Figure 2

<p>Cytotoxic effect of 5-FU on non-sorted and sorted (uPAR-positive and uPAR-negative populations) derived from SCLC cell lines. (A) 1×10⁴ cells (H211, H69AR, H1417) were placed in wells of a 48-well plate in triplicates and incubated for 72 hr in the presence of varying concentrations of 5-FU. (B) SCLC cell lines were FACS sorted after staining with anti-uPAR antibodies and were plated at the same seeding density (4×10³/well of 96-well plate) and treated with 5-FU at 0, 10, 100, 200 µg/ml for 72 hr. Cell survival was evaluated after adding Guava ViaCount reagent and counting viable and dead cells. Only viable cells were included in data analysis, and 100% viability was defined as number of viable cells cultured in absence of 5-FU. Statistical analysis (2-way ANOVA) of uPAR(+) and uPAR(−) data sets revealed significant differences among viability of uPAR(+) and uPAR(−) cells (<i>P</i> = 0.0002, 0.0027, 0.0008 for H211, H69AR, H1417 cells, respectively). The data points represent averages±SD of three independent experiments.</p

Figure 4

<p>Colony-forming activity of uPAR-positive and uPAR-negative cells derived from SCLC cell lines. (A) H1417- derived, uPAR-positive sorted cells formed multiple colonies in methylcellulose media, while uPAR-negative cells from the same sorts displayed little or no clonogenic activity. (B) Graphical representation of colony-forming ability of uPAR-positive and uPAR-negative cells at different plating densities 3000, 1000, 100 cells/6-well plate (H1417, H69AR, H211). (C) Distribution of uPAR-positive cells in the colonies derived from sorted uPAR-positive cells grown in methylcellulose media. A total of 20 cell colonies from the H1417 cell line were analyzed.</p

Figure 5

<p>Expression of CD44 and MDR1 on uPAR-positive and uPAR-negative cells. (A) FACS analysis of, H211, H69AR and H1417 SCLC cell lines double-labeled with uPAR-FITC and CD44-PE, MDR1-PE. The percentages of cells expressing CD44 and MDR1 were calculated separately for uPAR-positive and uPAR-negative cells. (B) Fluorescent microscopic analysis of double-labeled and FACS-sorted cells. Examples of uPAR-FITC/CD44-PE double-labeling (a,b,c) and uPAR-FITC/MDR1-PE double-labeling (d,e,f). (Bf-inset) H1417 cell line stained with mouse IgG isotype control-PE (red), isotype control-FITC (green) and DAPI (blue).</p

Figure 3

<p>Cytotoxic effect of cisplatin and etoposide on non-sorted cells derived from SCLC cell lines. (A) SCLC cell lines (non-sorted) treated with cisplatin, etoposide at concentrations 0, 3, 10, 100 µg/ml or their combinations (cisplatin and etoposide at final concentrations of 10 µg/ml, 100 µg/ml) for 72 hr. Cell survival was evaluated after addition of Guava ViaCount reagent and counting of both surviving and dead cells using Guava ViaCount software. Data were normalized as 100% viability of cells cultured in absence of drugs. Error bars indicate standard deviation of triplicate cultures (results of three independent experiments). (B) After treatment cisplatin and etoposide, viable adherent cells were detached by trypsin treatment and were stained with anti-uPAR-FITC antibodies and percentage of uPAR-positive cells was determined by FACS analysis. Sample with mouse IgG isotype control antibody was used to set the value of the FACS gate, which was applied to all samples stained with uPAR-FITC.</p

Retention of FE-Pro label in HB1.F3.CD NSCs.

<p>Data is displayed as means +/− SD of Prussian blue positive iron-loaded NSCs (% of total cell number). The data were obtained from 5 random fields of each independently labeled triplicate sample at 24, 48 and 96 h post-labeling.</p

Labeling efficiency of FE-Pro.

<p>(A) Light microscopy images of Prussian blue-stained non-labeled and FE-Pro-labeled NSCs at 24, 48 and 96 hours after labeling. (B) Electron micrographs of Fe-Pro-labeled NSCs. (C) Higher magnification image of outlined area in (B). Red arrows point to internalized FE-Pro complex in membrane-bound organelles. (D–E) T2-weighted MR images of labeled (L), non-labeled (N), and an equal mixture (M) of NSCs grown in soft agar. Each phantom contained three different total numbers of NSCs (1×10⁴, 1×10⁵ and 5×10⁵) each in 500 µl of 20% DMEM and 0.8% agar. Coronal view (D) and axial view at 5×10⁵ (E. left) and 1×10⁵ (E. right) of the phantoms. Decrease in T2-w signal strength correlated with the number of labeled cells in the phantom. (F) Graph of T2-w signal intensity vs. number of labeled NSCs. Data were extracted from 5 random fields of each corresponding phantom using ImageJ and shown as mean±SE. MRI conditions: 7.0 Tesla, Gradient-Echo sequence, voxel size = 0.09 mm³, TR/TE = 5402.5/90 ms. Scale bars = 50 µm (A), 2 µm (B) and 200 nm (C).</p

Cellular viability of FE-Pro-labeled NSCs.

<p>(A) Cellular biomass normalized to non-labeled NSC cell growth at day 1 as measured by absorbance of protein-bound sulforhodamine B (SRB) at 570 nm. Data are mean±SE of triplicate samples and were analyzed using paired t-test between non-labeled vs. each FE-Pro dosage. P<0.05 was considered statistically significant. (B) Representative FACS plots showing the viable and apoptotic cell populations at 24 hours post-label and before sub-culturing. (C–D) Bar graphs showing the percentage of healthy cells at days 1, 4 and 8 for non-labeled NSCs (C), and FE-Pro-labeled NSCs (D) after sub-culturing passage at each time point. (E): Confocal images of healthy FE-Pro labeled and non-labeled NSCs (left panel) and apoptosis-induced FE-Pro labeled and non-labeled NSCs (right panel) at Day 6 post-labeling. Staining: PI (red), YO-Pro-1 (green). A FE-Pro dosage of 50∶3 µg/ml was used for each labeled sample unless otherwise indicated. Abbreviations: FE-Pro, Ferumoxide-Protamine Sulfate complex; PI, propidium iodide; Magnification: 20×.</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

Functionality of FE-Pro labeled NSCs.

<p>(A) Results from Boyden chamber migration assays, showing inherent NSC migration towards conditioned media from U251 (media collected at 24 and 48 hours), UPN029, U87, and U87ffluc cell lines. P<0.05 was considered statistically significant. (B) Flow cytometry plot, showing expression of Cytosine Deaminase (CD) in non-labeled (red (isotype control) and green (anti-bCD)) and FE-Pro-labeled (brown (isotype control) and blue (anti-b-CD)) HB1.F3.CD cells. Abbreviations: HB1.F3.CD.FE-Pro, FE-Pro-labeled HB1.F3.CD NSCs; Anti-bCD, anti-bacterial CD primary antibody.</p

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