Molecular Heterogeneity and Immunosuppressive Microenvironment in Glioblastoma

Abstract

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with a poor prognosis, despite surgical resection combined with radio- and chemotherapy. The major clinical obstacles contributing to poor GBM prognosis are late diagnosis, diffuse infiltration, pseudo-palisading necrosis, microvascular proliferation, and resistance to conventional therapy. These challenges are further compounded by extensive inter- and intra-tumor heterogeneity and the dynamic plasticity of GBM cells. The complex heterogeneous nature of GBM cells is facilitated by the local inflammatory tumor microenvironment, which mostly induces tumor aggressiveness and drug resistance. An immunosuppressive tumor microenvironment of GBM provides multiple pathways for tumor immune evasion. Infiltrating immune cells, mostly tumor-associated macrophages, comprise much of the non-neoplastic population in GBM. Further understanding of the immune microenvironment of GBM is essential to make advances in the development of immunotherapeutics. Recently, whole-genome sequencing, epigenomics and transcriptional profiling have significantly helped improve the prognostic and therapeutic outcomes of GBM patients. Here, we discuss recent genomic advances, the role of innate and adaptive immune mechanisms, and the presence of an established immunosuppressive GBM microenvironment that suppresses and/or prevents the anti-tumor host response.

Keywords: astrocytes; brain tumor; glioblastoma; immunity; microenvironment; microglia.

Figure 1

Dissection of Mutational and Epigenetic GBM Subtype Classifications. Glioblastoma (GBM) is a highly heterogeneous disease with distinct, recurring molecular subtypes that differ in their associated expression profile, mutational signature, and epigenetic modifications. GBM can be classified into three main subtypes: the proneural (PN), mesenchymal (MSC), and classical (CL) subtype. PN gliomas tend to display an expression profile resembling oligodendrocytes, high levels of PDGFRα (due to amplifications or mutations) as well as characteristic mutations in IDH1. The latter leads to an epigenetic CpG island methylator phenotype (C-GIMP), which is associated with younger patients and a better prognosis. MSC subtype tumors, on the other hand, show a high rate of NF1 mutations which, in turn, promotes NF-κB activation and, thereby, aggressiveness, invasiveness, and myeloid recruitment. This translates into a therapy resistant phenotype for MSC gliomas with poorer survival compared to the other subtypes. The third subtype is the classical subtype, which preserves wild-type p53 expression, but shows over-expression and/or mutation of EGFR. Both MSC and CL tumor cells resemble (cultured) astrocytic gene expression profiles as well as epigenetically a G-CIMP low phenotype. The distinction between G-CIMP high and low is not only prognostically relevant (as G-CIMP high shows improved prognosis), but also predictively. Methylation of MGMT, which is observed in G-CIMP high tumors, in conjunction with 1p/19q deletion, has been shown to sensitize cells to TMZ treatment, leading to significantly improved survival.

Figure 2

Regulation of complement pathways in Glioblastoma: (A) C1 inactivator (C1-IA), also called C1 inhibitor (C1-Inh), binds covalently to the active site of C1r and C1s, blocking their function. It also dissociates C1r₂C1s₂ from C1, releasing C1q. This inactivation subsequently prevents the cleavage of C4 and C2 mediated classical pathway. C1-IA can also inhibit the function of MASP-1 and MASP-2 and prevent cleavage of C4 and C2 of the lectin pathway. (B) Endogenous or GBM synthesized Factor H (FH) and FH-like protein 1 (FHL-1) can successfully bind to GBM cell membrane. FH is a decay accelerating factor for C3 convertase. This plasma alternative pathway regulator FH binds with C3b in the convertase, displacing Factor Bb to inactivate the convertase. This FH-C3b also acts as a cofactor for cleavage of C3b by Factor I (FI) to yield the inactive product iC3b. CR1 allows FI to perform the second cleavage generating C3c and C3dg. Complement factor H related protein 5 (FHR5) secreted from GBM also exhibits functional activity similar to factor H. FHR5 functions as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase, thus inhibiting complement mediated lysis. (C) The membrane bound regulators such as CD59, CD55, and CD46 are also important for resisting complement attack on GBM cells. CD59 binds to C5b-8 complex and blocks the sites for C9 attachment, thus, preventing polymerization of C9 and inhibition of MAC formation. CD55 inhibits the formation and accelerates the decay of C3 and C5 convertase of alternative and classical pathway. CD46 causes inactivation of C3b and C4b deposited on the membrane.

Figure 3

Inflammatory Tumor Microenvironment of GBM and its Therapeutic Implications. Illustration of the interplay of innate and adaptive immune components within the glioma microenvironment. On the side of the innate immune system, tumor-associated macrophages (TAMs), mainly comprised of microglia and peripheral monocytes, are attracted by tumor cells, which release pro-inflammatory cytokines, matrix remodelers, and growth factors to aid tumorigenesis. Myeloid-derived suppressor cells (MDSCs) are also recruited by the tumor and potently suppress anti-tumor immunity. Alternative pathway molecules factor H (FH) and FH-like protein 1 of the complement system enhance immunosuppression and prevent complement-mediated lysis of the tumor cells. The adaptive immune system, on the other hand, is largely suppressed in its function through the recruitment of regulatory T cells (T_reg). These inhibit the action of cytotoxic T cells and dendritic cells, disturbing a competent anti-tumor immune response. Tumor cells also exert direct suppression of adaptive immunity through immune checkpoint expression, e.g., PD-L1 or CTLA-4. Therapeutically, this tumor-immune crosstalk can be targeted by inhibiting chemoattractants of pro-tumor immune cells, such as anti-CCL2 monoclonal antibody, by immune checkpoint inhibition, dendritic cell vaccination approaches or adoptive transfer of chimeric antigen receptor (CAR) T cells that target the glioma cells (red indicators).