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VEGF-A acts via neuropilin-1 to enhance epidermal cancer stem cell survival and formation of aggressive and highly vascularized tumors.

ABSTRACT: We identify a limited subpopulation of epidermal cancer stem cells (ECS cells), in squamous cell carcinoma, that form rapidly growing, invasive and highly vascularized tumors, as compared with non-stem cancer cells. These ECS cells grow as non-attached spheroids, and display enhanced migration and invasion. We show that ECS cell-produced vascular endothelial growth factor (VEGF)-A is required for the maintenance of this phenotype, as knockdown of VEGF-A gene expression or treatment with VEGF-A-inactivating antibody reduces these responses. In addition, treatment with bevacizumab reduces tumor vascularity and growth. Surprisingly, the classical mechanism of VEGF-A action via interaction with VEGF receptors does not mediate these events, as these cells lack VEGFR1 and VEGFR2. Instead, VEGF-A acts via the neuropilin-1 (NRP-1) co-receptor. Knockdown of NRP-1 inhibits ECS cell spheroid formation, invasion and migration, and attenuates tumor formation. These studies suggest that VEGF-A acts via interaction with NRP-1 to trigger intracellular events leading to ECS cell survival and formation of aggressive, invasive and highly vascularized tumors.

SUBMITTER: Grun D

PROVIDER: S-EPMC4959993 | biostudies-other | 2016 Aug

REPOSITORIES: biostudies-other

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Project description:BackgroundVascularized composite tissue allotransplantation (VCA) to replace limbs or faces damaged beyond repair is now possible. The resulting clear benefit to quality of life is a compelling reason to attempt this complex procedure. Unfortunately, the high doses of immunosuppressive drugs required to protect this type of allograft result in significant morbidity and mortality giving rise to ethical concerns about performing this surgery in patients with non-life-threatening conditions. Here we tested whether we could suppress anti-graft immune activity by using a safe β2 -adrenergic receptor (AR) agonist, terbutaline, to mimic the natural immune suppression generated by nervous system-induced signalling through AR.MethodsA heterotopic hind limb transplantation model was used with C57BL/6 (H-2b) as recipients and BALB/c (H-2d) mice as donors. To test the modulation of the immune response, graft survival was investigated after daily intraperitoneal injection of β2 -AR agonist with and without tacrolimus. Analyses of immune compositions and quantification of pro-inflammatory cytokines were performed to gauge functional immunomodulation. The contributions to allograft survival of β2 -AR signalling in donor and recipient tissue were investigated with β2 -AR-/- strains.ResultsTreatment with the β2 -AR agonist delayed VCA rejection, even with a subtherapeutic dose of tacrolimus. β2 -AR agonist decreased T-cell infiltration into the transplanted grafts and decreased memory T-cell populations in recipient's circulation. In addition, decreased levels of inflammatory cytokines (IFN-γ, IL-6, TNF-α, CXCL-1/10 and CCL3/4/5/7) were detected following β2 -AR agonist treatment, and there was a decreased expression of ICAM-1 and vascular cell adhesion molecule-1 in donor stromal cells.Conclusionsβ2 -AR agonist can be used safely to mimic the natural suppression of immune responses, which occurs during adrenergic stress-signalling and thereby can be used in combination regimens to reduce the dose needed of toxic immunosuppressive drugs such as tacrolimus. This strategy can be further evaluated for feasibility in the clinic.

Project description:Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a KD value of 0.71 μM. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity.

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VEGF-A acts via neuropilin-1 to enhance epidermal cancer stem cell survival and formation of aggressive and highly vascularized tumors.

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