Kai Kessenbrock

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s), collectively known as group 1 innate lymphoid cells (ILCs), are critical mediators of anti-tumor immunity, yet their role in brain metastasis remains poorly defined. Using single-cell RNA sequencing and functional depletion models, we show that group 1 ILCs fail to provide adequate tumor control in breast cancer brain metastasis (BCBM). In contrast to the liver, where diverse, functionally competent group 1 ILC populations facilitate excellent metastatic control, group 1 ILCs in BCBM show limited diversity and profound tissue-specific dysfunction such that their depletion had no effect on tumor burden despite modest parenchymal infiltration. We attribute this group 1 ILC dysfunction to severely restricted IL-15 availability, with IL-15Rα protein detected at 500-fold lower concentration in the brain than in the liver. Local IL-15 complex administration restored group 1 ILC accumulation, cytotoxicity, and tissue residency, significantly reducing brain metastatic burden independent of T cells. Intracranial delivery of IL-15-secreting induced pluripotent stem-cell-derived (iPSC)-NK cells, already in clinical testing for non-small cell lung cancer, significantly reduced tumor burden. Collectively, these findings reveal how tissue-specific differences in cytokine availability shape group 1 ILC population diversity and disease response, establishing localized IL-15 signaling as a compelling strategy to unlock innate immunity against intracranial malignancies. One Sentence Summary: Group 1 ILCs, including NK cells and ILC1s, can be pharmacologically enhanced with IL-15 to improve control of breast cancer brain metastasis.

Mammary gland development during puberty requires tightly coordinated epithelial proliferation, lineage specification, and branching morphogenesis, processes critically regulated by growth factor signaling. While epidermal growth factor receptor (EGFR) signaling is essential for ductal development, how its activity is quantitatively controlled within mammary epithelial cells (MECs) remains incompletely understood. Here, we identify Rasgrp1, a Ras guanine nucleotide exchange factor, as a key modulator of EGFR signaling in the mammary epithelium. Using Rasgrp1-deficient mice, primary MEC assays, and organoid models, we demonstrate that loss of Rasgrp1 leads to elevated EGFR–Ras–PI3K–AKT and mTORC1-S6 signaling, resulting in enhanced proliferative capacity and aberrant EGF-driven branching. Transcriptomic analysis of organoids reveals that EGF signaling suppresses Wnt/R-spondin-dependent stem-cell gene programs, suggesting that excessive EGFR activity disrupts stem cell maintenance. In vivo, Rasgrp1 deficiency causes impaired ductal elongation, persistent terminal end buds, and increased epithelial proliferation, indicating a breakdown in the spatial and temporal coordination of mammary morphogenesis. Together, our findings establish Rasgrp1 as a signaling rheostat that dampens EGFR pathway activity to support coordinated mammary gland development. These results highlight the importance of precise signaling calibration in epithelial development and suggest broader implications for Ras pathway regulation in tissue homeostasis and disease.

Despite the major roles of choroid plexus epithelial cells (CPECs) in brain homeostasis and repair, their developmental lineage and diversity remain undefined. In simplified differentiations from human pluripotent stem cells, derived CPECs (dCPECs) display canonical properties and dynamic motile multiciliated phenotypes that interact with Aβ uptake. Single dCPEC transcriptomes over time correlate well with human organoid and fetal CPECs, while pseudotemporal and cell cycle analyses highlight the direct CPEC origin from neuroepithelial cells. In addition, time series analyses define metabolic (type 1) and ciliogenic dCPECs (type 2) at early timepoints, followed by type 1 diversification into anabolic-secretory (type 1a) and catabolic-absorptive subtypes (type 1b) as type 2 cells contract. These temporal patterns are then confirmed in independent derivations and mapped to prenatal stages using human tissues. In addition to defining the prenatal lineage of human CPECs, these findings suggest dynamic models of ChP support for the developing human brain.

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8⁺ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin ( Spp1 ). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3 ⁺ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3 ⁺ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3 ⁺ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal Spp1 as a major regulator of macrophage and stromal progenitor interactions.

The adult human breast is comprised of an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue^1,2,3. Although most previous studies have focused on the breast epithelial system^4,5,6, many of the non-epithelial cell types remain understudied. Here we constructed the comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics study profiled 714,331 cells from 126 women, and 117,346 nuclei from 20 women, identifying 12 major cell types and 58 biological cell states. These data reveal abundant perivascular, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Spatial mapping using four different technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide a reference of the adult normal breast tissue for studying mammary biology and diseases such as breast cancer.

SUMMARY: The adult human breast comprises an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue. While previous studies have mainly focused on the breast epithelial system, many of the non-epithelial cell types remain understudied. Here, we constructed a comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics data profiled 535,941 cells from 62 women, and 120,024 nuclei from 20 women, identifying 11 major cell types and 53 cell states. These data revealed abundant pericyte, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Our spatial mapping using three technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells in the ducts and lobules, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide an unprecedented reference of adult normal breast tissue for studying mammary biology and disease states such as breast cancer.

Women with germline BRCA1 mutations (BRCA1^+/mut) have increased risk for hereditary breast cancer. Cancer initiation in BRCA1^+/mut is associated with premalignant changes in breast epithelium; however, the role of the epithelium-associated stromal niche during BRCA1-driven tumor initiation remains unclear. Here we show that the premalignant stromal niche promotes epithelial proliferation and mutant BRCA1-driven tumorigenesis in trans. Using single-cell RNA sequencing analysis of human preneoplastic BRCA1^+/mut and noncarrier breast tissues, we show distinct changes in epithelial homeostasis including increased proliferation and expansion of basal-luminal intermediate progenitor cells. Additionally, BRCA1^+/mut stromal cells show increased expression of pro-proliferative paracrine signals. In particular, we identify pre-cancer-associated fibroblasts (pre-CAFs) that produce protumorigenic factors including matrix metalloproteinase 3 (MMP3), which promotes BRCA1-driven tumorigenesis in vivo. Together, our findings demonstrate that precancerous stroma in BRCA1^+/mut may elevate breast cancer risk through the promotion of epithelial proliferation and an accumulation of luminal progenitor cells with altered differentiation.

Recent advances in single-cell transposase-accessible chromatin using a sequencing assay (scATAC-seq) allow cellular heterogeneity dissection and regulatory landscape reconstruction with an unprecedented resolution. However, compared to bulk-sequencing, its ultra-high missingness remarkably reduces usable reads in each cell type, resulting in broader, fuzzier peak boundary definitions and limiting our ability to pinpoint functional regions and interpret variant impacts precisely. We propose a weakly supervised learning method, scEpiLock, to directly identify core functional regions from coarse peak labels and quantify variant impacts in a cell-type-specific manner. First, scEpiLock uses a multi-label classifier to predict chromatin accessibility via a deep convolutional neural network. Then, its weakly supervised object detection module further refines the peak boundary definition using gradient-weighted class activation mapping (Grad-CAM). Finally, scEpiLock provides cell-type-specific variant impacts within a given peak region. We applied scEpiLock to various scATAC-seq datasets and found that it achieves an area under receiver operating characteristic curve (AUC) of ~0.9 and an area under precision recall (AUPR) above 0.7. Besides, scEpiLock’s object detection condenses coarse peaks to only ⅓ of their original size while still reporting higher conservation scores. In addition, we applied scEpiLock on brain scATAC-seq data and reported several genome-wide association studies (GWAS) variants disrupting regulatory elements around known risk genes for Alzheimer’s disease, demonstrating its potential to provide cell-type-specific biological insights in disease studies.