The Role Of TPX2 In Promoting Macrophage Polarization In Lung Adenocarcinoma Via The NF-κB/M-CSF Axis

The targeting protein for Xklp2(TPX2)is a microtubule-associated protein involved in multiple biological processes including nuclear functions and spindle assembly during mitosis.Besides its role in normal cellular mitosis,TPX2 has been found upregulated in a variety of cancers such as lung,pancreatic,esophageal,liver,and breast cancers,and is closely related to the classification of pathological grade and clinical stage,local infiltration,and metastasis of tumors.At present,research on the role of TPX2 in promoting the occurrence and development of lung adenocarcinoma(LUAD)predominantly concentrates on the malignant biological behaviors of LUAD.Conversely,there is less literature addressing its impact on the tumor microenvironment of LUAD.Advancements in cancer immunotherapy highlight the complex interplay between genetically mutated tumor cells and stable stromal cells within the tumor microenvironment(TME),driving the activation and reprogramming of stromal cells.This interaction crucially shapes immune profile of the tumor.The association between TPX2 and the abundant tumor-associated macrophages(TAMs)within the TME is increasingly recognized.In addition to directly promoting tumor invasion,proliferation,and distant metastasis,TPX2,as an oncogene,significantly impacts tumor behavior through its close relationship with TAM polarization.TAMs not only directly participate in cancer cell proliferation,survival,invasion,and metastasis but also act as immunosuppressive cells enabling lung cancer cells to evade immune responses.TAMs are emerging as crucial targets in cancer therapy,given their role in shifting between the tumor-promoting M2 state and the tumor-suppressing M1 state,a process vital for the effectiveness of immunotherapy.Various factors secreted by LUAD cells,such as interleukin-6(IL-6),interleukin-10(IL-10),macrophage colony-stimulating factor(M-CSF),and granulocyte-macrophage colony-stimulating factor(GM-CSF),affect macrophage polarization.Research indicates that in LUAD,M-CSF as a downstream target gene is an important factor affecting the polarization of CD163~+macrophages.However,the effects and mechanisms of TPX2on macrophage polarization remain unclear,as most research is limited to cellular studies in the lab,without supportive data from in vivo animal experiments or histopathological validation in LUAD.This study aims to initially screen for oncogenes related to tumor immunity in LUAD,determine the relevance of TPX2 with macrophages in the TME;clarify the effect of TPX2 on macrophage polarization and preliminarily investigate the mechanisms promoting macrophage polarization;and observe the expression of TPX2,M-CSF,and CD163 using the tumor xenograft animal model and human lung cancer pathological tissues,to provide basic research on TPX2 influencing macrophage polarization and thereby promoting malignant progression of tumors,and to identify new targets for immune cell therapy in clinical LUAD.Methods1.Identification of key genes involved in LUAD(1)Key genes linked with LUAD were screened using WGCNA,PPI,and Cox regression analysis.By ESTIMATE and ss GSEA analyses,the correlation of key genes with TIME and TIICs was further examined.(2)The expression of TPX2 in A549,H1975,H1299,PC-9,and HBE cell lines was determined by the RT-q PCR and Western Blot.A549 and H1975 cells were transfected with lentivirus TPX2-sh RNA,and TPX2 expression was assessed by RT-q PCR and Western Blot to identify target cells with the highest silencing effectiveness.2.Mechanism of TPX2 effect on macrophage polarization in LUAD(1)The optimal duration of M0 macrophages obtained from PMA-treated THP1cells were determined by cell morphology and adherence rate;RT-q PCR was used to detect the expression of related genes in M0 macrophages to screen the optimal concentration.(2)M0 macrophages were cultured with supernatants from LUAD,Vehicle and TPX2-sh RNA-2 groups to observe the morphological changes.The expression of CD80,CD163,and the CD80/CD163 combination on macrophages was assessed using flow cytometry and Western blot analysis,and the chemotaxis of THP1 cells was detected by transwell methodology.(3)Western blot and immunofluorescence(IF)were used to detect the expression of M-CSF,the downstream target gene of LUAD,Vehicle and TPX2-sh RNA-2 cells,and the secretion of M-CSF in the supernatants of the three groups was detected by ELISA.DEGs of A549/Vehicle and A549/TPX2-sh RNA-2 cells were identified by RNA whole-genome sequencing analysis,and functionally enriched pathways were analyzed by GO,KEGG,and GSEA;Western Blot was applied to identify the expression of macrophage polarization-related pathway proteins NF-κB p65,p-NF-κB p65,p38 MAPK,p-p38 MAPK,p44/42 MAPK,p-p44/42 MAPK after silencing TPX2in LUAD cells;TPX2,NF-κB p65 and p-NF-κB p65 were identified using IF.(4)The levels of NF-κB p65,p-NF-κB p65 and M-CSF were determined by Western blot after the NF-κB pathway was activated.3.TPX2,M-CSF,and CD163 expression in animal xenograft tumors and human LUAD tissues(1)The effect of TPX2 on xenograft tumors growth were observed in BALB/c A nude mice.(2)TPX2,M-CSF,and CD163 in xenograft tumor tissues were examined using immunohistochemistry(IHC).IF was used to detect the co-expression of CD163 and F4/80.(3)TPX2,M-CSF,and CD163 in LUAD tissues were examined using IHC and the correlation of TPX2,M-CSF,and CD163 in LUAD tissues was analyzed.Results1.Identification of key genes associated with LUAD(1)The core gene TPX2 of LUAD was successfully screened by bioinformatics methods.The analysis results showed that the stromal score,immune score,and ESTIMATE score of TPX2 high expression group were lower than those of the corresponding low expression group,and TPX2 was highly correlated with a variety of immune infiltrating cells including macrophages.These results indicate that TPX2exhibits high expression levels in LUAD and is closely linked to the TME and macrophage.(2)The results of TPX2 gene and protein expression in HBE and 4 LUAD cell lines showed that TPX2 was highly expressed in A549 and H1975 cells.Therefore,three sequences of TPX2-sh RNA were transfected into these two cells,and the silencing efficiency of TPX2-sh RNA-2 group was the best.The above results showed that we successfully obtained cell lines after silencing TPX2,which lays a foundation for studying the effect of TPX2 on macrophages.2.The effect and mechanism of TPX2 effect on macrophage polarization in LUAD(1)Three concentrations of PMA were used to screen THP1 for different duration,and the results showed that 24 h group had the highest cell adhesion rate.RT-q PCR was used to detect the m RNA expression of CD68,TNF-αand TGF-β1 in the cells of each concentration group after 24 hours of PMA treatment,and the expression of CD68and TGF-β1 was significantly increased at 50 ng/m L.These results suggested that the optimal induction conditions were 50 ng/m L and 24 h.(2)Compared with the LUAD group and Vehicle group,the morphology of macrophages treated with the supernatant of TPX2-sh RNA-2 group tended to be longer and fusiform,and most of the pseudopodia were at the poles.Flow cytometry analysis revealed a decreased trend of CD163~+macrophages in the supernatant of A549/TPX2-sh RNA-2 cells but there was no significant statistical difference,while the proportion of CD163~+macrophages in H1975/TPX2-sh RNA-2 cell supernatant decreased.However,the ratio of CD80~+M1/CD163~+M2 was significantly increased in both cells.Western Blot results showed that the expression of CD163 protein in macrophages treated with the supernatant of TPX2-sh RNA-2 group decreased,the expression of CD80 protein remained largely unchanged,while the CD80/CD163 ratio exhibited a significant increase.Compared with the TPX2-sh RNA-2 group,the number of THP1cells passing through the porous filter membrane was significantly increased in the presence of the supernatant of LUAD and Vehicle groups.These results suggested that TPX2 silencing inhibited the polarization of M0 macrophages to CD163~+M2macrophages,thereby increasing the ratio of CD80~+M1/CD163~+M2 and reducting the chemotaxis of macrophages.(3)The mechanism of the effect of TPX2 on macrophage polarization:Western Blot showed that compared with the control group,the expression of M-CSF protein in the TPX2-sh RNA-2 group was decreased.The IF expression of M-CSF was decreased in the TPX2-sh RNA-2 group.ELISA results confirmed significantly lower M-CSF secretion in the supernatant of this group compared to the control.The signal pathways were screened by RNA whole genome sequencing,and the genes differentially expressed upon silencing TPX2 in A549 cells predominantly exhibited enrichment in the TNF signaling pathway,NF-κB signaling pathway,and MAPK signaling pathway.Compared with the A549 group and the Vehicle group,the expression of p-NF-κB p65protein and the ratio of p-NF-κB p65/NF-κB p65 in the TPX2-sh RNA-2 group decreased.The expression level of p-p38 MAPK and the ratio of p-p38 MAPK to p38MAPK both exhibited an increase,whereas the expression level of p-p44/42 MAPK remained largely unaltered.Further validation in H1975 cells indicated a decrease in NF-κB p65 protein expression,p-NF-κB p65 levels,and the phosphorylated-to-total NF-κB p65 ratio within the TPX2-sh RNA-2 group.IF results showed that the expression of p-NF-κB p65 immunofluorescence in TPX2-sh RNA-2 group was significantly decreased after TPX2 silencing in A549 and H1975 cells.Finally,the protein expression of p-NF-κB p65,NF-κB p65 and M-CSF after activation of NF-κB pathway showed that A549/TPX2-sh RNA-2 cells were treated with PMA at different concentrations of 100 n M,500 n M and 1μM.The expression of p-NF-κB p65/NF-κB p65 and M-CSF increased with the concentration of 100 n M and 500 n M.These results indicated that silencing TPX2 inhibited the phosphorylation of NF-κB and the expression of its downstream gene M-CSF.After activation of NF-κB signaling pathway,M-CSF expression increased,and the inhibitory effect of TPX2 silencing on M-CSF expression could be reversed.3.TPX2,M-CSF,and CD163 expression in animal xenograft tumors and human LUAD tissues(1)A549/Vehicle and A549/TPX2-sh RNA-2 cells were subcutaneously inoculated into nude mice to create a lung adenocarcinoma xenograft model.Compared with A549/Vehicle group,the tumor weight and volume of A549/TPX2-sh RNA-2group showed a significant reduction after 20 days.HE staining showed that compared with the TPX2-sh RNA-2 group,the tumor cells in the control group showed adenoid structure or arranged in rows,and the tumor cells were large and irregular in size,with obvious nucleoli,vacuolated nuclei,nuclear division,and capillary proliferation in the stroma.(2)IHC analysis of TPX2,M-CSF and CD163 expression in xenograft tumor tissues of BALB/c A nude mice showed that TPX2 was primarily observed in the nuclei of cancer cells,while a portion of the positive cells demonstrated expression in the cytoplasm,characterized by tan granules.M-CSF was mainly expressed in the cell membrane of cancer cells,and CD163 was mainly expressed in the stroma.Compared with the control group,the expression of TPX2 protein in tumor tissues of TPX2-sh RNA-2 group was significantly decreased,and the expression levels of M-CSF and CD163 in TPX2 knockdown group were also significantly decreased.IF staining for CD163 and F4/80 demonstrated co-expression of these markers on the membranes of macrophages.(3)Further analysis of the expression of TPX2,M-CSF and CD163 in human LUAD tissues showed that TPX2 expression was detected in LUAD tissues across various differentiation levels,predominantly localized in the nuclei of cancer cells.Additionally,a subset of positive cells exhibited cytoplasmic expression,characterized by tan granules.The results indicate that the lower the degree of tumor differentiation,the higher the expression density of TPX2-positive cells;in tumors larger than 3 cm and in the lymph node metastasis group,the expression is higher than in the corresponding control groups.Furthermore,TPX2 expression in lung cancer tissues was markedly higher than in adjacent normal lung tissues.M-CSF was mainly expressed in the cell membrane of cancer cells,and the lower the degree of tumor differentiation,the higher the expression of M-CSF.M-CSF and CD163 expression were higher in cancer tissues than in normal lung tissues,with CD163 density increasing alongside tumor differentiation.The expression of CD163 was higher in the group with tumor size>3 cm and lymph node metastasis than that in the control group.(4)Correlation analysis of TPX2,M-CSF,and CD163 expression in LUAD tissues:There was a positive correlation between TPX2 and M-CSF,M-CSF and CD163,and TPX2 and CD163.In conclusion,this study verified the relationship among TPX2,NF-κB,M-CSF and CD163 through in vivo and in vitro experiments and human LUAD tissues and revealed the key factors and related signaling pathways in the process of TPX2 affecting macrophage polarization,which lays the foundation for further mechanism research.At the same time,it points out the direction for our future research work.The study on the phenotype and mechanism of macrophage polarization in LUAD provides theoretical and experimental basis for the exploration of immune cell therapy for LUAD.Conclusions1.TPX2,as a key oncogene in LUAD,promotes the progression of the disease through the polarization of CD163~+M2 macrophages.2.The NF-κB/M-CSF axis may play a role in the TPX2-induced polarization of macrophages towards CD163~+M2 macrophages.3.The promoting effect of TPX2 on macrophage polarization may become a new target for clinical cell therapy in LUAD.