A Positive Feedback Loop Between Tumor Associated Macrophages And Cancer Cells Promotes Cholangiocarcinoma Progression And Chemoresistance

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Tumor-associated macrophages induce cholangiocarcinoma chemoresistance to gemcitabineObjective To establish a tumor-associated macrophage model and identify the phenotype,examine the effect of tumor-associated macrophages on chemoresistance of cholangiocarcinoma cells,and verify their effects on the regulation of the apoptosis.Methods Human monocyte cell line THP-1 cells were induced to differentiate to tumorassociated macrophages(TAM)by the classical protocol,and the expression of corresponding markers were verified by RT-PCR and flow cytometry.cholangiocarcinoma chemoresistance to gemcitabine induced by TAM was detected by CCK8,flow cytometry and WB.At last,to verify the effect of TAM on apoptosis of cholangiocarcinoma cells.Results The CD11 b was highly expressed in all macrophage phenotypes.Furthermore,the expression of CD80 and IL12 increased significantly in M1 macrophages,and the expression of CD206 and IL10 was high in M2 macrophages.The CCK8 results showed that TAM conditioned medium(CM)significantly reduced the sensitivity of cholangiocarcinoma cells to gemcitabine.The flow cytometry results found that chemoresistance was mediated by reducing apoptosis induced by gemcitabine.In addition,WB analysis showed that TAM-CM inhibited the activation of the caspase-3 in cholangiocarcinoma cells.Conclusion Tumor-associated macrophage models have been successfully established.TAM-CM inhibited the activation of the caspase pathway to mediate chemotherapy resistance in cholangiocarcinoma cells.Part ? a PKC?/NF-?B signaling pathway mediates tumor-associated macrophage-induced cholangiocarcinoma resistanceObjective To investigate the mechanism of cholangiocarcinoma chemoresistance induced by tumor-associated macrophages via a PKC?.Methods human cholangiocarcinoma cell lines stably overexpressing and low expressing a PKC? were established.WB was used to detect the expression of NF-?B,P62 and Nuclear NF-?B in these cell lines.The dual luciferase reporter system validates NF-?B activation,soft agar assays were performed to detect the transformed growth of these cells treatment as indicated.WB was used to detect the activation of a PKC? and NF-?B in cholangiocarcinoma which was resistance to gemcitabine mediated by TAM-CM.WB and dual luciferase reporter system was used to explore whether P62 participated in the a PKC?/NF-?B signaling pathway.Amino acid mutant construction and IP experiments verified the mechanism of a PKC? regulating NF-?B by P62.Results In vitro,when the expression of a PKC? was up-regulated in human cholangiocarcinoma cells,and the expression of P-a PKC?,P-NF-?B and NF-?B was significantly increased.The dual luciferase reporter system also confirmed the activation of NF-?B.When a PKC? was suppressed,the expression of the above molecules were decreased.when a PKC? was overexpressed,the ability of transformed growth was enhanced,while NF-?B inhibitor(PDTC)reversed this phenomenon.The activation of a PKC?,NF-?B was enhanced in cholangiocarcinoma cells which were resistance to gemcitabine mediated by TAM-CM;Silencing of P62 expression in human cholangiocarcinoma cells reduced NF-?B activation.After mutation of PB1 domain,the binding of a PKC? to P62 was inhibited,and the activation of NF-?B was reduced.Conclusions In vitro,a PKC? can promote the transformed growth of cholangiocarcinoma cells by regulating the NF-?B into the nucleus;tumor-associated macrophages induce the resistance of cholangiocarcinoma cells through a PKC?/NF-?B signaling pathway;There is a binding relationship between a PKC? and P62 in cholangiocarcinoma cells through the PB1 domain,and they can regulate the activation of NF-?B.Part ? TAM induced cholangiocarcinoma cells epithelial-mesenchymal transition via a PKC?/NF-?B signaling pathwayObjective The second part of the experiment suggested that a PKC?/NF-?B signaling pathway was involved in in the regulation of tumor-associated macrophages to cholangiocarcinoma cells.Therefore,we further investigate whether a PKC?/NF-?B signaling pathway is involved in tumor-associated macrophage-induced epithelialmesenchymal transition in cholangiocarcinoma cells.Methods q RT-PCR and ELISA were used to detect the expression and secretion of TGF?1 in different macrophages.The luciferase reporter system was used to detect the level of NF-?B activity in CCA cells after treatment with or without TGF?1,a TGF-?R inhibitor or with a neutralizing anti–TGF-?1 antibody.WB was performed to determine the activation of a PKC? and NF-KB in CCA cells transfected with Controlsi RNA or a PKC?-si RNA cells after TGF-?1 treatment for the indicated times.The activation of a PKC? and NF-KB was dected in CCA cells transfected with a PKC? c DNA or control-c DNA cells after treated with conditioned media from THP1-derived M2 macrophages alone or with LY2157299,anti-TGF-?1 neutralizing antibody,PDTC.Co-focusing to verify whether TGF?1 promotes NF-?B nuclear import.Immunofluorescence,WB were performed to detect whether TAM induced cholangiocarcinoma cells EMT and explored the mechanism.Scratch and invasion experiments verified the mechanism of cholangiocarcinoma EMT induced by TAM.a PKC?-mediated TAM-induced proliferation and metastasis of cholangiocarcinoma was detected in vivo by constructing a subcutaneous xenograft and lung metastasis model in nude mice,and P-a PKC? and F4/80(macrophage)expression were detected by immunohistochemistry.Results The expression of TGF?1 in TAM was significantly increased compared with other phenotype macrophages.TGF?1 could induce NF-?B activation and was inhibited by TGF?1 neutralizing antibody or TGF? receptor inhibitor.Along with the TGF?1 treatment time increased,the activation of a PKC?/NF-?B in cholangiocarcinoma cells enhanced continuously.The peak time is about 5 hours later,and all effects were attenuated when a PKC? was low expression.Confocal scanning showed that TGF?1 promoted NF-?B entry into the nucleus,and this effect was inhibited by a PKC?-si RNA or PDTC.The expression of P-a PKC? and P-NF-?B was increased in cholangiocarcinoma cells cultured with TAM-CM,and P-a PKC?,P-NF-?B expression was significantly enhanced when a PKC? was overexpressed.This effect can be reversed by PDTC or TGF?1 inhibitor.IHC and WB showed that expression of epithelial marker E-cad was decreased and Vimentin was increased when the CCA cells cultured with M2-CM,all the above effects can be reversed when treatmented with PDTC or TGF?1 inhibitor.Meanwhile,the ability of invasion,migration and proliferation was enhanced under the treatment of TAM-CM,and this effect could be inhibited by PDTC or TGF?1 inhibitor.In vivo,tumor volume and the number of lung metastases was significantly increased in nude mice xenografts and lung metastasis models constructed by cholangiocarcinoma cell lines transfected with a PKC?-c DNA respectively.When macrophages were cleared,the volume of the xenograft tumors were reduced and the number of lung metastases was reduced too.The expression of P-a PKC? in the xenografts and lung metastases decreased along with the macrophage clearanced.Conclusions a PKC? mediates TGF?1-induced NF-?B activation.TGF?1 secreted by TAM induced cholangiocarcinoma cells EMT via a PKC?/NF-?B signaling pathway and promoted tumor growth and metastasis.Part ? CCL5 secreted by a PKC?-mediated mesenchymal like CCA cells recruitmented and induced macrophage polarizationObjective To further explore the interaction between the CCA cells and TAM and the molecular mechanism of recruitment of macrophages by a PKC?-mediated mesenchymal like CCA cells.Methods Raybio microarray was used to analyze the differential immunological factors in culture supernatants of a PKC?-mediated mesenchymal like CCA cells compared with normal cells;RT-PCR and ELISA for detection of CCL5 expression in mesenchymal CCA cells and verification of the effect of PDTC.Transient transfection of wild-type or mutant CCL5 promoter luciferase reporter plasmids,and then to analysis the relative fluorescence intensity of cholangiocarcinoma cells treated with TGF?1 and / or TNFa.Flow cytometry and RT-PCR were used to detect TAM-related surface marker and cytokines expression of THP-1 treated with CM of Mesenchymal-like CCA cells or CCL5,and detected the effect of treatment with PDTC or CCL5 neutralizing antibody.WB was performed to detect the influences on the classical signaling pathway in THP-1 cells treated as described above.Subsequently,Cell migration assay was used to detect the chemotactic effect of CCA cells on macrophages by coculturing a PKC?-mediated mesenchymal-like cholangiocarcinoma cells with THP-1 cells,and a PKC?-si RNA,PDTC and CCL5 neutralizing antibody were used to detect the effects on the recruitment of macrophages.At last,subcutaneous xenografts of nude mice was performed to detect whether a PKC?-CCL5 signaling mediates TAM infiltration and modulates CCA progression.Results CCL5 was significantly increased in the CM of a PKC?-mediated mesenchymal like CCA cells,whereas PDTC could reduce CCL5 expression.Both TGF?1 and TNFa could enhance the activition of NF-k B,while TGF?1 can further enhance fluorescence intensity based on TNFa.After treatment with CM of mesenchymal-like CCA cells,the expression of CD206 and TGF?1 was significantly increased in macrophages.WB results showed a significant increase in macrophages' P-STAT3 expression,whereas PDTC or anti-CCL5 treatment could attenuat these effects.Mesenchymal-like CCA cells made a significant recruitment effect on macrophages,while a PKC?-si RNA,PDTC and CCL5 neutralizing antibodies can eliminate this effect.In vivo,nude mouse tumor model constructed by CCA cell lines transfected with a PKC?-c DNA,the volume of the tumor reduced when CCL5 receptor was inhibited.IHC experiments indicated that when the CCL5 receptor was inhibited by Maraviroc,the recruiment of macrophages was weakened and the expression of P-a PKC? was decreased.Conclusions CCL5 promoted the activation and chemotactic migration of macrophages by a PKC? mediated mesenchymal-like CCA cells.The a PKC?-CCL5 signaling recruimented TAM infiltration and modulated CCA progression.Part ? Nano-liposome combined with a PKC?-si RNA and gemcitabine for the treatment of cholangiocarcinomaObjective To design a liposome system for combining with a PKC?-si RNA and gemcitabine,to explore the synergy anti-tumor effect in cholangiocarcinoma.Methods In cooperation with the school of Pharmacy,Tongji Medical College,the liposomes were synthesized according to the previous study of thin-film dispersed hydration method.The positive-charged liposomes adsorbed negative-charged si RNA by electrostatic interaction at w/w(weight liposome/weight si RNA)ratio of more than 200:1 in RNase free H2 O to form final liposomes complex of Gemcitabine/a PKC?-si RNA-L.Zeta PALS determined the physical and chemical characterization.Anchorage-independent experiment to detect the effect of various drugs on the transformed growth of CCA cells.In the subcutaneous tumor mice model,we detected the anti-tumor effects of various preparations by injecting different drug formulations through the tail vein once a week.IHC verificated the effect of Gemcitabine/a PKC?-si RNA-L on the a PKC?/NF-?B pathway and TAM.Results The liposome system was successfully prepared.In vitro experiments confirmed that Gemcitabine/a PKC?-si RNA-L significantly inhibited the transformed growth of CCA cells compared with other preparations.In the nude mouse model,the co-delivered liposome group significantly inhibited the volume of subcutaneous xenografts.IHC showed that gemcitabine induced NF-?B activation and macrophage aggregation,whereas a PKC?-si RNA treatment attenuated these effects.Conclusions Nano-liposomes combined with a PKC?-si RNA and gemcitabine have a good synergistic anti-tumor effect.Targeting a PKC? can inhibit tumor growth and enhance the sensitivity of gemcitabine chemotherapy.