Dual-biomarkers encoding tumor-activatable minicircles with scaffold/matrix attachment region motif for sensitive and sustained blood and urine-based cancer detection

BACKGROUND: Liquid biopsy has been used for decades to detect cancer; however, endogenous biomarkers have limitations that can lead to misdiagnosis and hinder early detection. We previously developed tumor-activatable nonviral minicircles driven by a tumor-specific survivin promoter (pSURV), which successfully identified tumor-bearing mice. Nevertheless, minicircles are a type of episomal vector and are often lost in fast-dividing cancer cells. Our goal was to develop a new version of these tumor-activatable minicircles to spot early-stage cancer with enhanced and prolonged signals. METHODS: We constructed pSURV-driven hCG and Gluc nonviral minicircles, with and without M18 scaffold/matrix attachment region (S/MAR) motif. These were tested in multiple cancer cell lines in culture and then administered intravenously into healthy, subcutaneous, and orthotopic tumor-bearing mice. We measured hCG and Gluc levels in blood and urine and used Gluc signals to localize tumors. RESULTS: We found that hCG and Gluc are ideal synthetic biomarkers due to minimal background signals and wide detection ranges. Moreover, minicircles containing the M18 S/MAR motif produced enhanced and prolonged signals in both cell culture and tumor-bearing mice. CONCLUSIONS: Our study demonstrates that tumor-activatable minicircles encoding hCG and Gluc are ideal for cancer liquid biopsy. The M18 S/MAR motif significantly elevates synthetic biomarker expressions and maintains signals for a longer duration in vivo. This new version of tumor-activatable minicircles allows for the detection of smaller tumors with a wider time window for sampling and subsequent assays.