Extracellular vesicles (EVs) are an exciting mode of intercellular communication that function in nearly every aspect of health and disease. Many characteristics of these particles make them particularly attractive to inform development of diagnostics and therapeutics. In health, EVs are active players in maintenance and cell signaling, functions which could be exploited for use as therapeutics or drug delivery vehicles, while in disease, EVs are often co-opted to contribute to disease progression, making them a potential target for therapeutics and a source of diagnostic markers. In parallel to our understanding of EVs’ functional diversity, there is also a growing understanding of EVs’ compositional heterogeneity both between EVs from different sources (i.e. interpopulation heterogeneity) and within EVs from a single cell source (i.e. intrapopulation heterogeneity). This heterogeneity consists of differences in chemical compositions, such as protein and nucleic acid expression, and physical characteristics, such as size and density, which can both exist across a spectrum or in distinct subpopulations. This compositional heterogeneity appears to be directly tied to EVs’ role in intercellular communication, allowing for diverse functional signaling dependent on these features. Yet, the contributions of this heterogeneity to both native function and its impact on our use of these particles in the clinic remains understudied. To this end, the objectives of this work were to identify how EV heterogeneity may contribute to disease progression and how we can exploit heterogeneity to develop more effective EV-based diagnostics and therapeutics.
To address the contribution of EV interpopulation heterogeneity to disease progression, the functions of EVs from healthy and diseased cells were compared in the context of multiple diseases. The specific targeting of EVs from breast cells known to metastasize to the brain was compared with EVs from breast cancer without organotropic metastasis and EVs from a non-cancer cell line using a novel assay combining neurons, astrocytes, and microglia. This work examined the difference in inflammatory response dependent on cell disease state and probed how inclusion of microglia altered uptake. In a similar vein, EVs from osteosarcoma cells with high metastatic potential were compared to EVs from a less metastatic cell line to understand how their interaction with macrophages may contribute to formation of the metastatic niche. This work identified potential differences within these EVs from highly metastatic cells including differential protein expression that may have contributed to the driving of macrophages to a more tumor-permissive phenotype. Finally, EVs were examined for contribution to feline calicivirus infection. Specifically, we utilized iodixanol gradient to separate EVs from co-isolated virions and reported on the contribution of each to infection. Together this work examined how interpopulation heterogeneity of EVs and their co-isolates between normal and diseased cells is co-opted during disease progression.To address the impact of EV intrapopulation heterogeneity in development of diagnostics and therapeutics, we examined EV subpopulations in the context of each. First, we reported on EV subpopulations from ovarian cancer, identifying specific subpopulations of EVs that offer lower and higher sensitivity to cancer-associated biomarkers. Second, we developed methods for quantifying heterogenous EV engineering, showing that certain methods produce different ratios of native to engineered EVs in the final product. Finally, in a systematic review, we probed the field of EV-based clinical trials focusing on the degree of incorporation of intrapopulation heterogeneity in trial design, highlighting that this is an underutilized resource. Together this work highlights how ignoring intrapopulation EV heterogeneity can weaken our use of EVs while examining how direct use of these subpopulations may allow for much more effective use of EVs in the clinic.
