| Objective:To explore the molecular underlying mechanism of the Nlrp3 inflammasome in macrophages to mediate the colorectal cancer liver metastasis.Methods:1.To detect the role of CRC in macrophages:To collect the supernatant of SW480(SW480 conditioned media or SW480 CM),and then exposed to macrophages.To confirm whether the SW480 CM have any effect on promoting macrophages migration by using transwell migration experiment;After 48h incubation with SW480 CM;the activation of Nlrp3 inflammasome in macrophages were detected by immunofluorescence,and the expression of Nlrp3 and IL-1β were detected by QPCR and Western blot;ELISA KIT were used to detected the IL-1βsecretion of macrophages.2.To detect the role of macrophages-CRC co-culture:To collect the supernatant of macrophages-CRC co-culture(co-culture conditioned media or co-culture CM),and then exposed to colorectal cancer cells(SW480).To confirm whether the co-culture CM could further promote the CRC migration.After 48h incubation with co-culture CM,the expression of epithelial-mesenchymal transition(EMT)marker and the MMPs change were detected by QPCR and Western blot.3.To detect the role of IL-1β in CRC:CRC were exposed to different concentrations of recombinant IL-1β.To confirm whether the IL-1β have any effect on promoting CRC migration by using transwell migration experiment,and the expression of EMT marker and MMPs were detected by QPCR.4.The inhibitor of Nlrp3 or caspase-1 were added to co-culture system,while the Nlrp3 or caspase-1 knockout mice were used.Transwell migration experiment were used to verify whether Nlrp3 signalling pathway paly an important role in CRC migraton.5.In vivo experiment:To generate the model of liver metastasis,5 × 105 MC38 cells in 100 μl Hank’s balanced salt solution(HBSS)were injected into the spleen of each mice(Nlrp3-/-and WT mice).21 days after tumor cell inoculation,mice were euthanized and the liver were collected.The liver was immediately stored in 4%paraformaldehyde,then embedded and sliced for immunohistochemical analysis or hematoxylin and eosin stain.Results:1.Macrophages were demonstrated to migrate toward tumor cells,and the activation of Nlrp3 inflammasome and increase of IL-1β secretion had found in macrophage,after treated with CM of CRC.2.We found that macrophages could further promote the CRC migration,and up-regulated expression levels of claudin-1,Zeb1,vimentin MMP-2,9 in CRC after treated with co-culture CM.3.We exposed CRC to different concentrations of recombinant IL-1β,resulted in a significant increase in migration,while the upregulation of mesenchymal markers and MMP3 also occurred in CRC.4.Nlrp3 and caspase-1 antagonists,which could suppress the promotion of CRC migration.Similarly,bone marrow derived macrophages(BMDM)from Nlrp3-/-or caspase-1-/-mice,conformed to a decrease in IL-1β secretion and reductions of MC38 migration compared with BMDM from WT mice.5.Nlrp3-/-or caspase-1-/-mice leads to decreased migration to liver in mouse models of liver metastasis.Conclusions:In vitro study,it was found that there were a lot of macrophages with higher Nlrp3 expression located around the human colorectal cancer tissue.Macrophages were demonstrated to migrate toward tumor cells,then induce them into EMT,meanwhile,they also could enhance the expression levels of MMP2,3,9 in CRC.The communication between macrophages and CRC activated the Nlrp3 inflammasome,which lead to IL-1β secretion in macrophage,the IL-1β could contribute to tumor cell migration and metastasis.The CRC migration and metastasis were suppressed by Nlrp3 signalling blocking in vitro and in vivo.These findings demonstrated that the crosstalk between macrophages and CRC via Nlrp3 signaling plays vital roles in CRC migration and metastasis,the results of this study might provide a new alternatively treatment for this devastating disease. |
