Evaluation of the circulating angiome in cancer: Translational assessment of a 44-plex angiogenesis biomarker panel

Angiogenesis, the formation of new blood vessels from existing vasculature, is a hallmark of tumor progression, enabling malignant growth, invasion, and metastasis. Tumor-associated angiogenesis leads to the formation of leaky, poorly perfused vessels and localized hypoxia, fostering metabolic stress, immune evasion, and resistance to therapies. Vascular normalization through targeting of VEGF, Ang-2, and PDGF pathways can restore perfusion, enhance immune infiltration, and improve response. Although anti-angiogenic agents are widely used, no predictive biomarker identifies individuals that are likely to benefit. Previously, biomarker development for anti-angiogenic therapies focused on few vascular factors and did not capture the complexity of angiogenic signaling. To address this, we developed a 44-marker angiogenesis panel enabling high-throughput profiling of pro- and anti-angiogenic mediators relevant to oncology and vascular biology. Forty-four biomarkers encompassing established pro- and anti-angiogenic mediators (e.g., VEGF-A, Ang-2, Tie-2, IL-6) were literature-curated. Antibody pairs for each assay were optimized for the electrochemiluminescence detection-based MESO SCALE DISCOVERY (MSD) U-PLEX® platform. Double-spun platelet-poor EDTA plasma samples from Duke University was tested to identify a common diluent. Analytical characterization included LLOD/LLOQ, precision, spike recovery, and dilution linearity/parallelism determinations. The interclass correlation coefficient (ICC) and biological variance were evaluated using longitudinally collected plasma from 28 participants (27-79 y.o.), incorporating diurnal (morning and afternoon) draws over two months. Pharmacodynamic biomarker modulation was explored using retrospective plasma samples from two NCI studies -Cabozantinib + Panitumumab (NCT02008383) and Bevacizumab (NCT00416637). All assays met the predefined criteria, with >90% of assays achieving intra-/inter-assay precision of <15% CV. A single diluent supported multiplex compatibility. Dynamic ranges spanned 3-5 orders of magnitude, with analytical sensitivity for low and high abundance markers ranging from 0.06 pg/mL (IL-6) to 67.4 pg/mL (vWF), demonstrating sub-pg/mL detection. ICC analyses revealed good to excellent variable marker stability across the panel over two months. Retrospective analysis of both sets of NCI study samples demonstrated an association between (a) baseline markers and outcomes (OS, PFS) and (b) pharmacodynamic responses and expected treatment-induced changes, highlighting the value of the biomarker panel. This 44-plex panel demonstrates robust analytical performance, sensitivity, and reproducibility on the MSD® U-PLEX platform. The assay supports harmonized angiogenesis profiling, enabling standardized angiogenesis biomarker assessment in oncology research.