INTERNATIONAL CONGRESS OF
CancerGenetics
Examination Binding and Specificity of New Luciferase-Affiprobe to Breast Cancer Cells
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Farnaz Karbasi,1,*
1. University of Isfahan
- Introduction: Engineered affiprobes that detect a particular target by target-dependent signal, play an important role in biomedical sciences including breast cancer tracking. A novel bioluminescent affiprobe turned into designed through genetically fusing Z08699 with the Renilla luciferase variation C- SRLuc8 for monitoring HER3-expressing cells. In this study, its functionality was investigated in vitro using HER3-positive cell lines.
- Methods: MCF-7 cell line (HER3 positive human breast cancer adeno carcinoma cell line) and HS578T cell line (HER3 negative human breast cancer carcinoma cell line) were obtained from ATCC and cultured in RPMI 1640, 10 % fetal bovine serum (FBS) in a humidified environment at 37◦C. To prevent enzymatic removal of surface receptors during culturing, pre-cultivated cells were detached by EDTA-sodium salt for a maximum of 5 min. To assess the expression degree of HER3, flow cytometry analyses were conducted using a Fluorescence-Activated Cell Sorting (FACS) flow cytometer (BD Biosciences, USA). To start with, cells were discriminated based on their forward scatter (FSC) and side scatter (SSC) properties to exclude small debris and non-cellular particles. Then, cells have been classified with a PE-conjugated antibody unique to HER3/ERBB3 (Sinobiological, China). Then, 5 thousand MCF-7 cells and HS578T cells from gated populace have been analyzed for HER3 expression. To evaluate the expression levels of the HER3 receptor, flow cytometry analyses were performed. The HER3-positive MCF- 7 cell line and the HER3-negative HS578T cell line were analyzed by discriminating cells based on their FSC and SSC properties. Following the establishment of the gates, the cells were labeled with a PE-conjugated anti-HER3 antibody to assess cell surface expression of the HER3 receptor and were subjected to flow cytometry studies.
- Results: The examination showed that 71.9 ±6.3 % of MCF-7 cells were successfully labeled with the anti-HER3 antibody. This finding confirms that MCF-7 cells express detectable levels of the HER3 receptors on their surface. the fluorescence intensity of both unstained and PE-conjugated anti-HER3 antibody-labeled HS578T cells was minimal in the positive region, indicating that HS578T cells show off negligible expression of HER3 receptors.
- Conclusion: It can be concluded that C-SRLuc8 in fusion to the Z08699 affibody did not interfere with the HER3- binding ability of the affibody. Moreover, the results show that C-SRLuc8‒Z08699 specifically binds to the cells expressing HER3.
- Keywords: Recombinant protein, Bioluminescent, Biosensor, Luciferase, Cancer detection
