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Extracellular vesicles (EVs) have attracted great attention as promising intracellular drug delivery carriers. While the endocytic pathways of small EVs (sEVs, <200 nm) have been reported, there is limited understanding of large EVs (lEVs, >200 nm), despite their potential applications for drug delivery. Additionally, the low yield of EVs during isolation remains a major challenge in their application. Herein, we aimed to compare the endocytic pathways of sEVs and lEVs using MIA PaCa-2 pancreatic cancer cell-derived EVs as models and to explore the efficiency of their production. The cellular uptake of EVs by MIA PaCa-2 cells was assessed and the pathways were investigated with the aid of endocytic inhibitors. The yield and protein content of sEVs and lEVs from the Integra CELLine culture system and the conventional flasks were compared. Our findings revealed that both sEVs and lEVs produced by the Integra CELLine system entered their parental cells via multiple routes, including caveolin-mediated endocytosis, clathrin-mediated endocytosis, and actin-dependent phagocytosis or macropinocytosis. Notably, caveolin- and clathrin-mediated endocytosis were more prominent in the uptake of sEVs, while actin-dependent phagocytosis and macropinocytosis were significant for both sEVs and lEVs. Compared with conventional flasks, the Integra CELLine system demonstrated a 9-fold increase in sEVs yield and a 6.5-fold increase in lEVs yield, along with 3- to 4-fold higher protein content per 1010 EVs. Given that different endocytic pathways led to distinct intracellular trafficking routes, this study highlights the unique potentials of sEVs and lEVs for intracellular cargo delivery. The Integra CELLine proves to be a highly productive and cost-effective system for generating EVs with favourable properties for drug delivery. [ABSTRACT FROM AUTHOR]

Beta7-regulated adhesive associations between lymphocytes and endothelial cells determine the gut-associated lymphoid tissue formation. In mice deficient for the beta7 integrin subfamily of adhesion molecules (alpha4beta7 and alphaEbeta7), the development of lymphocytes remains normal. However, the number of intra-epithelial lymphocytes of the intestine (IELs) decreases in these mice. This occurs because alpha4beta7 regulates prothymocyte migration from bone marrow to thymus, and alphaEbeta7 immobilizes a number of IELs.

Abstract Background The phosphatidylinositol-3-kinase (PI3K-PKB), mitogen activated protein kinase (MEK-ERK) and the mammalian target of rapamycin (mTOR- p70S6K), are thought to regulate many aspects of tumour cell proliferation and survival. We have examined the utilisation of these three signalling pathways in a number of cell lines derived from patients with metastatic malignant melanoma of known PIK3CA, PTEN, NRAS and BRAF mutational status. Methods Western blotting was used to compare the phosphorylation status of components of the PI3K-PKB, MEK-ERK and mTOR-p70S6K signalling pathways, as indices of pathway utilisation. Results Normal melanocytes could not be distinguished from melanoma cells on the basis of pathway utilisation when grown in the presence of serum, but could be distinguished upon serum starvation, where signalling protein phosphorylation was generally abrogated. Surprisingly, the differential utilisation of individual pathways was not consistently associated with the presence of an oncogenic or tumour suppressor mutation of genes in these pathways. Conclusion Utilisation of the PI3K-PKB, MEK-ERK and mTOR-p70S6K signalling pathways in melanoma, as determined by phosphorylation of signalling components, varies widely across a series of cell lines, and does not directly reflect mutation of genes coding these components. The main difference between cultured normal melanocytes and melanoma cells is not the pathway utilisation itself, but rather in the serum dependence of pathway utilisation.

BIRC5/Survivin is known as a dual cellular functions protein that directly regulates both apoptosis and mitosis in embryonic cells during embryogenesis and in cancer cells during tumorigenesis and tumor metastasis. However, BIRC5 has seldom been demonstrated as a direct macroautophagy/autophagy regulator in cells. ATG7 expression and ATG12–ATG5-ATG16L1 complex formation are crucial for the phagophore elongation during autophagy in mammalian cells. In this study, we observed that the protein expression levels of BIRC5 and ATG7 were inversely correlated, whereas the expression levels of BIRC5 and SQSTM1/p62 were positively correlated in normal breast tissues and tumor tissues. Mechanistically, we found that BIRC5 negatively modulates the protein stability of ATG7 and physically binds to the ATG12–ATG5 conjugate, preventing the formation of the ATG12–ATG5-ATG16L1 protein complex in human cancer (MDA-MB-231, MCF7, and A549) and mouse embryonic fibroblast (MEF) cells. We also observed a concurrent physical dissociation between BIRC5 and ATG12–ATG5 (but not CASP3/caspase-3) and upregulation of autophagy in MDA-MB-231 and A549 cells under serum-deprived conditions. Importantly, despite the fact that upregulation of autophagy is widely thought to promote DNA repair in cells under genotoxic stress, we found that BIRC5 maintains DNA integrity through autophagy negative-modulations in both human cancer and MEF cells under non-stressed conditions. In conclusion, our study reveals a novel role of BIRC5 in cancer cells as a direct regulator of autophagy. BIRC5 may act as a "bridging molecule", which regulates the interplay between mitosis, apoptosis, and autophagy in embryonic and cancer cells. ACTA1: actin; ATG: autophagy related; BIRC: baculoviral inhibitor of apoptosis repeat-containing; BAF: bafilomycin A1; CQ: chloroquine; CASP3: caspase 3; HSPB1/Hsp27: heat shock protein family B (small) member 1/heat shock protein 27; IAPs: inhibitors of apoptosis proteins; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PLA: proximity ligation assay; SQSTM1/p62: sequestosome 1; siRNA: small interfering RNA [ABSTRACT FROM AUTHOR]