Chemerin Enhances Mesenchymal Features Of Glioblastoma By Establishing Autocrine And Paracrine Networks In A CMKLR1-Dependent Manner

Objective:Glioblastoma(GBM)is the most common and aggressive primary brain tumor.During GBM progression and recurrence,mesenchymal subtype GBM is the most stabilized molecular subtype which could be transformed from other GBM subtypes.GBM with higher mesenchymal feature has a more potent invasion ability and treatment resistance which finally leads to therapy failure and tumor recurrence.The molecular subtype features can be characterized as a dynamic condition in response to intrinsic(accumulating genetic and epigenetic abnormalities)and extrinsic(therapeutic stress)factors.Therefore,clarifying the mechanisms governing mesenchymal features may provide valuable clues for curing this intractable GBM subtype.Tumor-associated macrophages(TAMs)have been deemed as the major cellular component within GBM microenvironment.It has been reported that TAM infiltration is associated with the mesenchymal subtype and can even enhance mesenchymal features.In turn,mesenchymal transformed cells can recruit more TAMs to facilitate their mesenchymal subtype.Therefore,there is an interactive network between TAMs and mesenchymal GBM which boosts the malignancy of GBM.However,up to now,how TAMs become the major immune cells facilitating mesenchymal features in GBM,and the exact molecular network between their interaction requires further investigation.Methods:1.To explore the features of microenvironment in mesenchymal subtype GBM,GBM patients with transcriptomic data from TCGA and CGGA database were analyzed.The composition of immune cells was decoded by the CIBERSORT algorithms.2.To investigate the relation between TAMs and mesenchymal features,the GSVA and ss GSEA algorithm was used to estimate mesenchymal status and CIBERSORT and x Cell algorithms was used to evaluate TAM enrichment in CGGA and TCGA GBM cohorts.Results were further validated using samples of GBM patients.Genes upregulated in mesenchymal GBMs and had positive relation with TAM enrichment were selected.RARRES2,which encoded chemerin,was as a prognostic mediator to facilitate the crosstalk between mesenchymal GBM and TAMs.3.Using CGGA and TCGA GBM cohorts and single-cell sequencing data to investigate chemerin’s clinical characters and its producing cell types.Real-time PCR,ELISA,immunohistochemical staining,and flow cytometry were used to confirm the findings in in-house GBM samples.4.Lentivirus was used to establish chemerin overexpressed or knockdown GSCs,and by RNA sequencing,migration/invasion analysis,Real-time PCR,and Western blotting,we investigated chemerin’s autocrine effect on mesenchymal features enhancement in GBM cells.5.Using Real-time PCR,immunoprecipitation,and Western blotting analysis to investigate the role of CMKLR1 in chemerin mediated effect in GBM cells,and the suppressive effect of chemerin on K48/K27 chain dependent ubiquitin-proteasomal degradation of CMKLR1.6.From blocking or rescue experiments in multi-step Transwell co-culturing scheme,we investigated infiltration,polarization,and pro-mesenchymal ability of TAMs by using Real-time PCR,Western blotting,flow cytometry,and migration assay.7.Using Western blotting to investigate whether NF-κB signaling was indispensable for chemerin to elicit pro-mesenchymal effect.8.Using survival analysis,flow cytometry,immunohistochemical staining,Real-time PCR and Western blotting to investigate the effect of chemerin and chemerin-CMKLR1 axis blockade therapy on mesenchymal status,TAM infiltration,and anti-tumor immune environment in orthotopic xenograft mice models.Results:1.TAM infiltration is positively associated with mesenchymal status in GBM:Mesenchymal subtype GBM was enriched by the most abundant immune cell populations,and mesenchymal GBM could be characterized by M0 and M2 subtype TAM enrichment.As a feature of mesenchymal GBM,TAM enrichment score and infiltration could be a sensitive and accurate measurable indicator to predict mesenchymal subtype GBM.2.Chemerin has the potential to mediate TAMs and mesenchymal GBM crosstalk:According to gene selection procedure,RARRES2,which encoded chemerin,was identified as a prognostic mediator having possibility to facilitate the crosstalk between TAMs and mesenchymal subtype GBM.3.Chemerin is a malignant indicator mainly expressed by mesenchymal GBM cells:Both m RNA and protein expression of chemerin were the highest in tumor tissues and peripheral blood of GBM patients,as compared with that of LGG patients or healthy donors.Patients with higher RARRES2 expression tended to suffer reduced overall survival time in independent GBM cohorts.Higher chemerin expression also implied an increased likelihood of suffering with mesenchymal subtype GBMs and increased mesenchymal features.Moreover,chemerin was preferentially expressed in high mesenchymal status GBM cells.4.Chemerin enhances mesenchymal features in an autocrine manner:RNA sequencing analysis showed higher mesenchymal features in r Chemerin stimulated GSCs.In chemerin overexpressed GSCs,the migratory\invasive abilities and protein expression of mesenchymal markers were significantly increased,while chemerin knockdown GSCs had the opposite results.5.Chemerin facilitates its effect on mesenchymal promotion by inhibiting CMKLR1ubiquitin-proteasomal degradation:CMKLR1 is the main receptor for chemerin to transmit its stimulation signals,and its expression was also enriched in mesenchymal subtype GBM.Moreover,we found that RARRES2 indicated poor outcomes in patients with high CMKLR1 expression,but had limited prognostic implications in low CMKLR1 expression patients.Knocking down possible receptors for chemerin(CMKLR1,GPR1,and CCRL2)in GSCs showed that CMKLR1 was the only receptor mediating chemerin’s effect in mesenchymal enhancement.The pulldown assay of CMKLR1 showed that overexpression or knockdown of chemerin induced a significant downregulation or upregulation of CMKLR1 ubiquitination,respectively.Moreover,we found K48/K27-linked ubiquitin chain-mediated proteasome pathway mediated degradation of CMKLR1 in GBM.6.GBM-derived chemerin enhances the recruiting,M2 polarization,and mesenchymal-promoting effect of TAMs:Overexpressing chemerin in GSCs significantly increased their TAMs recruiting ability and encouraged the M2 polarization of TAMs.Treatment of chemerin knockdown GBM cells with recombinant chemerin can rescue the decreasing of TAM infiltration and M2 subtype TAM induction.Influence of TAMs in promoting mesenchymal markers expression in GBM cells was significantly improved after co-culturing TAMs with chemerin overexpressed GSCs,while chemerin knockdown GSCs weakened this effect.7.Chemerin positively regulates mesenchymal features and TAM infiltration to promote GBM progression in vivo:Chemerin overexpressed GBM orthotopic models had significantly shortened survival time and smaller tumor volume,and both higher mesenchymal features and TAM infiltration.8.Chemerin activates NF-κB signaling to enhance mesenchymal features in GBM cells and influence biological function of TAMs:RNA sequencing and publicly available data showed that chemerin was positively related to NF-κB signaling activation.Chemerin could increase NF-κB signaling activation in a time-dependent manner,and NF-κB signaling inhibition suppressed mesenchymal markers expression in chemerin overexpressed GSCs.Moreover,NF-κB signaling was also the only significantly changed pathway which was consistent in all the GSCs co-cultured TAMs.9.Targeting chemerin/CMKLR1 axis decreases mesenchymal features and increases mouse survival:Neutralization chemerin,knockdown CMKLR1,and a CMKLR1 inhibitor,α-NETA,treatment effectively abrogated chemerin-mediated mesenchymal features enhancement in GBM cells,decreased TAM recruiting and M2 polarization.Intraperitoneal injection ofα-NETA could significantly inhibit the growth of tumors and prolonged the survival time of orthotopic tumor-bearing mice.Decreased expression of mesenchymal markers and NF-κB signaling activation were observed inα-NETA-treated chemerin overexpressed orthotopic tumors.Treatment withα-NETA also reduced TAM infiltration and suppressed M2 polarization of TAMs.Meanwhile,the immune microenvironment afterα-NETA treatment was featured by enriched IFN-γ~+/TNF-α~+effective CD8~+and CD4~+T cells,while less PD-1~+exhausted CD8~+and CD4~+T cells.These results demonstrate that chemerin/CMKLR1 axis blockade effectively suppresses the chemerin-mediated mesenchymal promoting network and improves anti-tumor immune environment.Conclusions:Our study suggests chemerin as a novel mediator of GBM-TAM interaction,which was featured by its autocrine and paracrine effects in establishing a mesenchymal promoting network.Chemerin/CMKLR1 axis blockade can eliminate the pro-mesenchymal effects of chemerin,which indicates a novel promising therapeutic target in GBM.