Brain metastases are the most common tumor of the brain with a dismal prognosis. A fraction of patients with brain metastasis benefit from treatment with immune checkpoint inhibitors (ICI) and the degree and phenotype of the immune cell infiltration has been used to predict response to ICI. However, the anatomical location of brain lesions limits access to tumor material to characterize the immune phenotype. Here, we characterize immune cells present in brain lesions and matched cerebrospinal fluid (CSF) using single-cell RNA sequencing combined with T cell receptor genotyping. Tumor immune infiltration and specifically CD8+ T cell infiltration can be discerned through the analysis of the CSF. Consistently, identical T cell receptor clonotypes are detected in brain lesions and CSF, confirming cell exchange between these compartments. The analysis of immune cells of the CSF can provide a non-invasive alternative to predict the response to ICI, as well as identify the T cell receptor clonotypes present in brain metastasis.

Background GRP94 is a glucose-regulated protein critical for survival in endoplasmic reticulum stress. Expression of GRP94 is associated with cellular transformation and increased tumorigenicity in breast cancer. Specifically, overexpression of GRP94 predicts brain metastasis (BM) in breast carcinoma patients with either triple negative or ErbB2 positive tumors. The aim of this study was to understand if microenvironmental regulation of GRP94 expression might be a hinge orchestrating BM progression. Methods GRP94 ablation was performed in a BM model BR-eGFP-CMV/Luc-V5CA1 (BRV5CA1) of breast cancer. In vitro results were validated in a dataset of 29 metastases in diverse organs from human breast carcinomas and in BM tissue from tumors of different primary origin. BM patient-derived xenografts (PDXs) were used to test sensitivity to the therapeutic approach. Results BMs that overexpress GRP94 as well as tumor necrosis factor receptor-associated factor 2 are more resistant to glucose deprivation by induction of anti-apoptotic proteins (B-cell lymphoma 2 and inhibitors of apoptosis proteins) and engagement of pro-survival autophagy. GRP94 ablation downregulated autophagy in tumor cells, resulting in increased BM survival in vivo. These results were validated in a metastasis dataset from human patients, suggesting that targeting autophagy might be strategic for BM prevention. Indeed, hydroxychloroquine treatment of preclinical models of BM from PDX exerts preventive inhibition of tumor growth (P < 0.001). Conclusions We show that GRP94 is directly implicated in BM establishment by activating pro-survival autophagy. Disruption of this compensatory fueling route might prevent metastatic growth.

T cell bispecific antibodies (TCBs) are engineered molecules that bind both the T cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2-7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacological characteristics and potent anti-tumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft (PDX) models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.