Anticancer Drugs Cause Release Of Exosomes With Heat Shock Proteins From Human Hepatocellular Carcinoma Cells That Elicit Effective Natural Killer Cell Antitumor Responses In Vitro

【OBJECTIVE】Exosomes are actively released into the extracellular environmentfrom a wide range of living cells via endosomal vesicles/multivesicularbodies (MVBs) pathway by fusion with the plasma membrane, and specialized30-100nanometer (nm)-sized lipid-rich membrane-bound microvesicles. Theexosomal protein composition varies with the cell types from which theyare derived, and accounting for their biological functions. In recentyears, many studies have demonstrated that tumor-derived exosomes (Tex),carrying a variety of immune-related proteins, played an important rolein the host anti-tumor immune response and immune regulation. Tex areexpected to become a novel subcellular tumor vaccine for immunotherapyof a variety of human malignancies. Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. Dysfunction of immunesurveillance in tumor-bearing host resulted in ineffective induction ofanti-tumor immune responses, and was closely related to the high rate ofrecurrence and poor prognosis of HCC. Membrane-bound or extracellularlocalized heat shock proteins (HSPs) have been found to provide dangersignals for the host's cellular immune system. HSPs not only enhance theimmunogenicity of cancer cells, but also induce anti-tumor immunity byactivating natural killer (NK) cells. The relationship between HSPs andtumour immunotherapy has become a research hotspot in recent years.The purpose of the present study was to investigate whether anticancerdrugs may significantly increase exosome secretion by hepatocellularcarcinoma cells, and efficiently up-regulate the surface expression ofHSPs on exosomes. Based on the above results, the biological activitiesof HSPs-bearing exosomes were investigated through influencing NK cellfunctions. These results not only were expected to provide suitablesources of tumor vaccines for HCC immunotherapy, but also providedtheoretical and experimental basis for more in-depth clinicalapplications of HCC chemoimmunotherapy.【METHODS】1. The in vitro growth inhibitory effect of anticancer drugs on the humanhepatocellular carcinoma (HepG2and PLC/PRF/5) cell lines was measuredby the MTT assay.2. Cell culture supernatants were harvested at different time points fromHepG2and PLC/PRF/5cells, exposured to100%test drug concentration(TDC) of paclitaxel, carboplatin, etoposide, irinotecan hydrochloride,heat shock or control, and the concentration of HSP60, HSP70and HSP90in each sample were measured using the ELISA Kit. 3. Conditioned culture medium was collected from HepG2cells, andsubjected to differential centrifugation to obtain the purifiedexosomal pellet. The morphology of exosome was examined bytransmission electron microscopy (TEM), and the exosomal marker CD63was analysed by Western blot.4. We isolated exosomes from cell culture supernatants produced by HepG2cells under both basal and stress-induced conditions at different timepoints. The amount of exosomes was quantified via the determinationof AChE enzymatic activity, HSP60, HSP70and HSP90expression inexosomes were assessed by Western blot, and these molecules expressionon the surface of exosomes were analyzed by flow cytometry.5. NK cells were incubated either with low-dose IL-2alone or with IL-2in combination with exosomal protein for4days, which were isolatedfrom HepG2cells under100%TDC paclitaxel or carboplatin treatment.NK cell-mediated cytotoxic function was analyzed using a standardlactate dehydrogenase (LDH) release assay, granzyme B production wasmeasured by ELISA kit, and the cell surface density of several NK cellreceptors was examined by flow cytometry.【RESULTS】1. The human hepatocellular carcinoma (HepG2and PLC/PRF/5) cell linesshowed remarkably higher sensitivity to paclitaxel and etoposide, andexhibited resistance to irinotecan hydrochloride, carboplatin andmitomycin.2. HSP60, HSP70and HSP90secretion were generally higher afteranticancer drugs treatment, reaching statistical significance (P＜0.05). Moreover, our results demonstrated that HepG2and PLC/PRF/5cells secreted higher levels of HSP60, HSP70and HSP90treated with hepatocellular carcinoma cell-resistant anticancer drugs comparedwith cells exposed to hepatocellular carcinoma cell-sensitiveanticancer drugs.3. A pure exosomal population was present with typical cup-shapedmorphology, surrounded by a two-layer lipid membrane, and varying insize between30nm-100nm, the majority around70nm-90nm. Besidesmorphology and size, we confirmed the presence of exosomes through itsspecific marker by Western blot with anti-CD63antibody.4. There was an increase in exosome release by HepG2cells understress-induced conditions compared with cells under basal condition(P＜0.05). Moreover, hepatocellular carcinoma cell-resistantanticancer drugs could enhance exosome release to a higher degree thanhepatocellular carcinoma cell-sensitive anticancer drugs. HepG2cell-derived exosomes carried more HSP60, HSP70and HSP90undertreatment with anticancer drugs (P＜0.05), and hepatocellularcarcinoma cell-resistant anticancer drugs caused induction ofexosome-carried HSPs release at a level remarkably higher thanhepatocellular carcinoma cell-sensitive anticancer drugs. Elevatedlevels of HSP60, HSP70and HSP90were observed under stress-inducedconditions as compared to basal condition (P＜0.05), andhepatocellular carcinoma cell-resistant anticancer drugs seemed toenhance HSP60, HSP70and HSP90exosome-surface expression to a higherdegree than hepatocellular carcinoma cell-sensitive anticancer drugs.5. Contact of NK cells with the increased amount of HSPs-bearing exosomesaugmented cytolytic activity against K562target cells throughgranzyme B release, down-regulation of activating receptors CD69,NKG2D and NKp44, and up-regulation of inhibitory receptor CD94(P＜0.05). Carboplatin-treated HepG2cells-derived exosomes had astronger effect on NK cell function than paclitaxel-treated HepG2 cells-derived exosomes. Furthermore, the extent of cytotoxic effectof the HSPs-expressing exosomes was correlated with theirconcentrations, reaching a maximum effect at20μg/mL.【CONCLUSION】The in vitro growth inhibitory effect of anticancer drugs on the humanhepatocellular carcinoma (HepG2and PLC/PRF/5) cell lines was measuredby the MTT assay. Hepatocellular carcinoma cell-resistant anticancerdrugs (irinotecan hydrochloride and carboplatin) and hepatocellularcarcinoma cell-sensitive anticancer drugs (paclitaxel and etoposide)were selected for further investigation. In conclusion, our presentresults demonstrated that①HepG2and PLC/PRF/5cells secreted higherlevels of HSP60, HSP70and HSP90treated with hepatocellular carcinomacell-resistant anticancer drugs compared with cells exposed tohepatocellular carcinoma cell-sensitive anticancer drugs;②hepatocellular carcinoma cell-resistant anticancer drugs could enhanceexosome release to a higher degree than hepatocellular carcinomacell-sensitive anticancer drugs, and hepatocellular carcinomacell-resistant anticancer drugs seemed to enhance HSP60, HSP70and HSP90exosome-surface expression to a higher degree than hepatocellularcarcinoma cell-sensitive anticancer drugs;③carboplatin-treated HepG2cells-derived exosomes had a stronger effect on NK cell function thanpaclitaxel-treated HepG2cells-derived exosomes. HSPs-bearing exosomeshave potential values of application in HCC chemoimmunotherapy. Thesefindings not only were expected to provide safe and effective sources oftumor vaccines for HCC immunotherapy, but also provided theoretical andexperimental basis for more in-depth clinical applications of HCCchemoimmunotherapy.