Morphine stimulates cancer progression and mast cell activation and impairs survival in transgenic mice with breast cancer

Abstract

Background: Morphine stimulates angiogenesis and cancer progression in mice. We investigated whether morphine influences tumour onset, development, and animal model survival, and whether µ-opioid receptor (MOR), lymphangiogenesis, mast cell activation, and substance P (SP) are associated with the tumour-promoting effects of morphine.

Methods: Transgenic mice with a rat C3(1) simian virus 40 large tumour antigen fusion gene which demonstrate the developmental spectrum of human infiltrating ductal breast carcinoma were used. Mice were treated at different ages with clinically relevant doses of morphine or phosphate-buffered saline to determine the effect on tumour development and progression, and on mouse survival. Tumours were analysed for MOR, angiogenesis, lymphangiogenesis, SP, and mast cell activation by immunofluorescent- or laser scanning confocal-microscopy. Cytokine and SP levels were determined by enzyme-linked immunosorbent assay.

Results: Morphine did not influence tumour development when given before the onset of tumour appearance, but significantly promoted progression of established tumours, and reduced survival. MOR-immunoreactivity (ir) was observed in larger but not in smaller tumours. Morphine treatment resulted in increased tumour angiogenesis, peri-tumoural lymphangiogenesis, mast cell activation, and higher levels of cytokines and SP in tumours. SP-ir co-localized with mast cells and elsewhere in the tumours.

Conclusions: Morphine does not affect the onset of tumour development, but it promotes growth of existing tumours, and reduces overall survival in mice. MOR may be associated with morphine-induced cancer progression, resulting in shorter survival. Mast cell activation by morphine may contribute to increased cytokine and SP levels, leading to cancer progression and refractory pain.

Keywords: analgesics, opioid, morphine; angiogenesis, lymphangiogenesis; cancer; mast cells; μ-opioid receptor.

Fig 1

Influence of morphine on cancer outcomes in C3TAG mice. Tumour burden (weight and number per mouse) and survival of mice after treatments are shown. (a and b) Six-week-old mice were treated for 7 weeks. n=4 for PBS and 5 for MS. (c and d) Three-month-old mice were treated for 7 weeks with, either PBS, MS, NAL, or MS and NAL. n=6 per treatment. (e) Three-month-old mice with palpable mammary tumours were injected with either MS or PBS until they became moribund (considered end of survival). n=40 for PBS and 17 for morphine. The Kaplan–Meier curve for survival is shown. MS, morphine; NAL, naloxone; PBS, phosphate-buffered saline.

Fig 2

Higher MOR expression in growing tumours drives morphine-induced angiogenesis and lymphangiogenesis. (a) Cryosections of large and small tumours showing MOR-ir (green), vasculature (CD31-ir, red), and nuclei (DAPI, blue). Bottom row shows co-localization of MOR-ir with vasculature (yellow). Magnification ×1000; scale bar, 50 µm. (b). Three-month-old mice were treated with morphine or PBS for 7 weeks. Immunostaining of tumour sections co-stained for lymphatic vessel endothelium (LYVE-1, green), blood vessels (CD31, red), and cell nuclei (DAPI, blue) is shown. Magnification ×100; scale bar, 500 µm. Each image in (a) and (b) represents six different tumours.

Fig 3

Influence of morphine on mast cell activation in tumours of C3TAG mice. Three-month-old mice were treated with PBS or morphine for 7 weeks. (a) Toluidine blue-stained tumour sections showing intense degranulation in morphine-treated mouse tumour sections. Magnification ×900; scale bar, 200 µm. n=10 per image. (b) Number of mast cells enumerated using Toluidine blue staining is shown as mean (sem) from 10 different tumours. (c) Tumours sections were co-stained with mast cell markers FcεR1 (red) and c-Kit (blue), and CD31 (red, blood vessels). Intense mast cell degranulation is observed in the morphine group. Mast cells are closely associated with the vasculature (magenta) or are in close proximity to the vasculature. Magnification ×1200; scale bar, 20 µm. (d and e) Tryptase and β-hexosaminidase, markers of mast cell activation, were analysed in tumour lysates by ELISA. Each bar shows mean (sem) from 7 to 8 different tumours.

Fig 4

Morphine stimulates cytokine release in the tumours. Three-month-old mice were treated with PBS or morphine for 7 weeks. The concentrations of GM-CSF, RANTES, or IL-6 in tumour lysates were determined using ELISA assays. Each bar represents mean (sem) values from 5 to 8 different tumours.

Fig 5

Morphine stimulates SP release in the tumours. Three-month-old mice were treated with PBS or morphine for 7 weeks. (a) Tumour sections immunostained for CD31 (blood vessels, green), c-Kit (mast cells, red), and SP (blue) are shown. Lane 4 shows the co-localization of SP with mast cells on and near the vasculature (white). Each image in each row is from the same field of view. Magnification ×1000; scale bar, 25 µm. n=6 for each image. (b) SP concentration in whole tumour lysates determine by ELISA is shown as the mean (sem) from 7 different tumours. SP, substance P.

Fig 6

Model showing how morphine may stimulate a vicious cycle of SP release, pain, and inflammation. Morphine treatment stimulates mast cell activation in the tumours. Degranulating mast cells then release SP and tryptase, which may enhance peripheral/intra-tumoural nerve activation leading to pain. Activated nerve fibres may release more SP in turn, thus increasing neuro-inflammation and driving a vicious cycle of mast cell activation, inflammation, disease progression, and pain.