Cell line-derived xenograft models were established the back of immunodeficient mice (NCG or Balb/c nude), by subcutaneous injection of 5x10⁶ (MDA-MB-231) tumor cells, and treatmen with 2.5mg/kg ADC (via the tail vein, Qw&#423) after tumor volume about 100-300mm³. Determined tumor volume after the experiment, measured at day 28.

A total of 250-1500cells (PA-1) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (NCI-H446) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (HCC1187) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (NCI-N87) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (SK-CO-1) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (MDA-MB-231) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (COLO-678) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (HT-29) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (HCC827) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (SK-BR-3) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.

A total of 250-1500cells (MCF-7) were plated in 150ul cell culture medium per well of a 96-well flat plate in the appropriate growth media. After 3-5h, add ADC at different concentrations respectively. Set 2-4 replicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells. After 5-6 days of treatment (according to the cell growth rate to ensure sufficient cell division), pour off the culture medium, add CCK8 reaction solution (purchased from MCE, cat#HY - K0301), 100 uL per well. Incubate at 37°C until the expected color intensity is reached, and measure the cell viability (OD450nm) of each group.