Histone deacetylase (HDAC)-inhibitors (HDACis) are well characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome associated myeloid leukemia (DS-AMKL) blasts. Investigating the anti-leukemic function of HDACis revealed their transcriptional and posttranslational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mTOR activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA-damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells.

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In patients with glioblastoma, the average survival time with current treatments is short, mainly due to recurrences and resistance to therapy. This insufficient treatment success is, in large parts, due to the tremendous molecular heterogeneity of gliomas, which affects the overall prognosis and response to therapies and plays a vital role in gliomas’ grading. In addition, the tumor microenvironment is a major player for glioma development and resistance to therapy. Active communication between glioma cells and local or neighboring healthy cells and the immune environment promotes the cancerogenic processes and contributes to establishing glioma stem cells, which drives therapy resistance. Besides genetic alterations in the primary tumor, tumor-released factors, cytokines, proteins, extracellular vesicles, and environmental influences like hypoxia provide tumor cells the ability to evade host tumor surveillance machinery and promote disease progression. Moreover, there is increasing evidence that these players affect the molecular biological properties of gliomas and enable inter-cell communication that supports pro-cancerogenic cell properties. Identifying and characterizing these complex mechanisms are inevitably necessary to adapt therapeutic strategies and to develop novel measures. Here we provide an update about these junctions where constant traffic of biomolecules adds complexity in the management of glioblastoma. Graphical abstract

Extracellular vesicle (EV) secretion is a highly conserved evolutionary trait in all organisms in the three domains of life. The packaging and release of EVs appears to be a bulk-flow process which takes place mainly under extreme conditions. EVs participate in horizontal gene transfer, which supports the survival of prokaryotic and eukaryotic microbes. In higher eukaryotes, almost all cells secrete a heterogeneous population of EVs loaded with various biomolecules. EV secretion is typically higher in cancer microenvironments, promoting tumor progression and metastasis. EVs are now recognized as additional mediators of autocrine and paracrine communication in health and disease. In this context, proteins and RNAs have been studied the most, but extracellular vesicle DNA (EV-DNA) has started to gain in importance in the last few years. In this review, we summarize new findings related to the loading mechanism(s), localization, and post-shedding function of EV-DNA. We also discuss the feasibility of using EV-DNA as a biomarker when performing a liquid biopsy, at the same time emphasizing the lack of data from clinical trials in this regard. Finally, we outline the potential of EV-DNA uptake and its interaction with the host genome as a promising tool for understanding the mechanisms of cancer evolution.

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.