gms | German Medical Science

Objective: In the tumor microenvironment, ADAMs proteinases are important mediators of cell-cell communication between tumor cells and tumor-associated macrophages/microglia. One such proteinase, ADAM8, is highly expressed in glioblastomas and associated with a poor prognosis for GBM patients. The role of ADAM8 was explored in vitro and in vivo.

Methods: Knockdowns of ADAM8 (shRNA) in U87MG cells were generated. In addition, macrophages were cultured from ADAM8-deficient mice. In both cell types, the effect of ADAM8 knockdown was analysed with regard to cell migration, proliferation and to HUVEC based angiogenesis assays, followed by proteomic identification of potential ADAM8 substrates. For in vivo analyses, control and knockdown U87MG cells were injected stereotactically and tumor progress was monitored by MRT and histology.

Results: Angiogenesis of HUVEC cells monitored by tubulogenesis after addition of supernatants from either control or knockdown U87MG cells (100% for Ctrl vs. 69±23 %, p<0.01) and from ADAM8+/+ and ADAM8-/- macrophages (100% for ADAM8+/+ vs. 52±8%, p<0.001) was significantly reduced in the absence of ADAM8. In vivo analyses revealed a reduced tumor growth from ADAM8 knockdown cells, reflected by reduced angiogenesis visualized by CD31 staining (100% for Ctrl vs. 41±8%, p<0.01). Proteomic analysis of glioblastoma cell and macrophage supernatants revealed reduced levels of osteopontin (3±0.8-fold) in supernatants from ADAM8 knockdown cells and macrophages. The lack of angiogenic potential in ADAM8 knockdown glioblastoma cells and in ADAM8 deficient macrophages was overcome by exogenous addition of recombinant Osteopontin (10–100 ng/ml). Application of ADAM8 inhibitors in submicromolar concentrations caused a reduction in osteopontin release (3.5±0.7-fold) with concomitantly reduced glioma- and macrophage-derived angiogenesis suggesting that targeting ADAM8 in the GBM microenvironment reduces tumor angiogenesis.

Conclusion: ADAM8 is an important mediator of angiogenesis in the GBM microenvironment. Knockdown of ADAM8 in either GBM cells or in macrophages causes a significant reduction of angiogenesis, i.e. by affecting extracellular concentrations of osteopontin in the tumor microenvironment. Our results provide a basis for further studies to treat gliomas by application of ADAM8 inhibitory drugs.