Glioblastoma (GBM) is an aggressive and difficult-to-treat brain cancer. Advances in therapy are hindered by the complex nature of the disease and the challenges posed by the brain's unique environment. One area of increasing interest is the role of cholesterol metabolism in GBM progression. The brain has a high cholesterol content, primarily synthesized by astrocytes due to the blood-brain barrier. GBM cells exhibit a metabolic codependency on exogenous cholesterol uptake, making them vulnerable to cholesterol modulators. However, previous attempts to target cholesterol uptake or efflux, such as with the LXR-623 agonist, have been limited by adverse CNS events in clinical trials. Another promising strategy involves targeting tumor-infiltrating immune cells. Cholesterol homeostasis is crucial for immune cell function. For example, cholesterol accumulation in CD8 T cells leads to exhaustion, while tumor cells induce immune tolerance by depriving tumor-associated macrophages (TAMs) of cholesterol. Given GBM's unique cholesterol metabolism, it's reasonable to suspect a link between cholesterol metabolism and immune cell fate within the tumor microenvironment (TME). Pharmacological modulation of immune cell metabolism may offer targeted treatment, but the specific role of cholesterol in GBM TME immune cells remains unclear. This research investigates cholesterol's role in GBM progression and immune cell dysfunction. The study focuses on the effect of cholesterol accumulation in monocyte-derived TAMs on their phagocytic function. The researchers hypothesize that manipulating cholesterol efflux using ApoA1, a cholesterol reverse transporter, could restore TAM phagocytosis and antitumor immunity. The development and testing of an ApoA1-armed oncolytic adenovirus are also key components of this research.

Existing literature highlights the intricate relationship between cholesterol metabolism and cancer progression, particularly in glioblastoma. Studies have demonstrated the metabolic codependency of GBM cells on exogenous cholesterol uptake, making them susceptible to therapeutic interventions targeting cholesterol metabolism. The use of LXR agonists, like LXR-623, has shown promise in preclinical models by selectively killing GBM cells through the modulation of cholesterol uptake and efflux pathways. However, the clinical translation of these agents has been hampered by significant CNS side effects. The importance of cholesterol homeostasis in immune cell function is another well-established concept in cancer biology. Research has shown the detrimental effects of cholesterol accumulation in CD8+ T cells, leading to T cell exhaustion and impaired antitumor responses. Conversely, tumor cells can create an immunosuppressive environment by limiting cholesterol availability to TAMs, thereby suppressing their phagocytic activity. This research builds on this body of knowledge by investigating the specific roles of cholesterol metabolism in the GBM TME, focusing on the effects on TAMs and the consequential impact on antitumor immunity.

This study used a multi-pronged approach, combining in vivo experiments with in vitro analyses and computational modelling. **In vivo models:** The researchers utilized three orthotopic GBM models in male mice: a human GBM cell line (U251-MG), a mouse GBM cell line (GL261), and a rat GBM cell line (C6). These models allowed for the investigation of cholesterol metabolism in the context of a realistic tumor microenvironment. Interstitial fluid (IF) was collected from both normal brain tissue and tumor tissue to assess cholesterol levels. **Immune cell isolation and analysis:** Major immune cell subsets (macrophages, monocytes, lymphocytes) were isolated from tumor tissue and spleen. Flow cytometry was used to quantify cholesterol content in these cells and to assess the expression of relevant receptors (e.g., ABCA1, G1, Siglec-10, PD-1). Transmission electron microscopy was used to examine cell morphology and assess phagocytic function. **Cholesterol manipulation:** To investigate the impact of cholesterol efflux, ApoA1 was introduced either through genetic modification of GBM cells (creating ApoA1-expressing cell lines) or through direct treatment. The researchers also used inhibitors like DIDS (an ABCA1 inhibitor) to confirm the mechanisms involved. **Oncolytic adenovirus development:** An oncolytic adenovirus expressing ApoA1 (AdV^ApoA1) was engineered and tested in vivo. This aimed to combine the oncolytic effect of the virus with the cholesterol-modulating effects of ApoA1. **Immunodepletion experiments:** To determine the roles of different immune cell subsets in the observed effects, the researchers performed immunodepletion experiments in vivo, selectively depleting macrophages, CD8+ T cells, CD4+ T cells, or NK cells. **Metabolomics:** Cholesterol-targeted metabolomics was used to analyze the changes in the lipid profile of TAMs following ApoA1 treatment. This analysis identified changes in cholesterol and oxysterol levels, particularly a reduction in 7-ketocholesterol (7K-Cho). **Mitochondrial analysis:** RNA sequencing, qPCR analysis, SDS-PAGE, electron microscopy, and flow cytometry (JC-1 staining) were employed to assess the impact of cholesterol and 7K-Cho on mitochondrial translation, function, and mitophagy in macrophages. **Reporter assays:** Luciferase reporter assays were conducted in THP-1 macrophages to investigate the impact of cholesterol and 7K-Cho on the TNF signaling pathway. **Statistical analysis:** Appropriate statistical methods were used to analyze the data, with p < 0.05 considered statistically significant.

This study's key findings can be summarized as follows: 1. **Cholesterol Accumulation in TAMs:** The researchers found that cholesterol levels were significantly higher in TAMs within GBM tumors compared to peripheral macrophages, indicating a cholesterol reservoir in the TME. This accumulation was associated with impaired phagocytic function in TAMs. 2. **ApoA1 Restores TAM Phagocytosis:** Treatment with ApoA1 significantly enhanced cholesterol efflux from TAMs. This improved TAM phagocytosis in both in vitro and in vivo settings, demonstrated by increased phagocytosis of tumor cell debris. The enhanced phagocytosis was shown to be dependent on ABCA1, as it was blocked by the ABCA1 inhibitor DIDS. 3. **Immune Cell-Mediated Tumor Control:** The antitumor effects of ApoA1 were dependent on both macrophages and CD8+ T cells, as depletion of either cell type abolished the therapeutic benefit observed in vivo. In vitro coculture experiments confirmed that ApoA1 facilitated a synergistic interaction between TAMs and CD8+ T cells, resulting in increased tumor cell killing. The improved tumor killing was shown to not be due to the direct killing of tumor cells but rather the reprogramming of TAM function. 4. **7-Ketocholesterol and Mitochondrial Dysfunction:** Metabolomics revealed that ApoA1 treatment significantly reduced levels of 7K-Cho in TAMs. Further analysis demonstrated that 7K-Cho inhibited mitochondrial translation, resulting in mitochondrial dysfunction and impaired phagocytosis. ApoA1's restoration of phagocytosis was shown to be inhibited by treatment with 7K-Cho. 5. **TNF Signaling Pathway:** RNA sequencing of 7K-Cho-treated BMDMs revealed that the TNF signaling pathway was significantly affected. Further investigation showed that cholesterol and 7K-Cho inhibited TNF-α production and NF-κB activation, and this inhibition of the TNF-signaling pathway was critical to the observed impaired phagocytosis. ApoA1's improvement of phagocytosis was also inhibited by blocking TNF-α. 6. **Therapeutic Efficacy of AdV^ApoA1:** The oncolytic adenovirus expressing ApoA1 (AdV^ApoA1) demonstrated significant antitumor activity in multiple GBM models. AdV^ApoA1 treatment resulted in prolonged survival, reduced tumor growth, and improved body weight compared to the control virus. The complete response rate was impressive in certain models. 7. **Systemic Anti-tumor Immunity:** AdV^ApoA1 treatment triggered a systemic antitumor immune response, controlling tumor growth in both the injected and contralateral uninjected tumor sites of a subcutaneous model. This effect was mediated by CD8+ T cells and macrophages. The cured mice developed long-term tumor-specific immune memory.

This study provides compelling evidence for the importance of cholesterol metabolism in shaping the immunosuppressive TME in GBM. The findings demonstrate that cholesterol accumulation in TAMs leads to phagocytic dysfunction, contributing to disease progression. The successful manipulation of cholesterol efflux using ApoA1, both alone and as part of an oncolytic viral strategy, highlights a novel immunometabolic approach to GBM therapy. This approach overcomes the limitations of previous attempts to target cholesterol metabolism directly in GBM, avoiding the CNS toxicity observed with LXR agonists. The identification of 7K-Cho as a key mediator of mitochondrial dysfunction and impaired phagocytosis in TAMs provides a mechanistic explanation for the observed effects of ApoA1. This is a significant advance, moving beyond simply demonstrating an association between cholesterol metabolism and immune dysfunction to uncovering a specific underlying mechanism. The success of the ApoA1-armed oncolytic adenovirus further supports the therapeutic potential of this strategy, demonstrating a combined oncolytic and immunomodulatory effect. The finding that AdV^ApoA1 treatment triggers a systemic anti-tumor immune response is particularly noteworthy. This suggests that the approach may be effective even against disseminated disease, offering a potential treatment strategy beyond local tumor control.

This study demonstrates that targeting cholesterol metabolism in TAMs, specifically through ApoA1-mediated cholesterol efflux, is a promising immunometabolic approach for GBM therapy. The development of an ApoA1-armed oncolytic adenovirus represents a significant advancement in this area, combining oncolytic activity with immunomodulatory effects. Future research could focus on optimizing the delivery of ApoA1 and investigating its potential in other cancer types. Further exploration of the mechanisms through which 7K-Cho inhibits mitochondrial translation and TNF-α signaling is also warranted.

While this study presents strong evidence for the therapeutic potential of ApoA1-mediated cholesterol efflux, several limitations should be considered. Firstly, the study primarily used mouse models, and further investigation is needed to confirm the findings in human patients. Secondly, although the mechanism linking 7K-Cho to mitochondrial dysfunction was investigated, the precise molecular details of this process remain to be fully elucidated. Finally, the long-term effects of AdV^ApoA1 treatment and the durability of the induced immune memory require further investigation.