<scp> ^99m Tc </scp> ‐ <scp>HP</scp> ‐Ark2 <scp>SPECT</scp> / <scp>CT</scp> imaging reveals dynamic <scp>HER2</scp> regulation in trastuzumab resistance and its reversal by <scp>PI3K</scp> inhibition

Trastuzumab is a first-line treatment for human epidermal growth receptor 2 (HER2)-positive breast cancer, but resistance severely limits its efficacy, with underlying mechanisms involving HER2 expression still requiring further elucidation. This study aimed to investigate the dynamic changes in HER2 expression during the development and reversal of trastuzumab resistance, and to evaluate the utility of a novel HER2-targeting molecular probe, ^99mTc-HP-Ark2, for non-invasive, real-time monitoring of HER2 status and treatment response. Trastuzumab-resistant SK-BR-3R cells, established through chronic exposure of parental SK-BR-3 cells to increasing concentrations of trastuzumab, exhibited stable resistance (resistance index = 13.79), accompanied by significant upregulation of HER2 and phosphorylated PI3K (p-PI3K) expression. Treatment with the PI3K inhibitor LY294002 effectively inhibited p-PI3K activation and substantially reduced HER2 expression. Notably, the combination of LY294002 with trastuzumab led to a more pronounced downregulation of HER2 and restored trastuzumab sensitivity in SK-BR-3R cells. In vivo SPECT/CT imaging consistently showed significantly higher ^99mTc-HP-Ark2 uptake in SK-BR-3R tumors compared to SK-BR-3 tumors, which markedly decreased upon treatment with LY294002 alone or in combination with trastuzumab. Immunohistochemistry (IHC) analysis confirmed a strong positive correlation between changes in HER2 expression and tumor radioactive uptake trends. This study highlights the pivotal role of HER2 as a dynamic biomarker in trastuzumab resistance and supports the integration of molecular imaging into clinical decision-making for personalized therapeutic adjustments in HER2-positive breast cancer.