Glioblastoma (GBM) constitutes the most lethal primary brain tumor for which immunotherapy has provided limited benefit. The unique brain immune landscape is reflected in a complex tumor immune microenvironment (TIME) in GBM. Here, single cell sequencing of the GBM TIME revealed that microglia were under severe oxidative stress, which induced nuclear receptor subfamily 4 group A member 2 (NR4A2)-dependent transcriptional activity in microglia. Heterozygous Nr4a2 (Nr4a2+/-) or microglia-specific Nr4a2 (Nr4a2fl/flCx3cr1cre) genetic targeting reshaped microglia plasticity in vivo by reducing alternatively activated microglia and enhancing antigen presentation capacity for CD8+ T cells in GBM. In microglia, NR4A2 activated squalene monooxygenase (SQLE) to dysregulate cholesterol homeostasis. Pharmacological NR4A2 inhibition attenuated the pro-tumorigenic TIME, and targeting the NR4A2 or SQLE enhanced therapeutic efficacy of immune checkpoint blockade in vivo. Collectively, oxidative stress promotes tumor growth through NR4A2-SQLE activity in microglia, informing novel immune therapy paradigms in brain cancer.

<div>Abstract<p>Glioblastoma (GBM) constitutes the most lethal primary brain tumor for which immunotherapy has provided limited benefit. The unique brain immune landscape is reflected in a complex tumor immune microenvironment (TIME) in GBM. Here, single-cell sequencing of the GBM TIME revealed that microglia were under severe oxidative stress, which induced nuclear receptor subfamily 4 group A member 2 (NR4A2)–dependent transcriptional activity in microglia. Heterozygous <i>Nr4a2</i> (<i>Nr4a2</i>^+/−) or CX3CR1⁺ myeloid cell–specific <i>Nr4a2</i> (<i>Nr4a2</i>^fl/fl<i>Cx3cr1</i>^Cre) genetic targeting reshaped microglia plasticity <i>in vivo</i> by reducing alternatively activated microglia and enhancing antigen presentation capacity for CD8⁺ T cells in GBM. In microglia, NR4A2 activated squalene monooxygenase (SQLE) to dysregulate cholesterol homeostasis. Pharmacologic NR4A2 inhibition attenuated the protumorigenic TIME, and targeting the NR4A2 or SQLE enhanced the therapeutic efficacy of immune-checkpoint blockade <i>in vivo</i>. Collectively, oxidative stress promotes tumor growth through NR4A2–SQLE activity in microglia, informing novel immune therapy paradigms in brain cancer.</p>Significance:<p>Metabolic reprogramming of microglia in GBM informs synergistic vulnerabilities for immune-checkpoint blockade therapy in this immunologically cold brain tumor.</p><p><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-13-4-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 799</a></p></div>

Although metastasis is the major cause of death in cervical cancer, the mechanism of metastasis is still unclear. The mRNA expression and protein level of latent transforming growth factor beta binding protein 1 (LTBP1) were detected in tumor tissues and paracancerous tissues from in-house samples. Cell proliferation, cell cycle, migration, and in vivo metastasis were determined after LTBP1 was knocked down. Then, 13 drugs were screened, and the changes in cell apoptosis and proliferation and tumor metastasis were detected after drug treatment in shRNA cells. In our in-house samples, LTBP1 was lowly expressed in cervical cancer tissues. After LTBP1 knockdown, cell proliferation was increased, and the ability of in vitro migration and in vivo metastasis was enhanced. At the same time, the proportion of myeloid derived suppressor cells (MDSC) in situ increased, the proportion of T cells decreased, and transforming growth factor beta-1 (TGFβ1) signaling was activated. After carboplatin treatment, LTBP1 shRNA cell line apoptosis increased, metastasis in vivo was limited, and the proportion of MDSC in situ decreased. LTBP1 was lowly expressed in cervical cancer, and the inhibition of LTBP1 can improve the malignant degree of the tumor, and this process can be blocked by carboplatin.

<p>Table S4 describes list of literature-based immune marker panel selected to identity cell types of obtained clusters.</p>

<p>Table S3 describes primer information for qPCR analysis.</p>

<p>Supplemental Figure S4 describes that NR4A2-knockdown affects lipid metabolism and promotes antigen presentation of microglia.</p>

<p>Table S2 describes information of key research agents, data deposition and software versions.</p>

<p>Supplemental Figure S5 describes that genetic knockout of Nr4a2 in microglia reverses immunosuppressive state.</p>