Noninvasive enrichment of circulating tumor biomarkers in a mouse model of diffuse midline glioma using focused ultrasound.

Background: Diagnosing diffuse midline glioma (DMG) through invasive tissue biopsies is challenging due to the tumor's eloquent location at the pons. Liquid biopsies offer a promising noninvasive alternative; however, their limited detection sensitivity and lack of information on the source of biomarkers pose great challenges. This study aimed to evaluate the feasibility and safety of using focused ultrasound (FUS) with microbubbles to enrich circulating DMG tumor biomarkers in blood and cerebrospinal fluid (CSF) using a mouse model. Methods: Murine DMG cells, transfected with enhanced green fluorescent protein (EGFP) and firefly luciferase (Fluc) genes, were orthotopically injected into the mouse brain. Magnetic resonance imaging was used to guide FUS targeting of the DMG tumor. Droplet digital PCR assays were developed to detect circulating tumor DNA (ctDNA) and RNA (ctRNA) of EGFP and Fluc in blood and CSF samples collected after FUS. Results: FUS enhanced the plasma levels of EGFP ctRNA by 5.4-fold (p=0.0112), compared with liquid biopsy without FUS. CSF EGFP ctDNA was increased by 2.5-fold (p=0.0253), and Fluc ctDNA was increased by 2.6-fold (p=0.0253) with FUS. No brain tissue damage was associated with FUS sonication. Conclusions: This study demonstrated the feasibility and safety of FUS in enriching tumor biomarkers in blood and CSF in a mouse model of DMG. The enrichment ratio for circulating biomarkers depends on the source (plasma vs. CSF), analyte type (ctDNA vs. ctRNA), and individual marker (EGFP vs. Fluc). These findings support the potential future application of FUS to advance the diagnosis of DMG through liquid biopsy. Key points: ddPCR assays were developed to detect plasma and CSF ctDNA and ctRNA in a mouse model of DMGFUS with microbubbles enriched ctDNA and ctRNA in the blood and CSF of a mouse model of DMGThe enrichment ratio on circulating biomarkers is dependent on the source (plasma vs. CSF), analyte type (ctDNA vs. ctRNA), and individual marker (EGFP vs. Fluc). Importance of the study: Liquid biopsy via detecting circulating tumor biomarkers holds enormous clinical value in the diagnosis of diffuse midline glioma (DMG), where the eloquent location of the tumor poses significant risks to invasive surgical tissue biopsies. However, the effectiveness of liquid biopsy is hindered by its low sensitivity and the lack of information about the source of biomarkers. This study demonstrated that noninvasive and spatially targeted FUS treatment can release tumor-derived DNA and RNA into the blood and CSF in a mouse model of DMG. This study also found that the enrichment effect of FUS on circulating biomarkers depends on the source (plasma vs. CSF), analyte type (ctDNA vs. ctRNA), and individual marker (EGFP vs. Fluc). This study opens new avenues for advancing noninvasive DMG diagnosis through FUS-enhanced liquid biopsy (sonobiopsy).