Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution.

Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that contribute to intercellular communication by means of the cargo that they carry. To resolve EV heterogeneity and determine if cargo is partitioned into select EV populations, we developed a method named "EV Fingerprinting" that discerns distinct vesicle populations using dimensional reduction of multiparametric data collected by quantitative single-EV flow cytometry. EV populations were found to be discernible by a combination of membrane order and EV size, both of which were obtained through multiparametric analysis of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated into the lipid bilayer. Molecular perturbation of EV secretion and biogenesis through respective ablation of the small GTPase Rab27a and overexpression of the EV-associated tetraspanin CD63 revealed distinct and selective alterations in EV populations, as well as cargo distribution. While Rab27a disproportionately affects all small EV populations with high membrane order, the overexpression of CD63 selectively increased the production of one small EV population of intermediate membrane order. Multiplexing experiments subsequently revealed that EV cargos have a distinct, nonrandom distribution with CD63 and CD81 selectively partitioning into smaller vs larger EVs, respectively. These studies not only present a method to probe EV biogenesis but also reveal how the selective partitioning of cargo contributes to EV heterogeneity.

Exosomes, microvesicles, and other extracellular vesicles-a Keystone Symposia report.

Extracellular vesicles (EVs) are small, lipid-bilayer-bound particles released by cells that can contain important bioactive molecules, including lipids, RNAs, and proteins. Once released in the extracellular environment, EVs can act as messengers locally as well as to distant tissues to coordinate tissue homeostasis and systemic responses. There is a growing interest in not only understanding the physiology of EVs as signaling particles but also leveraging them as minimally invasive diagnostic and prognostic biomarkers (e.g., they can be found in biofluids) and drug-delivery vehicles. On October 30-November 2, 2022, researchers in the EV field convened for the Keystone symposium "Exosomes, Microvesicles, and Other Extracellular Vesicles" to discuss developing standardized language and methodology, new data on the basic biology of EVs and potential clinical utility, as well as novel technologies to isolate and characterize EVs.

A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells.

Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader-follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.

The importance of developing therapies targeting the biological spectrum of metastatic disease.

Great progress has been made in cancer therapeutics. However, metastasis remains the predominant cause of death from cancer. Importantly, metastasis can manifest many years after initial treatment of the primary cancer. This is because cancer cells can remain dormant before forming symptomatic metastasis. An important question is whether metastasis research should focus on the early treatment of metastases, before they are clinically evident ("overt"), or on developing treatments to stop overt metastasis (stage IV cancer). In this commentary we want to clarify why it is important that all avenues of treatment for stage IV patients are developed. Indeed, future treatments are expected to go beyond the mere shrinkage of overt metastases and will include strategies that prevent disseminated tumor cells from emerging from dormancy.

Modulation of cell adhesion and migration through regulation of the immunoglobulin superfamily member ALCAM/CD166.

In epithelial-derived cancers, altered regulation of cell-cell adhesion facilitates the disruption of tissue cohesion that is central to the progression to malignant disease. Although numerous intercellular adhesion molecules participate in epithelial adhesion, the immunoglobulin superfamily (IgSF) member activated leukocyte cell adhesion molecule (ALCAM), has emerged from multiple independent studies as a central contributor to tumor progression. ALCAM is an archetypal member of the IgSF with conventional organization of five Ig-like domains involved in homo- and heterotypic adhesions. Like many IgSF members, ALCAM is broadly expressed and involved in cellular adhesion across many cellular processes. While the redundancy of intercellular adhesion molecules (CAMs) could diminish the impact of any single CAM, consistent correlation between ALCAM expression and patient outcome for multiple cancers underscores its role in tumor progression. Unlike most oncogenes and tumor suppressors, ALCAM is neither mutated nor amplified or deleted. Experimental disruption of ALCAM-mediated adhesions implies that this IgSF member contributes to tumor progression through dynamic turnover of the protein at the cell surface. Since ALCAM is not frequently altered at the gene level, it appears to promote malignant behavior through regulation of its availability rather than its specific activity. These observations help explain its heterogeneous expression within malignant disease and the drastic changes in protein levels across tumor progression. To reveal how ALCAM contributes to tumor progression, we review regulation of its gene expression, alternative splicing, targeted proteolysis, binding partners, and surface shedding within the context of cancer. Studying ALCAM regulation has led to a novel understanding of the fine-tuning of cell adhesive state through the utilization of otherwise normal regulatory processes, which thereby enable tumor cell invasion and metastasis.

AXL Mediates Esophageal Adenocarcinoma Cell Invasion through Regulation of Extracellular Acidification and Lysosome Trafficking.

Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that is characterized by resistance to chemotherapy and a poor clinical outcome. The overexpression of the receptor tyrosine kinase AXL is frequently associated with unfavorable prognosis in EAC. Although it is well documented that AXL mediates cancer cell invasion as a downstream effector of epithelial-to-mesenchymal transition, the precise molecular mechanism underlying this process is not completely understood. Herein, we demonstrate for the first time that AXL mediates cell invasion through the regulation of lysosomes peripheral distribution and cathepsin B secretion in EAC cell lines. Furthermore, we show that AXL-dependent peripheral distribution of lysosomes and cell invasion are mediated by extracellular acidification, which is potentiated by AXL-induced secretion of lactate through AKT-NF-κB-dependent MCT-1 regulation. Our novel mechanistic findings support future clinical studies to evaluate the therapeutic potential of the AXL inhibitor R428 (BGB324) in highly invasive EAC.

Alternative splicing of ALCAM enables tunable regulation of cell-cell adhesion through differential proteolysis.

While many adhesion receptors are known to influence tumor progression, the mechanisms by which they dynamically regulate cell-cell adhesion remain elusive. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that a tunable mechanism of ectodomain shedding regulates its contribution to dissemination. To test this hypothesis, we examined an under-explored ALCAM splice variant (ALCAM-Iso2) and demonstrated that loss of the membrane-proximal region of ALCAM (exon 13) increased metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent membrane distal cleavage site in ALCAM-Iso2, which mediated a ten-fold increase in shedding, thereby decreasing cellular cohesion. Importantly, the loss of cohesion is not limited to the cell capable of shedding because the released extracellular domain diminished cohesion of non-shedding cells through disruption of ALCAM-ALCAM interactions. ALCAM-Iso2-dominated expression in bladder cancer tissue, compared to normal bladder, further emphasizes that ALCAM alternative splicing may contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of tumor cell adhesion is a locally tunable process.

Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues.

Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5 pathways were enriched in African American prostate tumors: the Small Cell Lung Cancer, Platelet-Amyloid Precursor Protein, Agrin, Neuroactive Ligand-Receptor Interaction, and Intrinsic pathways. The protein components of these pathways were either basement membrane proteins or coagulation proteins.

Differentially Expressed Genes and Signature Pathways of Human Prostate Cancer.

Genomic technologies including microarrays and next-generation sequencing have enabled the generation of molecular signatures of prostate cancer. Lists of differentially expressed genes between malignant and non-malignant states are thought to be fertile sources of putative prostate cancer biomarkers. However such lists of differentially expressed genes can be highly variable for multiple reasons. As such, looking at differential expression in the context of gene sets and pathways has been more robust. Using next-generation genome sequencing data from The Cancer Genome Atlas, differential gene expression between age- and stage- matched human prostate tumors and non-malignant samples was assessed and used to craft a pathway signature of prostate cancer. Up- and down-regulated genes were assigned to pathways composed of curated groups of related genes from multiple databases. The significance of these pathways was then evaluated according to the number of differentially expressed genes found in the pathway and their position within the pathway using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis. The "transforming growth factor-beta signaling" and "Ran regulation of mitotic spindle formation" pathways were strongly associated with prostate cancer. Several other significant pathways confirm reported findings from microarray data that suggest actin cytoskeleton regulation, cell cycle, mitogen-activated protein kinase signaling, and calcium signaling are also altered in prostate cancer. Thus we have demonstrated feasibility of pathway analysis and identified an underexplored area (Ran) for investigation in prostate cancer pathogenesis.