Hypoxia Induces The Formation Of Suppresive Immune Microenvironment In Glioblastoma

Glioma is the most common type of tumor in the central nervous system.The glioblastoma is a subtype of glioma with the highest malignancy and worst prognosis.The average survival time of glioblastoma patients is 16 months,even after treatment.The therapeutic outcome of conventional glioma treatments such as surgery,chemotherapy,and radiotherapy is not satisfying,which is largely contributed by the heterogeneity and complex immune microenvironment of glioblastoma.The immune microenvironment of glioblastoma consists of multiple immune cells,including tumor-associated macrophages(TAM),myeloid-derived suppressive cells(MDSC)and tumor-associated lymphocytes.The glioma-induced suppressive immune environment not only suppresses the local immune function but also the peripheral immune system.Like other solid tumors,glioma has a hypoxia microenvironment in the tumor core.The partial oxygen pressure is only around 1%in WHO grade IV glioma,compared to 2.5-10%in grade III glioma.The severe hypoxia is an important inducer of the suppressive immune microenvironment in glioma.TAM is a group of tumor-infiltrating immune cells derived from monocytes.They play essential roles in suppressing the immune system and promoting glioma progression.TAM can be divided into the M1 and M2 subtypes.The M2 TAM is identified by high expression of molecular markers like CD163,CD206,IL-10,IL-1ra,and CCL-22.M2 TAM can suppress the normal immune function by secreting immunosuppressive cytokines,and they are also involved in the chemotherapy resistance of glioma.Previous research demonstrated that hypoxia could modify the subtype and regulate the recruitment of TAM in tumors.It is important to identify the underlying mechanism of how hypoxia modulates the TAM in glioma.MDSC is another critical group of glioma-associated immune cells.Human MDSC is identified as CD11b+CD33+HLA-DR-and mouse MDSC is identified as Gr-1C+Cdllb+.Because the MDSC is derived from myeloid progenitor cells in the bone marrow and their differentiation has already begun in the peripheral immune system before entering the glioma,there exists certain long-distance glioma-MDSC interaction that induces MDSC differentiation.Exosomes are cell-derived small vesicles that can deliver microRNAs to target cells and subsequently alter the function of target cells.microRNAsare a group of 19-22 nucleotides non-coding RNAs that selectively bind to their target genes’3’UTR area and inhibit the translation of their target genes.microRNAs are important regulators of immune cell differentiation.Glioma-derived exosomes can pass the blood-brain barrier,enter the circulating system,and transfer microRNAs to target cells that are distant from the brain.Hypoxia has been reported to modify the content of tumor-derived exosomes.However,it remains unclear whether hypoxia alters the glioma-derived exosomes and further changes the differentiation of immune cells.In this study,we identified how glioma hypoxia modulates the different types of glioma-associated immune cells.First of all,we determined that hypoxia upregulates the expression of POSTN,which attracts more TAM to the hypoxic area.Second,hypoxia increases the expression of M-CSFR and TGF-β,which induce TAM to differentiate into the M2 subtype.Third,we illuminated that acriflavine suppresses tumor progression by inhibiting hypoxia-induced aggregation of M2 TAM in glioblastoma.Last,through microRNA sequencing in hypoxia-induced glioblastoma exosomes,we identified that hypoxia-induced mir-10 and mir-21 expression in glioblastoma exosomes promote the expansion and function of MDSC.In summary,this research determined how hypoxia shapes the suppressive immune environment in glioma and provides potential treatment targets in glioma management.Part I.Hypoxia induces the recruitment and M2 polarization of tumor-associated macrophage in glioma1.1 Objectives:(1)Investigate how glioblastoma hypoxia influences the recruitment and M2 polarization of tumor-associated macrophages.(2)Identify the underlying molecular mechanism through which glioma hypoxia drives the recruitment and M2 polarization of tumor-associated macrophages.(3)Identify drug(s)that inhibits glioma growth by targeting the hypoxia-induced infiltration of M2 tumor-associated macrophages in glioma.1.2 Materials and Methods:(1)Immunohistochemistry and Immunofluorescence straining at human glioma tissues and mouse glioma tissuesUsing immunohistochemistry and immunofluorescence staining to identify the distribution pattern of hypoxia(HIF-1α),tumor-associated macrophage(CDllb,CD206)POSTN,M-CSFR and TGF-P expression in paraffin-embedded tissue from human and mouse glioma tissues.(2)Identify the chemotactic characteristics of glioma cell culture supernatant and POSTNUsing the transwell migration assay to study chemotactic features of POSTN and normoxia/hypoxia stimulated glioma cell culture supernatants to tumor-associated macrophages.(3)Using in vitrotumor-associated macrophage induction assay from human blood-isolated monocytes to study the hypoxia-induced macrophage polarizationStimulate human blood-isolated monocytes with hypoxia or hypoxia-stimulated glioma cell culture medium to induce these monocytes to differentiate into tumor-associated macrophages.Using cell morphology,flow-cytometry,q-PCR,and ELISA to examine the M2 differentiation of these macrophages after different types of hypoxia stimulations.Identify how hypoxia stimulates the M2 polarization of macrophage in glioma.(4)Identify the molecular pathways of how hypoxia drives the recruitment and M2 polarization of tumor-associated macrophages.Using Western blot and q-PCR to examamine protein expression and gene expression,respectively,in U87 and U251 human glioblastoma cell lines,human blood monocytes differentiated macrophages and THP-1 differentiated macrophages(after miRNA transfection,cytokine stimulation or pathway inhibitors treatment).Determine the molecular pathwaysof hypoxia that drive the recruitment and M2 polarization of tumor-associated macrophages.(5)Orthotopically xenografted glioma mouse modelImplant nude mice with human U87 glioma cells to generate an in situ glioma model in mice.Treat the glioma-bearing mice with different doses of ACF during tumor development.Using MRI scan to examine tumor size and immunohistochemistry/immunofluorescence to study the tumor-associated macrophage infiltration after Acriflavine(ACF)treatment.1.3 Results:(1)A higher level of M2 tumor-associated macrophage infiltration and POSTN expression were found in the glioma hypoxia area.Usingthe expression of HIF-la as an indicator of the hypoxia area,we determined that the level of hypoxia is higher in human high-grade glioma compared to low-grade glioma.Moreover,we identified high levels of M2 tumor-associated macrophage infiltration and POSTN expression in the hypoxia regions in glioma tissue using immunofluorescence.The survival analysis showed that high levels of POSTN expression andM2 tumor-associated macrophage infiltration correlate with a poor prognosis in glioma patients.(2)Hypoxia increases the POSTN production in glioma cells which subsequently attracts more tumor-associated macrophageWe first identified that the glioma cells that express HIF-la also express POSTN in glioma tissue using immunofluorescence.Next,we validated in the U87 and U251 glioma cells that hypoxia increases POSTN production through the HIF-1α/TGF-a/RTK/PI3K pathway.In the macrophage transwell migration assay,we compared the normoxia-and hypoxia-stimulated glioma cell culture supernatant and found that hypoxia-stimulated cell supernatant attracts more macrophages.We found that increased POSTN levels in hypoxia-stimulated cell supernatant promote macrophage migration.(3)Hypoxia drives tumor-associated macrophage M2 polarization by upregulating the expression of TGF-βin glioma cells and M-CSFR in tumor-associated macrophagesUsing the cell morphology(Rod-like shape macrophage is M2 subtype),cell surface marker(CD 163),macrophage cytokines profile(IL-10 and CCL-22)and gene expression features as M2 subtype macrophage marker,we identified that hypoxia in glioma promotes the M2 polarization of tumor-associated macrophages in two ways.First,hypoxia directly promotes the M2 polarization by upregulating M-CSFR expression in tumor-associated macrophages.Secondly,hypoxia-stimulated glioma cell culture supernatant increases the M2 polarization by increasing TGF-β secretion from glioma cells.(4)The HIF-1α inhibitor ACF inhibits the M2 tumor-associated macrophage infiltration and glioma proliferation in an orthotopically xenografted glioma mouse modelIn our glioma mouse model,ACF treatment inhibited the M2 tumor-associated macrophage infiltration and reduced the tumor size.We determined that ACF inhibits the expression of POSTN and TGF-β in glioma cells and M-CSFR expression in tumor-associated macrophage by targeting HIF-la which subsequently reduces the tumor-associated macrophage attraction and M2 polarization.1.4 Conclusion:Hypoxia in glioma can promote the M2 tumor-associated macrophage infiltration in glioma in multiple ways.First of all,hypoxia attracts tumor-associated macrophage by increasing the POSTN secretion by glioma cells.Secondly,hypoxia can promote the M2 polarization by upregulating the M-CSFR expression on tumor-associated macrophages.Lastly,hypoxia-stimulated glioma cell culture supernatant increases the M2 polarization because hypoxia increases the TGF-β secretion by glioma cells.We also identified a novel anti-glioma drug ACF which targets the hypoxia-induced M2 tumor-associated macrophage infiltration.Part Ⅱ.Hypoxia-induced glioma exosome promotes the proliferation and immunosuppressive function of myeloid-derived suppressor cells by transferring miR-10a and miR-212.1 Objectives:(1)Investigate how hypoxia influences the size and amount of glioma-derived exosomes(2)Identify how hypoxia changes the content of miRNA in glioma-derived exosomes by exosome miRNA sequencing(3)Study the effect of normoxia-/hypoxia-stimulated glioma-derived exosomes on myeloid-derived suppressive cells.(4)Identify the underlying mechanism through which hypoxia-stimulated glioma-derived exosomes activate myeloid-derived suppressive cells.2.2 Materials and Methods:(1)Isolation and identification of glioma-derived exosomesGlioma-derived exosomes were isolated from conditioned medium collected from glioma cells cultured under normoxic or hypoxic conditions using gradient centrifugation.Electron microscopy,qNano and Western blot were used to identify the size,number,and markers of exosomes.(2)Glioma-derived exosome uptake assay(by myeloid-derived suppressive cells)Glioma-derived exosomes were labeled with PKH-67.The fluorescence-labeled exosomes were co-cultured with myeloid-derived suppressive cells in vitro orintravenously injected into mice.Then,the exosome uptake was detected by confocal microscopy and flow cytometry.(3)Using glioma-derived exosomes to induce differentiation and activation of myeloid-derived suppressive cells in vitro and in vivoIn the in vitro assay,we stimulated bone marrow cells with glioma-derived exosomes(Normoxia-or hypoxia-stimulated).In the in vivo assay,glioma-derived exosomes(Normoxia-or hypoxia-stimulated)were intravenously injected into mice.Then,the proliferation of myeloid-derived suppressive cells was measured by flow cytometry detecting Gr-1+CD 11 b+cells.The immunosuppressive function of myeloid-derived suppressive cells was studied by the CFSE cell proliferation assay kit,ELISA kit(IL-10 and TGF-β),ROS detecting kit,arginase detecting kit and ROS detecting kit.(4)miRNA sequencing in glioma-derived exosomes and the key miRNA screeningThe HiSeq2500 sequencing system was used to sequence the miRNA of normoxia and hypoxia glioma-derived exosomes.Then the top 20 miRNAs were expressed in mouse bone marrow cells(transfection)to study the differentiation and immunosuppressive function of myeloid-derived suppressive cells.The miRNAs were screened based on their ability to active myeloid-derived suppressive cells.(5)Bioinformatics prediction of the miRNA’s target genes and the luciferase reporter assayThe miRNA target gene databases(targetscan,etc.)were used to screen the myeloid-derived suppressive cell activation genes that miRNA-10a and miRNA-21 could directly regulate.And then candidates were verified by q-PCR and Western blot.Finally,myeloid-derived suppressive cells were co-transfected with miRNA-10a or miRNA-21 mimics and luciferase reporter plasmids containing the mutant or wild-type 3’UTR binding sites of the target genes.Double luciferase reporter assay was performed to determine the miRNA-10a and miRNA-21 direct target genes.(6)miRNA-10a and miRNA-21 downstream molecular pathway studyMyeloid-derived suppressive cells were treated with different types miRNAs,glioma exosomes and pathway inhibitors,the molecular pathwaysof miRNA-10a and miRNA-21 that drive myeloid-derived suppressive cell activation were determined using Western blot and q-PCR.(7)miRNA-10a or miRNA-21 knock-down glioma xenograft modelWe usedlentivirus to knock down the miRNA-10a or miRNA-21 expression in GL261 glioma cells.These modified GL261 cells were implanted into the brain ofC57BL/6 mice.T2-weighted MRI was used to image the mice’s brains after glioma cell implantation.Then,the proliferation of myeloid-derived suppressive cells in mice was measured by flow cytometry detecting the Gr-1+CD1 1b+cells.The immunosuppressive function of myeloid-derived suppressive cells was studied usingthe CFSE cell proliferation assay kit,ELISA kit(IL-10 and TGF-β),ROS detecting kit,arginase detecting kit and ROS detecting kit.2.3 Results:(1)Exosomes can be phagocytized by myeloid-derived suppressive cells in vitro and in vivo.We used the gradient centrifugation method to isolate high purity exosomes with an ultracentrifuge.The exosome diameter was around 100 nm and highly expressed TSG101 and CD9.Glioma-derived exosomes were labeled with PKH-67.The fluorescence-labeled exosome was co-cultured with myeloid-derived suppressive cells in vitro orintravenously injected into mice.The exosome uptake was confirmed by confocal microscopy and flow cytometry.(2)Hypoxia-inducible miRNA-10a and miRNA-21 in glioma exosomes promote the proliferation and immunosuppressive function of myeloid-derived suppressive cellsHypoxia glioma-derived exosomesare more potent in inducing the proliferation of myeloid-derived suppressive cells both in vitro(stimulate bone marrow cells to differentiate into myeloid-derived suppressive cells)and in vivo(intravenous injection of exosomes increases the fraction of myeloid-derived suppressive cells detected in the mouse spleen).Hypoxia glioma-derived exosomes are also a stronger inducer of the immunosuppressive function(suppressing lymphocytes proliferation,ROS,NO and arginase production)of myeloid-derived suppressive cells.According to the miRNA sequencing data in normoxia and hypoxia stimulated glioma-derived exosomes,we identified 20top-expressed miRNAs in hypoxia-vs normoxia-stimulated exosomes.We further identified miRNA-10a and miRNA-21 as the key miRNAs that drive the activation of myeloid-derived suppressive cells.Knocking down these 2 miRNAs in hypoxia stimulated glioma-derived exosomes inhibited their myeloid-derived suppressive cells promoting effect.(3)miRNA-10a or miRNA-21 knock-down glioma xenograft in mouse brain suppressed the proliferation and function of myeloid-derived suppressive cellsWe usedlentivirus to knock down miRNA-1Oa or miRNA-21 expression in GL261 glioma cells,and injected modified GL261 cells into the brain of C57BL/6 mice.T2-weighted MRI was used to image the mice’s brains after glioma cell implantation.We identified that the miRNAs knockdown in glioma reduced the proliferation and immunosuppressive function of myeloid-derived suppressive cells in this mouse model.(4)The hypoxia-stimulated glioma-derived exosomes induce the activation of myeloid-derived suppressive cells through the miRNA-10a/RORA/IκBα/NF-κBand miRNA-21/PTEN/PI3K/AKT pathwaysWe identified that the hypoxia-stimulated glioma-derived exosomes induce the activation of myeloid-derived suppressive cells through miRNA-10a/RORA/IκBα/NF-κB and miRNA-21/PTEN/PI3K/AKT pathways using luciferase reporter assay,Western blot and q-PCR.2.4 Conclusion:Our research identified that hypoxia can increase the secretion of exosomes from glioma cells and upregulate the expression of miRNA-10a or miRNA-21 in exosomes which subsequently promotes the proliferation and function of myeloid-derived suppressive cells through the miRNA-10a/RORA/IκBα/NF-κB and miRNA-21/PTEN/PI3K/AKT pathways.