Project description:In multiple myeloma (MM), abnormal plasma cells interact with bone marrow (BM) stromal cells and vascular cells among others. A part of the BM milieu is considered highly hypoxic, and myeloma cells in situ may be influenced by circumstances other than normoxia in vitro. Hence, we attempted to confirm the role of hypoxic MM-derived exosomes in the BM milieu. We established a novel hypoxia-resistant cell line, KMS-11-HR, derived from KMS-11 cells cultured for >4 months under hypoxia (1% O2), as a model of MM cells localizing in an extensively hypoxic milieu. We used KMS-11 cells and KMS-11-HR cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from KMS-11 cells (normoxia or hypoxia) and exosomes derived from KMS-11-HR cells (hypoxia-resistant sub-line) were used for validation of angiogeneic activity, such as tube formation assay. Exosomes derived from the KMS-11-HR cells significantly increased tube formation of HUVECs than those from KMS-11 cells. To identify intercellular and exosomal miRNAs specifically expressed in hypoxia-resistant cells, we assess the expression profiles of intercellular and extracellular miRNAs in KMS-11 cells and KMS-11-HR cells using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). KMS-11 cells and KMS-11-HR cells were cultured for 24 hours under hypoxic conditions (1% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).

Project description:In multiple myeloma (MM), abnormal plasma cells interact with bone marrow (BM) stromal cells and vascular cells among others. A part of the BM milieu is considered highly hypoxic, and myeloma cells in situ may be influenced by circumstances other than normoxia in vitro. Hence, we attempted to confirm the role of hypoxic MM-derived exosomes in the BM milieu. We established a novel hypoxia-resistant cell line, U266HR, derived from U266 cells cultured for >4 months under hypoxia (1% O2), as a model of MM cells localizing in an extensively hypoxic milieu. We used U266 cells and U266HR cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from U266 cells (normoxia or hypoxia) and exosomes derived from U266HR cells (hypoxia-resistant sub-line) were used for validation of angiogeneic activity, such as tube formation assay. Exosomes derived from the U266HR cells significantly increased tube formation of HUVECs than those from U266 cells. To identify intercellular and exosomal miRNAs specifically expressed in hypoxia-resistant cells, we assess the expression profiles of intercellular and extracellular miRNAs in U266 cells and U266HR cells using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). U266 cells were cultured for 24 hours under hypoxic conditions (1% O2) or normoxic conditions (20% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).

Project description:In multiple myeloma (MM), abnormal plasma cells interact with bone marrow (BM) stromal cells and vascular cells among others. A part of the BM milieu is considered highly hypoxic, and myeloma cells in situ may be influenced by circumstances other than normoxia in vitro. Hence, we attempted to confirm the role of hypoxic MM-derived exosomes in the BM milieu. We established a novel hypoxia-resistant cell line, RPMI8226HR, derived from RPMI8226 cells cultured for >4 months under hypoxia (1% O2), as a model of MM cells localizing in an extensively hypoxic milieu. We used RPMI8226 cells and RPMI8226HR cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from RPMI8226 cells (normoxia or hypoxia) and exosomes derived from RPMI8226HR cells (hypoxia-resistant sub-line) were used for validation of angiogeneic activity, such as tube formation assay. Exosomes derived from the RPMI8226HR cells significantly increased tube formation of HUVECs than those from RPMI8226 cells. To identify intercellular and exosomal miRNAs specifically expressed in hypoxia-resistant cells, we assess the expression profiles of intercellular and extracellular miRNAs in RPMI8226 cells and RPMI8226HR cells using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). RPMI8226 cells were cultured for 24 hours under hypoxic conditions (1% O2) or normoxic conditions (20% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).

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