Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition

Abstract

Extracellular shed vesicles (ESVs) facilitate a unique mode of cell cell communication wherein vesicle uptake can induce a change in the recipient cell’s state. Despite the intensity of ESV research, currently reported data represent bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo, and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

A Tale of Two Glutaminases: Homologous Enzymes with Distinct Roles in Tumorigenesis

“…A novel role of GLS in cancer is in the formation of microvesicles or oncosomes, nontraditional secretory vesicles which mediate signaling between cancer cells and their environment [98]. Production of microvesicles by cultured cancer cells can be slowed or halted via glutaminase inhibition [99].…”

Glutamine Metabolism in Cancer: Understanding the Heterogeneity

“…These fibroblasts boost glutamine synthesis from atypical sources to feed adjacent cancer cells [57]. Nevertheless, tumors that utilize macropinocytosis or uptake extracellular vesicles to acquire glutamine may still require glutaminase and exhibit sensitivity to its inhibition [18, 58]. …”

Decorrelation correction for nanoparticle tracking analysis of dilute polydisperse suspensions in bulk flow

“…For example, the study of small molecule inhibitors that suppress microvesicle production are greatly benefited by accurate size measurements of dilute, polydisperse suspensions [33]. Similarly, microdevices using size-based separation methods to nanoparticle populations for downstream assaying must use accurate cutoff diameters in the design process [49][50][51].…”

“…Accurate measurement of very low numbers of large particles in a bath of smaller particles is an important practical concern for NTA, especially for the characterization of tumor-derived extracellular vesicles, which are dilute, polydisperse and measured in applied bulk flow [33,34]. Importantly, the large-diameter portion of the PSD has typically been reported as a small tail of the distribution or a small secondary peak [37], and thus these particles are likely strongly affected by the biasing effect described herein.…”

“…De-drifting in nanoparticle tracking analysis (NTA) of highly disperse suspensions necessarily uses ensembles of particles with different diffusive mobility. Improved measurement accuracy could be influential for characterizing the PSD of biological extracellular vesicles, where dilute, polydisperse particle suspensions are commonly measured in applied fluid flow [33,34]. In addition, de-drifting in passive particle tracking microrheology of complex media with heterogeneous viscoelastic properties may require ensembles of monodisperse probes that are localized to regions with uniform collective motion but different rheological properties.…”