Summary of patient treatment. Before the development of malignant ascites, the patient received three lines of anti‐HER2 treatment for metastatic disease and following disease progression. FEC, fluorouracil, epirubicin, and cyclophosphamide; TH, docetaxel and trastuzumab; PD, progressive disease

Characteristics of the CK‐MB‐1 cell line. (A) Western blotting confirmed HER2 positivity, ER/PR negativity, and no expression of EGFR in CK‐MB‐1 cells. (B) There was no loss of PTEN protein expression in CK‐MB‐1 cells. (C) HER2 gene amplification was detected by FISH. Orange signals were HER2 genes and green signals were chromosome 17 centromeres. (D) Sanger sequencing of CK‐MB‐1 cells did not reveal common PIK3CA mutation in exons 9 and 20. EGFR, epidermal growth factor receptor; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PIK3CA, phosphoinositide‐3‐kinase, catalytic, alpha; PR, progesterone receptor; PTEN, phosphatase and tensin homolog

Antiproliferative effects of different drugs in three trastuzumab‐resistant cell lines. MDA‐MB‐453, HCC1954, and CK‐MB‐1 cells were treated with one anti‐HER2 monoclonal antibody, two chemotherapies, and five tyrosine kinase inhibitors targeting HER2, PI3K, or AKT. These drugs were less effective against the proliferation of CK‐MB‐1 cells than against the other two trastuzumab‐resistant cell lines. HER2, human epidermal growth factor receptor 2; PI3K, phosphoinositide‐3‐kinase

The zebrafish xenograft model of the CK‐MB‐1 cell line. Tg(fli1: EGFP) transgenic zebrafish were microinjected with PBS and CK‐MB‐1 cells (n = 4). Images of zebrafish were captured at 0, 1, and 4 dpi. CK‐MB‐1 cells were labeled with red fluorescence, in contrast to the green fluorescence from zebrafish. Migration and distant metastasis of CK‐MB‐1 cells to the tails of zebrafish could be observed at 4 dpi. There was no obvious deformity of zebrafish in the CK‐MB‐1 group in comparison with the findings in the PBS group. dpi: days post‐injury; PBS, phosphate‐buffered saline

Xenograft mouse models of the CK‐MB‐1 cell line. (A) IHC staining revealed that CK‐MB‐1 xenografts maintained the same ER‐negative/HER2‐positive profile of the parental cell line in the ASID mouse model. (B) NOD‐SCID mice carrying CK‐MB‐1 xenografts were intraperitoneally injected with PBS or trastuzumab (n = four per group). The sizes of xenografts were recorded and plotted according to the protocol. Each plot was the mean of xenografts, and error bars represented the SEM. Xenografts were harvested on the next day after the eighth injection. The final tumor volume of two treatment groups was statistically analyzed using the Mann–Whitney U test. P values less than 0.05 were considered statistically significant. (C) Harvested xenografts were photographed and arranged in the order of final tumor size in each treatment group. (D) HER2 staining remained positive regardless of treatment in every xenograft harvested from NOD‐SCID mice. ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemical; n.s., not significant; PBS, phosphate‐buffered saline; SEM, standard error of the mean