Dicer Deficiency In Glioblastoma Associated Macrophages Inhibits Glioblastoma Growth

Glioblastoma(GB)is the most common and lethal primary brain tumor.The risk of recurrence of GB is high,whereas the overall survival of GB patient remains badly short,these evidence proves a gloomy prognosis of GB.The maintenance and progression of GB is influenced by the tumor microenvironment of GB,within which glioma-associated macrophages(GAMs)share the most common population of non-transformed cells in GB.It is reported that GAMs displayed pro-tumoral phenotype,promoted the proliferation,invasive of GB cell,vascularization,and the self-renewal of Glioma stem cells(GSCs).As a result,promoted GB growth.However,the phagocytosis function and inflammatory-activation potential of GAMs are poorly inactivated,so a comprehensive understanding of GB immune microenvironment,as well as the mechanism involved in regulating GAM phenotype,is crucial for uncovering the causation for impaired immunotherapies towards GB,and prolonged the survival of GB patients.By applying 10×genomics single cell sequencing,scientists got to know that different GAM sub-populations with distinct phenotypes were involved in GB microenvironment,including Sepp1-positive GAM,hypoxic GAM,proliferated GAM,IFN-producing GAM and phagocytotic GAM.Sepp1-positive GAM and hypoxic GAM were highly expressed antiinflammatory-related genes such as Mrc1 and Arg1,suggesting their pro-tumoral potential.IFN-producing GAM and phagocytotic GAM displayed IFN signature genes and were capable of phagocytosis,indicating their anti-tumoral potential.The evidence above indicated that increasing the percentage of anti-tumoral GAM sub-population,or downregulating the percentage of pro-tumoral GAM sub-population could be a new GB therapeutic strategy.Dicer is a member of RNase protein family,plays key roles in the clipping of pre-miRNAs and thus promoting the maturation of miRNAs.miRNAs inhibit the transcription of multiple genes through binding to the 3’-UTR of m RNA.In macrophages,Dicer could regulate the content of miR-125 and let-7b,further regulate the inflammatory activation of macrophage.It is reported that knockout macrophage Dicer promoted macrophages trans-differentiated into classically activated macrophages,promoting the expression of IFNγ of macrophage,as a result,activated T cells and further inhibited tumor growth.However,the function of Dicer in regulating other functions of macrophages is not clear.In this study,we carried out10×genomics single cell sequencing on GB tissues acquired from Dicer WT group and macrophage conditional Dicer knockout group,to investigate the effect of deleting GAM Dicer on the constitution of GAM sub-populations.We discovered that Dicer knockout in GAM increased the percentage of IFN-producing GAM and phagocytotic GAM sub-clusters,and decreased proliferated GAM,Sepp1-positive GAM and hypoxic GAM sub-clusters to inhibit the growth of GB and prolong survival.This study provides a new insight of deleting macrophage Dicer on GAM phenotype.The main finding and significance of this study are listed as follows:1.The effect of knocking out macrophage Dicer on GB growth: 1)knocking out macrophage Dicer prolonged survival of xenograft-bearing transgenic mice,and inhibited the growth of murine GB;2)knocking out macrophage Dicer increased the percentage of NK cell population,T cell population and DC population in GB,decreased the proliferated macrophage population;3)knocking out macrophage Dicer increased the percentage of anti-tumoral like GAM sub-clusters,whereas decreased the percentage of pro-tumoral like GAM sub-clusters.2.The effect of knocking out macrophage Dicer on GAM function: 1)knocking out macrophage Dicer significantly inhibited the proliferation of GAMs,inhibited the S and G2 M phases of GAM;2)knocking out macrophage Dicer significantly enhanced phagocytosis of GAMs;3)knocking out macrophage Dicer promoted the inflammatory activation of GAMs;4)in macrophage Dicer knockout group,TNF-TNFR was enriched in the crosstalk between antitumoral GAM cluster and NK/T cell cluster;5)knocking out macrophage Dicer promoted the expression of IFN in T cells.3.the mechanism involved in Dicer regulating GAM proliferation and phagocytosis: 1)Dicer regulated GAM proliferation by miR-24-3p,mi2R-142-3p and miR-196a-5p;2)Dicer regulated GAM phagocytosis by miR-24-3p and miR-142-3p.4.the therapeutic significance of knocking out macrophage Dicer in GB:1)treatment of Dicer knockout monocytes significantly prolonged survival of GB-bearing mice;2)treatment of Dicer knockout monocytes promoted macrophages phagocyted GB cells 3)GAM Dicer knockout in combine with TMZ treatment significantly inhibited GB growth and prolonged the survival of GB-bearing mice.In summary,the conclusions of this study are listed as follows:1.Macrophage Dicer knockout prolonged the survival of xenograft-bearing transgenic mice and inhibited the growth of murine GB.2.Macrophage Dicer knockout increased the percentage of anti-tumoral like GAM subclusters and decreased the percentage of pro-tumoral like GAM sub-clusters.3.Macrophage Dicer knockout inhibited the proliferation of GAMs,promoted the phagocytotic function and inflammatory activation of GAMs through miR-24-3p and miR-142-3p.4.Dicer-knockout monocytes significantly prolonged survival of GB-bearing mice,GAM Dicer knockout in combine with TMZ treatment significantly inhibited GB growth and prolonged the survival of GB-bearing mice.Taken together,this study evaluated the effect and mechanism of macrophage Dicer knockout on GB growth,the constitution of GB microenvironment and GAM phenotype,facilitating the application of Dicer-deficient monocytes on tumor therapy.