CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10.

Brain metastasis (br-met) develops in an immunologically unique br-met niche. Central nervous system-native myeloid cells (CNS-myeloids) and bone-marrow-derived myeloid cells (BMDMs) cooperatively regulate brain immunity. The phenotypic heterogeneity and specific roles of these myeloid subsets in shaping the br-met niche to regulate br-met outgrowth have not been fully revealed. Applying multimodal single-cell analyses, we elucidated a heterogeneous but spatially defined CNS-myeloid response during br-met outgrowth. We found Ccr2+ BMDMs minimally influenced br-met while CNS-myeloid promoted br-met outgrowth. Additionally, br-met-associated CNS-myeloid exhibited downregulation of Cx3cr1. Cx3cr1 knockout in CNS-myeloid increased br-met incidence, leading to an enriched interferon response signature and Cxcl10 upregulation. Significantly, neutralization of Cxcl10 reduced br-met, while rCxcl10 increased br-met and recruited VISTAHi PD-L1+ CNS-myeloid to br-met lesions. Inhibiting VISTA- and PD-L1-signaling relieved immune suppression and reduced br-met burden. Our results demonstrate that loss of Cx3cr1 in CNS-myeloid triggers a Cxcl10-mediated vicious cycle, cultivating a br-met-promoting, immune-suppressive niche.

Targeting CXCL16 and STAT1 augments immune checkpoint blockade therapy in triple-negative breast cancer.

Chemotherapy prior to immune checkpoint blockade (ICB) treatment appears to improve ICB efficacy but resistance to ICB remains a clinical challenge and is attributed to highly plastic myeloid cells associating with the tumor immune microenvironment (TIME). Here we show by CITE-seq single-cell transcriptomic and trajectory analyses that neoadjuvant low-dose metronomic chemotherapy (MCT) leads to a characteristic co-evolution of divergent myeloid cell subsets in female triple-negative breast cancer (TNBC). Specifically, we identify that the proportion of CXCL16 + myeloid cells increase and a high STAT1 regulon activity distinguishes Programmed Death Ligand 1 (PD-L1) expressing immature myeloid cells. Chemical inhibition of STAT1 signaling in MCT-primed breast cancer sensitizes TNBC to ICB treatment, which underscores the STAT1's role in modulating TIME. In summary, we leverage single-cell analyses to dissect the cellular dynamics in the tumor microenvironment (TME) following neoadjuvant chemotherapy and provide a pre-clinical rationale for modulating STAT1 in combination with anti-PD-1 for TNBC patients.

Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes.

High dimensional compositional and transcriptional profiling of heterogeneous brain-infiltrating leukocytes can lead to novel biological and therapeutic discoveries. High-quality single-cell leukocyte preparations are a prerequisite for optimal single cell profiling. Here, we describe a protocol for epitope and RNA-preserving dissociation of adult mouse brains and subsequent leukocyte purification and staining, which is adaptable to homeostatic and pathogenic brains. Leukocyte preparation following this protocol permits exquisite single-cell surface protein and RNA profiling in applications including CyTOF and CITE-seq. For complete details on the use and execution of this protocol, please refer to Guldner et al. (2020) and Golomb et al. (2020).

Multi-modal Single-Cell Analysis Reveals Brain Immune Landscape Plasticity during Aging and Gut Microbiota Dysbiosis.

Phenotypic and functional plasticity of brain immune cells contribute to brain tissue homeostasis and disease. Immune cell plasticity is profoundly influenced by tissue microenvironment cues and systemic factors. Aging and gut microbiota dysbiosis that reshape brain immune cell plasticity and homeostasis has not been fully delineated. Using Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), we analyze compositional and transcriptional changes of the brain immune landscape in response to aging and gut dysbiosis. Discordance between canonical surface-marker-defined immune cell types and their transcriptomes suggest transcriptional plasticity among immune cells. Ly6C+ monocytes predominate a pro-inflammatory signature in the aged brain, while innate lymphoid cells (ILCs) shift toward an ILC2-like profile. Aging increases ILC-like cells expressing a T memory stemness (Tscm) signature, which is reduced through antibiotics-induced gut dysbiosis. Systemic changes due to aging and gut dysbiosis increase propensity for neuroinflammation, providing insights into gut dysbiosis in age-related neurological diseases.

Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor-resistant HER2-positive breast cancer.

Acquired resistance to targeted cancer therapy is a significant clinical challenge. In parallel with clinical trials combining CDK4/6 inhibitors to treat HER2+ breast cancer, we sought to prospectively model tumor evolution in response to this regimen in vivo and identify a clinically actionable strategy to combat drug resistance. Despite a promising initial response, acquired resistance emerges rapidly to the combination of anti-HER2/neu antibody and CDK4/6 inhibitor Palbociclib. Using high-throughput single-cell profiling over the course of treatments, we reveal a distinct immunosuppressive immature myeloid cell (IMC) population to infiltrate the resistant tumors. Guided by single-cell transcriptome analysis, we demonstrate that combination of IMC-targeting tyrosine kinase inhibitor cabozantinib and immune checkpoint blockade enhances anti-tumor immunity, and overcomes the resistance. Furthermore, sequential combinatorial immunotherapy enables a sustained control of the fast-evolving CDK4/6 inhibitor-resistant tumors. Our study demonstrates a translational framework for treating rapidly evolving tumors through preclinical modeling and single-cell analyses.