RESUMEN
Intercellular communication within the bone marrow niche significantly promotes leukemogenesis and provides protection of leukemic cells from therapy. Secreted factors, intercellular transfer of mitochondria and the receptor-ligand interactions have been shown as mediators of this protection. Here we report that tunneling nanotubes (TNTs)-long, thin membranous structures, which have been identified as a novel mode of intercellular cross-talk-are formed in the presence of stroma and mediate transfer of cellular vesicles from stroma to leukemic cells. Importantly, transmission of vesicles via TNTs from stromal cells increases resistance of leukemic cells to the tyrosine kinase inhibitor, imatinib. Using correlative light-electron microscopy and electron tomography we show that stromal TNTs contain vesicles, provide membrane continuity with the cell bodies and can be open-ended. Moreover, trans-SILAC studies to reveal the non-autonomous proteome showed that specific sets of proteins are transferred together with cellular vesicles from stromal to leukemic cells, with a potential role in survival and adaptation. Altogether, our findings provide evidence for the biological role of the TNT-mediated vesicle exchange between stromal and leukemic cells, implicating the direct vesicle and protein transfer in the stroma-provided protection of leukemic cells.
Asunto(s)
Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Mitocondrias/genética , Nanotubos/química , Transporte Biológico/genética , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Mesilato de Imatinib/química , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células del Estroma/ultraestructuraRESUMEN
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.
