Monitoring Src status after dasatinib treatment in HER2+ breast cancer with 89Zr-trastuzumab PET imaging

Abstract Background De novo or acquired resistance in breast cancer leads to treatment failures and disease progression. In human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer, Src, a non-receptor tyrosine kinase, is identified as a major mechanism of trastuzumab resistance, with its activation stabilizing aberrant HER2 signaling, thus making it an attractive target for inhibition. Here, we explored the causal relationship between Src and HER2 by examining the potential of 89Zr-trastuzumab as a surrogate imaging marker of Src activity upon inhibition with dasatinib in HER2+ breast cancer. Methods HER2+ primary breast cancer cell lines BT-474 and trastuzumab-resistant JIMT-1 were treated with dasatinib and assessed for expression and localization of HER2, Src, and phosphorylated Src (pSrc) (Y416) through western blots and binding assays. Mice bearing BT-474 or JIMT-1 tumors were treated for 7 or 14 days with dasatinib. At the end of each treatment, tumors were imaged with 89Zr-trastuzumab. The results of 89Zr-trastuzumab positron emission tomography (PET) was compared against tumor uptake of fluorodeoxyglucose (18F-FDG) obtained the day before in the same group of mice. Ex vivo western blots and immunohistochemical staining (IHC) were performed for validation. Results In BT-474 and JIMT-1 cells, treatment with dasatinib resulted in a decrease in internalized 89Zr-trastuzumab. Confirmation with immunoblots displayed abrogation of pSrc (Y416) signaling; binding assays in both cell lines demonstrated a decrease in cell surface and internalized HER2-bound tracer. In xenograft models, dasatinib treatment for 7 days (BT-474, 11.05 ± 2.10 % injected dose per gram of tissue %(ID)/g; JIMT-1, 3.88 ± 1.47 %ID/g)) or 14 days (BT-474, 9.20 ± 1.85 %ID/g; JIMT-1, 4.45 ± 1.23 %ID/g) resulted in a significant decrease in 89Zr-trastuzumab uptake on PET compared to untreated control (BT-474, 17.88 ± 2.18 %ID/g; JIMT-1, 8.04 ± 1.47 %ID/g). No difference in 18F-FDG uptake was observed between control and treated cohorts. A parallel decrease in membranous HER2 and pSrc (Y416) staining was observed in tumors post treatment on IHC. Immunoblots further validated the 89Zr-trastuzumab-PET readout. Positive correlation was established between 89Zr-trastuzumab tumor uptake versus tumor regression, pSrc and pHER2 expression. Conclusions 89Zr-trastuzumab can potentially assess tumor response to dasatinib in HER2+ breast cancer and could be used as a surrogate tool to monitor early changes in Src signaling downstream of HER2