Profiling of Integrin Isoforms on Lung‐Tropic Exosomes by Spectrally and Kinetically Multiplexed Single‐Molecule Imaging

Since the first report that integrins on exosomes can potentially dictate their organ-tropic transport, there have been great interest in obtaining a better understanding of this phenomenon. However, integrins have many isoforms, which are heterogeneously distributed among individual exosomes with relatively low abundances. As a result, it is difficult to profile their expression landscape at single exosome level and study their relationship with organ specificity. To overcome this limitation, a spectrally and kinetically multiplexed single-molecule imaging method is developed, which for the first time achieved simultaneously imaging of 12 exosomal proteins at an unparalleled single-copy resolution, allowing systematic profiling of integrins on individual exosomes. Using this method, integrin expression profile of various types of cellular exosomes are characterized. Using machine learning algorithms, clustering analysis is performed to identify key subpopulations of each type of exosomes. It is found that co-occurrence integrin α6 and the pairing β counterpart on single exosomes is essential for effective lung targeting. In contrast, exosomes with primary αv dimer or unpaired integrins show almost no lung targeting behavior. Overall, the method allows profiling of integrins on single exosomes with unprecedented resolution. The information gained can facilitate the development of exosome-based biopsies and therapies.