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Racchetti et al. Extracell Vesicles Circ Nucleic Acids 2023;4:44-58 https://dx.doi.org/10.20517/evcna.2023.03 Page 52

functional cytoplasm. Initially, autophagosomes are assembled by the growth of de-novo-formed double
membranes. Autophagosome persistence within the cells includes the integration of markers, fusion with
endosome components, and maturation completion [75,76] . Accurately regulated processes (not shown in
Figure 2) include the final steps of autophagosome interactions with MVB and exosomes. Moreover,
autophagosomes often fuse with lysosomes, and the ensuing mixed organelles are called autolysosomes.
Upon such fusion, the cargoes of autophagosomes are degraded. Their fragments are recycled via specific
pathways [77-79] .

Autophagy induces primarily protective and recycling effects. To confer adaptation to the ever-changing
environments, their interactions and fusions need to be tightly regulated. It is clear that various human
pathologies deregulate autophagy, and its modulations have significant therapeutic potential [80,81] . Three
forms of autophagosome exist, distinguished by their different size. The one operative in intercellular
communication is the large form called the macroautophagosome. Given its unique function considered in
the present review, the active macroautophagosome form is called by the general name of the
autophagosome. The properties of the latter are summarized in 5A, traffic, and exocytoses, direct and
mediated via endo-lysosomes; 5B, the interactions established by autophagosomes with MVBs and
exosomes; 5C, the establishment and regulation of protein secretion; and 5D, the effects of autophagosomes
in various diseases, especially in cancers.

Subsection 5A: Autophagosome trafficking and exocytoses. The autophagic machinery, adapted to unable
protein trafficking and UPS secretion [20,75,76] , operates with the expression of family markers such as LC3-II,
SQSTM1/p62, and core autophagy (ATG) proteins [75,76] . Of these markers, ATG9A concentrates in a
compartment comprising clusters of vesicles and tubules and participates in the movement of cell lines.
[9]
The pH-fluorine labeling technique revealed that ATG9A is distributed toward the migration front, with
protrusive activity triggered with clathrin adapter complexes. Therefore, ATG9A governs vesicular
[82]
trafficking, allowing the expansion of cell protrusion toward the extracellular matrix .
Autophagic exocytoses have been confirmed by the demonstration of specific markers among EVs [75-77,82] .
Moreover, the primary functions of autophagosomes, including their participation in cancers and UPS
secretion, discussed in the following subsections 5C and 5D, imply the existence of autophagosome cargoes
of exocytic vesicles released to the extracellular space [9,20,77] [Figure 2]. The properties and the details of
autophagocytic exocytosis, with the involvement of the small GTPase Rab27a and the SNARE protein
Sec22b, have been characterized several times . However, autophagic exocytosis needs to be further
[76]
investigated to circumvent the limitations of previous studies. An additional process, resulting in the release
of autophagic markers, could be due to the prefusion of autophagosomes with endo-lysosomes, the form of
lysosomes competent for exocytosis. Exocytoses of autophagosome/endo-lysosome fusions have been
reported [82-85] . Therefore, although limited in extent, the contribution of this indirect exocytosis [76,85] cannot
be excluded.

Subsection 5B: Autophagosome interactions with MVBs and exosomes. Alternatively to the direct fusion
with lysosomes, autophagosomes can establish interactions with MVBs named amphisomes (mentioned
[20]
in subsection 2A). The autophagosome/MVB interactions, established in the proximity of the plasma
membrane, are significant. They increase the frequency of MVB exocytoses, which in contrast are decreased
by the removal of autophagosomes and by their inhibitors [20,86,87] . This cooperation occurs in many types of
cells, including some plants .
[88]