Patients diagnosed with glioblastoma (GBM) have the most aggressive tumor progression and lethal recurrence. Research on the immune microenvironment landscape of tumor and cerebrospinal fluid (CSF) is limited. At the single-cell level, we aim to reveal the recurrent immune microenvironment of GBM and the potential CSF biomarkers and compare tumor locations. We collected four clinical samples from two patients: malignant samples from one recurrent GBM patient and non-malignant samples from a patient with brain tumor. We performed single-cell RNA sequencing (scRNA-seq) to reveal the immune landscape of recurrent GBM and CSF. T cells were enriched in the malignant tumors, while Treg cells were predominately found in malignant CSF, which indicated an inhibitory microenvironment in recurrent GBM. Moreover, macrophages and neutrophils were significantly enriched in malignant CSF. This indicates that they an important role in GBM progression. S100A9, extensively expressed in malignant CSF, is a promising biomarker for GBM diagnosis and recurrence. Our study reveals GBM’s recurrent immune microenvironment after chemoradiotherapy and compares malignant and non-malignant CSF samples. We provide novel targets and confirm the promise of liquid CSF biopsy for patients with GBM.

诊断为胶质母细胞瘤 (GBM) 的患者肿瘤进展最严重，复发率最高。对肿瘤和脑脊液（CSF）免疫微环境景观的研究有限。在单细胞水平上，我们的目标是揭示 GBM 的复发性免疫微环境和潜在的脑脊液生物标志物，并比较肿瘤位置。我们收集了两名患者的四个临床样本：一名复发性 GBM 患者的恶性样本和一名脑肿瘤患者的非恶性样本。我们进行了单细胞 RNA 测序 (scRNA-seq)，以揭示复发性 GBM 和 CSF 的免疫状况。T细胞在恶性肿瘤中富集，而Treg细胞主要在恶性脑脊液中发现，这表明复发性GBM中存在抑制性微环境。此外，恶性脑脊液中巨噬细胞和中性粒细胞显着富集。这表明它们在 GBM 进展中发挥重要作用。S100A9 在恶性脑脊液中广泛表达，是 GBM 诊断和复发的有前途的生物标志物。我们的研究揭示了放化疗后 GBM 复发的免疫微环境，并比较了恶性和非恶性脑脊液样本。我们提供新的靶点并证实液体脑脊液活检对 GBM 患者的前景。