Cell Type Architecture and Positional Gene Gradients in an Adult Animal at Subcellular Resolution

Metabolism underlies cell growth, survival, and function, yet its activities vary widely across cell types and tissue environments. Spatially resolving these processes in situ at single-cell resolution is essential to advance our understanding of cellular function and tissue physiology in health and disease. However, existing approaches are limited by either destructive workflows, insufficient spatial resolution and biochemical specificity, or lack of direct linkage to cell identity. Here, we present Raman Enhanced Delineation of Cell Atlases in Tissues (REDCAT), a multimodal all-optical platform that integrates stimulated Raman scattering, autofluorescence redox imaging, second harmonic generation, and high-plex immunofluorescence to co-map metabolic activities and cell types within the same tissue section. REDCAT achieves subcellular resolution profiling of protein, lipid, redox, and nuclear acid metabolism, together with extracellular matrix composition, in both FFPE and fresh-frozen human tissues. Applied to normal lymph nodes, REDCAT delineated distinct redox and lipid remodeling programs across germinal center B-cell zones and immune subsets, highlighting cell-type-specific metabolic specialization. In lymphoma, it revealed profound metabolic reprogramming, including extensive lipid accumulation, nuclear metabolic heterogeneity, and a transitional metabolic state associated with transformation from chronic lymphocytic leukemia to diffuse large B-cell lymphoma, thereby illuminating tumor evolution in situ . In human liver, REDCAT resolved cell-type-specific lipid droplet diversity and zonation-dependent nuclear metabolic gradients, uncovering new principles of spatial metabolic organization. By directly linking cell identity with spatial metabolic states at single-cell or subcellular resolution, REDCAT establishes a broadly applicable framework for studying immune function, tumor progression, and tissue physiology, and offers a new path to deciphering the metabolic basis of health and disease.

Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of plasma cells. Although therapeutic advances have been made to improve clinical outcomes and to prolong patients’ survival in the past two decades, MM remains largely incurable. Single-cell sequencing (SCS) is a powerful method to dissect the cellular and molecular landscape at single-cell resolution, instead of providing averaged results. The application of single-cell technologies promises to address outstanding questions in myeloma biology and has revolutionized our understanding of the inter- and intra-tumor heterogeneity, tumor microenvironment, and mechanisms of therapeutic resistance in MM. In this review, we summarize the recently developed SCS methodologies and latest MM research progress achieved by single-cell profiling, including information regarding the cancer and immune cell landscapes, tumor heterogeneities, underlying mechanisms and biomarkers associated with therapeutic response and resistance. We also discuss future directions of applying transformative SCS approaches with contribution to clinical translation.

Towards high resolution imaging of human airways with optical coherence microscopy

Spatial transcriptomics holds great promise for revolutionizing biology and medicine by providing gene expression profiles with spatial information. Until recently, spatial resolution has been limited, but advances in high-throughput in situ imaging technologies now offer new opportunities by covering thousands of genes at a single-cell or even subcellular resolution, necessitating databases dedicated to comprehensive coverage and analysis with user-friendly intefaces. We introduce the HISSTA database, which facilitates the archival and analysis of in situ transcriptome data at single-cell resolution from various human tissues. We have collected and annotated spatial transcriptome data generated by MERFISH, CosMx SMI, and Xenium techniques, encompassing 112 samples and 28 million cells across 16 tissue types from 63 studies. To decipher spatial contexts, we have implemented advanced tools for cell type annotation, spatial colocalization, spatial cellular communication, and niche analyses. Notably, all datasets and annotations are interactively accessible through Vitessce, allowing users to focus on regions of interest and examine gene expression in detail. HISSTA is a unique database designed to manage the rapidly growing dataset of in situ transcriptomes at single-cell resolution. Given its comprehensive data content and advanced analysis tools with interactive visualizations, HISSTA is poised to significantly impact cancer diagnosis, precision medicine, and digital pathology. HISSTA is freely accessible at https://kbds.re.kr/hissta/. The source code is available at https://doi.org/10.5281/zenodo.14904523.

Mass spectrometric characterization of elements and molecules in cell cultures and tissues

BackgroundThe development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy.ResultsHere we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo.ConclusionUsing this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

When California's US$3 billion Stem Cell Research and Cures Act, Proposition 71, was approved by voters in November 2004, the crucial paragraph did not mention embryonic stem cells (ES cells): There is hereby established a right to conduct stem cell research which includes research involving adult stem cells, cord blood stem cells, pluripotent stem cells, and/or progenitor cells. Pluripotent stem cells are cells that are capable of self-renewal, and have broad potential to differentiate into multiple adult cell types.1 Had the drafters somehow foreseen the international effort that would be spurred by Shinya Yamanaka and colleagues' breakthrough 2006 paper2 in Cell? Did they know that Yamanaka's and James Thomson's reprogramming teams3, 4 would announce in late 2007 that a quartet of transcription factors within the larger pool of embryo-specific genes was sufficient to reprogramme human skin cells to pluripotency? In 2004, the only known way to reset the genome of a cell from an adult animal was to transfer its nucleus to an egg and allow this to divide into an early embryo, and generating ES cells meant destroying the embryo. Unlike the original, differentiated cell, these ES cells would be pluripotent, capable of becoming any sort of cell in the body. Did the wording in Proposition 71 forecast or spur the innovation that patient-specific pluripotent stem cells might be possible without either embryo creation or embryo destruction?

In the United States and globally, prostate cancer (PCa) mortality rates are the highest among men of African descent. 24% of the disparities in PCa remains even when controlled for access to care and stage at presentation. Moreover, the tumor microenvironment plays an essential role in tumor progression, aggressiveness, therapeutic response, and patient outcomes. Additionally, the association of the genomic findings with patient ancestry and other characteristics, such as tumor biology and transcriptomic alterations, remains poorly understood. Here, we performed a multi-Omics approach (N=447) to unravel the complexity of tumor heterogeneity and understand disease progression & distinct tumor biology influenced by genetic ancestry. We performed Whole Exome (Normal/tumor paired) Sequencing matched with Methyl Seq and Whole transcriptomic Sequencing for three datasets of our African, African American Men (AAM), and European Men (EAM). Additionally, we ran Spatial NanoString high-plex GeoMx-DSP for a total of 118 treatment-naive PCa patients (around 500 ROIs). Simultaneously, we added matched whole transcriptome Sequencing for these patients. The cohort comprised 87 AAM, 3 unknown, and 28 EAM self-reported individuals. To verify the self-reported race, the genomic ancestry was qualified using genotype and Admixture analysis. To further validate differentially expressed genes at the protein level, we performed multi-plex histological staining of 40 markers to determine the spatial resolution and neighborhood clustering within the tumor and the microenvironment. In parallel, we performed scMultiOmics sequencing (scRNA & scATAC-Seq) at a single-cell resolution (6000 cells/sample at 25K reads per cell) from an additional 12 patients (9 AAM & 3 EAM). We reconstructed a 3D model of the distinct tumor microenvironment at subcellular resolution using serially sectioned hematoxylin and eosin- stained tissue sections (CODA technology). Our results demonstrate that patients who self-report as AAM or Nigerian are assigned to high African (> 70%) Ancestry with either Yoruba (Nigeria) and/or Bantu subpopulation in the Sub-Saharan area. Additionally, high African Ancestry patients are diagnosed at a younger age and show advanced pathology stages compared to patients with European Ancestry. AAM of Yoruba descent expresses significantly higher immune-inflammatory signatures (STAT/IFNG-signaling pathway) compared to the Nigerian-Yoruba subpopulation. Our scRNA-Seq analysis shows that AAM has suppressive myeloid cells infiltrate within the tumor cells. The infiltrations of these cells change with age, Gleason Grade, and pathology stage. Moreover, AAM-tumor cells (scATAC-Seq) have increased open Chromatin accessibility with STAT motifs enrichment (p-value;0.0001), which could be the driver regulators of the immune-suppressive signature as well as influence the upregulation of STAT/IFNG signature within African Ancestry patients. Our study provides new insight into how genetic ancestry impacts immune signatures in AAM/African and contributes to PCa racial disparities. Citation Format: Isra Elhussin, Ezra Baraban1, Ashley Kiemen, Tamara L Lotan, Cathy Handy Marshall, Emmanuel Antonarakis, Moray J Campbell, Melissa Davis, Michael Dixon, Isaac Kim, Stefan Ambs, Rick Kittles, Adam B Murphy, Clayton Yates. Integrative multi-omics profiling in patients of African descent diagnosed with prostate cancer reveals distinct tumor-promoting immunosuppressive niches at a single-cell level and spatial resolution [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C117.

Introduction and aims Atopic eczema is the most common inflammatory skin disease. Epidermal barrier dysfunction and the T helper (Th)2 immune axis are central to disease pathogenesis. In recent years, treatments targeting Th2 immune mediators [e.g. interleukin (IL)-4/IL-13 inhibitor dupilumab] have greatly expanded the therapeutic armamentarium. However, the observed interindividual heterogeneity in real-world outcomes of eczema treatment highlights our limited knowledge of drug action and response. To address this, we embedded a highly powered longitudinal single-cell multiomic and spatial transcriptomic profiling strategy within BEACON (n > 400), the world-first platform trial to compare effectiveness and tolerability of standard (methotrexate) and targeted (dupilumab, Janus kinase inhibitor) eczema drugs. Methods Our initial cohort consisted of 25 individuals (17 male patients; mean age 39.2 years) sampled at day 0, day 3, day 14, week 12 and week 24 of dupilumab therapy. We profiled circulating immune populations through single-cell transcriptome, surface proteome and T-cell receptor repertoire analysis of > 600 000 peripheral blood mononuclear cells. We also used 10 × Multiome and Xenium to profile chromatin accessibility and gene expression of matched eczema skin at single-cell resolution. Results We defined 20 immune populations in peripheral blood, including CD4+, CD8+, myeloid, natural killer and B cells. We observed drug-induced shifts in abundance and gene expression levels in each major cell type as early as 14 days into dupilumab treatment. We also identified dynamic shifts of key skin cell populations during early dupilumab treatment and spatially mapped these cell populations in eczema skin at subcellular resolution. Conclusions Our high-resolution multiomic therapeutics atlas, leveraging a unique clinical platform, maps changes of the skin and blood immune response induced by dupilumab treatment. It provides critical insight into the early effects of dupilumab therapy, which may open avenues for new target discovery and precision medicine approaches to treatment allocation and monitoring in eczema.

Recent technological advances in photonics are making intravital microscopy (IVM) an increasingly powerful approach for the mechanistic exploration of biological processes in the physiological context of complex native tissue environments. Direct, dynamic and multiparametric visualization of immune cell behavior in living animals at cellular and subcellular resolution has already proved its utility in auditing basic immunological concepts established through conventional approaches and has also generated new hypotheses that can conversely be complemented and refined by traditional experimental methods. The insight that outgrowing tumors must not necessarily have evaded recognition by the adaptive immune system, but can escape rejection by actively inducing a state of immunological tolerance calls for a detailed investigation of the cellular and molecular mechanisms by which the anti-cancer response is subverted. Along with molecular imaging techniques that provide dynamic information at the population level, IVM can be expected to make a critical contribution to this effort by allowing the observation of immune cell behavior in vivo at single cell-resolution. We review here how IVM-based investigation can help to clarify the role of cytotoxic T lymphocytes (CTL) in the immune response against cancer and identify the ways by which their function might be impaired through tolerogenic mechanisms.

Background Crohn's disease (CD) frequently results in fistula development in approximately 40% of patients due to sustained, transmural inflammation within the bowel wall. Despite advanced treatments, recurrence of fistulae affects one third of patients. Understanding its pathogenesis is crucial for targeted treatments, yet remains poorly defined. This study employs spatial transcriptomic and single-cell RNA-sequencing (scRNA-seq) technologies to characterise human fistulating CD tissue pathology. Methods Unbiased FFPE spatial transcriptomics (10x Visium) were applied to surgically resected, full-thickness (FT) tissue from 20 CD-associated fistulae cases and 15 controls. Subsequently, selected cases underwent further analysis using custom 500-plex MERFISH subcellular resolution spatial transcriptomics (Vizgen MERSCOPE). Additionally, a reference single-cell cohort encompassing patients with fistulating, stricturing, and inflammatory CD phenotypes, along with healthy controls, was generated. Optimisation of scRNA-seq for resected FT ileal tissue enabled the isolation and profiling of epithelial, immune, and stromal populations (10x Chromium). Computational analysis facilitated the spatial localisation of single-cell clusters enriched in fistula CD tissue, identifying key parameters linked to fistula development. Results The scRNA-seq data revealed diverse CD-specific cellular states adopted by intestinal fibroblasts, with IL11+ fibroblasts prominently expressing CD82, COL7A1, MMP1, CHI3L1, suggestive of pro-fibrotic signalling and regenerative morphogen pathways. This coincided with the expansion of a CD-specific subpopulation of pericytes (CCL19/CCL21) involved in leucocyte migration. Spatial profiling of CD fistula tracts by Visium and MERSCOPE unveiled an enrichment of key epithelial developmental transcription factors (e.g., GRHL3) and localised the pro-fibrotic signature displaying increased IL11 and MMP expression, along with perturbed WNT signalling within the fistula stroma. Active proliferation (HOPX, MKI67) was observed at the base of fistula tracts and within the stroma. Transcriptomic characterisation depicted a gradual loss in stem cell signature (LGR5, ASCL2, SMOC2) and supporting telocytes (POSTN) toward the leading edge of the fistula, signifying abnormal epithelium loss. Conclusion This pioneering study integrates multi-modal spatial transcriptomics and single-cell profiling to unveil the intricate cellular and molecular landscape in FT, fistulating CD pathology. Spatial analyses elucidate specific mechanisms involved in epithelial loss, stromal remodelling, and perturbed WNT signalling pathways within fistulating tissue, outlining a course of disrupted regenerative processes in fistulating CD.

From pathogens to microbiota: How Drosophila intestinal stem cells react to gut microbes

Dissecting human neurobiology at high resolution and with mechanistic precision requires a major leap in scalability, given the need for experimental designs that include multiple individuals and, prospectively, population cohorts. To lay the foundation for this, we have developed and benchmarked complementary strategies to multiplex brain organoids by pooling cells from different pluripotent stem cell (PSC) lines either during organoid generation (mosaic models) or before single-cell RNA sequencing (scRNA-seq) library preparation (downstream multiplexing). We have also developed a new computational method, SCanSNP, and a consensus call to deconvolve cell identities, overcoming current criticalities in doublets and low-quality cell identification. We validated both multiplexing methods for charting neurodevelopmental trajectories at high resolution, thus linking specific individuals’ trajectories to genetic variation. Finally, we modeled their scalability across different multiplexing combinations and showed that mosaic organoids represent an enabling method for high-throughput settings. Together, this multiplexing suite of experimental and computational methods provides a highly scalable resource for brain disease and neurodiversity modeling.

SummaryHuman induced pluripotent stem cells (hiPSCs) can differentiate into neurons and glia via neural progenitor cells and are widely used for neurogenic studies. However, directly visualizing the transition status during the neural differentiation of live cells is difficult. Here, targeting NEUROG2 (NGN2) and TUBB3 as markers of neurogenic cells and neurons, respectively, we established TUBB3EGFP/NGN2TagRFP dual-reporter hiPSCs using CRISPR-Cas9 technology. We induced the differentiation of cortical neurons from dual-reporter hiPSCs, successfully visualizing cell-fate conversion in two-dimensional (2D) culture and 3D organoids. The reporter cells were used to monitor drug effects to enhance neural induction, responses to gene knockdown, transplantation to the embryonic mouse brain, and live imaging at single-cell resolution. Notably, the earliest REELIN-positive neurons showed a distinctive migration pattern, and their production was accelerated by HES1-function loss. Together, these results demonstrate the potential for dual-reporter hiPSCs in therapeutic neural regeneration strategies and studies on human cortical development.

Hepatocellular carcinoma (HCC) is the most common liver cancer and arises from the malignant transformation of hepatocytes. HCC tumors often express genes critical for hepatocyte development but the regulatory mechanisms driving aberrant expression remain unclear. Recent evidence indicates that epigenetic alterations contribute to HCC progression with nearly 50% of cases associated with mutations in chromatin modifiers. Thus, it is imperative to understand how chromatin is altered in HCC and identify the DNA regulatory sequences associated with HCC tumorigenesis. Our study aims to map the chromatin landscape and transcriptome in HCC tumors using a multiomic approach at single cell resolution. Single-cell ATAC- and RNA-seq will be conducted in HCC tumors, matched controls and normal livers to identify regulatory sequences and their target genes critical for HCC tumor progression. We will characterize these regulatory sequences in the context of liver development using genetic manipulation and epigenomic datasets generated from human pluripotent stem cells differentiated to hepatic lineages. Our goal is to elucidate how epigenetic alterations in HCC drive cancer progression and how they relate to a dedifferentiated state. Citation Format: Miguel Ramirez, Tabea Stephan, Daivd Schaeffer, Pamela Hoodless. Defining chromatin alterations in liver cancer at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4412.