Melanoma exosome induction of endothelial cell GM-CSF in pre-metastatic lymph nodes may result in different M1 and M2 macrophage mediated angiogenic processes

Abstract

Angiogenesis is a key process in the preparation of lymph nodes for melanoma metastasis. Granulocyte macrophage colony stimulating factor (GM-CSF) induces hypoxia inducible factor 1 alpha (HIF-1α) in M1 or HIF-2α in M2 polarized macrophages. HIF-1α promotes neoangiogenesis while HIF-2α facilitates morphogenic normalization of neovasculature.

Melanoma exosomes induce GM-CSF expression by endothelial cells in vitro and HIF-1α expression in pre-metastatic lymph nodes in vivo. This suggest a relationship between melanoma exosome induced endothelial GM-CSF and macrophage mediated angiogenesis in lymph nodes. Theoretically, induction of endothelial cell derived GM-CSF by melanoma exosomes mediates different angiogenic functions in pre-metastatic lymph nodes depending on subcapsular sinus (SCS) macrophage polarity. To explore this hypothesis, experiments utilizing melanoma exosomes in a lymph node model are outlined. Despite their opposing immune functions, indirect melanoma exosome stimulation of M1 or M2 SCS macrophages via endothelial derived GM-CSF in lymph nodes may induce different although complementary pro-tumor angiogenic processes.

Introduction

Within tumor draining lymph nodes, melanoma derived soluble mediators such as vascular endothelial growth factor (VEGF) increase angiogenesis enabling tumor growth and survival [1]. Melanoma derived cytokines mediate immune suppression [2].

Previously, Mansfield et al. demonstrated that the microenvironment of dormant human lymph nodes is Th1 polarized [3]. Th1 lymphocytes release cytokines such as IFN-γ which polarize macrophages (Mφs) toward an anti-tumor M1 phenotype [4]. Recent evidence demonstrates Th2 inflammatory polarization within melanoma draining sentinel lymph nodes [5]. This is true for sentinel lymph nodes obtained from melanoma patients with or without metastasis [3]. Th2 cytokines such as interleukin 4 (IL-4), IL-5, and IL-10 polarize Mφs toward an immunosuppressive M2 phenotype in melanoma microenvironments [6]. Tumor draining factors are thought to be responsible for Th2 polarization in lymph nodes in the absence of lymph node associated melanoma cells [3].

Melanoma cells also produce exosomes. Exosomes are natural extracellular nanovesicles approximating 30–200 nm in size [7], [8], [9], [10]. Similar to soluble mediators, tumor exosomes can transmit pathogenic processes [11], [12]. Melanoma exosomes can re-program bone marrow progenitor cells toward a pro-vascular phenotype [13] or directly induce angiogenesis [11], [14]. They also contain pro-angiogenic and immunomodulatory factors [14] and can prepare lymph nodes for tumor metastasis [15].

One immunomodulatory strategy to treat melanoma is the administration of granulocyte-macrophage colony stimulating factor (GM-CSF) [6]. GM-CSF has long been known to facilitate proliferation, expansion and survival of granulocytes and Mφs. However, in recent years it has also been shown to increase maturation and expansion of dendritic cells (DCs). DCs present melanoma antigens resulting in activation of effector T lymphocytes and subsequent anti-tumor immune responses. In pre-clinical trials of patients with advanced melanoma, GM-CSF monotherapy was efficacious in inhibiting melanoma growth [16]. However, in prospective, randomized clinical trials, the efficacy of GM-CSF monotherapy was inconsistent. Conflicting findings suggested that GM-CSF might serve as a regulatory cytokine promoting both poorly understood pro- and anti-tumor immune responses [6].

In addition to its immunological roles, GM-CSF can induce angiogenesis via stimulation of endothelial cells and/or activation of Mφs to produce pro-angiogenic factors [17]. Given the interconnectedness between cytokine mediated immunological and angiogenic processes [17], the paradoxical nature of GM-CSF function may extend to its role in angiogenesis as well.