Konrad Gabrusiewicz

Characteristics of the alternative phenotype of microglia/macrophages and its modulation in experimental gliomas.

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b+ cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b+ cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas.

The Controversial Role of Microglia in Malignant Gliomas

Malignant gliomas contain stroma and a variety of immune cells including abundant activated microglia/macrophages. Mounting evidence indicates that the glioma microenvironment converts the glioma-associated microglia/macrophages (GAMs) into glioma-supportive, immunosuppressive cells; however, GAMs can retain intrinsic anti-tumor properties. Here, we review and discuss this duality and the potential therapeutic strategies that may inhibit their glioma-supportive and propagating functions.

Distinct roles of CSF family cytokines in macrophage infiltration and activation in glioma progression and injury response

Gliomas attract brain‐resident (microglia) and peripheral macrophages and reprogram these cells into immunosuppressive, pro‐invasive cells. M‐CSF (macrophage colony‐stimulating factor, encoded by the CSF1 gene) has been implicated in the control of recruitment and polarization of macrophages in several cancers. We found that murine GL261 glioma cells overexpress GM‐CSF (granulocyte–macrophage colony‐stimulating factor encoded by the CSF2 gene) but not M‐CSF when compared to normal astrocytes. Knockdown of GM‐CSF in GL261 glioma cells strongly reduced microglia‐dependent invasion in organotypical brain slices and growth of intracranial gliomas and extended animal survival. The number of infiltrating microglia/macrophages (Iba1+ cells) and intratumoural angiogenesis were reduced in murine gliomas depleted of GM‐CSF. M1/M2 gene profiling in sorted microglia/macrophages suggests impairment of their pro‐invasive activation in GM‐CSF‐depleted gliomas. Deficiency of M‐CSF (op/op mice) did not affect glioma growth in vivo and the accumulation of Iba1+ cells, but impaired accumulation of Iba1+ cells in response to demyelination. These results suggest that distinct cytokines of the CSF family contribute to macrophage infiltration of tumours and in response to injury. The expression of CSF2 (but not CSF1) was highly up‐regulated in glioblastoma patients and we found an inverse correlation between CSF2 expression and patient survival. Therefore we propose that GM‐CSF triggers and drives the alternative activation of tumour‐infiltrating microglia/macrophages in which these cells support tumour growth and angiogenesis and shape the immune microenvironment of gliomas. Copyright

Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

Distinctive pattern of cannabinoid receptor type II (CB2) expression in adult and pediatric brain tumors.

The efficacy of cannabinoids against high-grade glioma in animal models, mediated by two specific receptors, CB1 and CB2, raised promises for targeted treatment of the most frequent and malignant primary brain tumors. Unlike the abundantly expressed CB1, the CB2 receptor shows a restricted distribution in normal brain. Although brain tumors constitute the second most common malignancy in children and the prevalence of histological types of brain tumors vary significantly between the adult and pediatric populations, cannabinoid receptor expression in pediatric tumors remains unknown. In the present study, we compared the expression of the CB2 receptor in paraffin-embedded sections from primary brain tumors of adult and pediatric patients. Most glioblastomas expressed very high levels of CB2 receptors and the expression correlated with tumor grade. Interestingly, some benign pediatric astrocytic tumors, such as subependymal giant cell astrocytoma (SEGA), which may occasionally cause mortality owing to progressive growth, also displayed high CB2 immunoreactivity. The high levels of CB2 expression would predestine those tumors to be vulnerable to cannabinoid treatment. In contrast, all examined cases of embryonal tumors (medulloblastoma and S-PNET), the most frequently diagnosed malignant brain tumors in childhood, showed no or trace CB2 immunoreactivity. Our results suggest that the CB2 receptor expression depends primarily on the histopathological origin of the brain tumor cells and differentiation state, reflecting the tumor grade.

effect of mir-142-3p on the M2 Macrophage and therapeutic efficacy Against Murine Glioblastoma

BACKGROUND The immune therapeutic potential of microRNAs (miRNAs) in the context of tumor-mediated immune suppression has not been previously described for monocyte-derived glioma-associated macrophages, which are the largest infiltrating immune cell population in glioblastomas and facilitate gliomagenesis. METHODS An miRNA microarray was used to compare expression profiles between human glioblastoma-infiltrating macrophages and matched peripheral monocytes. The effects of miR-142-3p on phenotype and function of proinflammatory M1 and immunosuppressive M2 macrophages were determined. The therapeutic effect of miR-142-3p was ascertained in immune-competent C57BL/6J mice harboring intracerebral GL261 gliomas and in genetically engineered Ntv-a mice bearing high-grade gliomas. Student t test was used to evaluate the differences between ex vivo datasets. Survival was analyzed with the log-rank test and tumor sizes with linear mixed models and F test. All statistical tests were two-sided. RESULTS miR-142-3p was the most downregulated miRNA (approximately 4.95-fold) in glioblastoma-infiltrating macrophages. M2 macrophages had lower miR-142-3p expression relative to M1 macrophages (P = .03). Overexpression of miR-142-3p in M2 macrophages induced selective modulation of transforming growth factor beta receptor 1, which led to subsequent preferential apoptosis in the M2 subset (P = .01). In vivo miR-142-3p administration resulted in glioma growth inhibition (P = .03, n = 5) and extended median survival (miR-142-3p-treated C57BL/6J mice vs scramble control: 31 days vs 23.5 days, P = .03, n = 10; miR-142-3p treated Ntv-a mice vs scramble control: 32 days vs 24 days, P = .03, n = 9), with an associated decrease in infiltrating macrophages (R (2) = .303). CONCLUSIONS These data indicate a unique role of miR-142-3p in glioma immunity by modulating M2 macrophages through the transforming growth factor beta signaling pathway.

The antitumorigenic response of neural precursors depends on subventricular proliferation and age.

Glioblastomas, the most aggressive primary brain tumors, occur almost exclusively in adult patients. Neural precursor cells (NPCs) are antitumorigenic in mice, as they can migrate to glioblastomas and induce tumor cell death. Here, we show that the antitumor effect of NPCs is age‐dependently controlled by cell proliferation in the subventricular zone (SVZ) and that NPCs accumulating at a glioblastoma are diverted from their normal migratory path to the olfactory bulb. Experimentally induced cortical glioblastomas resulted in decreased subventricular proliferation in adult (postnatal day 90) but not in young (postnatal day 30) mice. Adult mice supplied fewer NPCs to glioblastomas and had larger tumors than young mice. Apart from the difference in proliferation, there was neither a change in cell number and death rate in the SVZ nor a change in angiogenesis and immune cell density in the tumors. The ability to kill glioblastomas was similar in NPCs isolated from young and adult mice. The proliferative response of NPCs to glioblastomas depended on the expression of D‐type cyclins. In young mice, NPCs express the cyclins D1 and D2, but the expression of cyclin D1 is lost during aging, and in adult NPCs only cyclin D2 remains. In young and adult cyclin D2‐deficient mice we observed a reduced supply of NPCs to glioblastomas and the generation of larger tumors compared with wild‐type mice. We conclude that cyclin D1 and D2 are nonredundant for the antitumor response of subventricular NPCs. Loss of a single D‐type cyclin results in a smaller pool of proliferating NPCs, lower number of NPCs migrating to the tumor, and reduced antitumor activity.

Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells.

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.

FGL2 as a Multimodality Regulator of Tumor-Mediated Immune Suppression and Therapeutic Target in Gliomas

BACKGROUND Fibrinogen-like protein 2 (FGL2) may promote glioblastoma multiforme (GBM) cancer development by inducing multiple immune-suppression mechanisms. METHODS The biological significance of FGL2 expression was assessed using the The Cancer Genome Atlast (TCGA) glioma database and tumor lysates analysis. The therapeutic effects of an anti-Fgl2 antibody and the role of immune suppression regulation by Fgl2 were determined in immune-competent, NOD-scid IL2Rgammanull (NSG), and FcɣRIIB-/- mice (n = 3-18 per group). Data were analyzed with two-way analysis of variance, log-rank survival analysis, and Pearson correlation. All statistical tests were two-sided. RESULTS In low-grade gliomas, 72.5% of patients maintained two copies of the FGL2 gene, whereas 83.8% of GBM patients had gene amplification or copy gain. Patients with high levels of FGL2 mRNA in glioma tissues had a lower overall survival (P = .009). Protein levels of FGL2 in GBM lysates were higher relative to low-grade glioma lysates (11.48±5.75ng/mg vs 3.96±1.01ng/mg, P = .003). In GL261 mice treated with an anti-FGL2 antibody, median survival was 27 days compared with only 17 days for mice treated with an isotype control antibody (P = .01). The anti-FGL2 antibody treatment reduced CD39(+) Tregs, M2 macrophages, programmed cell death protein 1 (PD-1), and myeloid-derived suppressor cells (MDSCs). FGL2-induced increases in M2, CD39, and PD-1 were ablated in FcɣRIIB-/- mice. CONCLUSIONS FGL2 augments glioma immunosuppression by increasing the expression levels of PD-1 and CD39, expanding the frequency of tumor-supportive M2 macrophages via the FcγRIIB pathway, and enhancing the number of MDSCs and CD39(+) regulatory T cells. Collectively, these results show that FGL2 functions as a key immune-suppressive modulator and has potential as an immunotherapeutic target for treating GBM.

Down-regulation of IKKβ expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma

Glioblastoma (GBM) is an aggressive malignancy associated with profound host immunosuppression. Microglia and macrophages infiltrating GBM acquire the pro-tumorigenic, M2 phenotype and support tumor invasion, proliferation, survival, angiogenesis and block immune responses both locally and systematically. Mechanisms responsible for immunological deficits in GBM patients are poorly understood. We analyzed immune/inflammatory gene expression in five datasets of low and high grade gliomas, and performed Gene Ontology and signaling pathway analyses to identify defective transcriptional responses. The expression of many immune/inflammatory response and TLR signaling pathway genes was reduced in high grade gliomas compared to low grade gliomas. In particular, we found the reduced expression of the IKBKB, a gene coding for IKKβ, which phosphorylates IκB proteins and represents a convergence point for most signal transduction pathways leading to NFκB activation. The reduced IKBKB expression and IKKβ levels in GBM tissues were demonstrated by qPCR, Western blotting and immunohistochemistry. The IKKβ expression was down-regulated in microglia/macrophages infiltrating glioblastoma. NFκB activation, prominent in microglia/macrophages infiltrating low grade gliomas, was reduced in microglia/macrophages in glioblastoma tissues. Down-regulation of IKBKB expression and NFκB signaling in microglia/macrophages infiltrating glioblastoma correlates with defective expression of immune/inflammatory genes and M2 polarization that may result in the global impairment of anti-tumor immune responses in glioblastoma.