Dissecting the biology of tumor-associated macrophages using lentiviral vectors and nanobodies

Scriptie/masterproef: Master's Thesis

Uittreksel

Type 2 tumor-associated macrophages (M2) play a role in tumor progression. Therefore, several strategies are investigated to modulate M2. These are often not selective and delivered systemically. As such they are accompanied by difficulties to reach M2 and systemic toxicity. Consequently, there is a need for selective and local M2 targeting. Since M2 are characterized by expression of macrophage mannose receptor (MMR, CD206), we propose to use MMR as a bull’s eye. We applied the nanobody (Nb) display technology to generate lentiviral vectors (LVs) coated with an MMR-specific Nb (MMR-LVs). Broad tropism (VSV.G-LVs) and Nb BCII10 displaying LVs (BCII10-LVs) served as controls. We transduced spleen cells of wild type and MMRKO mice in vitro with Thy1.1 encoding LVs, showing selective transduction of MMR+ CD11b+ cells by MMR-LVs using flow cytometry. We next injected Firefly luciferase encoding LVs intravenously in wild type and MMRKO mice, confirming selective transduction with MMR-LVs using bioluminescence imaging. However, using flow cytometry we failed to show transduction of MMR+ CD11b+ cells upon intra-tumoral injection of Thy1.1 encoding MMR-LVs. As a considerable percentage of tumor cells were transduced with VSV.G-LVs, we propose to use VSV.G-LVs encoding Nb MMR fused to a protein of interest to transform tumor cells into factories producing Nb-fusion proteins. Western blot data show secretion of Nb-fusion proteins upon in vitro transduction of cells with VSV.G-LVs encoding Nb MMR or BCII10 fused to wasabi green. We further investigated whether active second mitochondria-derived activator of caspases (tSMAC) induces macrophage death and as such can be used to deplete M2. Although annexin-V staining showed induction of RAW264.7 macrophage death upon transduction with tSMAC encoding VSV.G-LVs, we were unable to induce cell death in primary and in vitro generated macrophages. In contrast, ELISA showed that these produce higher levels of IL-6 and VEGF. These results warrant further investigation into the use of LVs, Nb MMR and tSMAC as tools to specifically modulate M2.
Datum Prijs18 jun 2015
TaalEnglish
Toekennende instantie
  • Vrije Universiteit Brussel
BegeleiderKarine Breckpot (Promotor) & Cleo Goyvaerts (Promotor)

Keywords

  • macrophages
  • Nanobodies
  • Lentiviral Vector
  • cancer
  • Immunology
  • Gene Therapy

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