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Mechanisms driving immunosuppression in chronic myeloid leukemia are mostly unknown. We show that leukemic extracellular vesicles (EVs) target lymphocytes and amplify suppressive function of thymic regulatory T cells, by driving expression of Foxp3 transcription factor. This could facilitate expansion of leukemic cells outside the bone marrow, leading to blast crisis.

Extracellular vesicles (EVs) are small, membranous particles that have recently emerged as one the most important mediators of intercellular communication. They can contain a variety of proteins, lipids, and nucleic acids and thus are responsible for modulation of multiple biological processes, including immune response and regulation of immune cells. Immunomodulatory activity of different EVs can be reliably assessed using EVs isolated from cell culture conditioned medium and added to in vitro or ex vivo cultures of immune cells. This article describes protocols for isolation of EVs from cell culture supernatants by differential ultracentrifugation and density gradient centrifugation. It also provides tools and protocols that enable characterization and validation of isolated particles, as well as analysis of interactions between EVs of interest and different subpopulations of human immune cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of extracellular vesicles by differential ultracentrifugation Basic Protocol 2: Isolation of extracellular vesicles by density gradient centrifugation Support Protocol 1: Imaging of extracellular vesicles using transmission electron microscopy Support Protocol 2: Detection of extracellular vesicle protein markers by Western blotting Support Protocol 3: Measurement and counting of extracellular vesicles by nanoparticle tracking analysis Basic Protocol 3: Analysis of extracellular vesicle uptake or association by different subpopulations of lymphocytes in vitro.

The unique bone marrow microenvironment is created by stromal cells and such physical conditions as hypoxia. Both hypoxia and interactions with stromal cells have a significant impact on the biology of leukemia cells, changing their sensitivity to antileukemic therapies. Thus, it is crucial to introduce biological systems, which enable the investigation of leukemia-stroma cross-talk and verification of novel therapies effectiveness under such bone marrow niche-mimicking conditions. Here, we have established an experimental setup based on the hypoxic co-culture of stromal cells with different cell lines derived from various leukemia patients. Flow cytometry enables simultaneous fluorescent tracking of viable cells and analysis of fundamental cellular processes, also to monitor the basal vital state of cells in the hypoxic co-culture. This is critically important, as the stromal cells deliver a big variability of signals to protect leukemia cells and provide drug resistance. Therefore, keeping stromal cells at the healthy state is crucial during experimental procedures. In the proposed studies, viability, apoptosis, proliferation, ROS production, and mitochondrial membrane potential were monitored in both cell types, which were separated on the basis of the fluorescence of a cell tracker. We have shown that the proposed hypoxic co-culture conditions do not affect basal live parameters of stromal cells, indicating the relevance of proposed model. Finally, we utilized this experimental setup to monitor the stroma-mediated protection of leukemia cells from the imatinib-induced cell death, which contributes to the leukemia progression and development of therapy resistance. Altogether, we recommend such flow cytometric strategy as an elementary screen of the vital state of stromal cells, which should be performed when using the co-culture hypoxic models. The proposed approach can also be broadly used for other studies of the leukemia-stroma cross-talk and of the part played by the leukemic microenvironment in drug screening studies.

CNS endothelial cells (CNS-ECs), one of the main non-neural CNS cell populations, play a vital role in physiology, pathology, and regeneration of the nervous system. Therefore, there is an urgent need to enhance our knowledge on their biology to elucidate mechanisms responsible for the blood brain barrier function in normal and pathological conditions, interaction between brain endothelium and neural stem cells in the neurogenic niche, the paracrine processes in the brain and spinal cord, etc. Here, we described a novel, simple, and efficient protocol for isolation of endothelial, vessel-forming cells from the murine CNS, which is based on Sca-1 expression. Using this newly described protocol we were able to detect and sort viable, highly pure CNS-ECs with minimal contamination by cells of non-endothelial origin. This method will increase the availability of CNS-ECs for in vitro research. Moreover, we compared phenotype of CNS-ECs isolated from neonatal mice and adult intact and injured brain looking for the cells of endothelial precursor characteristic, such as those found in the bone marrow and circulating in the bloodstream after organ injuries. We have found that neonatal brain capillaries contain proportion of endothelial precursor cells (Sca-1(+) , CD45(-) , c-Kit(+) ). Such precursors were also found in adult brain cortex, both in intact and injured brain. Finally, we discuss several crucial technical issues concerning CNS tissue preparation for flow cytometry analysis and cell sorting as well as nonspecific antibody binding caused by inflammatory microglia/macrophages which should be avoided in order to reliable isolation of pure CNS cells for downstream procedures including cell transplantation-based translational studies.

Curcumin, the major component of the spice turmeric, used in 50- micro M concentration, induced cell death in multidrug-resistant CEM(P-gp4) and LoVo(P-gp4) cells as well as in their sensitive counterparts as assessed by TUNEL method and morphological observation. In all cells induced to undergo cell death with curcumin, there was no caspase-3 activation because only the unprocessed form of caspase-3 was observed using immunoblotting.