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. 2014 Apr;184(4):1073-1084.
doi: 10.1016/j.ajpath.2013.12.019. Epub 2014 Feb 1.

Morphine inhibits migration of tumor-infiltrating leukocytes and suppresses angiogenesis associated with tumor growth in mice

Lisa Koodie  1 , Hongyan Yuan  2 , Jeffery A Pumper  3 , Haidong Yu  2 , Richard Charboneau  4 , Sundaram Ramkrishnan  3 , Sabita Roy  5
Affiliations

Morphine inhibits migration of tumor-infiltrating leukocytes and suppresses angiogenesis associated with tumor growth in mice

Lisa Koodie et al. Am J Pathol. 2014 Apr.

Abstract

Tumor cells secrete factors that stimulate the migration of peripheral blood leukocytes and enhance tumor progression by affecting angiogenesis. In these studies, we investigated the effect of morphine, a known immunosuppressant, on leukocyte migration and recruitment to conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells. Our results indicate that morphine treatment reduced the migration and recruitment of tumor-infiltrating leukocytes into Matrigel plugs and polyvinyl alcohol sponges containing conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells when compared with placebo. A reciprocal increase in peripheral blood leukocytes was observed at the time of plug or sponge removal in morphine-treated mice. Decreased angiogenesis was observed in conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells Matrigel plugs taken from morphine-treated wild-type mice when compared with placebo but was abolished in morphine-treated μ-opioid receptor knockout mice. In addition, in vitro studies using trans-well and electric cell substrate impedance sensing system studies reveal for the first time morphine's inhibitory effects on leukocyte migration and their ability to transmigrate across an activated endothelial monolayer. Taken together, these studies indicate that morphine treatment can potentially decrease leukocyte transendothelial migration and reduce angiogenesis associated with tumor growth. The use of morphine for cancer pain management may be beneficial through its effects on angiogenesis.

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Figures

Figure 1
Figure 1
Effect of morphine on leukocyte infiltration into a solid tumor microenvironment. A: Graph shows morphine decreased tumor growth when compared with placebo treatment (n = 5 per group) observed during 21 days. H&E-stained LLC tumor sections taken from two distinct placebo-treated (B) and morphine-treated (C) mice on day 21. Fluorescent images show frozen tumor sections stained for CD31-positive endothelial cells (red) counterstained with DAPI [nuclear stain (blue)] from placebo-treated (D) and morphine-treated (E) mice. F: Morphometric analysis showing relative blood vessel density, length, and branching was determined using reindeer plug-in functions for Adobe Photoshop. The infiltration of Gr1+ neutrophils (green) from placebo-treated (G) and morphine-treated (H) mice are shown, as well as F4/80+ macrophages (red) from placebo (J) and morphine (K) merged with corresponding DAPI. Corresponding relative fluorescent units (RFU) between groups are shown (I) and (L). ∗P < 0.05, ∗∗P < 0.01 (A); P < 0.011 (I); P < 0.025 (L). Scale bar = 100 μm (B and C). Original magnification ×4 (B and C); ×20 (D and E); ×10 (G, H, J, and K).
Figure 2
Figure 2
A: Effect of morphine on leukocyte infiltration. LLC cells secrete chemotactic factors as determined using an ELISA. LLC-CM–induced leukocyte infiltration as seen in H&E-stained Matrigel plugs (inset) taken from placebo- and morphine-treated mice (B) (n = 5 per group); leukocyte accumulation near blood vessels (arrows) at the plug periphery (C) and cell nuclei using DAPI stain on sections showing plug centers (D). E: Graph shows relative hemoglobin levels of Matrigel plugs removed on day 7 and determined using a standard curve from WT and MORKO mice (n = 4 per group) implanted with either placebo or morphine. F: Fluorescent captured images of CD31-PE–stained (red) Matrigel plug (containing 100 ng/mL of VEGF) sections taken from WT and MORKO placebo and morphine pellet implanted mice. G: Quantification of blood vessel density using morphometric analysis. ∗P < 0.05; ∗∗∗P < 0.005. Scale bars: 100 μm (B); 20 μm (C and D). Original magnification ×4 (B); ×20 (C and D).
Figure 3
Figure 3
Effect of morphine on leukocyte migration toward tumor-derived factors using a PVA sponge assay. A: Whole-body bioluminescent captured images showing infiltration of Luc-expressing BM cells (injected i.p.) into PVA-LLC-CM–containing sponges (arrow) from placebo (top row) and morphine pelleted mice (bottom row), n = 4 per group). B: Graph shows the percentage of total infiltrating leukocytes into PVA-LLC-CM sponges after 24 hours of implantation in placebo- and morphine-treated mice. Graphs show the relative percentages of Gr1+ neutrophils (C) and Ly6C+ monocytes (D) on day 1 and day 5 (E and F) from placebo- and morphine-treated mice (n = 5 per group). G: Graph shows the relative percentage of leukocytes that infiltrated LLC-CM Matrigel plugs (inset). H: The corresponding peripheral blood levels at the time of plug removal as determined using flow cytometry (n = 5 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
Figure 4
Figure 4
Effect of morphine on leukocyte migration into LCC-derived conditioned media–containing PVA sponge assay in mice. A: Graphs show the fold change in RLU. Luminescence was collected as a measure of Luc+BM infiltrating preimplanted LLC-CM-PVA sponges in WT and MORKO Luc-expressing mice (n = 4 per group) (∗P < 0.25). Graphs show the relative migration of WT Luc+BM cells isolated from placebo- and morphine-treated Luc-positive mice and injected via tail vein into non-Luc MORKO host mice (n = 4 per group) (∗P < 0.05) (B); the relative migration of naive MORKO Luc+BM cells in drug pretreated WT mice (n = 6 per group) (∗P < 0.005) (C); percent change in normalized TER from time on addition of naive human PLB985 cells to vehicle and morphine (1.0 μmol/L) pretreated HUVECs (D); and saline- or morphine-pretreated huPLB985 cells to human TNF-α (100 ng/mL every 2 hours)–pretreated huMVECs (E). LPS induces increase in ICAM-1 on HUVECs as determined using flow cytometry (∗P < 0.05) (F).
Figure 5
Figure 5
A: Schematic shows the working model for the effect of morphine on leukocyte migration. Morphine suppresses leukocyte infiltration to reduce angiogenesis associated with tumor growth. Leukocytes normally circulate freely in peripheral blood. B: Once an infection or injury occurs, damaged and stressed cells secrete a host of cytokines and chemokines that alter cell motility and promote slow rolling of activated leukocytes for firm binding, adhesion, and transmigration through the receptive endothelium. Data suggest that morphine alters leukocyte and endothelial cell expression of cell adhesion molecules necessary for effective TEM, leading to either slowed and/or delayed infiltration into the tumor tissue.
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