Characterization of exosome surface proteins related to colon cancer using proximity barcoding assay to profile single-exosomes

Colorectal cancer (CRC) is a common disease with high incidence around the world. A large body of evidence reveals exosomes play vital roles in regulating CRC proliferation, metastasis, and prognosis. It is generally acceptable that exosomes are heterogeneous in size, membrane composition, content, and function. Proximity barcoding assay (PBA) is a mass spectrometry-free approach that can resolve exosome surface proteomes at a single exosome level. For the first time, we have initiated a systematic investigation to profile and cluster the CRC-related exosomes in vitro using PBA. The study goals at classifying exosome subtypes secreted from CRC cell lines and clarifying differences in exosome subtypes among cancer cell lines.

A total of 9 CRC and 2 gastric cancer (GC) cell lines were cultured and the corresponding exosomes were harvested through differential ultracentrifugation. After assessment with nanoparticle tracking analysis and transmission electron microscopy, the prepared exosomes were loaded onto microtiters followed by adding PBA probes with 262 antibodies. Then the mixture incubated with RCA products was put into a PCR machine to amplify the tag signals. The MGI T7 sequencer was employed for acquiring sequence and readout information. The sequencing data was treated by EVisualizer, and the treated data was further statistically evaluated by GraphPad Prism.

The exosomes measured by PBA in individual cancer cell lines varied very much, from 0.2 to 5.2 million. Of 262 exosome biomarkers, only 27 proteins were identified in the exosomes of these cell lines if a cutoff set at S/N >= 30. To inquire about whether a cell line secrets similar exosomes or not, the qualitative and quantitative surface proteins in the exosomes from individual cell lines were clustered under unsupervised mode using Partitioning Around Medoids. Of these CRC cell lines, 5 exhibited simply hierarchical subgroups of exosomes with 2-4 clusters, while the other cell lines had exosome clusters around 20. The next question was whether CRC cell lines shared similar exosome subgroups. There were 5 proteins generally detected in these exosomes, EPCAM (58.7%), CD151 (50.0%), ITGB1 (46.7%), ITGA6 (27.2%) and ITGB4 (23.9%). Thus, the patterns of their abundance distribution could decide the exosome subgroups. For instance, based on the expression level of CD151, the exosomes in CRC cell lines were broadly divided into two categories, while the other four exosome biomarkers were decisive factors to several exosome subgroups. With the same approach, the single exosomes from two GC cell lines were examined as well, indicating that CD151 was consistently identified in these exosomes, however, THBD and EGFR were the key proteins to define the exosome subgroups of GC cell lines. This study, therefore, offers the convincing evidence that the CRC cell lines indeed share some exosome subgroups, leading to a hypothesis of exosome subgroups involved in cancer metastasis and therapy.