  Extracellular Vesicles (EVs) carry cell-derived proteins that confer
functionality and selective cell uptake. However, whether proteins are packaged
stochastically or co-enriched within individual EVs, and whether co-enrichment
fluctuates under homeostasis and disease, has not been measured. EV abundance
and protein global relative expression have been qualified by bulk analysis.
Meanwhile, co-enrichment is not directly accessible via bulk measurement and
has not been reported for single EV analysis. Here, we introduce the normalized
index of co-enrichment (NICE) to measure protein co-enrichment. NICE was
derived by (i) capturing EVs based on the expression of a membrane-bound
protein, (ii) probing for the co-expression of a second protein at the
population level - EV integrity underwrites the detection of single EV
co-expression without the need to resolve single EVs - and (iii) normalizing
measured values using two universal normalization probes. Axiomatically, NICE =
1 for stochastic inclusion or no overall co-enrichment, while for positive and
negative co-enrichment NICE > 1 or < 1, respectively. We quantified the NICE of
tetraspanins, growth factor receptors and integrins in EVs of eight breast
cancer cell lines of varying metastatic potential and organotropism,
combinatorially mapping up to 104 protein pairs. Our analysis revealed protein
enrichment and co-expression patterns consistent with previous findings. For
the organotropic cell lines, most protein pairs were co-enriched on EVs, with
the majority of NICE values between 0.2 to 11.5, and extending from 0.037 to
80.4. Median NICE were either negative, neutral or positive depending on the
cells. NICE analysis is easily multiplexed and is compatible with microarrays,
bead-based and single EV assays. Additional studies are needed to deepen our
understanding of the potential and significance of NICE for research and
clinical uses.
