MCF7, MDA-MB-453, A-431, H1781, H1819, Calu-3, BT-474, SKBR3, and NCI-N87 cell lines were purchased from the ATCC. MCF7, MDA-MB-453, and A-431 were cultured in DMEM; H1781, H1819, Calu-3, and NCI-N87 in RPMI-1640; BT-474 in ATCC HybriCare Medium with 1.5 g/L sodium bicarbonate; SKBR3 in the ATCC-formulated McCoy’s 5a Medium Modified. To make the complete medium, all media were supplemented with 10% FBS and 1% streptomycin/penicillin. All cells used in this study were periodically tested negative for Mycoplasma using the Mycoplasma Plus PCR Primer Kit (Agilent). All patient-derived cancer cells or tumor spheroids in this study were collected and studied according to the Declaration of Helsinki and DFCI IRBs. DFCI 429 was established from a pleural effusion sample. Next-Generation Sequencing confirmed de novo HER2 amplification. DFCI315 and DFCI359 were characterized to harbor HER2 exon20 V777_G778insGSP and HER2 exon19 755_757LREdelinsRP, respectively (23 (link)). Xenograft tumors were processed for short-term ex vivo as previously described (23 (link)). The spheroids in a range of 40 to 70 μmol/L were resuspended in type I collagen (Corning) and loaded into the DAX-1 3-D cell culture chip (AIM Biotech). After solidification, the media were applied to the outer channels with indicated drugs.