Person:

Rozenblatt-Rosen, Orit

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

ABSTRACTSynovial sarcoma is an aggressive mesenchymal neoplasm, driven by the SS18-SSX fusion, and characterized by immunogenic antigens expression and exceptionally low T cell infiltration levels. To study the cancer-immune interplay in this disease, we profiled 16,872 cells from 12 human synovial sarcoma tumors using single-cell RNA-sequencing (scRNA-Seq). Synovial sarcoma manifests antitumor immunity, high cellular plasticity and a core oncogenic program, which is predictive of low immune levels and poor clinical outcomes. Using genetic and pharmacological perturbations, we demonstrate that the program is controlled by the SS18-SSX driver and repressed by cytokines secreted by macrophages and T cells in the tumor microenvironment. Network modeling predicted that SS18-SSX promotes the program through HDAC1 and CDK6. Indeed, the combination of HDAC and CDK4/6 inhibitors represses the program, induces immunogenic cell states, and selectively targets synovial sarcoma cells. Our study demonstrates that immune evasion, cellular plasticity, and cell cycle are co-regulated and can be co-targeted in synovial sarcoma and potentially in other malignancies.

Single cell RNA-sequencing revealed extensive transcriptional cell state diversity in cancer, often observed independently from genetic heterogeneity, raising the central question of how malignant cell states are encoded epigenetically. To address this, we performed multi-omics single-cell profiling – integrating DNA methylation, transcriptome, and genotyping within the same cells – of diffuse gliomas, tumors governed by defined transcriptional cell state diversity. Direct comparison of the epigenetic profiles of distinct cell states revealed key switches for state transitions recapitulating neurodevelopmental trajectories, and highlighted dysregulated epigenetic mechanisms underlying gliomagenesis. We further developed a quantitative framework to measure cell state heritability and transition dynamics based on high resolution lineage trees directly in human samples. We demonstrated heritability of malignant cell states, with key differences in hierarchal vs. plastic cell state architectures in IDH-mutant glioma vs. IDH-wildtype glioblastoma, respectively. This work provides a novel framework anchoring transcriptional cancer cell states in their epigenetic encoding, inheritance and transition dynamics.

Th17 cells are heterogenous, consisting of non-pathogenic Th17 cells (npTh17) that contribute to tissue homeostasis and pathogenic Th17 cells (pTh17) that mediate tissue inflammation. Here, we characterized regulatory pathways underlying Th17 heterogeneity and discovered substantial differences in the chromatin landscape of npTh17 and pTh17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTh17 cells share accessible chromatin programs with Tregs, whereas pTh17 cells exhibit features of both npTh17 cells and Th1 cells. Integrating single-cell ATAC-seq and single-cell RNA-seq, we inferred self-reinforcing and mutually exclusive regulatory networks controlling the different cell states and predicted transcription factors (TFs) regulating Th17 cell pathogenicity. We validated that BACH2 promotes immunomodulatory npTh17 programs and restrains pro-inflammatory Th1-like programs in Th17 cells in vitro and in vivo, Furthermore, human genetics implicate BACH2 in multiple sclerosis. Overall, our work identified regulators of Th17 heterogeneity as potential targets to mitigate autoimmunity.