Prior to high resolution spatial technologies, sequencing insight into tissue architecture was only achievable by conducting bulk sequencing on tumor specimens that had been micro sampled based on pre-defined histopathological regions. The Ivy Glioblastoma Atlas Project collected over 100 samples from histopathological tumor regions for RNA-sequencing, providing unparalleled insight, at the time, into niche-specific transcriptional and genetic profiles in GBM (Puchalski et al., 2018). In particular, this project identified signaling pathways unique to each niche, such as cellular stress response and inflammatory response in the perinecrotic region and cell migration and immune activation in the perivascular region. While this data continues to be useful today, the emergence of high resolution spatially resolved sequencing tools allow for detailed and in-depth analysis of tumor tissue at cellular resolution. Going beyond the classically defined histopathological regions in GBM, spatial technologies have uncovered a vast degree of knowledge relating to the localization and co-localization of malignant and non-malignant cells, and their related interactions within specific niches (Table 2; Figure 2).

In a pioneering study, Ravi et al. combine spatial transcriptomics, MALDI-MSI, and IMC to describe five spatially and transcriptionally distinct malignant states in patient tumors–regional oligodendrocyte progenitor cell-like (OPC), regional neuronal progenitor cell-like (NPC), radial glia, reactive immune, and reactive hypoxia (Ravi et al., 2022a). Aside from their transcriptional profiles, these states differ in their metabolic profiles, chromosomal variations, and co-localization with non-neoplastic cell types. Notably, both reactive immune and reactive hypoxia are associated with increased TAM and T cell abundance, especially PD1+ T cells, demonstrating that these states reside in regions of immune infiltration (Figure 2C). Additionally, the reactive hypoxia state utilizes glycolysis and amino sugar metabolism more so than other states. Given the dependence of anti-tumor lymphocytes on glucose metabolism (Ho et al., 2015; Siska et al., 2016), this hints at a potential immune-suppressive role of reactive hypoxia cells which may monopolize glucose in the TME.

A subsequent study conducted by Zheng et al. identified five spatially distinct tumor cell states–NPC, OPC, reactive astrocyte, mesenchymal-like (MES), and a subset of MES cells termed MES-hypoxia (Zheng et al., 2023). In line with the previous study, tumor cell states tend to fall under one of two umbrellas: neurodevelopment or astrocyte/mesenchymal-like. In comparison, both the NPC and OPC states from Zheng et al. resembled the regional NPC and spatial OPC states from Ravi et al., respectively, while both the reactive astrocyte and MES states had overlap with the reactive immune state (Figure 1). Notably, the MES-hypoxia state, which closely resembles the reactive hypoxia state, is associated with a worse prognosis in patients. While there are transcriptional similarities between these spatial cell states and the cell states previously defined by scRNA-seq (Figure 1) (Ravi et al., 2022a; Al-Dalahmah et al., 2023; Zheng et al., 2023), these spatially distinct states are fundamentally characterized with respect to their local microenvironment and are therefore influenced by surrounding cells and nutrients, providing an added dimension critical for exploring tumor-immune interactions.

Spatially resolved omics have also helped elucidate unique tissue regions, composed of both neoplastic and non-neoplastic cell types, within GBM patient specimens. Ren and colleagues described four tissue regions–tumor core, invasive niche, vascular niche, and hypoxic niche - using spatial transcriptomics that corresponded to enrichment of specific cell states (Ren et al., 2023). For example, astrocyte-like and radial glia-like GBM cells localized in the invasive niche while OPC-like GBM cells were more prominent in the tumor core suggesting that astrocyte-like and radial glia-like cells may be more involved in tumor invasion and progression (Figure 2A). A study by Al-Dalahmah et al. also characterized distinct tissue states using spatial transcriptomics. Of the three identified states, two were somewhat homogeneous in composition with one predominantly enriched in non-neoplastic brain cells and the other predominantly enriched in CNV-positive tumor cells, reminiscent of the cellular tumor niche (Wen et al., 2020). A more heterogeneous third state was enriched for astrocyte/mesenchymal-like tumor cells, reactive astrocytes, macrophages, and T cells and was associated with shortened patient survival (Al-Dalahmah et al., 2023), likely representing aspects of both the perivascular and perinecrotic niches (Wen et al., 2020). In another study, a combination of Nanostring GeoMx, CosMx and Xenium elucidated heterogeneous cell neighborhoods in GBM. Two major niches conserved across patients and spatial platforms were a brain-intrinsic environment, marked by astrocytic- and oligodendrocytic-like tumor cells and microglia, and a brain-extrinsic niche consisting of peri-vascular enrichment of mesenchymal-like cells, monocytes, T cells, and neutrophils (Figure 1) (Moffet et al., 2023).

Aside from transcriptomics, distinct tissue regions have also been uncovered using spatial proteomics. By using a combination of MALDI-MSI proteomics and shotgun proteomics, Duhamel et al. described three unique tissue regions (Duhamel et al., 2022). These regions were predominantly distinct from histopathological niches, though with some similarities. One region resembled both the perinecrotic and perivascular niches and had increased expression of immune-related proteins while another region embodied traits of both the perivascular niche and cellular tumor and was enriched for tumorigenic proteins. The third included both infiltrating tumor and cellular tumor and expressed neurodevelopmental and synaptic transmission proteins. Interestingly, these proteomic tissue states appear to align, molecularly, with the transcriptional tissue states defined by Al-Dalahmah et al.; however, a direct comparison has not been done. Notably, several region-specific proteins were determined to be prognostic markers for patients. In particular, ANXA11, a protein involved in tumor proliferation and invasion in other cancers (Liu et al., 2015; Hua et al., 2018), was highly expressed in the neurodevelopmental region and correlated with poor patient prognosis. A separate study by Shekarian et al. unveiled seven distinct tissue states via CODEX spatially resolved proteomics technology (Goltsev et al., 2018; Shekarian et al., 2022). Researchers demonstrated enrichment of an adaptive immune state, characterized by infiltrating lymphocytes and M2-like macrophages, and two vascular-related states in the tumor core and tumor periphery, respectively. Additionally, they determined that the tumor core has increased cellular density and heterogeneity compared to the more homogeneous cellular composition in the tumor periphery. Given what is known about the effect of TAM interactions on tumor cell plasticity (Ye et al., 2012; Hara et al., 2021), one can predict that this tumor core heterogeneity may result from increased interactions facilitated by higher immune cell infiltration and cellular density.

Spatially resolved omics can also provide insight into the diversity and consequences of cell-cell interactions within tumor regions. Spatial transcriptomics and proteomics have associated invading brain tumor cells with non-neoplastic glial cells in the invasive tumor edge (Wang et al., 2022). Spatial transcriptomics have also shown that cancer-associated fibroblasts (CAFs), a subset of non-neoplastic tumor-resident fibroblasts, also co-localize with tumor cells and support GBM progression (Jain et al., 2023). Specifically, CAFs are in close proximity to mesenchymal-like and stem-like GBM cells, endothelial cells, and TAMs within the perivascular niche (PVN) (Figure 2B). Furthermore, CAFs are recruited to the PVN by GSCs where they promote a malignant GSC phenotype characterized by HIF1a activation, epithelial-to-mesenchymal transition and increased cell proliferation. Notably, intracranial implantation of CAFs with GSCs in mice enhanced tumorigenesis relative to tumors formed by GSCs alone, emphasizing the potent pro-tumoral effects of CAFs in GBM.

Regarding the immune TME, spatial transcriptomics and proteomics have shown that tumor cells co-localize with both exhausted CD8⁺ T cells (Ravi et al., 2022b) and a variety of myeloid cells (Coy et al., 2022; Shekarian et al., 2022; Al-Dalahmah et al., 2023; Jain et al., 2023; Sattiraju et al., 2023). Interactions between tumor cells and myeloid cells have a well-established role in promoting an immune-suppressive TME and multiple scRNA-seq studies have revealed mechanisms whereby TAMs can drive a mesenchymal transition in GBM tumor cells (Hara et al., 2021; Xiong et al., 2022). One study using spatial transcriptomics and metabolomics revealed that activated TAMs surrounding the hypoxic niche produce creatine to support the growth of nearby tumor cells in an otherwise low-nutrient environment, highlighting the importance of TAM interactions for tumor cell survival (Figure 2C) (Rashidi et al., 2024). Conversely, a spatial transcriptomics study demonstrated that hypoxic tumor cells promote an immune-suppressive phenotype in TAMs through induction of CCL8 and IL1B cytokines (Figure 2C) (Sattiraju et al., 2023). An additional spatial study based on MALDI-MSI and CycIF discovered that co-localization of CD39⁺ myeloid cells and CD73⁺ tumor cells results in increased extracellular adenosine, an immune-regulatory molecule (Figure 2C) (Coy et al., 2022). Notably, CD73 expression in tumor cells is correlated to HIF1a expression and enriched in the perinecrotic niche. This link between the tumor cell hypoxic response and immune-suppression is corroborated by evidence that HIF1a activation in tumor cells results in a mesenchymal shift and expression of immunosuppressive genes and is linked with poor patient survival and tumor recurrence (Joseph et al., 2015; Sattiraju et al., 2023). In this same study, Sattiraju and colleagues elegantly showed that adaptive immunity is necessary for generation of hypoxic tumor zones which in turn work to suppress the anti-tumor immune response. Furthermore, obstructing communication between hypoxic tumor cells and TAMs reduces hypoxia frequency, suggesting that generation of hypoxic regions is at least partially dependent upon tumor-TAM interactions. Together, these data highlight the pro-tumor impact of immune infiltration and hypoxic conditions on anti-tumor immunity and tumor cell phenotypes.

Based on the inevitability of tumor recurrence and high degree of inherent plasticity in GBM, understanding how the spatial landscape of GBM changes over time and in response to therapy will go a long way toward advancing therapeutic development for recurrent GBM. A study by van Hooren et al. revealed an increase in monocyte-derived macrophages (MDMs) at time of recurrence in GBM (van Hooren et al., 2023). Van Hooren et al. used spatially resolved IMC to demonstrate MDM, regulatory T cell (Treg) and PD1-high CD8⁺ T cell enrichment in recurrent GBM, resulting in increased MDM-CD8⁺ T cell and Treg-CD8⁺ T cell interactions and enhanced immune-suppressive activity. Spatial changes in response to immunotherapy, specifically anti-CD47 and anti-PD1, have also been explored by Shekarian et al. In their study, treatment of patient tumor explants with single or combinatorial immunotherapy resulted in increased CD4⁺ and CD8⁺ T cell infiltration and M1-like macrophage presence, as determined by CODEX spatial technology (Shekarian et al., 2022). Immunotherapy-treated explants had markedly high levels of interferon-gamma, especially those treated with anti-PD1, suggesting functional activation of infiltrating lymphocytes. Notably, explants with low interferon-gamma levels post-treatment had a higher abundance of M2-like macrophages and enhanced expression of checkpoints on T cells, suggesting that a prevalence of immune-suppressive TAMs may impair immunotherapy-induced T cell activation. While valuable, particularly in studying immune responses to immunotherapy treatment, these studies omitted analysis of the effects on the neoplastic cell populations. Given what we know about tumor cell and TME interactions impacting tumorigenesis and immune regulation, improving our understanding of tumor cell phenotype transitions in the context of neighboring non-neoplastic cells will be crucial for development of effective therapeutics.

One potential avenue for augmenting efficacy of immunotherapy in GBM involves restricting tumor-supportive cell-cell interactions, especially those that induce immunosuppressive phenotypes. For example, spatial analyses have demonstrated both CAF-tumor cell and TAM-tumor cell interactions that promote tumor progression and immune-suppression in association with hypoxia (Coy et al., 2022; Jain et al., 2023; Sattiraju et al., 2023). As mentioned in previous sections, these interactions involve various signaling mechanisms and involve GBM cells residing in the hypoxic niche. CAF-mediated induction of a hypoxic response, mesenchymal shift, and expression of immune-suppressive genes in GSCs relies on osteopontin and HGF signaling (Jain et al., 2023). Alternatively, tumor cell-TAM interactions can involve TGF-b and oncostatin signaling or creatine metabolism to support immunosuppressive TAMs and tumor cells, respectively (Ye et al., 2012; Hara et al., 2021; Rashidi et al., 2024). Inhibiting these signaling mechanisms would be expected to decrease immune-suppression and potentially increase anti-tumor T cell infiltration and/or functionality (Figure 3A).

Another approach to boost the anti-tumor immune response is to target immunosuppressive cell types directly (Figure 3B). Malignant cell states that modulate the immune TME, such as reactive hypoxia (Ravi et al., 2022a) or MES-hypoxia (Zheng et al., 2023) cells which express immune-suppressive factors and correlate spatially with immune-suppressive TAMs, are a promising therapeutic target. Approaches to effectively inhibit these states remain unclear. One option could be to identify and target molecular pathways downstream of the hypoxic response, such as dysregulated DNA methylation, the mesenchymal shift, cytokine signaling in TAMs, or molecular drivers of the reactive hypoxia or MES-hypoxia tumor cell states (Joseph et al., 2015; Johnson et al., 2021; Ravi et al., 2022a; Chanoch-Myers et al., 2022; Sattiraju et al., 2023). Similarly, data on the unique metabolic profiles identified using MALDI-MSI (Ravi et al., 2022a) may be leveraged to identify targetable metabolic vulnerabilities in hypoxic GBM. Another option would be to target potent immunosuppressive effectors individually, such as with a CD73 inhibitor like α,β-methylene-ADP (APCP) (Cho et al., 2006), or collectively by inhibiting a common upstream driver like TGF-β signaling (Johnson ALJK et al., 2022). As previously mentioned, spatial proteomics revealed that tumor cell expression of CD73 is strongly correlated to TAM co-localization, hypoxia, and immune-suppression. Therefore, inhibiting CD73 could impair pro-tumor adenosine signaling in the perinecrotic niche and enhance the anti-tumor immune response.

Due to the abundance of pro-tumor myeloid cells within GBM, especially in recurrent GBM, TAMs have become a novel focus for developing therapies. These attempts have involved direct inhibition of pro-immunosuppressive mechanisms, such as CSF1R, KDM6B, or TREM2 inhibition (Przystal et al., 2021; Goswami et al., 2023; Sun et al., 2023), and TAM reprogramming to an anti-tumor state (Zhang et al., 2016; Chryplewicz et al., 2022; Zhang et al., 2023). Several of these approaches have proven successful in preclinical studies whereby TAM reprogramming increases tumor cell phagocytosis and augments checkpoint inhibitor therapy (Przystal et al., 2021; Chryplewicz et al., 2022; Goswami et al., 2023; Sun et al., 2023). An interesting facet of TAM reprogramming yet to be explored is the impact on tumor hypoxia. Since pro-tumor TAMs are interdependent, both spatially and mechanistically, with hypoxia, one would hypothesize that polarizing TAMs to an anti-tumor state may reduce the immunosuppressive TAM-tumor cell interactions that occur under hypoxic conditions.

Another feature of GBM that can be exploited based on spatial understanding is the hypoxic niche. As discussed previously, hypoxia is a potent regulator of cell states in GBM, promoting mesenchymal and stem-like phenotypes in tumor cells and a pro-tumor, immune-suppressive phenotype in TAMs (Joseph et al., 2015; Dirkse et al., 2019; Johnson et al., 2021; Sattiraju et al., 2023). Furthermore, tumor cell states and tumor-immune interactions in the hypoxic niche facilitate tumor growth and suppress anti-tumor immunity (Sattiraju et al., 2023; Rashidi et al., 2024). These tumor-supporting, hypoxia-mediated cell states and mechanisms, discovered using single-cell and spatial technologies, represent new and promising therapeutic opportunities in GBM.

Aside from directly targeting interactions and cell states in the tumor, hypoxic conditions can be therapeutically exploited or reversed using hypoxia-activated cytotoxic agents such as evofosfamide (Evo or TH-302) or agents that increase intra-tumoral oxygen delivery like fluorocarbons, respectively (Teicher et al., 1997; Johnson et al., 2009; Murayama et al., 2012; Anduran et al., 2022) (Figures 3C, D). This would allow for specific targeting of cells residing in hypoxic tumor regions, including immunosuppressive GBM and myeloid cells, in a manner that spares normal brain tissue. Evo has been tested in other cancers where it augmented anti-PD1 therapy in preclinical studies (Jayaprakash et al., 2018; Wang et al., 2023). Translation into clinical trials demonstrated that Evo was well-tolerated and resulted in stable disease status in most patients (Hegde et al., 2021). In GBM, in vivo use of Evo −/+ radiation reduced tumor burden, eliminated hypoxia-responsive tumor cells, and reduced the presence of pseudopalisades, a morphological feature indicative of hypoxia surrounding necrotic regions (Sattiraju et al., 2023). Alone, Evo increased vascular density while simultaneously disrupting TAM infiltration and reducing TAM abundance near hypoxia. Beyond the lab setting, a phase II clinical trial tested Evo in combination with bevacizumab, an FDA-approved anti-angiogenic therapy, failed to have a significant impact on overall patient survival compared to a historical control (Brenner et al., 2021). Based on the promising pre-clinical data (Sattiraju et al., 2023), Evo warrants further investigation in the context of GBM, especially in the absence of bevacizumab. Additional studies exploring the specifics of how Evo impacts the anti-tumor immune response and whether it augments GBM immunotherapy could provide valuable insight into methods for targeting hypoxia-mediated immune suppression.

Alternative to exploiting hypoxia, reversing hypoxia via oxygen transporter drugs has been tested in GBM and effectively sensitizes tumor cells to chemoradiation (Teicher et al., 1997; Murayama et al., 2012). This can be achieved through fluorocarbons which efficiently deliver oxygen to hypoxic regions in high volumes through passive diffusion (Johnson et al., 2009). Notably, the fluorocarbon known as dodecafluoropentane emulsion (DDFPe) has been tested against newly-diagnosed and recurrent GBM in clinical trials where it was well-tolerated and effectively reversed tumor hypoxia (Lickliter et al., 2023). Furthermore, the study demonstrated a trend towards increased patient survival and is being tested further in a phase II/III trial [NCT02189109]. The effects of DDFPe have not been explored in pre-clinical GBM studies, highlighting a gap in knowledge with the potential to provide insight into DDFPe’s impact on hypoxia-mediated malignant cell states and suppressed anti-tumor immune response.

The prevalence of immunosuppressive cell types and interactions within and surrounding the hypoxic niche suggests that pinpoint targeting of these cells, pro-tumor interactions, or hypoxia itself may augment the anti-tumor immune response and enhance efficacy of immunotherapy against GBM. A byproduct of disrupted vasculature and consequently decreased oxygen availability, hypoxia is fundamentally dependent on tissue architecture. Therefore, high-resolution spatially resolved technologies, like those discussed in previous sections, are critical to understanding the clinical impact of hypoxia-mediated cell phenotypes and identifying viable therapeutic targets. Multi-omic spatial approaches are especially advantageous in that they allow for in-depth analysis of cell interactions, which can elucidate new mechanisms of hypoxia-mediated anti-tumor immune suppression, further expanding upon those previously identified.

Overall, spatially resolved studies have already revealed several attractive therapeutic targets in GBM (Figure 3). Moving forward, additional spatial approaches paired with robust preclinical testing of novel therapeutics will be critical to advance the field of GBM therapy. Treatments aimed at exploiting hypoxia or targeting hypoxia-mediated immunosuppressive cell states, hold particular promise for GBM and could ultimately be combined with current immunotherapies like checkpoint inhibitors or CAR-T cell therapy to synergistically impair tumor progression and extend patient survival.