Fig. 6
Inhibition of US28-mediated GBM-growth by US28-specific nanobodies. a US28-mediated accumulation of 3H-labeled inositol phosphates (IP) in US28-expressing HEK239T cells treated with either CCL5 alone (black squares), monovalent US28 nanobody alone (10−6 M, open blue circle), monovalent nanobody with CCL5 (10−6.5 M, filled blue circle), bivalent US28-nanobodies alone (concentration range, open red circles) or bivalent nanobody with CCL5 (10−6,5 M, filled red circle). CCL5 was added at 10−7.5 M. IP levels were plotted in percentage of basal US28-mediated IP accumulation (dashed line). b US28-induced NF-κB reporter gene activation upon treatment with Biv. US28-Nb (red) or Mono US28-Nb (blue) in HEK239T cells, plotted in percentage of basal US28-mediated NF-κB activation (dashed line). c Inhibition of US28-mediated growth of U251-iUS28 spheroids by bivalent US28-Nbs (US28-Nb, 10−7 M), plotted in percentages as mean ± SEM (n = 6 spheroids per group). d Secretion of VEGF from U251–iUS28 spheroids upon treatment with bivalent US28-Nbs (10−7 M), as detected using ELISA. e T1 weighed MRI scan of a brain of one of the mice carrying an orthotopic doxycycline-induced U251-iUS28 tumor before (left) and after (right) intravenous administration of the contrast agent gadolinium. The impaired blood–brain barrier function this tumor model is visualized by extravasation of gadolinium from the blood vasculature into the tumor (red arrow). f, g Growth of orthotopic GBM tumors in mice upon treatment with half-life extended (HLE) bivalent US28-nanobodies (US28-NbHLE, red circles), or an irrelevant nanobody (Irr.-NbHLE, open black circles). Nanobody treatment (500 μg/injection, 3 times a week, black arrows) was started upon tumor take and stratification of the mice (day 11). Pooled data of n = 2 with 6 mice per group per experiment. ***P < 0.001, *P < 0.05 (unpaired t test)
