Ultrasensitive and High-Resolution Protein Spatially Decoding Framework for Tumor Extracellular Vesicles.

Proteins localized on the surface or within the lumen of tumor-derived extracellular vesicles (EVs) play distinct roles in cancer progression. However, quantifying both populations of proteins within EVs has been hampered due to the limited sensitivity of the existing protein detection methods and inefficient EV isolation techniques. In this study, the eSimoa framework, an innovative approach enabling spatial decoding of EV protein biomarkers with unmatched sensitivity and specificity is presented. Using the luminal eSimoa pipeline, the absolute concentration of luminal RAS or KRASG12D proteins is released and measured, uncovering their prevalence in pancreatic tumor-derived EVs. The pulldown eSimoa pipeline measured absolute protein concentrations from low-abundance EV subpopulations. The eSimoa assays detected EVs in both PBS and plasma samples, confirming their applicability across diverse clinical sample types. Overall, the eSimoa framework offers a valuable tool to (1) detect EVs at concentrations as low as 105 EV mL-1 in plasma, (2) quantify absolute EV protein concentrations as low as fM, and (3) decode the spatial distribution of EV proteins. This study highlights the potential of eSimoa in identifying disease-specific EV protein biomarkers in clinical samples with minimal pre-purification, thereby driving advancements in clinical translation.

Integrated pipeline for ultrasensitive protein detection in cancer nanomedicine.

Although nanotechnologies have attractive attributes in cancer therapy, their full potential has yet to be realized due to challenges in their translation to clinical settings. The evaluation of cancer nanomedicine efficacy in preclinical in vivo studies is limited to tumor size and animal survival metrics, which do not provide adequate understanding of the nanomedicine's mechanism of action. To address this, we have developed an integrated pipeline called nanoSimoa that combines an ultrasensitive protein detection technique (Simoa) with cancer nanomedicine. As a proof-of concept, we assessed the therapeutic efficacy of an ultrasound-responsive mesoporous silica nanoparticle (MSN) drug delivery system on OVCAR-3 ovarian cancer cells using CCK-8 assays to evaluate cell viability and Simoa assays to measure IL-6 protein levels. The results demonstrated significant reductions in both IL-6 levels and cell viability following nanomedicine treatment. In addition, a Ras Simoa assay (limit of detection: 0.12 pM) was developed to detect and quantify Ras protein levels in OVCAR-3 cells, which are undetectable by commercial enzyme-linked immunosorbent assays (ELISA). These results suggest that nanoSimoa has the potential to guide the development of cancer nanomedicines and predict their behavior in vivo, making it a valuable tool for preclinical testing and accelerating the development of precision medicine if its generalizability is confirmed.