Glioblastoma multiforme (GBM) is the most common malignant primary brain tumour, representing ~45% of all incidences of glioma (Ostrom et al., 2014, 2020), with an annual prevalence of ~5 people per 100,000 of the population (Omuro and DeAngelis, 2013). GBM is highly aggressive and carries a very poor prognosis, with a median survival of ~15 months post diagnosis, and a 5-year survival rate of 5% (Ostrom et al., 2014, 2020).

Treatment options are typically limited to surgical removal of as much of the tumour mass as is safe, followed with radiotherapy, and concomitant chemotherapy using alkylating agents such as temozolomide (TMZ) (Stupp et al., 2005, 2017; Weller et al., 2017). However, TMZ, the standard-of-care, provides only marginal benefit, and is effective in less than half of GBM patients (Kamson and Grossman, 2021). Crucially, patients also acquire resistance to TMZ over time and/or in recurrent incidences of GBM (Hanif et al., 2017). Recently, an alternative strategy inhibiting tumour neoangiogenesis using bevacizumab, a humanised monoclonal antibody raised against vascular endothelial growth factor A (VEGF-A), showed no significant improvement on patient survival (Chinot et al., 2014; Gilbert et al., 2014; Kim et al., 2018), indicating the need for improved therapeutics.

The heterogenous, or ‘multiforme’, nature of GBM contributes to therapeutic failure. Whilst the neoplasms associated with the disease typically share characteristics of high invasiveness, high vascularisation, and feature hypoxic/necrotic cores (Perry and Wesseling, 2016), they exhibit substantial macroscopic, microscopic, genetic, and epigenetic discrepancies, at both the inter-and intra-tumour level (Holland, 2000).

Diverse subsets of progenitor glioblastoma stem cells (GSCs), present in and around the tumour microenvironment, comprise some of the key regulators of GBM heterogeneity, driving tumour initiation, proliferation, invasiveness, renewal, and resistance to radio/chemotherapy (Bao et al., 2006; Chen et al., 2012; Lathia et al., 2012; Llaguno et al., 2015; Kim et al., 2021; Zheng et al., 2021). Recently, studies have used single-cell RNA sequencing, lineage tracing, and bulk genetic profiling to characterise the GSC subpopulations driving tumour heterogeneity, and a consensus is emerging that incorporates four distinct subtypes: (1) neural-progenitor-like cells (NPCs); (2) oligodendrocyte-progenitor-like cells (OPCs); (3) mesenchymal-like cells (MES); and (4) astrocytic cells, particularly reactive astrocytes that respond to insults in the tumour microenvironment (Patel et al., 2014; Neftel et al., 2019; Couturier et al., 2020; Zheng et al., 2023). A schematic of the various GSC subpopulations and antibody markers used to identify them are summarised in Figure 1. We hypothesise that identifying drug targets common across all four GSC subtypes may offer novel and effective approaches to improve therapeutic outcome in a greater number of patients.

The apelin receptor is a class A G protein-coupled receptor (GPCR), first identified based on its sequence homology with the angiotensin II type 1 receptor (O'Dowd et al., 1993), that binds two endogenous peptide ligands, apelin and elabela/Toddler (ELA) (Tatemoto et al., 1998; Chng et al., 2013; Pauli et al., 2014). Whilst only ELA acts as a key driver of proper heart formation during embryogenesis, both ligands are present in the adult cardiovascular system, where they induce potent vasodilatation and positive cardiac inotropic responses through action on the receptor present in endothelia and cardiomyocytes, respectively, (Szokodi et al., 2002; Kleinz and Davenport, 2004; Kleinz et al., 2005; Japp et al., 2008, 2010; Maguire et al., 2009; Pauli et al., 2014; Yang et al., 2017).

The apelin receptor has also garnered attention as a complex target in various cancers (Read et al., 2019; Masoumi et al., 2020), including GBM (Ivanov et al., 2022). ELA has been shown to be elevated in ovarian clear cell carcinoma (Yi et al., 2017), and, importantly, in glioma and GBM patients, where high ELA expression is directly associated with lower patient survival (Ganguly et al., 2019). Apelin was identified as a central regulator of endothelium-dependent tumour initiation and maintenance in GBM cells with stem-like properties, and the apelin receptor antagonist, MM54, was used to successfully reduce tumour expansion and lengthen survival in a GBM xenograft mouse model (Harford-Wright et al., 2017; Harford-Wright and Gavard, 2018). As further proof-of-principle, MM54 attenuates the pro-tumourigenic effects of apelin in a mouse melanoma lung metastasis model (Berta et al., 2021), suggesting that apelin receptor blocking therapy is not necessarily limited to brain cancers.

Apelin signalling has also been shown to initiate the formation of new blood vessels by driving vascular endothelial cells toward a pro-angiogenic state, in both physiological and cancerous states (Kälin et al., 2007; Yang et al., 2016; Helker et al., 2020; Berta et al., 2021). Crucially, both apelin peptide and the receptor are upregulated in GBM patient samples, and, in an orthotopic mouse model of GBM, apelin was required for tumour vascularisation, where apelin depletion significantly reduced tumour size and increased survival of tumour-bearing mice (Frisch et al., 2020). A consensus is emerging that pharmacological inhibition of the apelin receptor might offer beneficial effects on survival, either through direct actions on stem cell populations or indirectly through anti-angiogenesis or both, in various forms of cancer, including GBM (Mastrella et al., 2019; Read et al., 2019).

The expression of the apelin receptor and its endogenous ligands has not been mapped in the diverse stem cell populations in GBM that drive tumour initiation, renewal, and resistance to current therapies. Here, we used semi-quantitative fluorescence microscopy to test our hypothesis that the three key components of the apelin signalling axis (the receptor, apelin, and ELA) are expressed in progenitor GPCs, and the subsequent MES, NPC, and OPC lineages in surgical samples of primary GBM tissue from patients. Additionally, we assessed expression in microvascular proliferations in the tumour environment. Our data confirm the presence of the apelin receptor and its cognate ligands and support the potential of the apelin receptor as a drug target that is shared across the critical cell types that drive and maintain GBM.

GBM is characterised by pathological hallmarks, such as hypoxic/necrotic cores and aberrant microvascular proliferations (Perry and Wesseling, 2016), which we were consistently observed in the human tissue samples used in this study (Figure 2A). We were also able to show that these GBM samples typically expressed Ki-67 and vimentin (markers of proliferation and potential migration, respectively) at higher levels than in HBC (Figures 2B,C,D), as might be expected in cancerous tissue.

The apelin receptor has emerged as a potential therapeutic target in various cancers, including GBM (Read et al., 2019; Masoumi et al., 2020; Ivanov et al., 2022). Apelin signalling is known to drive formation of new blood vessels in both physiology and disease (Kälin et al., 2007; Yang et al., 2016; Helker et al., 2020; Berta et al., 2021), and it has recently been shown that apelin receptor knockdown or receptor inhibition with a peptide antagonist reduces vascularisation and angiogenesis in GBM (Mastrella et al., 2019).

In this study we visualised and measured intense immunofluorescence with antisera to apelin receptor and its two endogenous ligands in GBM tissue. These were chosen as they have been extensively validated by other researchers as indicated in Table 1. We used adjacent sections with primary antisera omitted to measure autofluorescence, although a limitation is that the small samples available precluded using additional isotype controls.

We have shown that blood vessels in GBM tissue express the apelin receptor (Figure 3A), expanding on previous findings showing expression of apelin peptide in GBM tumour blood vessels (Harford-Wright et al., 2017). In our study, vascular expression of the apelin receptor was significantly higher in GBM versus HBC (Figures 3B,C), identifying an upregulation of the receptor in the vasculature in the cancerous state. Overall, the data confirm that the apelin receptor is present as a potential vascular anti-angiogenic drug target in GBM.

The pathological role of the apelin receptor and its endogenous peptide ligands, apelin and ELA, in cancers such as GBM may well extend beyond angiogenesis. In this study, we used a panel of cell markers to assess expression of the apelin signalling axis in the four distinct GSC lineages – NPCs, OPCs, MES, and astrocytes – that have emerged as the key drivers of GBM proliferation, maintenance, invasion, and therapeutic resistance (Patel et al., 2014; Neftel et al., 2019; Couturier et al., 2020; Zheng et al., 2023).

First, co-staining with CD133 showed that both the apelin receptor and apelin peptide were expressed in GSCs (Figures 4A,B,E), the cells from which the three lineages originate. Interestingly, however, we did not observe expression of ELA peptide in these cells (Figures 4C,E). Apelin peptide expression has been demonstrated in GSCs previously and apelin receptor inhibition has been shown to blunt GSC invasion (Mastrella et al., 2019). Our work is important in providing evidence that this blunting effect on pro-invasiveness might be mediated directly through apelin receptor expression in GSCs.

Next, co-staining with CD24, NG2, CD44, and GFAP isolated NPCs, OPCs, MES, and astrocytic cells, respectively. In each of these cell subtypes, we observed significant levels of apelin receptor, apelin peptide, and ELA peptide (Figures 5, 6, 7, 8; Table 2). It is interesting that ELA was present in the three lineages and astrocytes but not the originating GSCs, and further work will need to be done to characterise whether ELA plays a role as a potential phenotypic switch in stem cell differentiation in the brain. Nevertheless, our data demonstrate that the apelin receptor and its ligands are commonly present across the key GBM cell subtypes.

In terms of future work and perspectives, we suggest that it would be of great interest to further employ in vivo xenograft models (Harford-Wright et al., 2017; Harford-Wright and Gavard, 2018), or use vascularised brain organoid models (Sun et al., 2022) that incorporate the necessary stromal cells in the microenvironment, such as macrophages, to investigate the function of the apelin receptor in GBM and other gliomas. These brain tumour models could also act as ideal platforms for the screening of potential drugs targeting the apelin signalling axis.

[Pyr¹] apelin infused systemically into volunteers (Japp et al., 2008) has been shown to be safe and well tolerated, even at the highest concentration of 135 nmol/min for 120 min (Nyimanu et al., 2019). A similar safety profile was also reported for the G-protein biassed agonist MM07 (Brame et al., 2015) and the small molecule agonist (Winkle et al., 2023) as well as for [Pyr¹] apelin in obesity (Castan-Laurell et al., 2008) and patients with heart failure (Japp et al., 2010). The results with agonists demonstrate apelin is limited to two principal actions in humans in health and disease: modest vasodilatation within the first hour of systemic infusion and a beneficial increase in cardiac output that is maintained for up to six hours in patients with heart failure (Barnes et al., 2013).

Our therapeutic hypothesis is that the apelin receptor contributes to the development and progression of glioblastoma, and supported by results from animal models, apelin antagonist may act in combination with current standard-of-care to improve the treatment of this condition. Although the precise mechanism of action has to be determined, we have shown all cell types present in GBM that have been implicated in tumour progression, express the apelin receptor. We further hypothesise that an antagonist would be expected to be effective against all these cell types. The apelin antagonist MM54 has been shown to be effective in a mouse models of GBM (Harford-Wright et al., 2017) and a mouse melanoma lung metastasis model (Berta et al., 2021). MM54 has been tested for potential toxic effects in vivo using mice. MM54 was administered in vivo at the same concentration (2 mg/kg) that was effective in slowing the progression of glioblastoma patient-derived cells with stem-like properties. There were no adverse changes to cardiac frequency, blood pressure or glycaemia, or pathological changes in the heart, kidney, aorta, or liver. The results showed MM54 was safe and effective in reducing tumour expansion and lengthening the survival of intracranially xenografted mice (Harford-Wright et al., 2017). To date there have been no clinical trials of apelin antagonists in humans. It is not yet known if apelin contributes to vascular tone or maintenance of cardiac output but unwanted side effects, such as increase in blood pressure, if they occur, may be reduced by existing anti-hypertensive therapies.

In summary, whilst we have not examined molecular mechanisms underlying apelin receptor signalling in GBM in this study, we present a descriptive analysis of receptor and ligand expression in the vasculature, GSCs, and the four key cell types that drive GBM tumour proliferation, invasiveness, maintenance, and resistance to current therapies. The demonstration of the presence of the apelin receptor and ligands in the diverse GSC and astrocytic populations holds therapeutic promise as a common druggable target in an otherwise heterogenous microenvironment. Targeting the apelin receptor may present a new treatment option, which blunts GSC proliferation and pathological angiogenesis, to synergise with current therapies such as TMZ to improve patient outcome in GBM.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies were conducted in accordance with the local legislation and institutional requirements. The patients provided their written informed consent to participate in this study. The human samples used in this study were acquired from surgical samples of human tissue that were collected with ethical approval and informed consent (GREF G11970; REC 05/Q0104/142) by the Human Research Tissue Bank and Cambridge Brain Bank.

TW: Conceptualization, Writing – original draft, Writing – review & editing, Formal analysis, Investigation, Methodology. PN: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. RK: Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. KS: Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. AP: Formal analysis, Writing – original draft, Writing – review & editing. KA: Formal analysis, Writing – original draft, Writing – review & editing. JM: Formal analysis, Writing – original draft, Writing – review & editing, Conceptualization, Funding acquisition, Supervision. AD: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing, Resources.