A novel,rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+monocytes within 48 hours of in vitro culture

AIM:Dendritomas formed by fusing cancer cells to dendriticcells have already been applied to clinical treatment trialof several types of cancers.Dendritic cells for the fusion inmost trials and experiments were from blood monocytesin standard 7-d protocol culture,which requires 5-7 d ofculture with granulocyte-macrophage-colony-stimulatingfactor(GM-CSF)and interleukin-4(IL-4),followed by 2-3 dof activation with a combination of proinflammatorymediators such as tumor necrosis factors(TNFα),interleukin-1β(IL-1β),interleukin-6(IL-6)and prostaglandin E2(PGE2).One study showed that mature monocyte-derived dendriticcells could be obtained within 48 h of in vitro culture withthe same protocol as standard 7-d culture and referred toas FastDCs.Here we aimed to fuse human hepatocellularcarcinoma cell line HCCLM3 cells with mature monocyte-derived dendritic cells within 48 h of in vitro culture(FastDC).METHODS:HCCLM3 cells were cultured in RPMI 1640 with150 mL/L fetal calf serum(FCS).CD14+monocytes fromhealthy human peripheral blood were purified with MACSCD14 isolation kit and cultured in six-well plates in freshcomplete DC medium containing RPMI-1640,20 mL/Lheat inactivated human AB serum,2 mmol/L L-glutamine,100 μg/mL gentamicin,1 000 U/mL GM-CSF and 500 U/mLIL-4 for 24 h,then proinflammatory mediators such as TNFα(1000 U/mL),IL-1β(10 ng/mL),IL-6(10 ng/mL)and PGE2(1μg/mL)were supplemented for another 24 h,and thusmature FastDCs were generated.HCCLM3 cells and FastDCswere labeled with red fluorescent dye PKH26-GL and greenfluorescent dye PKH67-GL respectively.After the redfluorescent-stained HCCLM3 cells were irradiated with50 Gy,FastDCs and irradiated HCCLM3 cells were fused in500 mL/L polyethylene glycol(PEG)+100 mL/L dimethylsulfoxide(DMSO)to generate novel dendritomas.The FastDCs and novel dendritomas were immunostained with anti-CD80,anti-CD86,anti-CD83,anti-HLA-DR mAbs andanalyzed by fluorescence-activated cell sorting(FACS).Novel dendritomas were nucleus-stained with Hoechst33258 and analyzed by confocal laser scanning microscopy.RESULTS:Mature FastDCs with highly expressed surfacemarkers CD80,CD86,CD83 and HLA-DR were generatedwithin 48 h in vitro.Novel dendritomas with dual red-greenfluorescence were constructed fast and successfully,andFACS analysis showed that the fusion efficiency was 24.27%and the novel dendritomas expressed the same activationmarkers as FastDCs.Confocal laser scanning microscopyanalysis showed representative images of dendritomas.CONCLUSION:Dendritomas can be formed fast withmature FastDCs from healthy human peripheral bloodmonocytes(PBMC)by incubation with GM-CSF and IL-4 for24 h and by activation with proinflammatory mediators foran additional period of 24 h.Owing to shorter time requiredfor in vitro DCs development,the generation of these noveldendritomas reduced labor and cost.This rapid methodfor formation of dendritomas may represent a new strategyfor immunotherapy of hepatocellular carcinoma.