Microglial TLR9: Plausible Novel Target for Therapeutic Regime Against Glioblastoma Multiforme

Abstract

An understanding of pattern recognition receptors (PRRs) and immunomodulatory approach based on activation of these receptors has provided insights critical for the management of neurological health disorders. Toll-like receptors (TLRs) are one of the most widely explored PRRs and have been exploited in the recent past for development of novel immunomodulatory therapeutic agents. Glioblastoma multiforme is characterized by significant infiltration of resident microglia and expresses all the members of the TLR family. The present report is focused on exciting findings pertaining to probable implications of TLR9 activation by unmethylated CG sequences for novel therapeutic intervention against glioblastoma multiforme, which could be a discrete step toward the effective management of neurological health issues.

Glioblastoma multiforme (GBM) represents the most precarious form of brain tumors originating mostly de novo or from a preceding astrocytoma. Owing to its malignancy, mitotic and microvascular proliferation, GBM fulfills all the criteria for categorization among the most deadly grade of brain tumors (Grade IV) according to the World Health Organization classification. Brain tumors along with other nervous system cancers impinge on 6.4 per 100,000 men and women per year with a poor survival rate of 32.6% (National Cancer Institute, Brain cancer stats factsheet; Louis et al. 2016). GBMs accounting for 60% of all brain tumors can be subdivided into primary and secondary glioblastoma. Histologically indistinguishable, both the GBMs bear distinct genetic and epigenetic signposts. The hallmarks of secondary glioblastoma are associated with isocitrate dehydrogenase (IDH) mutation, whereas wild type or hypermethylated IDH is an orthodox molecular marker of primary GBMs. Additional, hallmark alterations associated with glioblastoma comprises of mutation within epidermal growth factor receptor (EGFR) gene, phosphatase and tensin homolog (PTEN and TERT) promoters and platelet-derived growth factor A (PDGFA/PDGFRa). The amplification of EGFR and over expression of mouse double minute 2 (MDM2) along with deletion of p16 and loss of heterozygosity (LOH) of chromosome 10q and 19q has also been observed in GBM patients (Hanif et al. 2017). All these observations indicate that both the primary and secondary GBMs display great deal of heterogeneity both at inter- and intratumoral level thereby rendering the early diagnosis, characterization and treatment quite challenging.

Clinical presentation of the disease is associated with frequent headaches, focal neural deficit along with cognitive impairment and seizures depending upon the localization of tumor. Computed tomography (CT) and magnetic resonance imaging (MRI) are primary techniques employed for diagnosing the same. Optimal surgical resection of tumor underlies the core frontier for treating GBM. Owing to its infiltrative character followed by limitations in achieving complete surgical resection, the median survival time of patients undergoing surgical interventions along with chemotherapy constituted by temozolomide remains only about 12–15 months (Cantrell et al. 2019). Moreover, the selectively permeable shield provided by blood brain barrier to tumor cells, their internal resistance to the induction of cell death, and poor dependence on lone, targetable oncogenic pathways altogether enforce challenges for systemic therapy in GBM patients. As brain is an immunologically dynamic organ, the immunotherapeutic approaches are considered to be “a ray of hope” owing to its potency of stimulating or remodeling the tumor microenvironment that could probably surpass several side effects associated with chemotherapeutical regimes (Thomas et al. 2012). Considering the findings of immunomodulation-centric research on GBM, we surmise that targeting of toll-like receptors (TLRs) could be focused upon as a plausible novel therapeutic regime for treating GBM cases.

The tumor microenvironment (TME) in GBM is not immunologically inert and represents a complex heterogeneity in the localization mainly constituted by vascular, glial as well as infiltrating and resident immune cells within the tumor proximity. Both the glioblastoma-associated microglial (GAMs) and macrophages are worth of special mention as they play a fundamental role in adopting pro-inflammatory state and providing cytotoxic responses against microbial intruders (Hanisch 2002), as well as participate in tumor surveillance (Jaiswal et al. 2010). The polarization of these pro-inflammatory M1 phenotype cells (pro-inflammatory) to M2 myeloid (anti-inflammatory) cells during pathological conditions leads to tissue remodeling, angiogenesis and immunosuppressive state thereby promoting tumor progression (Hambardzumyan et al. 2016). Tumor-derived IL-4, IL-10, hyaluronic acid and M-CSF exposure triggers this transformation of GAMs into M2 phenotype (Komohara et al. 2008). Adoptions of lineage tracing methods and specific markers have contributed substantially in mapping phenotype specific role of GAMs constituted by microglia and bone marrow-derived macrophages (BMDMs) (Sevenich 2019). During initiation of tumors, in an attempt to restore homeostasis, microglia are further aided by infiltrating BMDMs resulting in a combination that may account for one-third of tumor mass. Thereby these GAMs play a pivotal role in promoting tumorigenesis and conceive immunosuppressive TME (Roesch et al. 2018). Thus, targeting these immune cells to deflate pro-tumorigenic and/or immunosuppressive environment could provide potential therapeutical leads. At their associated tumorigenic sites, GAMs funnel neo-GAMs, by virtue of the presence of chemoattractants, such as HGF, MCP1/CCL2, and SDF1; along with cytokines, viz. M-CSF and GM-CSF, these neo-GAMs are further unmasked to the anti-inflammatory milieu (Russo and Cappoli 2018).

In close resemblance to their peripheral counterparts, microglia are reported to express pathogen recognition receptors (PRRs), which are critical mediators in recognizing pathogen-associated molecular patterns (PAMPs) associated with broad delineation of intruders. Among these PRRs, the family of toll-like receptors (TLRs) has special relevance owing to its ability of sculpting the immune response toward any external and/or internal stress. Among 10 TLRs known till date in humans, TLRs 1, 2, 4, 5, 6 and 10 are expressed on the cell surface where they bind to various molecules derived from microbes as ligands (Kawai and Akira 2009). The intracellular early endosomal compartment includes TLR3, TLR7, TLR8, and TLR9 which intrinsically recognize foreign nucleic acids. Amidst these intracellular TLRs, TLR9 recognizes dsDNA of microbial origin, synthetic CpG ODNs and has been extensively evaluated for its role of mediating inflammation in a vast array of diseases. TLR9 activation depends on the binding of unmethylated CG sequences with the ectodomain of the receptor, eventually culminating in the expression of NFκB, which further confers a pro-inflammatory milieu within the tissue.

Rat glioblastoma cells (C6) resemble human GBM as they share histopathological features as well as elevated mitotic index, nuclear polymorphism, and tumor necrosis foci; these features make C6 cells useful for in vitro therapeutic screening of novel drug/targets for GBM (Giakoumettis et al. 2018). However, delivery of TLR9 agonists within the endosomal compartment is a critical issue that hampers proficient receptor–ligand interaction because of aggregation of short DNA sequences, eventually resulting in the precipitation of the TLR9 ligand (Tiwari et al. 2020). Recent trends in drug delivery have substantiated the use of a polymer, schizophyllan (SPG), derived from the fungus, Schizophyllum commune, for delivering therapeutic payloads at an organelle level. The distinctive structural features of SPG, especially its hydrophobic cavity, potentiate the encapsulation of short DNA sequences, making it safe from endosomal degradation. SPG is a representative of the β-glucan family, having 1,6-β-monoglucosyl branches at every third 1,3-β-glucosyl residue on the main chain and possesses a profound array of documented anti-tumor, antimicrobial, and hepatoprotective properties.

Focusing on the attributes of C6 cells along with SPG carriers, we surmise the plausible role of TLR9 in remodeling the TME from the M2 to the M1 type. SPG used to deliver therapeutic payload was reduced to the nanoscale and could efficiently cross the BBB. During in vitro assessment, the immunosuppressive nature of C6 cells was evident from the intracellular levels of the IL-4 cytokine. However, post treatment, C6 cells displayed augmented intracellular levels of signature pro-inflammatory cytokines, namely IFN-γ and IL-1β, making the M2-to-M1 phenotypic shift evident. The use of SPG as a carrier for a TLR9 agonist (CpG ODN 1826) enhanced its delivery, which was confirmed from the higher intracellular levels of the aforementioned cytokines. These engineered nanovehicles also induced oxidative stress at different dilutions. Moreover, post-treatment evaluation of C6 cells also showed the efficacy of these nanovehicles, encapsulating the TLR9 agonist, in inducing apoptosis (Tiwari et al. 2020). Although recently a robust approach for identifying novel therapeutic regimes against GBM focusing on innate immune response has been successful in delineating several probable targets, exploration of microglial TLR9 for remodeling of the immunosuppressive phenotype into the desired inflammatory type remains largely unexplored. In this regard, the synthesis of SPG nanovehicles, encapsulating a TLR9 agonist, that possess the ability to cross the BBB elucidates an effective strategy to target the GAM TLR9 to cautiously regulate inflammation attributed to tumor regression. Further enhancement of therapeutic potency of these nanocarriers can be attained by using standard drugs in a combinatorial approach.

The challenges addressed by using TLRs as immunotherapeutic agents against glioblastoma are not only defined to immunosuppressive environment around the tumor but also includes the enhanced inflammation thereby worsening the disease. The feeble translation of animal experimental results into clinical findings has emerged as a major concern. Scientific reports related to the Phase I and II clinical trials of CpG ODN administration do not correlate with the animal studies (Abarca-Merlin et al.2019). The increased expression of TLR9 has been associated with the enhanced invasion and progression of tumor routed through activation of STAT 3 (Kortylewski et al. 2006) and IFN-γ-mediated enhanced expression of CCL2/CCL5 (Lin et al. 2009).

Consequently, a cautious activation of TLR9 is highly imperative specifically in the case of glioblastoma. So far the drug delivery approaches, viz. carbon nanotubes, have somewhat assisted to overcome these issues generating a ray of hope toward development of novel promising therapeutic agent against glioblastoma.

Funding

Bajpai P and Tiwari RK would extend their gratitude toward Science Engineering and Research Board (SERB), New Delhi, India for financially assisting this study (Grant No: YSS/2014/000106).

Compliance with Ethical Standards

Conflict of interest

The authors of present communication declare no competing conflict of interest.