. D. The magnitude of tumor cell invasion into pancreas adjacent to the primary tumor was quantified by ex vivo bioluminescence imaging after surgical resection of the primary tumor. Luciferase activity: ×10⁶ p/sec.

Figure 4. Beta-adrenergic signaling is sufficient to accelerate pancreatic cancer progression

A. Tumor progression was tracked using non-invasive bioluminescence imaging in mice treated with isoproterenol (iso) vs. placebo (control). Y-axis: fold change over day 0 luciferase activity. B. Representative images of tumor-specific bioluminescence in mice treated with iso vs control. Luminescence scale: p/sec/cm2/sr. C. Primary tumor mass was determined on day 42 after tumor cell injection in mice treated with iso vs control. D. The magnitude of tumor cell dissemination into pancreas adjacent to the primary tumor was quantified by ex vivo imaging after surgical resection of the primary tumor. Luciferase activity: ×10⁶ p/sec. E. The frequency of metastasis-bearing mice was determined at 42 days after tumor cell injection.

Figure 5. Beta-adrenergic signaling induced pancreatic cancer cell invasion

A. Immunostaining for β1-adrenoceptor (β1AR) and β2-adrenoceptor (β1AR, red) on Panc-1 pancreatic cancer cells (Upper and middle panel, blue: DAPI, inset: isotype control, scale bar: 5 µm) and archived human pancreatic cancer (Lower panel, scale bar: 100 µm). B. Quantitative RT-PCR analyses of ADRB1 and ADRB2 expression on Panc-1 cells, normalized to RPL30 expression. C. cAMP accumulation in Panc-1 cells presented as percent relative to maximal stimulation by 10 µM forskolin. D. Matrigel invasion by Panc-1 cells treated with increasing concentration of isoproterenol (0, 0.01–10 µM), or with 10 µM propranolol ± 10 µM isoproterenol. E. Quantitative RT-PCR analyses of tumor cell (T) or stromal cell (S) MMP2 and MMP9 expression in primary pancreatic tumors.