Skip to main content
. 2024 May 21;30(15):3298–3315. doi: 10.1158/1078-0432.CCR-23-3110

Figure 5.

Figure 5.

Anti-EGFR ADC reduces breast cancer cell activities and demonstrated bystander killing effects. A, MTT viability assay following 96-hour treatment with SNS-032, cetuximab, isotype ADC or ADC. Top, EGFR-high breast cancer models. Bottom, EGFR-low breast cancer and immune cell models. B, Cell viability assessment of MDA-MB-468 showed that the addition of SNS-032 did not re-sensitize cells to EGFR inhibition by cetuximab alone, whereas reduced cancer cell viability was detected only when SNS-032 was conjugated to cetuximab as an ADC, suggesting that inhibition of cancer cell viability was induced by ADC internalization and subsequent drug release within cancer cells. SNS-032 efficacy improved by conjugating in the ADC instead of treating it as a free drug. C, Reduction of surface EGFR expression using siRNA, and cell viability comparisons between parental and knockdown cells after 96 hours of ADC treatment (10 nmol/L). D, Time-lapse measurement of cell confluency for cells treated with isotype control (10 nmol/L), cetuximab, and ADC (1 or 10 nmol/L) using Incucyte live-cell microscopy (representative phase images of EGFR-high MDA-MB-468). Scale bar, 0.2 mm. E, TNBC spheroids in Matrigel were treated with 10 nmol/L cetuximab, ADC, or isotype controls, and confluence measured for 7 days using Incucyte. ADC-treated MDA-MB-231 and MDA-MB-468 showed reduced spheroid growth, whereas the ADC showed less potent effects on CAL51. Scale bar, 0.5 mm. F, MDA-MB-468 were transduced with a lentiviral expression vector encoding a mCherry fluorescent protein tag (mChery-MDA-MB-468). Bystander killing effects of the ADC were accessed in co-cultures of high and low EGFR-expressing cells in a one-to-one ratio. High EGFR-expressing mCherry-MDA-MB-468 and either low EGFR-expressing MCF7 or CAL51 cells were plated alone as mono-culture or co-culture, and treated with 1 nmol/L ADC or unconjugated-isotype control antibody. Cell count was measured by Incucyte after washing at 120 hours. Scale bar, 0.5 mm. P values determined by two-tailed unpaired t test of three independent experiments.