Fig. 4
Knockdown of FLT3 via αCD33-mAB-P/P-nanocarrier leads to significantly decreased colony and tumor growth of MV4-11 cells and of colony formation of primary AML blasts. A Internalization of αCD33-mAB-P/P-Alexa488-control-siRNA into MV4-11 cells. B Western blot for FLT3 of FLT3-ITD-mutant AML cell line MV4-11. Expression of FLT3 was suppressed upon αCD33-mAB-P/P-FLT3-siRNA treatment in contrast to control-siRNA in MV4-11 with β-actin as control. C Colony formation assay of FLT3-ITD mutated cell lines MV4-11 (n = 3). There is a significant decrease in colony growth due to αCD33-mAB-P/P-FLT3-siRNA treatment in contrast to control (cntr)- or DNMT3A-siRNA carriers, respectively. Means plus SD of three independent experiments. 2-sided T-test: *p < 0.02. D Schematic overview of in vivo i.p. treatment after s.c. injection of 1 × 107 MV4-11 cells in CD1-nude mice. Mice were treated with PBS, αCD33-mAB-P/P-control (cntr)-siRNA or αCD33-mAB-P/P-FLT3-ITD-siRNA three times weekly. E Tumor growth curves of MV4-11 transplants. Growth of MV4-11 tumors was significantly inhibited due to αCD33-mAB-P/P-FLT3-siRNA treatment in contrast to control-siRNA carrier treatment or PBS treatment. F Flow cytometric analysis of CD33-expression on the surface of AML patient #751 (driven by DNMT3A-R882H and FLT3-ITD mutations) cells. G The αCD33-mAB-P/P nanocarrier is able to internalize Alexa488-siRNA into patient blasts. Left: Fluorescence microscopy; right: Quantification via flow cytometry of non-treated (upper panels) and αCD33-mAB-P/P-Alexa488-control-siRNA internalized AML patient cells (lower panels). H Colony formation capacity of primary AML blasts. Cells were pre-incubated with the antibody-siRNA complex, resuspended in methylcellulose and cultivated for 8—12 days. Colonies were stained with INT, counted and photographed. There is a significant decrease in colony growth upon αCD33-mAB-P/DNMT3A-siRNA and αCD33-mAB-P/P-FLT3-siRNA treatment in contrast to control-siRNA carrier. Significance: *p < 0.05, 2-tailed T-test. Means plus SD of 3 independent replicates. α, anti