Fig. 1: Generation and characterization of resistant cells.

a Schematic showing the assay of HER2-TCB in cocultures. b Cocultures of PBMCs with parental BT474 or resistant BT-R cells were treated with different concentrations of HER2-TCB (PBMC:target cell ratio 1:1) for 72 h. Then, viable cells were quantified by flow cytometry using EpCAM as a marker. c Schematic showing in vivo treatment with HER2-TCB. Totally, 10⁷ BT474 or BT-R cells were injected orthotopically into NSG mice. When tumors reached ~200 mm³ (dark background), 10⁷ PBMCs were injected i.p. Then animals were treated i.v. with 0.125 mg/kg HER2-TCB as indicated (red arrows). d Tumor volumes are represented as averages ± SD (standard deviation) (n = 4 per arm). e Schematic showing the HER2-CAR assay. f Parental BT474 cells or resistant BT-R cells were cocultured with different ratios of CAR T cells for 48 h. Cell numbers were calculated and expressed as in (b). g Schematic showing in vivo treatment with HER2-CAR T cells. Mice were injected with BT474 or BT-R cells as in (c). When tumors reached ~200 mm³, 3 × 10⁶ HER2-CAR T-positive cells were injected i.p. h Tumor volumes are represented as averages ± SD (BT474, n = 6; BT-R, n = 8). i Levels of HER2, normalized to BT474 cells, were determined by Western Blot. j Cells were stained with HER2-TCB and analyzed by flow cytometry. Results were normalized to BT474 cells. k Cells were stained with antibodies against the indicated factors. Results are presented as the MFI of staining in BT474 and BT-R cells. l, Levels of 80 cytokines and growth factors were measured using a commercial array in the media conditioned by cocultures of BT474 cells and PBMCs or BT-R cells and PBMCs treated with HER2-TCB. Results are presented as the fold change of BT-R relative to BT474 cells. b **p = 0.005, *p = 0.02, **p = 0.006. f ***p < 0.001. k *p = 0.04, two-tailed t test. d, h **p < 0.01, ***p < 0.001, two-way analysis of variance (ANOVA) and Bonferroni correction. Data are presented as mean ± SD of four (b, f, j, k), or three (i) independent experiments. Source data are provided as a Source Data file.