Exosomes Take Disease Modeling to the Next Level: From the Cage to the Plate

Historically, the most common methods for studying a disease process have involved developing an animal model and analyzing tissue histology and biochemistry for alterations in protein expression, but a small development in cell biology is poised to make a big impact on the way we think about and study diseases.  Exosomes are small (<120 nm), membrane-bound vesicles that are released by cells into the interstitial fluid between and surrounding cells.  Because of the way that they are formed, being pinched off from endosomes and fused with the plasma membrane, exosomes contain a sampling of the DNAs, RNAs, and proteins found in their cell of origin, including cell surface markers.1 By isolating the exosomes, typically from a liquid biopsy, it’s possible to analyze the constituent macromolecules found in the exosomes to draw conclusions about the health of their cells, organs, or organisms of origin.2  This means, rather than harvesting a mouse’s kidney, cutting serial sections or running a Western blot, and staining with a phenotypic marker of disease, it’s possible to culture disease-specific cell lines, isolate exosomes from conditioned cell culture medium, and profile the proteins, DNAs, and RNAs found therein.3  Higher throughput, lower cost, and human-relevant, disease-specific details are all reasons to consider exploring the world of exosomes. 

Exosomes in Disease

Just as exosomes are released by most, if not all, healthy human cells, they are also released by unhealthy, stressed, and diseased cells.  It follows, then, that the cargo contained inside exosomes released by such cells would be reflective of that unhealthy, stressed, or diseased state.  Indeed, liquid biopsies have confirmed that circulating exosomes from patients with certain diseases can contain aberrant proteins4-6 and disease-promoting nucleic acids.7, 8 These states have even been demonstrated to be transmitted from organism to organism in the wake of exosome transfusion.6

During the transition from health to disease, the constituent macromolecules found within exosomes shift to reflect the altered homeostasis of disease burden9, 10, while the number of exosomes released increases;11 a phenomenon caused by increased intracellular sodium in, what may be, an attempt at signaling cellular distress to nearby cells. Therefore, isolating and profiling exosomes from cells or tissues of a known pathology can shed light not only on the biological function of these cellular messengers, but on the identities of possible therapeutic targets that were previously undefined.12

Recapitulating Disease In Vitro

While it’s true that some exosome disease research is conducted directly from patient-derived fluid samples (urine, saliva, blood, etc.), there is another method for analyzing the exosomal profile of various diseases.  Cell lines in culture have been demonstrated to release exosomes into the culture medium, and evidence points to the comparability of cell-line derived exosomes and patient-derived exosomes for similar diagnoses.13-17 Cell lines harvested from patients with a particular disease appear to maintain their exosome profile over time, consistently releasing exosomes of the same composition.  Several diseases’ exosome profiles have been characterized,18-23 allowing the extrapolation from cell line-released exosomes to liquid biopsy-derived exosomes, and enabling the use of non- or minimally invasive fluid samples as diagnostic tests for exosome biomarkers of disease.

Cell lines are derived from tissue samples, usually in the wake of a surgical tissue biopsy or resection of diseased tissue. Some disease-specific cell lines are capable of surviving in culture for extended periods due to the spontaneous immortalization exhibited by tumor cells, but cells may be immortalized in the laboratory by inducing the expression of an oncogene, like E1A.  Normal or healthy cell lines are created in this way.  Primary cell lines, on the other hand, are not immortal, and are only capable of dividing a handful of times before either senescing or undergoing cell death. For this reason, primary cells are not an ideally reproducible source of exosomes for study in culture, because their preparation cannot be adequately controlled and replicated.  Best practices dictate the comparison of sufficiently similar cell lines, such that only the disease state should be influencing exosome profile; compare exosomes produced by a melanoma cell line with exosomes produced by a melanocyte cell line, not a colon cancer cell line, for the most interpretable data. 

Engineered cell lines can be exploited to generate exosomes by modifying an existing cell line and altering or specifying its exosomes’ cargoes, using a variety of molecular biology and genome editing techniques.  Examples of using modified cell lines to produce custom exosomes include conjugation of fluorescent or bioluminescent proteins to follow exosome trafficking and altering the sequence of cargo protein, DNA, or RNA for the downstream study of exosomal function.24

Phenotypic Analysis: The Magic of Markers

Generating and isolating exosomes is only the first step to characterizing disease in vitro.  The next step involves profiling the isolated exosomes with a series of phenotypic markers, to confirm the exosome’s cell of origin, and to compare the alterations in the profile of the disease-state cell line with profile of the normal-state cell line.  The phenotypic markers used to characterize the exosomes’ profiles are antibodies that recognize proteins found in the exosome’s lipid bilayer and vesicular compartment. 

While there is no consensus on a panel of markers that can confirm the identity of an exosome, there are several markers that are used to identify exosomes.  Markers like CD9, C81, MHC Class 2 are some of the commonly accepted markers of exosomes.  Exosome identification can also be a process of exclusion by excluding microvesicles postitive for GM130 and calnexin, which are derived from portions of the cell of origin that are not involved in exosome biogenesis.  For specific cell-of-origin details, exosomes may also be stained for lineage- or cell line-specific markers to specifically identify the cell type from which they were released.

Imaging flow cytometry is a common method for the analysis of phenotypically identified exosomes, which can confirm the size and shape of the exosome, but exosomes may be analyzed by immunofluorescence, electron microscopy, Western blotting, or ELISA.  Polymerase chain reaction (PCR) may also be used to characterize the nucleic acid component of exosomes.

Characterizing disease with cell line-derived exosomes offers several advantages over traditional methods of disease-state analysis, including higher throughput, lower cost, and direct applicability to human disease.  For additional information about exosomes in disease research, read about exosomes as nanomaterials for drug delivery.

References:

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