NSCs target breast carcinoma and can deliver anti-HER2 antibody <i>in vivo</i>.

<p>Confocal fluorescence micrographs of tumor sections from MCF7/HER2 xenografts. First three panels in the upper row show the presence of CM-DiI-labeled red NSCs (HB1.F3, HB1.F3.Ad-H2IgG, HB1.F3.Lenti-H2IgG, respectively) in tumors 4 days after intravenous injection. The fourth panel of the upper row shows tumor with no red NSCs in mice treated with trastuzumab alone (sporadic small red dots not associated with cells are visible as autofluorescence background). Middle row shows tumor sections stained with FITC-conjugated anti-human IgG (green). Bar, 50 µm. Insets are 2× magnification (Bar, 20 µm). Bottom row shows confirmation of the presence or absence of HB1.F3 NSCs within tumors by PCR detection of a DNA amplicon (293 bp) of the v<i>-myc</i> transgene, a unique identifier of the HB1.F3 cell line.</p

NSC-secreted human IgG specifically binds HER2.

<p>Transfected NSCs were co-cultured with CM-DiI-labeled (red) MCF7 (<b>A</b>) or MCF7/HER2 cells (<b>B</b>) and stained with FITC-conjugated anti-human IgG (green) and DAPI (blue). Arrows indicate NSCs expressing human IgG that does not bind to adjacent MCF7 control cells (<b>A</b>) or areas of NSC-secreted human IgG bound to MCF7/HER2 target cells (<b>B</b>). Bar, 50 µm. Flow cytometric analysis of BT474, MCF7/HER2, or MCF7 target cells after incubation with supernatant from HB1.F3.H2IgG, HB1.F3.Adeno-H2IgG, or HB1.F3.Lenti-H2IgG (<b>C</b>), followed by incubation with FITC-conjugated anti-human IgG (blue histograms). Red histograms on each graph show target cells incubated with supernatant from unmodified HB1.F3 NSCs as a negative control.</p

NSC-secreted anti-HER2 antibody is functionally equivalent to trastuzumab.

<p>Flow cytometric analysis (<b>A</b>) of MCF7, MCF7/HER2, and BT474 cells labeled with F3-IgG, trastuzumab, or a human IgG isotype control antibody. Graphs show mean fluorescence intensity (MFI) of labeled cells. Inhibition of cell proliferation (<b>B</b>) of MCF7, MCF7/HER2, or BT474 cells treated for 6 days with F3-IgG, trastuzumab, or isotype control antibody. Graphs show proliferation as a percentage of untreated cells.</p

<i>In vitro</i> migration of NSCs to breast carcinoma conditioned media.

<p>Migration of parental NSCs and anti-HER2-transfected HB1.F3 NSCs to breast tumor-conditioned media in an <i>in vitro</i> chemotaxis assay. In this assay, bovine serum albumin (BSA) was used as a negative control for chemotaxis. Both parental and transfected NSC lines preferentially migrated to MCF7/HER2 compared to negative control (2% BSA) (<i>p</i><0.01).</p

Expression of human IgG in NSCs.

<p>Fluorescence micrograph of parental HB1.F3 NSCs (<b>A</b>), HB1.F3.H2IgG (<b>B</b>), HB1.F3.Adeno-H2IgG NSCs (<b>C</b>), and HB1.F3.Lenti-H2IgG (<b>D</b>). Cells were stained with anti-human IgG (green) and DAPI nuclear stain (blue). Expression was confirmed by intracellular flow cytometry of fixed and permeabilized NSCs (<b>E</b>). Fluorescence of fixed and permeabilized parental NSCs (indicated by gray histogram) was used to set the marker for each graph.</p