Renal Cell Carcinoma-derived Mesenchymal Stem Cells Induce Macrophage Polarization To M2 Type By Activating NF-kB Pathway And Promote Migration And Invasion Of Renal Cancer

Objectives Renal cancer is one of the most common malignancies of the urinary system.Macrophage(M0)is an important composition of the tumor microenvironment,and the polarization from M0 to M2 plays an important role in tumor progression.Mesenchymal stem cells(MSCs)are a group of multifunctional adult stem cells that can be differentiated into two subtypes under proper stimuli,MSC1 and MSC2,Our previous study found that advanced renal cell carcinoma can promote migration of MSCs by secreting high levels of IL-8;It is well known that MSCs have a wide range of sources with easy acquisition,which are considered as an ideal cell therapy target.If we can elucidate the molecular mechanism of MSCs in regulating macrophage polarization in renal cell carcinoma,and further aim to reverse macrophage polarization by targeting MSCs,it is expected that the renal cancer microenvironment could be remodeled via reversing macrophage function by targeting MSCs,which will be of great therapeutic significance for renal cell carcinoma at present;The main research purpose of this article is to further explore the role of MSCs on tumor-associated macrophages in the renal cancer microenvironment,the regulatory role of the NF-k B signaling pathway in this process,and the effects on the invasion and migration capabilities of renal cancer;Method We isolated mesenchymal stem cells from kidney cancer tissues and routinely identified them and passed them to the third generation for subsequent experiments;In order to explore tumor-associated macrophages,we treated THP-1 cells with150 n MPMA for 24 hours to obtain M0 type TAMs,M0 cells were exposed to a culture solution containing 10 pg / ml LPS for 24 h to obtain M1 macrophages.M0 macrophages were cultured to a culture solution containing 20 ng / ml IL-13 for 24 h to obtain M 2 macrophages;M0 macrophages were seeded in the lower chamber and RCC-MSC cells were seeded in the upper chamber through a 6-well plate compartment.Observe the cell morphology of macrophages after co-cultured under a microscope,detect the expression of M0 macrophage markers and the activation of NF-k B signal pathway after co-cultured by WB,and then using PDTC,a specific inhibitor of NF-k B signal pathway,to detect Whether the above polarization process is suppressed;In order to investigate the effect of RCC-MSC on renal cancer in the microenvironment of renal cancer,renal cancer cell 786-O was co-cultured with M0 macrophages or RCC-MSC cells,wound healing assay was used to detect tumor cell migration after co-cultured,transwell assay was used to detect the change of tumor cell invasion ability after co-cultured.In order to investigate the effect of MSC cells on M2 macrophages,we exposed B-MSCs to a medium containing 10 pg / ml LPS for 24 hours to obtain MSC-1,we co-cultured M2 macrophage with different numbers of MSC-1 cells,and then WB was used to detect changes in M2 macrophage surface markers.Results Flow cytometry was used to identify the extracted cells,indicating that CD105 and CD73 were highly expressed,while CD34 and CD45 were lowly expressed on cell surface;Thp-1 cells were treated with 150 n MPMA for 24 h,showing that cells changed from suspension growth to adherent growth with pseudopodia.WB assay suggested that the treated cells had high expression of macrophage markers,including CD36,CD71,CD68,and low expression of monocyte markers CD14.Based on M0 macrophages,M0 cells were exposed to culture medium containing 10pg/ml LPS for24 h to induce M1 macrophages with high expression of CD86,TNF-α,IL-1β.M0 macrophages were cultured in a medium containing 20ng/ mli-13 for 24 h to induce M2-type macrophages with high expression of CD206,CD163 and arg-1.M0 macrophages were co-cultured with RCC-MSCs for 2 days,followed by WB validation,showing that with the increasing number of MSCs,the expression of CD206,CD163,ARG-1,NF-k B signaling pathway-specific protein p50,p65 on M0 macrophages was gradually increased,while the expression of IKBa(inhibitory protein of NF-k B pathway)was gradually decreased.Moreover,the above process was significantly inhibited by NF-k B signaling pathway specific inhibitor PDTC.Afterwards,RCC cells were co-cultured with macrophage M0,or RCC-MScs,or both of them for 48 h.Transwell assay further indicated that the number of tumor cells crossing the chamber in the MSCS +M0 group was higher than that in the other groups;similarly,the wound healing assay revealed that the number of tumor cells in wound region of MSCS +M0 group was higher than that in the other groups.After co-culture of M2 macrophages with MSC-1,the expression of CD206,CD163,and Arg-1 gradually decreased,and the expression of CD86,TNF-α,and IL-1β gradually increased Conclusion As a result,RCC-MSCs could induce macrophage polarization to M2 type by activating NF-KB pathway,and further promote the progression of RCC.In addition,MSCs could reverse the polarization of M2 type macrophages.