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. 2023 Oct 20;12(20):2491.
doi: 10.3390/cells12202491.

Isolation and Characterization of Cow-, Buffalo-, Sheep- and Goat-Milk-Derived Extracellular Vesicles

Monisha Samuel  1   2 , Rahul Sanwlani  1   2 , Mohashin Pathan  1   2 , Sushma Anand  1   2 , Ella L Johnston  2   3 , Ching-Seng Ang  4 , Maria Kaparakis-Liaskos  2   3 , Suresh Mathivanan  1   2
Affiliations

Isolation and Characterization of Cow-, Buffalo-, Sheep- and Goat-Milk-Derived Extracellular Vesicles

Monisha Samuel et al. Cells. .

Abstract

Milk is a complex biological fluid that has high-quality proteins including growth factors and also contains extracellular vesicles (EVs). EVs are a lipid bilayer containing vesicles that contain proteins, metabolites and nucleic acids. Several studies have proposed that EVs in cow milk can survive the gut and can illicit cross-species communication in the consuming host organism. In this study, we isolated and characterized extracellular vesicles from the raw milk of the four species of the Bovidae family, namely cow, sheep, goat and buffalo, that contribute 99% of the total milk consumed globally. A comparative proteomic analysis of these vesicles was performed to pinpoint their potential functional role in health and disease. Vesicles sourced from buffalo and cow milk were particularly enriched with proteins implicated in modulating the immune system. Furthermore, functional studies were performed to determine the anti-cancer effects of these vesicles. The data obtained revealed that buffalo-milk-derived EVs induced significantly higher cell death in colon cancer cells. Overall, the results from this study highlight the potent immunoregulatory and anti-cancer nature of EVs derived from the milk of Bovidae family members.

Keywords: bovine milk EVs; cancer; extracellular vesicles; milk EV proteome; milk extracellular vesicles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Isolation and characterization of MEVs from various species of the bovidae family. (a) Schematic representation of the differential centrifugation coupled with the ultracentrifugation-based method used to isolate and purify MEVs from various species. (b) Western blot analysis of various fractions of increased density obtained after OptiPrep density gradient centrifugation probed for EV-enriched proteins Alix and TSG101. Representative images of three biological replicates.
Figure 2
Figure 2
Biophysical characterization of EVs isolated from CoM, BM, GM and SM samples. (a) Representative TEM images of EVs isolated using OptiPrep density gradient centrifugation revealed vesicles with a morphology similar to EVs. (b) NTA revealed peak EV diameters at 115, 105, 135 and 105 nm for CoM, BM, GM, and SM samples, respectively. (c) Average peak EV diameter for individual biological replicates was compared for the samples. Data are representative of three biological replicates.
Figure 3
Figure 3
Proteomics analysis of EVs isolated from CoM, BM, GM and SM samples. (a) A four-way Venn diagram of proteins distributed between CoM, BM, GM and SM EVs revealing 331 proteins common to all four samples. (b) Heatmap showing enrichment of Rab proteins in CoM, BM, GM and SM samples. The scaled expression of each protein, denoted as the row Z-score, is plotted in the red–black–green colour scale. High expression levels are indicated in red and low expression levels are shown in green. The higher abundance of Rab family proteins was observed in the BM samples. (c) Heatmap depicting enrichment of EV proteins. EV-enriched protein TSG101 was comparatively highly enriched in CoM samples, whereas other EV proteins (VPS and CHMP) involved in the budding of these vesicles are more abundant in COMM and BM samples.
Figure 4
Figure 4
EVs from BM are enriched with proteins implicated in immune response. (a) Functional enrichment analysis using FunRich revealed that various proteins associated with innate immune response, inflammatory response and cytokine secretion were highly enriched in EVs isolated from BM. * Denotes p < 0.05. (b) Histogram representing top five biological pathways for proteins exclusively present in BM and SM samples.
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