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

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. 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. 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. 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.