Study Of Mechanism Of Coculture Mediated Diffuse Large B Cell Lymphoma Cell Line Toledo Transforming To Lymphomatic Stem Like Cells

Part I Coculture mediated diffuse large B cell lymphoma cell line Toledo transforming to lymphomatic stem like cellsObjective:The aim is to explore whether cocultured with stromal cells can induce diffuse large B cell lymphoma cell line Toledo to transform to lymphomatic stem like cells.Methods:Coculture system comprising murine bone marrow stromal cell line OP9and diffuse large B cell lymphoma cell line Toledo was constructed. The impact on proliferation of Toledo by cocuture system was investigated by MTT and CFSE. The impact on the chemosensitivity of Toledo to doxorubicin was evaluated by MTT and Annexin-V/PI methods. The impact on cell cycle distribution was measured by Brdu/7-AAD method. The influence on clonogeneity of Toledo was evaluated using methycellulose based semi-solid culture medium. The expression of CD44,CD90,CD27and CD133on Toledo was determined after being cocultured with OP9.The transcripts of BMI1and HES1were measured by real time RT-PCR.Results:The results from MTT assay showed that Toledo from coculture system had higher OD value at48h(t=3.161, P=0.010) and72h(t=2.662, P=0.024) after being cocultured; CFSE assay showed that Toledo from coculture system had lower MFI at24h (t=-27.613, P=0.000)、48h (t=-15.058, P=0.000) and72h (t=-29.874, P=0.000). After treatment with doxorubicin for48h, cells were analyzed by MTT assay and the results showed that Toledo from coculture system had higher survival rate [(54.80±1.88)%] than those without being cocultured with OP9[(34.58±3.69)%](t=13.127, P=0.000); the results of Annexin-V/PI assay showed that doxorubicin had induced (8.84±0.10)%of apoptosis within Toledo from coculture system, much lower than those without cocultured with OP9[(58.15±1.17)%](t=-73.042, P=0.000). More lymphoma cells from coculture system[(80.30±1.26)%]entered the G0/G1phase of cell cycle than those without being cocultured with OP9[(44.88±2.01)%](t=25.840, P=0.000). Toledo from coculture system had higher clone forming rate[(0.41±0.05)%] than those without being cocultured with OP9[(0.03±0.01)%](t=14.125, P=0.000). There were few lymphoma cells that expressed CD44[(0.39±0.05)%]、CD90[(0.07±0.01)%]、 CD27[(0.04±0.01)%] or CD133[(0.02±0.01)%]. But after being cocultured with OP9, the expression of CD133was significantly increased [(2.59±0.49)%](t=-9.083, P=0.001). The transcripts of BMI1(t=3.053, P=0.038) and HES1(t=3.100, P=0.036) were increased at the same time.Conclusion:After being cocultured with OP9, Toledo had acquired higher proliferative capacity and clonogenic potential. They were more resistant to doxorubicin. There were higher ratio of Toledo that entered G0/G1phase of cell cycle after being cocultured with OP9. Toledo from coculture system upregulated the expression of CD133and the transcripts of BMI1and HES1. Diffuse large B cell lymphoma cell line Toledo had a tendency to transform to lymphomatic stem like cells after being cocultured with murine bone marrow stromal cells. Part Ⅱ The mechanism of coculture mediated diffuse large B cell lymphoma cell line Toledo transforming to lymphomatic stem like cellsObjective:To explore the mechanism of coculture mediated diffuse large B cell lymphoma cell line Toledo transforming to lymphomatic stem like cells.Methods:The expression of p-STAT3was detected by Western Blot. AG490was added to inhibit STAT3activation when the coculture system was constructed. The impact on the proliferative capacity of Toledo by AG490was evaluated by CFSE. The impact on the chemosensity to doxorubicin by AG490was evaluated by Annexin-V/PI assay. The influence on cell cycle distribution of Toledo by AG490was investigated by PI and Brdu/7-AAD assays. The influence on clonogenic potential of Toledo was investigated by methylcellulose based semi-solid culture medium. The impact on the expression of CD133was detected using flow cytometry. The influence on the transcripts of BMI1and HES1mRNA was detected using real time RT-PCR.Results:The expression of p-STAT3was present in lymphoma cells from coculture system and it was absent in Toledo without being cocultured with OP9. After AG490was added into the coculture system, the expression of p-STAT3was obviously inhibited. The results of CFSE assay showed that Toledo from coculture system with AG490had higher MFI than those without AG490at24h(t=-14.822, P=0.000)、48h(t=-12.954, P=0.000) and72h(t=-14.041, P=0.000). Doxorubicin had induced (16.07±1.46)%of apoptosis within Toledo from coculture system with AG490, much higher than those from coculture system without AG490[(8.15±0.51)%](t=-8.871, P=0.001). The results of PI assay indicated that less lymphoma cells from coculture system with AG490[(56.09±2.87)%] entered G0/G1phase of cell cycle than those from coculture system without AG490[(68.04±1.64)%](t=2.733, P=0.032). Brdu/7-AAD assay showed similar results that less lymphoma cells entered G0/G1phase of cell cycle after AG490was added into the coculture system(t=60.015, P=0.000). Toledo from coculture system with AG490had lower clone forming rate[(0.18±0.06)%] than those from coculture system without AG490[(0.44±0.05)%](t=5.534, P=0.005). The expression of CD133[(0.79±0.03)%] was downregulated with the adding of AG490into coculture system (t=37.427, P=0.000). The transcripts of BMI1(t=4.455, P=0.011) and HES1(t=3.529, P=0.024)were downregulated at the same time.Conclusion:Cocultured with murine bone marrow stromal cell line OP9mediated activation of STAT3in diffuse large B cell lymphoma cell line Toledo. AG490could inhibit coculture induced STAT3activation. Toledo from coculture system with AG490had lower proliferative capacity, drug resistance and clonogenic potential. Less lymphoma cells entered into G0/G1phase of cell cycle. The expression of stem cell marker CD133and the transcripts of BMI1and HES1were downregulated with the addition of AG490into coculture system. Inhibition of STAT3by AG490prevented Toledo from transforming to lymphomatic stem like cells. Lymphomatic stem like cells transformation was dependent on the activation of STAT3.