The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern's NanoSight NS300 or Particle Metrix' ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP‐IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze‐thaw cycle‐dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1‐fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% D_TEM range: 79.5–134.3) compared to ZetaView (% D_TEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP‐IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.

The biology of solid tumors is strongly determined by the interactions of cancer cells with their surrounding microenvironment. In this regard, pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) represents a paradigmatic example for the multitude of possible tumor–stroma interactions. PDAC has proven particularly refractory to novel immunotherapies, which is a fact that is mediated by a unique assemblage of various immune cells creating a strongly immunosuppressive environment in which this cancer type thrives. In this review, we outline currently available knowledge on the cross-talk between tumor cells and the cellular immune microenvironment, highlighting the physiological and pathological cellular interactions, as well as the resulting therapeutic approaches derived thereof. Hopefully a better understanding of the complex tumor–stroma interactions will one day lead to a significant advancement in patient care.

Epidemiological studies have shown a dramatic increase in the incidence and the prevalence of allergic diseases over the last several decades. Environmental triggers including risk factors (e.g., pollution), the loss of rural living conditions (e.g., farming conditions), and nutritional status (e.g., maternal, breastfeeding) are considered major contributors to this increase. The influences of these environmental factors are thought to be mediated by epigenetic mechanisms which are heritable, reversible, and biologically relevant biochemical modifications of the chromatin carrying the genetic information without changing the nucleotide sequence of the genome. An important feature characterizing epigenetically-mediated processes is the existence of a time frame where the induced effects are the strongest and therefore most crucial. This period between conception, pregnancy, and the first years of life (e.g., first 1000 days) is considered the optimal time for environmental factors, such as nutrition, to exert their beneficial epigenetic effects. In the current review, we discussed the impact of the exposure to bacteria, viruses, parasites, fungal components, microbiome metabolites, and specific nutritional components (e.g., polyunsaturated fatty acids (PUFA), vitamins, plant- and animal-derived microRNAs, breast milk) on the epigenetic patterns related to allergic manifestations. We gave insight into the epigenetic signature of bioactive milk components and the effects of specific nutrition on neonatal T cell development. Several lines of evidence suggest that atypical metabolic reprogramming induced by extrinsic factors such as allergens, viruses, pollutants, diet, or microbiome might drive cellular metabolic dysfunctions and defective immune responses in allergic disease. Therefore, we described the current knowledge on the relationship between immunometabolism and allergy mediated by epigenetic mechanisms. The knowledge as presented will give insight into epigenetic changes and the potential of maternal and post-natal nutrition on the development of allergic disease.

Extracellular vesicles released by tumor cells (T-EVs) are known to contain danger-associated molecular patterns (DAMPs), which are released in response to cellular stress to alert the immune system to the dangerous cell. Part of this defense mechanism is the heat shock protein 70 (HSP70), and HSP70-positive T-EVs are known to trigger anti-tumor immune responses. Moreover, extracellular HSP70 acts as an immunogen that contributes to the cross-presentation of major histocompatibility complex (MHC) class I molecules. However, the release of DAMPs, including HSP70, may also induce chronic inflammation or suppress immune cell activity, promoting tumor growth. Here, we summarize the current knowledge on soluble, membrane-bound, and EV-associated HSP70 regarding their functions in regulating tumor-associated immune cells in the tumor microenvironment. The molecular mechanisms involved in the translocation of HSP70 to the plasma membrane of tumor cells and its release via exosomes or soluble proteins are summarized. Furthermore, perspectives for immunotherapies aimed to target HSP70 and its receptors for cancer treatment are discussed and presented.

Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed–Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.

Extracellular vesicles released by tumor cells contribute to the reprogramming of the tumor microenvironment and interfere with hallmarks of cancer including metastasis. Notably, melanoma cell-derived EVs are able to establish a pre-metastatic niche in distant organs, or on the contrary, exert anti-tumor activity. However, molecular insights into how vesicles are selectively packaged with cargo defining their specific functions remain elusive.