The utilization of single-cell sequencing (scRNA-seq) has represented a significant advancement in the study of gliomas. Glioma, a prevalent and highly heterogeneous brain tumor, often poses challenges to conventional cancer research methodologies. Traditional approaches typically involve analyzing the average characteristics of a large population of tumor cells, overlooking both intercellular and intracellular variabilities. In contrast, scRNA-seq technology enables a comprehensive exploration of cellular heterogeneity by scrutinizing the gene expression of individual cells. Furthermore, by examining the gene expression regulatory network among different cell types, scRNA-seq can unveil the mechanisms underlying interactions between tumor cells and immune cells, endothelial cells, and other cellular components within the tumor microenvironment. The single-cell assay for transposase-accessible chromatin (scATAC-seq) can help us to understand the transcriptional and regulatory processes of cells at the genomic level, reveal the sites of different regulatory factors, and analyze the gene information from the perspective of epigenetics. scATAC-seq has great potential for applications in open chromatin mapping, cell differentiation, and development, disease pathogenesis, tumor microenvironment, biomarkers, etc. (Buenrostro et al., 2015). Single-cell sequencing requires the dissociation of cells, which results in the loss of information about the original location of the cells in the tissue, so it is difficult to map detailed cellular maps and relationships between cells at the level of tissue architecture. This is where the spatial transcriptome can be used to help understand the cellular and organizational functions of multicellular organisms, mapping cells from their physiological, morphological, and anatomical contexts, as well as their spatial architecture (Jung et al., 2022). This enhanced understanding of the cellular composition and interactions in gliomas holds promise for advancing precision medicine approaches and developing targeted therapies for this complex and challenging cancer.

Therefore, we integrated several publicly available scRNA-seq GBM datasets for data processing and normalization. Data from Synapse database (https://synapse.org/singlecellglioma) (Johnson et al., 2021). We used a total of 11 glioma patients with a total cell count of 55,089. The total number of Astrocytes, Microglial cells, macrophages, T cells, Endothelial cells, and Oligodendrocytes after our clustering and annotation (Figure 1). We found that Microglial cells, Macrophages, and Oligodendrocytes occupied the majority (Figure 1). The number of immune T-cells was very low, probably due to confinement to the bone marrow (Chongsathidkiet et al., 2018). Gliomas are commonly referred to as “cold tumors” due to their immunosuppressive microenvironment, which impedes the infiltration and function of immune cells that could potentially target and eradicate tumor cells. The glioblastoma microenvironment is a complex milieu comprising various elements that interact dynamically to influence tumor development and progression. This microenvironment encompasses tumor cells, the extracellular matrix (ECM), blood vessels, and a diverse array of immune cells, including monocytes, macrophages, mast cells, microglia, neutrophils, and T cells (Broekman et al., 2018). As in Figure 2, the Blood-brain barrier and tumor microenvironment are closely related. Immune cells play an important role in tumor immune escape and anti-tumor immune response. Macrophages from the hematopoietic system are located in the tumor tissue due to the attraction of chemical factors secreted by tumor cells. They play an important role in inflammation and TME, which can not only promote tumor growth and spread but also exert an anti-tumor effect (Mei et al., 2023).

In essence, the tumor microenvironment (TME) of gliomas comprises an intricate network of various cellular and non-cellular elements that interact and collectively govern tumor growth and advancement. A comprehensive comprehension of the evolving dynamics within the tumor microenvironment and the diverse immune milieu within a genetic framework is crucial for the advancement of novel therapeutic approaches for tumors.

Hierarchical clustering analysis of inferCNV and CopyKAT results have identified the majority of vascular endothelial and mural cells as diploid non-tumor cells. This suggests that in glioblastoma (GBM), tumor cells derived from neural stem/precursor cells tend to maintain their neural lineage restriction and are less likely to differentiate into non-neural lineage cells. The analysis indicates that different malignant cells within GBM converge into four distinct cell states: neural progenitor cell-like (NPC-like), oligodendrocyte progenitor cell-like (OPC-like), astrocyte-like (AC-like), and mesenchymal stromal cell-like (MES-like) states. These cell states exhibit potential for plasticity, allowing for transitions between different states within the tumor microenvironment (Hara et al., 2021). These types of tumor cells play a crucial role in driving clonal evolution, tumor progression, and treatment resistance (Greenwald et al., 2024). Studies have shown that AC-like tumor cells are associated with poor prognosis in patients (Zheng et al., 2023). He dispersion and connection of AC-like cells with other transcriptional subtypes are associated with a reduced risk (Greenwald et al., 2024). At the same time, NPC-like cells couple with neurons in the infiltrating regions, mimicking the migration of normal NPCs to developing neurons (Greenwald et al., 2024). Pseudotime analysis revealed the dynamic transition of tumor cells from pro-neural to mesenchymal subtypes (Xiong et al., 2022). Urthermore, patients with higher MES-like scores always correlate with worse prognosis compared to those with lower MES-like scores (Xiong et al., 2022).

Glioblastoma stem cells (GSCs) and the immunosuppressive tumor microenvironment are two important factors contributing to glioblastoma genesis, treatment resistance, and recurrence (Pang et al., 2022). It has been found and characterized that GSCs-derived TFPI2 can regulate the maintenance of tumor stem cell stemness and the migration and immunosuppression of microglia in the tumor microenvironment through different mechanisms, respectively (Pang et al., 2023). The tumor mesenchymal-like state is associated with increased abundance and cytotoxicity of tumor-infiltrating T cells (Hara et al., 2021). A large number of MES subtypes correlate with macrophage abundance (Wang et al., 2017). In glioblastoma, the MES status of macrophages and cancer cells may also represent a therapeutic opportunity, as they are associated with high levels of MHC-I and MHC-II as well as a high number of T-cells, favoring a cytotoxic state, which may affect the response to immunotherapy (Hara et al., 2021). Infiltration of glioma-associated macrophages and microglia (GAMs) is usually triggered by chemokines secreted by GBM cells. Simultaneous environment-dependent tumor-immune symbiotic interactions promote tumor progression and treatment resistance in GBMs (Pang et al., 2022). Immunosuppressive GAM releases different cytokines and growth factors, such as IL-6, IL-11, IL-1β, IL-10, and TGF-β1, to promote GBM progression by activating pre-tumor signaling in GBM cells (Ye et al., 2012; Bloch et al., 2013; Zhang et al., 2018; Li et al., 2021; Liu et al., 2021; Ochocka et al., 2021).

In conclusion, the interactions between tumor cells and immune cells within the tumor microenvironment play a crucial role in driving the growth, infiltration, and metastasis of gliomas. These interactions also impact the immune status of the tumor microenvironment and influence the response to therapeutic interventions. By gaining a deeper understanding of these complex interaction mechanisms, researchers can identify novel therapeutic targets and develop more effective treatment strategies for gliomas. This knowledge can ultimately lead to improved outcomes for patients with glioma and other types of cancer.

T-cell immunity plays an important role in the glioma TME (Figure 4). However, the anti-tumor function of T cells is often limited due to the immune escape mechanisms and immunosuppressive environment of gliomas (Ravi et al., 2022). The limitations of GBM immunotherapy are intricately linked to the deficiency of T-cells within the brain tumor microenvironment. A comprehensive analysis combining single-cell transcriptome and spatial transcriptome sequencing has shed light on the role of interleukin-10 (IL-10) secreted by myeloid cells in driving T-cell dysfunction within the GBM microenvironment. This IL-10-mediated T-cell dysfunction ultimately promotes tumor cell growth and suppresses the immune response against the tumor. Understanding the mechanisms by which IL-10 and myeloid cells contribute to T-cell deficiency in GBM can provide valuable insights for developing targeted immunotherapies that aim to overcome these barriers and enhance the efficacy of immune-based treatments for GBM (Ravi et al., 2022). It was demonstrated that after α-PD-1 treatment, regulatory T cells (Tregs) with upregulated CD103 + lipid metabolism accumulated in the tumor microenvironment and suppressed the immune checkpoint blockade response by inhibiting CD8 + T cell activation (van Hooren et al., 2023). Treg cell targeting triggers the formation of tertiary lymphoid structures, enhances CD4⁺ and CD8⁺ T cell frequency and function, and unleashes radio immunotherapeutic efficacy (van Hooren et al., 2023). The spatial distribution of tumor-associated myeloid cells (TAM) coincided with hypoxic regions in GBM while targeting hypoxic sites disrupted the spatial distribution of TAM (Sattiraju et al., 2023). One study identified a population of CD4⁺ T cells that highly express IL-8 (Liu et al., 2023). Blocking IL-8 was able to reverse the immunosuppressive microenvironment of the tumor and enhance the therapeutic effect of immune checkpoint blockade (Liu et al., 2023) (Figure 5).