Human cancer cell lines were obtained from the American Type Culture Collection (CEA5-positive: SK-CO-1, MKN-45, LS174T; CEA5-negative MDA-MB-231) and maintained according to standard culture conditions (SK-CO-1, MKN-45: 37 °C, 5% CO2, 95% humidity; LS174T, MDA-MB-231: 37 °C, 10% CO2, 95% humidity). SK-CO-1 cells were cultured in Minimum Essential Medium Eagle (Sigma-Aldrich, FG0325) including l-alanylglutamine supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate. LS174T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, FG0435) including l-alanyl-glutamine supplemented with 10% FBS, 1 mM sodium pyruvate, and 1% (v/v) nonessential amino acid 100× mix (Sigma-Aldrich, M7145). MDA-MBA-231 cells were cultured in DMEM including l-alanylglutamine supplemented with 10% FBS and 1 mM sodium pyruvate. MKN-45 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich, FG1215) medium including l-alanylglutamine supplemented with 10% FBS and 1 mM sodium pyruvate. For subculturing, adherent cells were detached by the addition of TrypLE Express (1×) (Gibco, 12604-013), diluted with fresh medium, and transferred into a new culturing flask. For the evaluation of exatecan and exatecan conjugates, 90 uL of viable cell (vc) suspension was seeded into 96-well plates (SK-CO-1 2,500 vc/well, MKN-45 500 vc/well, LS174T 2,500 vc/well, MDA-MB-231 1,500 vc/well) followed by incubation (37 °C, 5 or 10% CO2) in a humid chamber overnight. Test compounds were formulated at a 10-fold starting concentration, prepared in serial dilution (1:4), and 10 uL was added to cells for each dilution in triplicates. The cell viability was determined after 6 days using the CellTiter-Glo reagent (Promega, G7573) according to the manufacturer's instructions. Luminescence values were measured by using a Varioskan plate reader (Thermo Fisher) and normalized to the luminescence of nontreated cells. Dose-response curves were fitted using the log (inhibitor) vs response variable slope 4-parameter fitting function of GraphPad Prism (GraphPad Software, Inc.). Data are shown as % relative cell viability versus molar compound concentration. Error bars indicate the standard deviation (SD) of triplicates. Geometric mean values of IC50's derived from multiple experiments were calculated.

Human cancer cell lines were obtained from the American Type Culture Collection (CEA5-positive: SK-CO-1, MKN-45, LS174T; CEA5-negative MDA-MB-231) and maintained according to standard culture conditions (SK-CO-1, MKN-45: 37 °C, 5% CO2, 95% humidity; LS174T, MDA-MB-231: 37 °C, 10% CO2, 95% humidity). SK-CO-1 cells were cultured in Minimum Essential Medium Eagle (Sigma-Aldrich, FG0325) including l-alanylglutamine supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate. LS174T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, FG0435) including l-alanyl-glutamine supplemented with 10% FBS, 1 mM sodium pyruvate, and 1% (v/v) nonessential amino acid 100× mix (Sigma-Aldrich, M7145). MDA-MBA-231 cells were cultured in DMEM including l-alanylglutamine supplemented with 10% FBS and 1 mM sodium pyruvate. MKN-45 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich, FG1215) medium including l-alanylglutamine supplemented with 10% FBS and 1 mM sodium pyruvate. For subculturing, adherent cells were detached by the addition of TrypLE Express (1×) (Gibco, 12604-013), diluted with fresh medium, and transferred into a new culturing flask. For the evaluation of exatecan and exatecan conjugates, 90 uL of viable cell (vc) suspension was seeded into 96-well plates (SK-CO-1 2,500 vc/well, MKN-45 500 vc/well, LS174T 2,500 vc/well, MDA-MB-231 1,500 vc/well) followed by incubation (37 °C, 5 or 10% CO2) in a humid chamber overnight. Test compounds were formulated at a 10-fold starting concentration, prepared in serial dilution (1:4), and 10 uL was added to cells for each dilution in triplicates. The cell viability was determined after 6 days using the CellTiter-Glo reagent (Promega, G7573) according to the manufacturer's instructions. Luminescence values were measured by using a Varioskan plate reader (Thermo Fisher) and normalized to the luminescence of nontreated cells. Dose-response curves were fitted using the log (inhibitor) vs response variable slope 4-parameter fitting function of GraphPad Prism (GraphPad Software, Inc.). Data are shown as % relative cell viability versus molar compound concentration. Error bars indicate the standard deviation (SD) of triplicates. Geometric mean values of IC50's derived from multiple experiments were calculated.

Human cancer cell lines were obtained from the American Type Culture Collection (CEA5-positive: SK-CO-1, MKN-45, LS174T; CEA5-negative MDA-MB-231) and maintained according to standard culture conditions (SK-CO-1, MKN-45: 37 °C, 5% CO2, 95% humidity; LS174T, MDA-MB-231: 37 °C, 10% CO2, 95% humidity). SK-CO-1 cells were cultured in Minimum Essential Medium Eagle (Sigma-Aldrich, FG0325) including l-alanylglutamine supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate. LS174T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, FG0435) including l-alanyl-glutamine supplemented with 10% FBS, 1 mM sodium pyruvate, and 1% (v/v) nonessential amino acid 100× mix (Sigma-Aldrich, M7145). MDA-MBA-231 cells were cultured in DMEM including l-alanylglutamine supplemented with 10% FBS and 1 mM sodium pyruvate. MKN-45 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich, FG1215) medium including l-alanylglutamine supplemented with 10% FBS and 1 mM sodium pyruvate. For subculturing, adherent cells were detached by the addition of TrypLE Express (1×) (Gibco, 12604-013), diluted with fresh medium, and transferred into a new culturing flask. For the evaluation of exatecan and exatecan conjugates, 90 uL of viable cell (vc) suspension was seeded into 96-well plates (SK-CO-1 2,500 vc/well, MKN-45 500 vc/well, LS174T 2,500 vc/well, MDA-MB-231 1,500 vc/well) followed by incubation (37 °C, 5 or 10% CO2) in a humid chamber overnight. Test compounds were formulated at a 10-fold starting concentration, prepared in serial dilution (1:4), and 10 uL was added to cells for each dilution in triplicates. The cell viability was determined after 6 days using the CellTiter-Glo reagent (Promega, G7573) according to the manufacturer's instructions. Luminescence values were measured by using a Varioskan plate reader (Thermo Fisher) and normalized to the luminescence of nontreated cells. Dose-response curves were fitted using the log (inhibitor) vs response variable slope 4-parameter fitting function of GraphPad Prism (GraphPad Software, Inc.). Data are shown as % relative cell viability versus molar compound concentration. Error bars indicate the standard deviation (SD) of triplicates. Geometric mean values of IC50's derived from multiple experiments were calculated.

Human cancer cell lines were obtained from the American Type Culture Collection (CEA5-positive: SK-CO-1, MKN-45, LS174T; CEA5-negative MDA-MB-231) and maintained according to standard culture conditions (SK-CO-1, MKN-45: 37 °C, 5% CO2, 95% humidity; LS174T, MDA-MB-231: 37 °C, 10% CO2, 95% humidity). SK-CO-1 cells were cultured in Minimum Essential Medium Eagle (Sigma-Aldrich, FG0325) including l-alanylglutamine supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate. LS174T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, FG0435) including l-alanyl-glutamine supplemented with 10% FBS, 1 mM sodium pyruvate, and 1% (v/v) nonessential amino acid 100× mix (Sigma-Aldrich, M7145). MDA-MBA-231 cells were cultured in DMEM including l-alanylglutamine supplemented with 10% FBS and 1 mM sodium pyruvate. MKN-45 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich, FG1215) medium including l-alanylglutamine supplemented with 10% FBS and 1 mM sodium pyruvate. For subculturing, adherent cells were detached by the addition of TrypLE Express (1×) (Gibco, 12604-013), diluted with fresh medium, and transferred into a new culturing flask. For the evaluation of exatecan and exatecan conjugates, 90 uL of viable cell (vc) suspension was seeded into 96-well plates (SK-CO-1 2,500 vc/well, MKN-45 500 vc/well, LS174T 2,500 vc/well, MDA-MB-231 1,500 vc/well) followed by incubation (37 °C, 5 or 10% CO2) in a humid chamber overnight. Test compounds were formulated at a 10-fold starting concentration, prepared in serial dilution (1:4), and 10 uL was added to cells for each dilution in triplicates. The cell viability was determined after 6 days using the CellTiter-Glo reagent (Promega, G7573) according to the manufacturer's instructions. Luminescence values were measured by using a Varioskan plate reader (Thermo Fisher) and normalized to the luminescence of nontreated cells. Dose-response curves were fitted using the log (inhibitor) vs response variable slope 4-parameter fitting function of GraphPad Prism (GraphPad Software, Inc.). Data are shown as % relative cell viability versus molar compound concentration. Error bars indicate the standard deviation (SD) of triplicates. Geometric mean values of IC50's derived from multiple experiments were calculated.