(P 19) EXTRACELLULAR VESICLE CLICK MAGBEADS: A MULTIFACETED LIQUID BIOPSY PLATFORM FOR PEDIATRIC SARCOMAS

Objective: There is a pressing need for developing new diagnostic tools for the pediatric sarcoma community that enable more precise and timely detection of a patient's disease progression prior to radiographic evidence of treatment failure or disease recurrence. Using osteosarcoma (OS) as a clinical model, we demonstrate a liquid biopsy approach capable of simultaneously assessing systemic tumor burden via isolation of tumor-derived extracellular vesicles (EVs) and quantifying the activity of proteases used by OS to promote tumor invasion and dissemination. Isolating and characterizing sarcoma-derived EVs for specific protease activity profiling is expected to offer an opportunity for precision cancer diagnosis and treatment monitoring.

Methods: Given the crucial role of EV-mediated transport of active proteases in tumor progression, we hypothesized that profiling OS EV protease activity signatures can be utilized to detect disease progression in OS patients. To evaluate this hypothesis, we developed and synergistically integrated two platform technologies (Figure 1): i) EV Click MagBeads for OS EV enrichment -that leverage click chemistry-mediated targeting of three OS-specific markers (i.e., B7H3, GPNMB, and LRRC15); and ii) Fluorescence resonance energy transfer (FRET)-based peptide cleavage assays that quantify the activity of OS-associated active proteases, including MMP-14 and MMP-2/9, which play a crucial role in modulating OS progression and metastasis. The detection capabilities of this integrated OS EV Protease Activity liquid biopsy platform were validated in both artificial plasma samples and in samples obtained from a small OS patient cohort (n=30) undergoing treatment at the UCLA Pediatric Bone and Soft Tissue Sarcoma Program.

Results: The concentration dependence of the OS EV protease activity of artificial plasma samples was evaluated using GPNMB as an OS-specific marker for EV isolation. MMP-14 activity from captured GPNMB(+)-OS EVs was measured via the FRET-based peptide cleavage assay and a linear titration curve (R2 = 0.987) was observed. We next preliminarily assessed whether our OS EV MMP-14 Activity Assay (Figure 2) could differentiate OS patients with known metastatic disease from localized OS patients and healthy donors. Using MMP-14 as a representative OS-associated active protease, we observed elevated activity in OS EV samples obtained from the metastatic OS patients, represented by the AUROC of 0.92 (healthy vs. localized OS) and 0.83 (localized vs metastatic OS). No significant difference was observed between the whole plasma from healthy donors and OS patients without EV enrichment.

Our preliminary studies further applied the OS EV Protease Activity assay to determine whether dynamic changes in protease activity could be correlated to treatment response. Serial samples taken before the initiation of treatment and up to 9 weeks after the first blood draw were assessed from two OS patients. Radiographic findings from routine surveillance imaging from these patients are consistent with the dynamic changes observed in the corresponding MMP-14 activity on GPNMB(+)-OS EVs.

Conclusion: Liquid biopsy of sarcoma-derived EVs represents a promising approach to systemically and repeatedly assess the activity of key proteases of the tumor, aiding in the diagnosis, prognostication, and detection of disease progression. This multifaceted OS EV Protease Activity liquid biopsy assay is rapidly translatable, offering pediatric oncologists a sensitive noninvasive solution for dynamic monitoring of treatment response and detecting disease progression in OS patients. This capability could enable pediatric oncologists to make more informed and effective alterations to their management quickly and with higher confidence.