Surface exosome integrin profiling to predict organotropic metastasis of breast cancer

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PROJECT SUMMARY It is well established that tumors modify microenvironments in distant organs before the arrival of cancer cells. These predetermined microenvironments, termed ‘pre-metastatic niches’, encourage the outgrowth of the incoming cancer cells and make metastasis sucessful. Recent studies have shown that tumors send exosomes with distinct integrins to prepare pre-metastatic niches in distant organs, with 64 and 61 associated with lung metastasis, v5 associated with liver metastasis, and IIb3 with brain metastasis (Hoshino et al., Nature 2015, 527, 329). These findings point to the possibility that the integrin profiles of plasma exosomes from cancer patients could be used to predict metastasis and organotropic dissemination. To establish whether this knowledge can be put to practice use, further evaluation is required. To evaluate exosomal biomarkers in clinical samples and use it in the clinical settings, facile but high sensitivity and high specificity technologies are needed but currently lacking. This is because exosomes are small (lower than 200 nm) and the tumor-derived exosomes are mixed with a vast background of non-tumor exosomes from various tissues and hematopoietic cells, which makes quantitative detection of exosomal biomarkers in clinical samples challenging. The goal of this project is to validate and extend the depth of exosomal integrins for metastasis prediction by providing single exosome landscapes that were previously lumped into bulk assays. We propose a facile dual imaging single vesicle technology (DISVT) that is capable of detecting targeted surface protein markers on individual exosomes and quantifying the target-specific vesicle subtypes in plasma samples. The DISVT captures exosomes directly from diluted plasma onto a multi-well gold chamber slide, localize the individual exosomes with membrane dye and laser excited fluorescence imaging, and detect surface proteins of interests using light scattering gold nanoparticles and dark field imaging at single particle level. Using human epidermal growth factor receptor 2 (HER2)-positive breast cancer as the disease model, our preliminary studies have shown that this DISVT, but not traditional enzyme-linked immunosorbent assay, can detect BC at early-stage, opening the possibilities to probe and quantify exosomal biomarkers at single vesicle level for clinical use. Using DISVT, we propose to quantitatively characterize integrin-carrying exosomes in plasma samples from locally-advanced BC patients who developed metastatic recurrence within three to five years of diagnosis and compare with a group of patient controls and healthy control. We will characterize eight integrin markers based on their potential for organotropic metastasis prediction. If successful, this project will 1) lead to a facile and robust SVT that can be used for basic vesicle research and clinical use, and 2) largely expand our understanding of the clinical value of exosomal integrins for the prediction of BC metastasis and organotropism by providing a map of single exosome integrin profiles for locally-advanced, advanced, and early-stage patients. The outcomes may accelerate the translation of exosomal biomarkers for cancer diagnostics, prognostics, and monitoring.