Discovery of Large Extracellular Vesicles Blebbisomes and Their Functional Study

Immunology Cell Biology Oncology Life Sciences Medicine
extracellular vesicles Blebbisomes intercellular communication mitochondria immune checkpoint proteins

Academic Background

Extracellular vesicles (EVs) are crucial mediators of intercellular communication, carrying proteins, lipids, and genetic information to participate in various physiological and pathological processes. However, our understanding of EVs remains incomplete, particularly regarding the study of exceptionally large EVs. To address this gap, researchers have explored a novel type of exceptionally large EVs—Blebbisomes. Blebbisomes exhibit unique “blebbing” behavior and contain various functional organelles, such as mitochondria and multivesicular endosomes, but lack a distinct nucleus. This discovery opens new avenues for research in intercellular communication, especially in the fields of cancer immune evasion and cell-autonomous communication.

Source of the Paper

This paper was co-authored by Dennis K. Jeppesen, Zachary C. Sanchez, and other researchers from Vanderbilt University Medical Center and Vanderbilt University School of Medicine. It was published in Nature Cell Biology in March 2025, titled “Blebbisomes are large, organelle-rich extracellular vesicles with cell-like properties,” with the DOI 10.1038/s41556-025-01621-0.

Research Process and Results

1. Discovery and Characterization of Blebbisomes

While studying vesicles released by cancer cells, researchers observed a type of exceptionally large EVs with diameters up to 20 micrometers, which exhibited continuous “blebbing” behavior. They named these vesicles Blebbisomes. Using differential interference contrast microscopy (DIC) and scanning electron microscopy (SEM), researchers found that the formation mechanism of Blebbisomes differs from traditional vesicle release. They are separated from cells through cellular retraction events rather than direct vesicle budding. Additionally, Blebbisomes contain functional mitochondria that generate ATP, supporting their continuous blebbing activity.

Key Results:
- The average diameter of Blebbisomes is 10 micrometers, with a maximum of 20 micrometers.
- Fluorescence microscopy and super-resolution microscopy (iSIM) confirmed that Blebbisomes contain functional mitochondria.
- Experiments showed that Blebbisomes form through the contractile mechanism of non-muscle myosin IIb.

2. Proteomic Analysis of Blebbisomes

To further investigate the composition of Blebbisomes, researchers purified them using high-resolution density gradient fractionation and performed proteomic analysis. The results revealed that Blebbisomes contain a large number of proteins associated with mitochondria, endoplasmic reticulum, Golgi apparatus, and ribosomes, but have lower levels of nuclear membrane proteins compared to cells. Additionally, proteins related to immune evasion, such as CD47 and CD73, were detected in Blebbisomes.

Key Results:
- The protein composition of Blebbisomes resembles that of cells but lacks nuclear membrane proteins.
- Blebbisomes contain various organelle-related proteins, such as VDAC2 (mitochondria), Calreticulin (endoplasmic reticulum), and TGN protein 2 (Golgi apparatus).
- Immunoblot analysis showed that Blebbisomes contain immune evasion-related proteins like CD47 and CD73.

3. Distinction Between Blebbisomes and Other Large EVs

Researchers compared Blebbisomes with another type of large EVs—Large Oncosomes. The results showed that Large Oncosomes do not exhibit “blebbing” behavior and have dysfunctional mitochondria. Moreover, Blebbisomes contain more proteins related to the endoplasmic reticulum and Golgi apparatus, while Large Oncosomes have lower levels of these proteins.

Key Results:
- Large Oncosomes do not exhibit “blebbing” behavior and have dysfunctional mitochondria.
- Blebbisomes contain more proteins related to the endoplasmic reticulum and Golgi apparatus.

4. Organelle Composition of Blebbisomes

Using transmission electron microscopy (TEM) and immunofluorescence, researchers found that Blebbisomes contain various organelles, including mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, multivesicular endosomes, and autophagosomes. Additionally, Blebbisomes contain RNA, further indicating their independent cellular functions.

Key Results:
- TEM images revealed that Blebbisomes contain mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and multivesicular endosomes.
- Fluorescence in situ hybridization (FISH) confirmed the presence of RNA in Blebbisomes.

5. Uptake and Secretion Functions of Blebbisomes

Researchers discovered that Blebbisomes can uptake other EVs and secrete exosomes and microvesicles. Through fluorescence labeling experiments, researchers observed internalized EVs within Blebbisomes, and immunoblot analysis confirmed that Blebbisomes can secrete exosomes and microvesicles.

Key Results:
- Blebbisomes can uptake other EVs and secrete exosomes and microvesicles.
- Immunoblot analysis showed that EVs secreted by Blebbisomes contain Syntenin-1 (exosome marker) and Annexin A1 (microvesicle marker).

6. In Vivo Presence of Blebbisomes and Immune Checkpoint Proteins

Researchers observed the release of Blebbisomes in three-dimensional collagen matrices and zebrafish embryos, and isolated Blebbisomes from mouse bone marrow. Additionally, cancer-derived Blebbisomes were found to contain various immune checkpoint proteins, such as PD-L1, PD-L2, B7-H3, VISTA, and HLA-E, which may play a role in cancer immune evasion.

Key Results:
- Blebbisomes were observed in three-dimensional collagen matrices and zebrafish embryos.
- Cancer-derived Blebbisomes contain immune checkpoint proteins such as PD-L1, PD-L2, B7-H3, VISTA, and HLA-E.

Conclusion and Significance

Blebbisomes are a novel type of exceptionally large EVs with unique “blebbing” behavior and various functional organelles. They can not only uptake other EVs but also secrete exosomes and microvesicles, indicating their important role in intercellular communication. Moreover, Blebbisomes contain multiple immune checkpoint proteins, which may play a critical role in cancer immune evasion. This discovery provides new perspectives for research in intercellular communication and cancer immunotherapy.

Research Highlights

  1. Discovery of Novel EVs: Blebbisomes are the largest EVs discovered to date, exhibiting unique “blebbing” behavior.
  2. Functional Organelles: Blebbisomes contain functional mitochondria, endoplasmic reticulum, Golgi apparatus, and other organelles.
  3. Uptake and Secretion Functions: Blebbisomes can uptake other EVs and secrete exosomes and microvesicles.
  4. Immune Checkpoint Proteins: Cancer-derived Blebbisomes contain multiple immune checkpoint proteins, which may play a significant role in cancer immune evasion.

Additional Valuable Information

Researchers also found that Blebbisomes form through the contractile mechanism of non-muscle myosin IIb, and their formation is independent of the ESCRT (Endosomal Sorting Complex Required for Transport) complex. This discovery provides new insights into the biogenesis mechanism of Blebbisomes.