Mast Cell Accumulation in Glioblastoma with a Potential Role for Stem Cell Factor and Chemokine CXCL12

Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf−/− mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma

Glioma as an Ecosystem : Studies of Invasion, Onco-miR Addiction and Mast Cell Infiltration

Despite recent advances in oncology and extensive research efforts, gliomas remain essentially incurable. Glioblastoma multiforme (GBM, WHO grade IV) is the most common glioma and may arise de novo or progress from a lower-grade lesion. GBM is characterized by invasive growth, aberrant angiogenesis and necrosis. The heterogeneity of GBM is further complicated by the contribution of the inflammation that is facilitated by immune cells that reside in and infiltrate this immuno-privileged organ. One of the cells types present in the tumor microenvironment are mast cells (MC) that accumulate in the tumor in a grade-dependent manner. GBM cells secrete a plethora of cytokines acting as chemoattractants in MC recruitment and to a lesser degree induce MC proliferation in situ. Expression of one of the cytokines secreted by GBM cells - macrophage migration inhibitory factor (MIF) - correlates with MC accumulation in vivo. GBM cells invade the surrounding parenchyma making complete resection impossible. Here, migration was studied with the focus on RAP1 and its negative regulator RAP1GAP. Activation of RAP1 signaling by lentiviral silencing of RAP1GAP lead to decrease in cell migration and a shift in expression of SOX2 and GFAP, presumably enhancing stem cell phenotype. MicroRNAs are small non-coding RNAs known to regulate the mRNA network. miR-21 is highly overexpressed in the majority of cancers including GBM. Its expression is strictly regulated during embryonic development of the brain. SOX2 is co-regulated with miR-21 demarcating a cell population with neural/glial progenitor/stem cell properties. In an experimental mouse model, expression of miR-21 can be sustained by forced expression of PDGF-BB leading to gliomagenesis. GBM cells seem to be addicted to oncogenic properties of miR-21 as its knockdown leads to extensive apoptosis. This observation combined with the fact that miR-21 is absent in the normal adult mammalian brain suggest miR-21 to be an excellent therapeutic target. Effects of conventional therapy (surgery combined with radiochemotherapy) on prolonging patient survival have reached a plateau. New effective personalized therapeutic modalities need to be designed and implemented. Targeting the tumor microenvironment as well as cell intrinsic properties like invasive potential, stemness and onco-miR addiction studied in this thesis will hopefully lead to efficient disruption of GBM’s aberrant ecosystem

Glioma as an Ecosystem : Studies of Invasion, Onco-miR Addiction and Mast Cell Infiltration

Despite recent advances in oncology and extensive research efforts, gliomas remain essentially incurable. Glioblastoma multiforme (GBM, WHO grade IV) is the most common glioma and may arise de novo or progress from a lower-grade lesion. GBM is characterized by invasive growth, aberrant angiogenesis and necrosis. The heterogeneity of GBM is further complicated by the contribution of the inflammation that is facilitated by immune cells that reside in and infiltrate this immuno-privileged organ. One of the cells types present in the tumor microenvironment are mast cells (MC) that accumulate in the tumor in a grade-dependent manner. GBM cells secrete a plethora of cytokines acting as chemoattractants in MC recruitment and to a lesser degree induce MC proliferation in situ. Expression of one of the cytokines secreted by GBM cells - macrophage migration inhibitory factor (MIF) - correlates with MC accumulation in vivo. GBM cells invade the surrounding parenchyma making complete resection impossible. Here, migration was studied with the focus on RAP1 and its negative regulator RAP1GAP. Activation of RAP1 signaling by lentiviral silencing of RAP1GAP lead to decrease in cell migration and a shift in expression of SOX2 and GFAP, presumably enhancing stem cell phenotype. MicroRNAs are small non-coding RNAs known to regulate the mRNA network. miR-21 is highly overexpressed in the majority of cancers including GBM. Its expression is strictly regulated during embryonic development of the brain. SOX2 is co-regulated with miR-21 demarcating a cell population with neural/glial progenitor/stem cell properties. In an experimental mouse model, expression of miR-21 can be sustained by forced expression of PDGF-BB leading to gliomagenesis. GBM cells seem to be addicted to oncogenic properties of miR-21 as its knockdown leads to extensive apoptosis. This observation combined with the fact that miR-21 is absent in the normal adult mammalian brain suggest miR-21 to be an excellent therapeutic target. Effects of conventional therapy (surgery combined with radiochemotherapy) on prolonging patient survival have reached a plateau. New effective personalized therapeutic modalities need to be designed and implemented. Targeting the tumor microenvironment as well as cell intrinsic properties like invasive potential, stemness and onco-miR addiction studied in this thesis will hopefully lead to efficient disruption of GBM’s aberrant ecosystem

Glioma as an Ecosystem : Studies of Invasion, Onco-miR Addiction and Mast Cell Infiltration

Despite recent advances in oncology and extensive research efforts, gliomas remain essentially incurable. Glioblastoma multiforme (GBM, WHO grade IV) is the most common glioma and may arise de novo or progress from a lower-grade lesion. GBM is characterized by invasive growth, aberrant angiogenesis and necrosis. The heterogeneity of GBM is further complicated by the contribution of the inflammation that is facilitated by immune cells that reside in and infiltrate this immuno-privileged organ. One of the cells types present in the tumor microenvironment are mast cells (MC) that accumulate in the tumor in a grade-dependent manner. GBM cells secrete a plethora of cytokines acting as chemoattractants in MC recruitment and to a lesser degree induce MC proliferation in situ. Expression of one of the cytokines secreted by GBM cells - macrophage migration inhibitory factor (MIF) - correlates with MC accumulation in vivo. GBM cells invade the surrounding parenchyma making complete resection impossible. Here, migration was studied with the focus on RAP1 and its negative regulator RAP1GAP. Activation of RAP1 signaling by lentiviral silencing of RAP1GAP lead to decrease in cell migration and a shift in expression of SOX2 and GFAP, presumably enhancing stem cell phenotype. MicroRNAs are small non-coding RNAs known to regulate the mRNA network. miR-21 is highly overexpressed in the majority of cancers including GBM. Its expression is strictly regulated during embryonic development of the brain. SOX2 is co-regulated with miR-21 demarcating a cell population with neural/glial progenitor/stem cell properties. In an experimental mouse model, expression of miR-21 can be sustained by forced expression of PDGF-BB leading to gliomagenesis. GBM cells seem to be addicted to oncogenic properties of miR-21 as its knockdown leads to extensive apoptosis. This observation combined with the fact that miR-21 is absent in the normal adult mammalian brain suggest miR-21 to be an excellent therapeutic target. Effects of conventional therapy (surgery combined with radiochemotherapy) on prolonging patient survival have reached a plateau. New effective personalized therapeutic modalities need to be designed and implemented. Targeting the tumor microenvironment as well as cell intrinsic properties like invasive potential, stemness and onco-miR addiction studied in this thesis will hopefully lead to efficient disruption of GBM’s aberrant ecosystem

miRNA-21 is developmentally regulated in mouse brain and is co-expressed with SOX2 in glioma

Background MicroRNAs (miRNAs) and their role during tumor development have been studied in greatdetail during the last decade, albeit their expression pattern and regulation during normaldevelopment are however not so well established. Previous studies have shown that miRNAsare differentially expressed in solid human tumors. Platelet-derived growth factor (PDGF)signaling is known to be involved in normal development of the brain as well as in malignantprimary brain tumors, gliomas, but the complete mechanism is still lacking. We decided toinvestigate the expression of the oncogenic miR-21 during normal mouse development andglioma, focusing on PDGF signaling as a potential regulator of miR-21. Methods We generated mouse glioma using the RCAS/tv-a system for driving PDGF-BB expression ina cell-specific manner. Expression of miR-21 in mouse cell cultures and mouse brain wereassessed using Northern blot analysis and in situ hybridization. Immunohistochemistry andWestern blot analysis were used to investigate SOX2 expression. LNA-modified siRNA wasused for irreversible depletion of miR-21. For inhibition of PDGF signaling Gleevec(imatinib mesylate), Rapamycin and U0126, as well as siRNA were used. Statisticalsignificance was calculated using double-sided unpaired Student´s t-test. Results We identified miR-21 to be highly expressed during embryonic and newborn braindevelopment followed by a gradual decrease until undetectable at postnatal day 7 (P7), thiscorrelated with SOX2 expression. Furthermore, miR-21 and SOX2 showed up-regulation andoverlapping expression pattern in RCAS/tv-a generated mouse brain tumor specimens. Uponirreversible depletion of miR-21 the expression of SOX2 was strongly diminished in bothmouse primary glioma cultures and human glioma cell lines. Interestingly, in normalfibroblasts the expression of miR-21 was induced by PDGF-BB, and inhibition of PDGFsignaling in mouse glioma primary cultures resulted in suppression of miR-21 suggesting thatmiR-21 is indeed regulated by PDGF signaling. Conclusions Our data show that miR-21 and SOX2 are tightly regulated already during embryogenesisand define a distinct population with putative tumor cell of origin characteristics. We believethat miR-21 is a mediator of PDGF-driven brain tumors, which suggests miR-21 as apromising target for treatment of glioma

Glioma-derived plasminogen activator inhibitor-1 (PAI-1) regulates the recruitment of LRP1 positive mast cells

Glioblastoma (GBM) is a high-grade glioma with a complex microenvironment, including various inflammatory cells and mast cells (MCs) as one of them. Previously we had identified glioma grade-dependent MC recruitment. In the present study we investigated the role of plasminogen activator inhibitor 1 (PAI-1) in MC recruitment.PAI-1, a primary regulator in the fibrinolytic cascade is capable of forming a complex with fibrinolytic system proteins together with low-density lipoprotein receptor-related protein 1 (LRP1). We found that neutralizing PAI-1 attenuated infiltration of MCs. To address the potential implication of LRP1 in this process, we used a LRP1 antagonist, receptor-associated protein (RAP), and demonstrated the attenuation of MC migration. Moreover, a positive correlation between the number of MCs and the level of PAI-1 in a large cohort of human glioma samples was observed. Our study demonstrated the expression of LRP1 in human MC line LAD2 and in MCs in human high-grade glioma. The activation of potential PAI-1/LRP1 axis with purified PAI-1 promoted increased phosphorylation of STAT3 and subsequently exocytosis in MCs.These findings indicate the influence of the PAI-1/LRP1 axis on the recruitment of MCs in glioma. The connection between high-grade glioma and MC infiltration could contribute to patient tailored therapy and improve patient stratification in future therapeutic trials.De två första författarna delar förstaförfattarskapet.</p

Glioma-derived plasminogen activator inhibitor-1 (PAI-1) regulates the recruitment of LRP1 positive mast cells

Glioblastoma (GBM) is a high-grade glioma with a complex microenvironment, including various inflammatory cells and mast cells (MCs) as one of them. Previously we had identified glioma grade-dependent MC recruitment. In the present study we investigated the role of plasminogen activator inhibitor 1 (PAI-1) in MC recruitment.PAI-1, a primary regulator in the fibrinolytic cascade is capable of forming a complex with fibrinolytic system proteins together with low-density lipoprotein receptor-related protein 1 (LRP1). We found that neutralizing PAI-1 attenuated infiltration of MCs. To address the potential implication of LRP1 in this process, we used a LRP1 antagonist, receptor-associated protein (RAP), and demonstrated the attenuation of MC migration. Moreover, a positive correlation between the number of MCs and the level of PAI-1 in a large cohort of human glioma samples was observed. Our study demonstrated the expression of LRP1 in human MC line LAD2 and in MCs in human high-grade glioma. The activation of potential PAI-1/LRP1 axis with purified PAI-1 promoted increased phosphorylation of STAT3 and subsequently exocytosis in MCs.These findings indicate the influence of the PAI-1/LRP1 axis on the recruitment of MCs in glioma. The connection between high-grade glioma and MC infiltration could contribute to patient tailored therapy and improve patient stratification in future therapeutic trials.De två första författarna delar förstaförfattarskapet.</p

Glioma-derived plasminogen activator inhibitor-1 (PAI-1) regulates the recruitment of LRP1 positive mast cells

Glioblastoma (GBM) is a high-grade glioma with a complex microenvironment, including various inflammatory cells and mast cells (MCs) as one of them. Previously we had identified glioma grade-dependent MC recruitment. In the present study we investigated the role of plasminogen activator inhibitor 1 (PAI-1) in MC recruitment.PAI-1, a primary regulator in the fibrinolytic cascade is capable of forming a complex with fibrinolytic system proteins together with low-density lipoprotein receptor-related protein 1 (LRP1). We found that neutralizing PAI-1 attenuated infiltration of MCs. To address the potential implication of LRP1 in this process, we used a LRP1 antagonist, receptor-associated protein (RAP), and demonstrated the attenuation of MC migration. Moreover, a positive correlation between the number of MCs and the level of PAI-1 in a large cohort of human glioma samples was observed. Our study demonstrated the expression of LRP1 in human MC line LAD2 and in MCs in human high-grade glioma. The activation of potential PAI-1/LRP1 axis with purified PAI-1 promoted increased phosphorylation of STAT3 and subsequently exocytosis in MCs.These findings indicate the influence of the PAI-1/LRP1 axis on the recruitment of MCs in glioma. The connection between high-grade glioma and MC infiltration could contribute to patient tailored therapy and improve patient stratification in future therapeutic trials.<p>De två första författarna delar förstaförfattarskapet.</p