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Abstract While triple-negative breast cancer (TNBC) is known to be heterogeneous at the genomic and transcriptomic levels, spatial information on tumor organization and cell composition is still lacking. Here, we investigate TNBC tumor architecture including its microenvironment using spatial transcriptomics on a series of 92 patients. We perform an in-depth characterization of tumor and stroma organization and composition using an integrative approach combining histomorphological and spatial transcriptomics. Furthermore, a detailed molecular characterization of tertiary lymphoid structures leads to identify a gene signature strongly associated to disease outcome and response to immunotherapy in several tumor types beyond TNBC. A stepwise clustering analysis identifies nine TNBC spatial archetypes, further validated in external datasets. Several spatial archetypes are associated with disease outcome and characterized by potentially actionable features. In this work, we provide a comprehensive insight into the complexity of TNBC ecosystem with potential clinical relevance, opening avenues for treatment tailoring including immunotherapy.

Abstract The respiratory system, including the lungs, is essential for terrestrial life. While recent research has advanced our understanding of lung development, much still relies on animal models and transcriptome analyses. In this study conducted within the Human Developmental Cell Atlas (HDCA) initiative, we describe the protein-level spatiotemporal organization of the lung during the first trimester of human gestation. Using high-parametric tissue imaging with a 30-plex antibody panel, we analyzed human lung samples from 6 to 13 post-conception weeks, generating data from over 2 million cells across five developmental timepoints. We present a resource detailing spatially resolved cell type composition of the developing human lung, including proliferative states, immune cell patterns, spatial arrangement traits, and their temporal evolution. This represents an extensive single-cell resolved protein-level examination of the developing human lung and provides a valuable resource for further research into the developmental roots of human respiratory health and disease.

Abstract Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3’ and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.

Abstract Upon infecting its vertebrate host, the malaria parasite initially invades the liver where it undergoes massive replication, whilst remaining clinically silent. The coordination of host responses across the complex liver tissue during malaria infection remains unexplored. Here, we perform spatial transcriptomics in combination with single-nuclei RNA sequencing over multiple time points to delineate host-pathogen interactions across Plasmodium berghei-infected liver tissues. Our data reveals significant changes in spatial gene expression in the malaria-infected tissues. These include changes related to lipid metabolism in the proximity to sites of Plasmodium infection, distinct inflammation programs between lobular zones, and regions with enrichment of different inflammatory cells, which we term ‘inflammatory hotspots’. We also observe significant upregulation of genes involved in inflammation in the control liver tissues of mice injected with mosquito salivary gland components. However, this response is considerably delayed compared to that observed in P. berghei-infected mice. Our study establishes a benchmark for investigating transcriptome changes during host-parasite interactions in tissues, it provides informative insights regarding in vivo study design linked to infection and offers a useful tool for the discovery and validation of de novo intervention strategies aimed at malaria liver stage infection.