gms | German Medical Science

Objective: In the tumor microenvironment, ADAMs as zinc-dependent metalloproteases are important mediators of tumor cell growth, cell migration and tumor angiogenesis. ADAM8 as a metalloprotease is highly expressed in ~54% of high-grade gliomas and ADAM8 expression levels are inversely correlated with patient prognosis. The role of ADAM8 was explored in vitro and in vivo by stereotactic injection of glioma cells expressing variable levels of ADAM8.

Method: Stable knockdowns of ADAM8 (shRNA) and respective control cells of U87MG were generated and cell clones were selected. The effect of the ADAM8 knockdown was assessed by scratch, migration, proliferation and in HUVEC based angiogenesis assays followed by proteomic identification of potential ADAM8 substrates. For in vivo analyses, control and knockdown U87MG cells were injected stereotactically into nude mice and tumor progress was monitored by MRT and histology.

Results: Compared to control cells, an ADAM8 knockdown caused in vitro a significant reduction in migration and invasion behaviour whilst cell proliferation was not affected. Moreover, HUVEC angiogenesis monitored by tubulogenesis after addition of supernatants from either control or knockdown cells was significantly reduced in the absence of ADAM8. In vivo analyses over 3 weeks observation time revealed a significantly reduced tumor growth from ADAM8 knockdown cells (8.3 ± 1.2-fold, p<0.001) in conjunction with reduced CD31 staining, a marker of angiogenesis. Proteomic analysis of glioma cell supernatants showed reduced levels of osteopontin and Interleukin-8 (IL-8) in supernatants from ADAM8 knockdown cells. The lack of angiogenic potential in ADAM8 knockdown cells was “rescued” by exogenous addition of recombinant Osteopontin and Interleukin-8. Application of ADAM8 inhibitors in submicromolar concentrations caused a similar reduction of glioma-derived angiogenesis suggesting that targeting ADAM8 in gliomas is a feasible therapeutic strategy.

Conclusions: Our results demonstrate that knockdown or inhibition of ADAM8 causes a significant reduction of tumor growth in vivo, affecting cellular migration, invasiveness and angiogenesis, hallmarks of high-grade gliomas. The pathophysiological effect of ADAM8 is given by influencing the extracellular concentrations of osteopontin and IL-8. Our results provide a basis for further studies to treat gliomas by application of ADAM8 inhibitory drugs.