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INTERNATIONAL CONGRESS OF

CancerGenetics

• Selective Detection of HER3-Positive Breast Cancer Cells Using a Novel Affibody-EGFP Biosensor

• Sahar Babaei Khorzoughi,^1,* Mojtaba Mortazavi,² Rahman Emamzadeh,³
  1. Graduate University of Advanced Technology
  2. Graduate University of Advanced Technology
  3. University of Isfahan
  
  
  
• Introduction: Breast cancer continues to be a leading cause of cancer-related death among women worldwide. Among the molecular drivers of tumor progression, HER3 (ErbB3) has gained attention due to its role in cancer cell survival, metastasis, and resistance to therapy. Even though HER3 has limited kinase activity on its own, it forms heterodimers with HER2 and other ErbB receptors, activating pathways like PI3K/AKT that promote oncogenic signaling. Elevated HER3 expression has been correlated with poor prognosis, making it an attractive biomarker for early detection and monitoring. Most current detection methods rely on antibodies, which can be expensive and difficult to produce. Affibodies, small engineered proteins, offer a promising alternative due to their high specificity, stability, and ease of production. When fused to a fluorescent protein such as EGFP, affibodies can act as sensitive biosensors capable of rapid detection of target cells. In this study, we tested a recombinant fusion protein combining the HER3-specific affibody Z08699 with EGFP. We evaluated its ability to selectively bind to HER3-positive breast cancer cells and investigated its potential as a practical diagnostic tool.
• Methods: The HER3-binding affibody-EGFP fusion protein was expressed in E. coli BL21(DE3) cells and purified using Ni-NTA affinity chromatography. Breast cancer cell lines with different HER3 expression levels were selected: BT-474 (HER3-positive) and MDA-MB-231 (HER3-negative). Cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified incubator. For binding studies, cells were incubated with various concentrations of the fusion protein for 20 minutes at 4°C and washed thoroughly to remove unbound protein. EGFP alone was used as a control to assess nonspecific binding. Fluorescence microscopy was employed to visualize the distribution of the probe on the cell surface, and flow cytometry was performed for quantitative analysis of binding specificity and intensity. To confirm selective binding, competitive assays were conducted by pre-incubating HER3-positive cells with excess unlabeled affibody before adding the fluorescent probe. A reduction in fluorescence indicated specific HER3-mediated interaction.
• Results: Fluorescence microscopy clearly showed bright cell-surface signals on BT-474 cells treated with the affibody-EGFP fusion protein, while MDA-MB-231 cells exhibited minimal background fluorescence. EGFP alone produced negligible signals, confirming that binding was driven by the affibody domain. Flow cytometry confirmed the microscopy observations. HER3-positive cells displayed a significant increase in fluorescence compared to negative controls, demonstrating both high sensitivity and specificity. Competitive binding experiments further validated that the interaction was HER3-specific, as pre-treatment with unlabeled affibody substantially decreased fluorescence in BT-474 cells. These results collectively indicate that the affibody-EGFP biosensor can efficiently discriminate HER3-positive from HER3-negative breast cancer cells in vitro. The probe remained stable under assay conditions and provided reproducible results across independent experiments.
• Conclusion: The HER3-targeting affibody-EGFP fusion protein functions as a sensitive and specific biosensor for detecting HER3-expressing breast cancer cells. Its small size, ease of production, and robust fluorescence signal make it a promising alternative to conventional antibody-based assays. This biosensor could potentially be used for early cancer diagnosis, monitoring treatment response, or stratifying patients for targeted therapies. Future studies should expand testing to additional cancer cell lines and explore integrating the probe into imaging or biosensing platforms, paving the way for practical applications in translational oncology.
• Keywords: HER3, Affibody, EGFP, Biosensor, Cancer detection