Immunotoxin inhibits CHO-K1^GD3+ and SK-Mel-28 cells colony formation. A

<p>) A schematical representation of the experimental procedure used in <b>B</b> and <b>C</b>. CHO-K1^GD3+(<b>B</b>) and SK-Mel-28 (<b>C</b>) cells (50–80 cells) were seed in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cultures were supplemented with 0.95 nM Saporin-Ab or 30 nM R24/0.95 nM Saporin-Ab and maintained at 37°C in a hummed atmosphere. Quantification of the colony area was performed every day, but only indicated at 7 and 10 days. Cells maintained only with medium were used as negative control (control). Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note the drastic inhibition of colony formation only in presence of R24/Saporin-Ab.</p

Selective cytotoxicity of R24-targeted immunotoxin on mouse B16^GD3+ melanoma cells. A

<p>) Wild-type B16 cells (B16^wt) and B16 cells genetically modified to express GD3 (B16^GD3+) grown on coverslips were incubated at 4°C to inhibit intracellular transport, then with R24 antibody for 45 min at 4°C, washed and fixed. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸ (45 min, 4°C; left panels). Cells were incubated with R24 for 45 min at 4°C and after washing temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. Then, cells were fixed and R24 antibody detection was carried out as indicated above (30 min, 37°C; right panels). <b>B</b>) B16^GD3+ cells transiently expressing Rab5-GFP or Lamp1-GFP were incubated with R24 for 45 min at 4°C. After washing, temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁵⁴³. Expression of Rab5 and Lamp1 was detected by the intrinsic fluorescence of GFP. In another set of experiments, uptake of Alexa Fluor⁶⁴⁷-transferrin (Tf) was monitored simultaneously with R24 endocytosis. In this case, R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸. Insets in merge panels (right column) show details at higher magnifications. In all experimental conditions, single confocal sections were taken every 0.8 µm parallel to the coverslip. The fluorescence micrographs shown are representative of three independent experiments. Scale bar: 10 µm. <b>C</b>) B16^wt and B16^GD3+ cells were cultured at 37°C for 72 h in 96-well plates at the indicated concentration of monoclonal antibody to GD3 (R24 antibody) in combination with goat antibody to mouse IgG (squares, black lines) or saporin conjugated goat antibody anti mouse IgG secondary antibody (circles, grey lines). The concentration of the secondary antibodies was 0.95 nM. As negative control (100% viability), B16^GD3+ cells were incubated only with culture medium. Cell viability was determined using the colorimetric MTT metabolic activity assay. Absorbance was measured at 595 nm using a multiplate reader. Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. Note that R24-targeted saporin selectively kills B16^GD3+ melanoma cells (** p<0.001, respect to control condition).</p

Targeted delivery of immunotoxin by R24 antibody inhibit the clonogenic growth of CHO-K1^GD3+ cells. A

<p>) A schematical representation of the experimental procedure used in (<b>B</b> and <b>C</b>). <b>B</b>) CHO-K1^GD3+ cells (50–80 cells) were grown in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cells were maintained at 37°C in a hummed atmosphere until cell colonies appeared (7 days, upper row. Colonies indicated with arrows). Then, cells were treated for 3 days (7+3 days, lower row) with 0.95 nM Saporin-Ab (Saporin-Ab, middle panel) or 30 nM R24/0.95 nM Saporin-Ab (R24/Saporin-Ab, right panel). CHO-K1^GD3+ cells maintained only with medium were used as negative control (control, left panel). The micrographs are representative of three independent experiments. <b>C</b>) Quantification of the colony area at 7 and 7+3 days at the different conditions indicated in <b>B</b>. Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note that the clonogenic growth of CHO-K1^GD3+ cells was severely affected only in presence of R24/Saporin-Ab (*** p<0.0001, respect to control condition at 7+3 days).</p

R24 antibody is specifically internalized in GD3-expressing cells. A

<p>) CHO-K1^GD3+, CHO-K1^WT (GD3-) and SK-Mel-28 cells grown on coverslips were incubated at 4°C to inhibit intracellular transport, then with R24 antibody for 45 min at 4°C, washed and fixed. R24 antibody was detected by using goat anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸ (45 min, 4°C; left panels). <b>B</b>) Cells were incubated with R24 for 45 min at 4°C and after washing temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. Then, cells were fixed and R24 antibody detection was carried out as indicated in <b>A</b> (30 min, 37°C; right panel). Single confocal sections were taken every 0.8 µm parallel to the coverslip. The fluorescence micrographs shown are representative of three independent experiments. Scale bar: 10 µm.</p

Selective delivery of saporin via R24 antibody drastically reduces the clonogenic growth of human SK-Mel-28 melanoma cells. A)

<p>SK-Mel-28 cells (50–80 cells) were grown in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cells were maintained at 37°C in a hummed atmosphere until cell colonies appeared (7 days). Then, cells were exposed for 3 days (7+3 days) to 0.95 nM Saporin-Ab or 30 nM R24/0.95 nM Saporin-Ab. Colonies are indicated with arrows. <b>B)</b> Quantification of the colony area was performed at 7 and 7+3 days. SK-Mel-28 cells maintained only with medium were used as negative control (control). Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note that the clonogenic growth of SK-Mel-28 cells was severely affected only in presence of R24/Saporin-Ab (***p<0.0001, respect to control condition at 7+3 days).</p