Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival

Pancreatic tumors are highly desmoplastic and poorly-vascularized, and therefore must develop adaptive mechanisms to sustain their survival under hypoxic condition. Extracellular vesicles (EV) play vital roles in pancreatic tumor pathobiology by facilitating intercellular communication. Here we studied the effect of hypoxia on the release of EVs and examined their role in adaptive survival of pancreatic cancer (PC) cells. Hypoxia promoted the release of EV in PC cell lines, MiaPaCa and AsPC1, wherein former exhibited a far greater induction. Moreover, a time-dependent, measurable and significant increase was recorded for small EV (SEV) in both the cell lines with only minimal induction observed for medium (MEV) and large EVs (LEV). Similarly, noticeable changes in size distribution of SEV were also recorded with a shift toward smaller average size under extreme hypoxia. Thrombospondin (apoptotic bodies marker) was exclusively detected on LEVs, while Arf6 (microvesicles marker) was mostly present on MEV with some expression in LEV as well. However, CD9 and CD63 (exosome markers) were expressed in both SEV and MEVs with a decreased expression recorded under hypoxia. Among all subfractions, SEV was the most bioactive in promoting the survival of hypoxic PC cells and hypoxia-inducible factor-1α stabilization was involved in heightened EV release under hypoxia and for their potency to promote hypoxic cell survival. Altogether, our findings provide a novel mechanism for the adaptive hypoxic survival of PC cells and should serve as the basis for future investigations on broader functional implications of EV.     

Extracellular vesicle‐mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling

Primary open‐angle glaucoma is a leading cause of irreversible blindness, often associated with increased intraocular pressure. Extracellular vesicles (EVs) carry a specific composition of proteins, lipids and nucleotides have been considered as essential mediators of cell‐cell communication. Their potential impact for crosstalk between tissues responsible for aqueous humour production and out‐flow is largely unknown. The study objective was to investigate the effects of EVs derived from non‐pigmented ciliary epithelium (NPCE) primary cells on the expression of Wnt proteins in a human primary trabecular meshwork (TM) cells and define the mechanism underlying exosome‐mediated regulation that signalling pathway. Consistent with the results in TM cell line, EVs released by both primary NPCE cells and NPCE cell line showed diminished pGSK3β phosphorylation and decreased cytosolic levels of β‐catenin in primary TM cells. At the molecular level, we showed that NPCE exosome treatment downregulated the expression of positive GSKβ regulator‐AKT protein but increased the levels of GSKβ negative regulator‐PP2A protein in TM cells. NPCE exosome protein analysis revealed 584 miRNAs and 182 proteins involved in the regulation of TM cellular processes, including WNT/β‐catenin signalling pathway, cell adhesion and extracellular matrix deposition. We found that negative modulator of Wnt signalling miR‐29b was abundant in NPCE exosomal samples and treatment of TM cells with NPCE EVs significantly decreased COL3A1 expression. Suggesting that miR‐29b can be responsible for decreased levels of WNT/β‐catenin pathway. Overall, this study highlights a potential role of EVs derived from NPCE cells in modulating ECM proteins and TM canonical Wnt signalling.     

A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumour and immune cell communication

Cancer-derived extracellular vesicles (EVs) have emerged as important mediators of tumour-host interactions, and they have been shown to exert various functional effects in immune cells. In most of the studies on human immune cells, EVs have been isolated from cancer cell culture medium or patients' body fluids and added to the immune cell cultures. In such a setting, the physiological relevance of the chosen EV concentration is unknown and the EV isolation method and the timing of EV administration may bias the results. In the current study we aimed to develop an experimental cell culture model to study EV-mediated effects in human T and B cells at conditions mimicking the tumour microenvironment. We constructed a human prostate cancer cell line PC3 producing GFP-tagged EVs (PC3-CD63-GFP cells) and developed a 3D heterotypic spheroid model composed of PC3-CD63-GFP cells and human peripheral blood mononuclear cells (PBMCs). The transfer of GFP-tagged EVs from PC3-CD63-GFP cells to the lymphocytes was analysed by flow cytometry and fluorescence imaging. The endocytic pathway was investigated using three endocytosis inhibitors. Our results showed that GFP-tagged EVs interacted with a large fraction of B cells, however, the majority of EVs were not internalised by B cells but rather remained bound at the cell surface. T cell subsets differed in their ability to interact with the EVs - 15.7–24.1% of the total CD3⁺ T cell population interacted with GFP-tagged EVs, while only 0.3–5.8% of CD8⁺ T were GFP positive. Furthermore, a fraction of EVs were internalised in CD3⁺ T cells via macropinocytosis. Taken together, the heterotypic PC3-CD63-GFP and PBMC spheroid model provides the opportunity to study the interactions and functional effects of cancer-derived EVs in human immune cells at conditions mimicking the tumour microenvironment.     

Extracellular matrix (ECM) stiffness and degradation as cancer drivers

Alteration in the density and composition of extracellular matrix (ECM) occurs in tumors. The alterations toward both stiffness and degradation are contributed to tumor growth and progression. Cancer-associated fibroblasts (CAFs) are the main contributors to ECM stiffness and degradation. The cells interact with almost all cells within the tumor microenvironment (TME) that could enable them to modulate ECM components for tumorigenic purposes. Cross-talks between CAFs with cancer cells and macrophage type 2 (M2) cells are pivotal for ECM stiffness and degradation. CAFs induce hypoxia within the TME, which is one of the key inducers of both stiffness and degradation. Cancer cell modulatory roles in integrin receptors are key for adjusting ECM constituents to either fates. Cancer cell proliferation, migration, and invasion as well as angiogenesis are consequences of ECM stiffness and degradation. ECM stiffness in a transforming growth factor-β (TGF-β) related pathway could make a bridge in the basement membrane, and ECM degradation in a matrix metalloproteinase (MMP)-related pathway could make a path in the TME, both of which contribute to cancer cell invasion. ECM stiffness is also obstructive for drug penetration to the tumor site. Therefore, it would be a promising strategy to make a homeostasis in ECM for easy penetration of chemotherapeutic drugs and increasing the efficacy of antitumor approaches. MMP and TGF-β inhibitors, CAF and M2 reprogramming toward their normal counterparts, reduction of TME hypoxia and hampering integrin signaling are among the promising approaches for the modulation of ECM in favor of tumor regression.     

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non–small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6β4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.     

Pro-inflammatory cytokines IL-6 and CCL2 suppress expression of circadian gene Period2 in mammary epithelial cells

Chronic inflammation is known to contribute to tumor initiation and cancer progression. In breast tissue, the core circadian gene Period (PER)2 plays a critical role in mammary gland development and possesses tumor suppressor function. Interleukin (IL)-6 and C-C motif chemokine ligand (CCL) 2 are among the most abundant cytokines in the inflammatory microenvironment. We found that acute stimulation by IL-6/CCL2 reduced PER2 expression in non-tumorigenic breast epithelial cells. Longer term exposure to IL-6/CCL2 suppressed PER2 to an even lower level. IL-6 activated STAT3/NFκB p50 signaling to recruit HDAC1 to the PER2 promoter. CCL2 activated the PI3K/AKT pathway to promote ELK-1 cytoplasm-to-nucleus translocation, recruit HDAC1 to the proximal PER2 promoter and facilitate DNMT3-EZH2-PER2 promoter association. Ectopic expression of PER2 inhibited IL-6 or CCL2 induced mammosphere forming ability and reduced sphere size indicating that PER2 repression in breast epithelial cells can be crucial to activate tumorigenesis in an inflammatory microenvironment. The diminished expression of PER2 can be observed over a time scale of hours to weeks following IL-6/CCL2 stimulation suggesting that PER2 suppression occurs in the early stage of the interaction between an inflammatory microenvironment and normal breast epithelial cells. These data show new mechanisms by which mammary cells interact with a cancerous microenvironment and provide additional evidence that PER2 expression contributes to breast tumorigenesis.