Stress-free single-cell transcriptomic profiling and functional genomics of murine eosinophils.

Eosinophils are a class of granulocytes with pleiotropic functions in homeostasis and various human diseases. Nevertheless, they are absent from conventional single-cell RNA sequencing atlases owing to technical difficulties preventing their transcriptomic interrogation. Consequently, eosinophil heterogeneity and the gene regulatory networks underpinning their diverse functions remain poorly understood. We have developed a stress-free protocol for single-cell RNA capture from murine tissue-resident eosinophils, which revealed distinct intestinal subsets and their roles in colitis. Here we describe in detail how to enrich eosinophils from multiple tissues of residence and how to capture high-quality single-cell transcriptomes by preventing transcript degradation. By combining magnetic eosinophil enrichment with microwell-based single-cell RNA capture (BD Rhapsody), our approach minimizes shear stress and processing time. Moreover, we report how to perform genome-wide CRISPR pooled genetic screening in ex vivo-conditioned bone marrow-derived eosinophils to functionally probe pathways required for their differentiation and intestinal maturation. These protocols can be performed by any researcher with basic skills in molecular biology and flow cytometry, and can be adapted to investigate other granulocytes, such as neutrophils and mast cells, thereby offering potential insights into their roles in both homeostasis and disease pathogenesis. Single-cell transcriptomics of eosinophils can be performed in 2-3 d, while functional genomics assays may require up to 1 month.

Identifying Spatial Co-occurrence in Healthy and InflAmed tissues (ISCHIA).

Sequencing-based spatial transcriptomics (ST) methods allow unbiased capturing of RNA molecules at barcoded spots, charting the distribution and localization of cell types and transcripts across a tissue. While the coarse resolution of these techniques is considered a disadvantage, we argue that the inherent proximity of transcriptomes captured on spots can be leveraged to reconstruct cellular networks. To this end, we developed ISCHIA (Identifying Spatial Co-occurrence in Healthy and InflAmed tissues), a computational framework to analyze the spatial co-occurrence of cell types and transcript species within spots. Co-occurrence analysis is complementary to differential gene expression, as it does not depend on the abundance of a given cell type or on the transcript expression levels, but rather on their spatial association in the tissue. We applied ISCHIA to analyze co-occurrence of cell types, ligands and receptors in a Visium dataset of human ulcerative colitis patients, and validated our findings at single-cell resolution on matched hybridization-based data. We uncover inflammation-induced cellular networks involving M cell and fibroblasts, as well as ligand-receptor interactions enriched in the inflamed human colon, and their associated gene signatures. Our results highlight the hypothesis-generating power and broad applicability of co-occurrence analysis on spatial transcriptomics data.

Fragment-sequencing unveils local tissue microenvironments at single-cell resolution.

Cells collectively determine biological functions by communicating with each other-both through direct physical contact and secreted factors. Consequently, the local microenvironment of a cell influences its behavior, gene expression, and cellular crosstalk. Disruption of this microenvironment causes reciprocal changes in those features, which can lead to the development and progression of diseases. Hence, assessing the cellular transcriptome while simultaneously capturing the spatial relationships of cells within a tissue provides highly valuable insights into how cells communicate in health and disease. Yet, methods to probe the transcriptome often fail to preserve native spatial relationships, lack single-cell resolution, or are highly limited in throughput, i.e. lack the capacity to assess multiple environments simultaneously. Here, we introduce fragment-sequencing (fragment-seq), a method that enables the characterization of single-cell transcriptomes within multiple spatially distinct tissue microenvironments. We apply fragment-seq to a murine model of the metastatic liver to study liver zonation and the metastatic niche. This analysis reveals zonated genes and ligand-receptor interactions enriched in specific hepatic microenvironments. Finally, we apply fragment-seq to other tissues and species, demonstrating the adaptability of our method.

Active eosinophils regulate host defence and immune responses in colitis.

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.

In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels.

Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

Differential regulation of ß-catenin-mediated transcription via N- and C-terminal co-factors governs identity of murine intestinal epithelial stem cells.

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/ß-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic ß-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of ß-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with ß-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune ß-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/ß-catenin output requires selective modulation of gene expression by transcriptional co-factors.

Senescence Reprogramming by TIMP1 Deficiency Promotes Prostate Cancer Metastasis.

Metastases account for most cancer-related deaths, yet the mechanisms underlying metastatic spread remain poorly understood. Recent evidence demonstrates that senescent cells, while initially restricting tumorigenesis, can induce tumor progression. Here, we identify the metalloproteinase inhibitor TIMP1 as a molecular switch that determines the effects of senescence in prostate cancer. Senescence driven either by PTEN deficiency or chemotherapy limits the progression of prostate cancer in mice. TIMP1 deletion allows senescence to promote metastasis, and elimination of senescent cells with a senolytic BCL-2 inhibitor impairs metastasis. Mechanistically, TIMP1 loss reprograms the senescence-associated secretory phenotype (SASP) of senescent tumor cells through activation of matrix metalloproteinases (MMPs). Loss of PTEN and TIMP1 in prostate cancer is frequent and correlates with resistance to docetaxel and worst clinical outcomes in patients treated in an adjuvant setting. Altogether, these findings provide insights into the dual roles of tumor-associated senescence and can potentially impact the treatment of prostate cancer.

TBX3 acts as tissue-specific component of the Wnt/ß-catenin transcriptional complex.

BCL9 and PYGO are ß-catenin cofactors that enhance the transcription of Wnt target genes. They have been proposed as therapeutic targets to diminish Wnt signaling output in intestinal malignancies. Here we find that, in colorectal cancer cells and in developing mouse forelimbs, BCL9 proteins sustain the action of ß-catenin in a largely PYGO-independent manner. Our genetic analyses implied that BCL9 necessitates other interaction partners in mediating its transcriptional output. We identified the transcription factor TBX3 as a candidate tissue-specific member of the ß-catenin transcriptional complex. In developing forelimbs, both TBX3 and BCL9 occupy a large number of Wnt-responsive regulatory elements, genome-wide. Moreover, mutations in Bcl9 affect the expression of TBX3 targets in vivo, and modulation of TBX3 abundance impacts on Wnt target genes transcription in a ß-catenin- and TCF/LEF-dependent manner. Finally, TBX3 overexpression exacerbates the metastatic potential of Wnt-dependent human colorectal cancer cells. Our work implicates TBX3 as context-dependent component of the Wnt/ß-catenin-dependent transcriptional complex.

Single-Molecule RNA FISH in Whole-Mount Organoids.

Single-molecule RNA fluorescent in situ hybridization (smFISH) enables the detection and quantification of single RNA molecules. Three-dimensional organoid cultures have emerged as versatile in vitro primary culture models that recapitulate many physiological features of their tissue of origin. Here we describe a protocol to visualize single RNA molecules in organoid cultures. Our method accommodates both a whole-mount staining workflow which requires spinning disk confocal microscopy, and a cryosectioning workflow which is compatible with widefield microscopy. Organoid smFISH enables to address various biological problems that range from the identification of cell types (e.g., via the intestinal stem cell marker Lgr5) to the quantification of RNA localization in an epithelium.