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1. Introduction

Oncolytic viral therapy or virotherapy is a form of immunotherapy showing promising results for cancers with poor prognosis [1]. In this approach, oncolytic viruses (OVs) are employed to kill tumor cells, while in parallel stimulating an anti-tumor immune response [2]. OVs exhibit either natural tropism to malignant cells or their genome is altered to confer them higher specificity for malignant cells [3]. Viruses from ten different families (Adenoviridae, Herpesviridae, Paramyxoviridae, Reoviridae, Retroviridae, Picornaviridae, Parvoviridae, Poxviridae, Rhabdoviridae, Alphaviruses) have thus far been utilized as oncolytic virus platforms in clinical trials for various cancer types [2].

One deadly type of cancer is glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor [4]. The standard treatment consists of maximal safe surgical resection followed by radiotherapy plus concomitant and adjuvant temozolomide chemotherapy. However, the median overall survival among all GBM patients is less than one year, and only 15 months in patients receiving complete standard treatment with 3-year survival being less than 10% [5,6]. In the past decades, numerous therapeutic approaches have been tested in clinical trials, with disappointing outcomes. The main obstacles in treating GBM include its infiltrative growth, its intrinsic resistance to chemo- and radiotherapy, its notorious intratumoral heterogeneity with dynamic changes in subclones facilitating treatment escape, its protected location behind the blood-brain-barrier and the immunological ‘cold’ microenvironment of these tumors. These hurdles to more conventional therapies, as well as the dismal prognosis of GBM patients, have encouraged scientists and clinicians to develop and evaluate the local application of various types of oncolytic viruses in this patient group. Table 1 summarizes the most commonly applies OVs in GBM trials. The OVs differ in their primary attachment molecules to host receptors as well as in the source of their tumor selectivity, which may be derived from a natural tropism to cancer cells or by genetic engineering.

Table 1. Characteristics of the most commonly used Oncolytic viruses (OVs) in glioblastoma multiforme (GBM) clinical trials.

In a recent review, Chiocca et al. summarized the findings [19] from all the recent GBM oncolytic virotherapy trials and illustrated that a subgroup of GBM patients responds exceptionally well to OV treatments, with survivors at 36-months, and with some patients exhibiting long term remission [20,21]. This phenomenon has also been observed in OV trials for other cancer types. For instance, a phase II clinical trial employing an oncolytic herpes simplex virus 1 for stage IIIC or IV melanoma showed 26% overall response [22].

These observations raise the question: would the responding patients have been the same individuals if they had been treated with any other OV, or are we looking at responders to a specific OV? In other words, is the elicited immune response a generalized one for all types of OVs, or does each OV elicit a specific anti-tumor immune response? The latter would suggest that response rates may be significantly increased if we are able to define which OV is best suited for a particular patient. Identification of robust predictive biomarkers for OV response would allow future design of stratified clinical trials employing multiple OV strains. The replication efficiency of the virus is thought to be of importance for generation of the subsequent inflammatory and anti-tumor responses. Moreover, host immune status is also expected to contribute to the efficacy of OV treatment. This review, therefore, focuses on tumor and host resistance mechanisms to viral infection, replication and oncolysis and discusses potential biomarkers that have previously been reported in relation to sensitivity or resistance to the most frequently employed OVs in preclinical and clinical GBM research.

2. Glioblastoma

2.1. Heterogeneity, Stem Cells and Therapy Resistance

Common molecular abnormalities involved in the evolution of glioblastomas include aberrations in the oncogenes (EGFR, PDGF and its receptors) and tumor suppressor genes (p16INK4a, p14ARF, PTEN, RB1, and TP53), which are often observed in other human cancers as well [23]. GBM is also characterized by inter-tumoral heterogeneity, which is highlighted by the classification of GBMs into three subgroups: proneural, classical and mesenchymal [24,25]. Each subtype is characterized by specific gene expression patterns and molecular abnormalities, resulting in different clinical treatment outcomes [25,26]. Proneural subtype has the most favorable prognosis among the three subtypes; aberrations in the isocitrate dehydrogenase 1 (IDH1) gene and the platelet-derived growth factor receptor A (PDGFRA) define this subgroup. The classical subgroup is characterized by the amplification of EGFR, lack of TP53 mutations and often with homozygous CDKN2A deletions [26]. Lastly, the mesenchymal subtype is the most aggressive and it is characterized by aberrations in the neurofibromin 1 (NF1) and PTEN genes [23]. It is also characterized by a pro-inflammatory environment compared with the other subtypes [27]. It was hypothesized that one underlying cause for this was the higher incidence of tumor-associated antigens (TAAs), however this could not be proven, as specific tumor antigens are expressed in each subtype [27]. Nevertheless, this classification has not led to altered or adapted treatment approaches [28,29].

Apart from intertumoral heterogeneity, intra-tumoral heterogeneity poses another therapeutic obstacle in treatment of GBM, allowing escape of subclones from (targeted) therapies and driving treatment resistance. This heterogeneity was captured by genome-wide and single cell RNA studies, which showed tumor cells with different transcriptional profiles within the same tumor [30,31]. In addition, it was shown that within the same tumor, different subtypes can coexist, highlighting the heterogeneity that characterizes GBM [31]. In another study, paired primary and recurrent tumor tissue samples were analyzed to determine the persistence of possible drug targets. The results showed that the molecular targets between primary and recurrent tumors changed by 90% [32]. This may explain the failure of drugs that target specific molecular mutations in GBM, such as the EGFR [33].

Eventually, most of the patients experience tumor relapse due to therapeutic resistance [29]. This therapeutic resistance is mainly attributed to glioblastoma stem cells (GSCs), which activate DNA repair mechanisms to promote survival after chemo- and radiotherapy [34]. Additionally, outgrowth of resistant subclones and downregulation of targeted molecules contribute to drug resistance. Furthermore, the highly infiltrative nature of GSCs makes total surgical resection of the tumor impossible [35]. The remaining and/or treatment-resistant clones will eventually generate functional vessels for the nutrient transport and develop tumor recurrence [34].

2.2. GBM Microenvironment: Local Immunosuppressive Mechanisms

Glioblastoma arises in the central nervous system (CNS) [36], which is an immunologically distinct site. In the past, the CNS was considered an immune privileged site, due to its unique properties. For instance, the blood brain barrier, which tightly regulates the transportation of the immune cells from the periphery to the CNS; the lack of antigen presenting cells in a non-inflamed state; and more importantly the lack of a classic lymphatic system [37–39]. The concept of CNS being immune privileged has now been revised. Recent studies have shown that antigens derived from the CNS can efficiently elicit an immune response [40]. More importantly, Louveau et al. [41] discovered a functional lymphatic system, parallel to the dural sinuses, a possible route of transportation of antigen-presenting cells to the deep cervical lymph nodes, where they can present CNS-derived antigens and prime T cells. These recent studies have provided evidence that CNS-derived antigens can mount a vigorous immune response, offering ground to investigate immunotherapy approaches for GBM.

The GBM environment is characterized by the high influx of tumor-associated macrophages (TAMs). In a non-inflamed state, the myeloid composition of the CNS consists of the tissue-resident macrophages that arise from the yolk sac, the microglia [42]. However, in GBM, the microenvironment is comprised mainly of a mixture of microglia and infiltrating monocytes from the periphery. Glioma cells produce a milieu of monocyte chemoattractant proteins along with other factors, leading to disruption of the blood-brain barrier and facilitating recruitment of monocytes from the periphery [43]. When monocytes arrive at the tumor site, glioma cells drive their polarization to an immunosuppressive M2 phenotype [44,45]. These M2-like TAMs promote tumor growth and migration as well as the immune invasion by hampering the adaptive immunity [44,46,47]. TAMs are the most abundant immune cell population in GBM and can consist up to 50% of the GBM tumor mass. Their importance in tumor growth is highlighted by the correlation between increased TAM numbers and worse prognosis in GBM patients; furthermore, TAM infiltration has been associated with the mesenchymal subtype of GBM, being the most aggressive one [48,49].

Another feature that facilitates the local immune suppression in GBM is T cell dysfunction. Severe T cell exhaustion is observed in GBM, which is characterized by upregulation of expression of co-inhibitory molecules like PD-1, LAG-3 and TIM-3 [50]. Furthermore, an increase in numbers of the regulatory T cells (Tregs), which can suppress the antigen-specific T cells, was found in high grade gliomas compared to low grade gliomas [51]. The recruitment of Tregs at the tumor site is mainly facilitated by the production of the attractant indoleamine 2,3 dioxygenase (IDO) by gliomas [52]. Another facet that contributes to the ‘’cold’’ tumor microenvironment is the relatively low mutational burden of GBM cells, associated with limited expression of neoantigens [53,54]. Taken together, GBM has all the characteristics of a tumor with low immunogenicity. The M2-like macrophages that are abundant at the tumor site, the dysfunctional T cells and the low neoantigen expression are some of the barriers that we need to overcome to design successful immunotherapies.

Considering all of the above, a therapeutic strategy that is not hindered by specificity for a single molecular target or differentiation state of tumor cells, that is delivered locally in a single surgical intervention, hence bypassing the BBB, that is self-perpetuating in its anti-tumor activity, and which can overcome the immune-suppressive tumor microenvironment, may offer opportunities for achieving therapeutic responses in glioblastoma patients. Oncolytic viruses offer such a treatment strategy.

This entry is adapted from the peer-reviewed paper 10.3390/cancers13040614