Abstract:

Objective The present study was designed to explore the inhibitory effects of the ADC drug Disitamab Vedotin （RC-48） on breast cancer cells with different HER-2 expression levels by utilizing a tumor organoid culture system. Methods Within the framework of the tumor organoid culture system， the breast cancer cell lines MCF-7 （characterized by low HER-2 expression， Luminal A subtype） and BT-474 （exhibiting high HER-2 expression， HER-2 positive subtype） were cultured independently and in various mixed ratios. The histological characteristics， as well as the expression levels and distribution of HER-2 in MCF-7 and BT-474 organoids， were analyzed via immunohistochemistry and immunofluorescence techniques. MCF-7 and BT-474 organoids were separately treated with Vedotin （RC-48）， Disitamab， and Monomethyl auristatin E （MMAE）. Additionally， a drug sensitivity test of Disitamab Vedotin （RC-48） was carried out on mixed MCF-7 and BT-474 cell ratios and on patient-derived breast cancer organoids， with the assessment conducted using the 3D-Glo method. Results In the tumor organoid culture system， immunohistochemistry and immunofluorescence analyses demonstrated that HER-2 was predominantly localized in the cell membrane. Specifically， BT-474 organoids exhibited robust HER-2 expression， while MCF-7 organoids displayed relatively low expression levels. When compared with MCF-7 organoids， RC48-ADC exerted a more pronounced inhibitory effect on BT-474 organoids， with IC₅₀ values of 109.7 μg/mL and 2.792 μg/mL， respectively. The co-culture model further confirmed the bystander effect of RC-48， revealing that the ratio of HER-2-positive to HER-2-negative cells significantly influenced drug efficacy. Additionally， treatment with RC-48 led to a reduction in HER-2 expression in breast cancer organoids with diverse HER-2 expression levels. Conclusions The tumor organoid model can accurately mirror drug sensitivity and bystander effects. Within this model， RC-48 effectively inhibited HER-2 highly-expressing breast cancer cells， augmented the killing effect through the bystander mechanism， and downregulated HER-2 expression， thereby suggesting its potential for targeting HER2-associated breast cancer.

Key words: tumor organoids, breast cancer, HER-2, ADC, bystander effect, inhibition