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. 2012 Oct 23;107(9):1498-505.
doi: 10.1038/bjc.2012.392. Epub 2012 Sep 6.

Macrophage migration inhibitory factor produced by the tumour stroma but not by tumour cells regulates angiogenesis in the B16-F10 melanoma model

E Girard  1 , C Strathdee, E Trueblood, C Quéva
Affiliations

Macrophage migration inhibitory factor produced by the tumour stroma but not by tumour cells regulates angiogenesis in the B16-F10 melanoma model

E Girard et al. Br J Cancer. .

Abstract

Background: Macrophage migration inhibitory factor (MIF) has been proposed as a link between inflammation and tumorigenesis. Despite its potentially broad influence in tumour biology and prevalent expression, the value of MIF as a therapeutic target in cancer remains unclear. We sought to validate MIF in tumour models by achieving a complete inhibition of its expression in tumour cells and in the tumour stroma.

Methods: We used MIF shRNA-transduced B16-F10 melanoma cells implanted in wild-type and MIF-/- C57Bl6 mice to investigate the effect of loss of MIF on tumour growth. Cytokine detection and immunohistochemistry (IHC) were used to evaluate tumours ex vivo.

Results: Macrophage migration inhibitory factor shRNA inhibited expression of MIF protein by B16-F10 melanoma cells in vitro and in vivo. In vitro, the loss of MIF in this cell line resulted in a decreased response to hypoxia as indicated by reduced expression of VEGF. In vivo the growth of B16-F10 tumours was inhibited by an average of 47% in the MIF-/- mice compared with wild-type but was unaffected by loss of MIF expression by the tumour cells. Immunohistochemistry analysis revealed that microvessel density was decreased in tumours implanted in the MIF-/- mice. Profiling of serum cytokines showed a decrease in pro-angiogenic cytokines in MIF-/- mice.

Conclusion: We report that the absence of MIF in the host resulted in slower tumour growth, which was associated with reduced vascularity. While the major contribution of MIF appeared to be in the regulation of angiogenesis, tumour cell-derived MIF played a negligible role in this process.

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Conflict of interest statement

All authors are or were Amgen Inc. employees and shareholders.

Figures

Figure 1
Figure 1
Inhibition of MIF mRNA and protein expression in B16-F10 mouse melanoma cells transduced a lentiviral construct expressing MIF-targeting shRNA. Pools of B16-F10 expressing a control or MIF shRNA were grown under standard conditions with or without 2.5 μg ml–1 doxycyclin for 48 h. (A) RNA was isolated and cDNA was synthesised. Primers against MIF and GAPDH (control) were used in quantitative PCR. Data are shown as ΔCt for each condition between MIF and GAPDH. (B) Protein was harvested from cell lysates and western blot was performed on 40 μg total protein. Blots were probed with an anti-MIF rabbit polyclonal antibody and an anti-β actin antibody.
Figure 2
Figure 2
MIF-regulated VEGF expression in vitro. (A) B16-F10 cells were grown 48 h in a normoxic (20% O2) environment with or without Doxycyclin. Protein was harvested from cell lysates and western blot was performed on 40 μg total protein. Blots were probed with antibodies against MIF, VEGF, or β-actin. (B) B16-F10 cells transfected with shRNA were grown 24 h in normoxic conditions after which plates were grown an additional 24 h under normoxic or hypoxic (0.5% O2) conditions. Cell supernatant was collected and VEGF secretion was evaluated by ELISA. (*P<0.05).
Figure 3
Figure 3
Growth inhibition of B16-F10 subcutaneous syngeneic tumours in MIF−/− C57Bl6 mice. (A) In all, 1 × 105 B16-F10 cells transduced with control or MIF shRNA in 50% matrigel matrix solution were implanted subcutaneously in the right flank of age-matched wild-type C57Bl/6 or MIF−/− C57Bl/6 congenic strain. After 7 days of tumour growth, mice were randomized by tumour size into treatment groups of either standard drinking water or water with 2 mg ml–1 doxycyclin with 5% sucrose. Groups not treated with doxycyclin are not shown. Tumours were measured with digital calipers three times per week for 2 weeks. (B) Protein lysates were made from tumours harvested at the termination of the study. Lysates were evaluated for MIF expression by ELISA. (C) A portion of each tumour harvested at the termination of the study was preserved in RNALater. RNA was isolated and cDNA was synthesised. Primers against MIF, VEGF, and GAPDH were used in quantitative PCR. Data are shown as ΔCt comparing the control tumours from wild-type mice to MIF shRNA tumours from wild-type mice and control or MIF shRNA tumours from MIF−/− mice.
Figure 4
Figure 4
Reduced vascular density of B16-F10 tumours growing in MIF−/− mice. (A) B16-F10 tumour grown in wild-type or MIF−/− mice were collected at termination of the in vivo study and preserved in formalin. The proportion of endothelial cells present in the tumour was evaluated by IHC staining of CD31. Positive staining is visualised as red chromogen. Note the brown colour is melanin pigment inherent in the tumour and phagocytised (SP) by tumour-associated macrophages (melanophages). This pigment tended to be more prominent in the tumours of MIF KO mice. (B) Quantitated vessel area of B16-F10 control tumours in wild-type and MIF−/− mice at the intermediate time point (day 13) and terminal time point (day 20). The area of CD31 positivity (vessel area) is expressed in microns per mm2 of tumour area (*P<0.05).
Figure 5
Figure 5
Expression of serum cytokines. (A) Serum was collected from all mice at termination of the study. Expression of VEGF in 50 μl undiluted mouse serum was evaluated by ELISA. (B) Serum collected at the intermediate and terminal time points was evaluated undiluted by Cytokine/chemokine luminex bead assay. The circulating levels of 4 of the 22 cytokines evaluated MIP-1α, KC, IL-1α, and IL-9 were found to be reduced in the serum of MIF−/− mice compared with wild-type mice bearing B16-F10 tumours (*P<0.05).
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