Mina LuQuing Xu

Metabolism is fundamental to cell function, yet its activities vary across tissue environments. Resolving these processes in situ at single-cell resolution is crucial for understanding physiology in health and disease. However, existing methods lack biochemical specificity or direct linkage to cell identity. Here we report a method, Raman Enhanced Delineation of Cell Atlases in Tissues (REDCAT), an all-optical platform integrating Raman scattering microscopy and high-plex immunofluorescence to co-map metabolism and cell types. REDCAT achieves subcellular profiling of protein, lipid, nuclear metabolites and redox metabolism in human tissues. In lymph nodes, it revealed cell-type-specific metabolic specialization. In lymphoma, REDCAT uncovered profound reprogramming and transitional states during tumor transformation. In the liver, it resolved zonation-dependent metabolic gradients. By linking cell identity to spatial metabolic states, REDCAT provides a framework for studying immunity and cancer, offering a path to deciphering the metabolic basis of disease.

Spatially resolved CRISPR screening in vivo has been limited to small perturbation panels and subsets of protein-coding RNAs. We present Perturb-DBiT, a method for co-sequencing of spatial total RNA whole transcriptomes and single guide RNAs (sgRNAs) on the same tissue section in situ. In a human cancer metastatic colonization model, we applied large (80,000+) sgRNA panels across tumor colonies in multiple consecutive tissue sections alongside their corresponding total RNA transcriptomes. We linked perturbations affecting long noncoding RNA covariation, microRNA–mRNA interactions and distinct amino acid-specific tRNA alterations to tumor migration and growth. By integrating transcriptional pseudotime trajectories, we further observed the impact of perturbations on clonal dynamics and cooperation. In an immune-competent syngeneic mouse model, investigation of the tumor immune microenvironment indicated distinct, synergistic effects on immune infiltration and suppression. Perturb-DBiT provides a spatially resolved comprehensive view of perturbation responses in complex tissues, including small and large RNA regulation, tumor proliferation, migration, metastasis and immune interactions.

While formalin-fixed paraffin-embedded (FFPE) samples are invaluable for human non-Hodgkin B-cell lymphoma translational research, effective methods for spatial profiling of chromatin accessibility and histone modifications in these tissues remain limited. Here, we introduce epi-Patho-DBiT, a platform that combines reverse crosslinking of FFPE tissues with spatially resolved assays for transposase-accessible chromatin using sequencing (spatial-FFPE-ATAC) or cleavage under targets and tagmentation (spatial-FFPE-CUT&Tag). Using spatial-FFPE-ATAC, we map epigenetic landscapes in mucosa-associated lymphoid tissue and follicular lymphoma, identifying chromatin variants linked to B-cell malignancy and resolving tumor karyotypes. Mitotic age inference reveals spatial tumor dynamics and uncovers cholesterol-mediated cell proliferation. Furthermore, spatial-FFPE-CUT&Tag elucidates genomic alterations during transformation of follicular lymphoma into diffuse large B-cell lymphoma and identifies DIP2C with dysregulated H3K4me3 and H3K27me3 levels. Unexpectedly, we observe elevated H3K27me3 occupancy at a chromosome 2 locus containing tumor-promoting genes, attributed to copy number amplification and thereby upregulation in transformed diffuse large B-cell lymphoma.

Spatially mapping the transcriptome and proteome in the same tissue section can profoundly advance our understanding of cellular heterogeneity and function. Here we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multimodal spatial omics approach combining sequencing-based spatial transcriptomics and multiplexed protein imaging on the same section, enabling both single-cell-resolution cell typing and transcriptome-wide interrogation of biological pathways. DBiTplus utilizes spatial barcoding and RNase H-mediated cDNA retrieval, preserving tissue architecture for multiplexed protein imaging. We developed computational pipelines to integrate these modalities, allowing imaging-guided deconvolution to generate single-cell-resolved spatial transcriptome atlases. We demonstrate DBiTplus across diverse samples including frozen mouse embryos, and formalin-fixed paraffin-embedded human lymph nodes and lymphoma tissues, highlighting its compatibility with challenging clinical specimens. DBiTplus uncovered mechanisms of lymphomagenesis, progression and transformation in human lymphomas. Thus, DBiTplus is a unified workflow for spatially resolved single-cell atlasing and unbiased exploration of biological mechanisms in a cell-by-cell manner at transcriptome scale.

Histopathology and molecular pathology are currently distinct diagnostic modalities for the most part, one revealing tissue morphology at cellular resolution and the other providing molecular measurements with limited or no spatial context. Projecting genome-scale molecular information onto histopathology images at single-cell resolution across whole tissue sections represents a long-sought goal for next-generation pathology. Here we present SuperFocus, a modality-agnostic computational platform that generates histopathology-integrated single-cell spatial multi-omics from spot-based spatial measurements acquired on the same or an adjacent section without requiring external reference data. SuperFocus combines constrained cascading imputation with feature-level and cell-level quality-control scores to reduce spurious predictions and quantify confidence. On a ground-truth spatial transcriptomics benchmark dataset, SuperFocus improves key accuracy metrics by 28-73% over existing methods. Across Patho-DBiT, spatial ATAC-RNA, spatial CITE-seq and Visium-MALDI-MSI (SMA) datasets, SuperFocus enables cell-resolved analyses of MALT lymphoma microenvironments, gene regulatory programs in human hippocampus, lipotoxic hepatocyte states in human MASH, and transcriptomic-metabolomic states linked to neurotransmission and neuroinflammation in Parkinsonian mouse brain. Overall, SuperFocus enables scalable whole-slide single-cell spatial multi-omics integrated with histopathology, bridging histology and genome-scale molecular profiling for next-generation molecular pathology.

Chronic myelomonocytic leukemia (CMML) is associated with high rates of transformation to acute myeloid leukemia (AML) and poor overall survival. Plasmacytoid dendritic cell (pDC) proliferations in bone marrow biopsies have been proposed to be prognostically significant in CMML. In this multi-institutional study, we quantified pDC clusters in bone marrow biopsies of patients with CMML by interferon regulatory factor 8 (IRF8) immunohistochemical staining performed in separate laboratories and demonstrated the association of pDC clusters with overall survival. Two hundred five newly diagnosed CMML cases were scored for the presence of prominent pDC clusters, which were detected in 37 cases (18%). The median overall survival (OS) was 15.1 months for patients with pDC clusters versus 30.1 months for those without pDC clusters (p = 0.021 log-rank test, Kaplan–Meier analysis). We propose using IRF8 immunohistochemistry in CMML bone marrow evaluation.

Primary effusion lymphoma (PEL) is a rare B‐cell lymphoma that is most often seen in immunocompromised patients with human herpesvirus‐8 (HHV‐8) and Epstein–Barr virus (EBV) infections. PEL typically affects body cavities with occasional involvement of other organs (extracavitary PEL). We present an unusual case of primary cutaneous extracavitary PEL without body cavity effusions. A 56‐year‐old man with uncontrolled HIV (CD4 count 50, viral load 264 copies/mL) presented with a 2‐month history of fever, cough, and progressive dyspnea. Physical examination revealed bilateral inner thigh erythema and induration. Laboratory evaluation showed an elevated EBV viral load. Imaging studies did not reveal body cavity effusions. Skin biopsy from the thigh demonstrated a deep dermal and subcutaneous infiltrate of large pleomorphic lymphoid cells. The atypical cells were CD45+, CD138+, CD3+, HHV8+, EBV in situ hybridization (ISH)+, CD20‐, CD79a‐ and PAX5 negative. Our case highlights a rare primary cutaneous manifestation of extracavitary PEL. It is important for dermatopathologists to be aware of the histopathological features of extracavitary PEL and its unusual immunophenotype (CD138+, negative for B‐cell markers with aberrant expression of T‐cell markers), which could represent a potential diagnostic pitfall. HHV‐8 and EBER ISH positivity is crucial for the diagnosis of cutaneous extracavitary PEL.

Background: Effusion-based lymphoma (EBL) is a rare and aggressive large B-cell lymphoma. It presents as a body cavity effusion without a solid mass, lacks HHV-8 association, and typically expresses CD20. Objectives: To better understand the biology of this entity, we performed transcriptomic profiling of eight EBL cases. Methods: We analyzed the cases with the NanoString PanCancer Immune Profiling Panel and compared the results with publicly available datasets representing follicular lymphoma (FL), mantle cell lymphoma (MCL), and large B-cell lymphoma (LBCL) subtypes. Results: Unsupervised clustering and differential expression analysis revealed that EBL cases cluster transcriptionally with the LBCL group. Lymphoma-specific signaling pathway enrichment (SignatureDB) predominantly identified non-germinal center (activated B-cell-type) pathways. In addition, KEGG pathway analyses revealed enrichment in specific inflammatory and immune response pathways that are associated with B-cell lymphoma development in the setting of chronic inflammation, including those linked to Toll-like receptor and NF-κB signaling. Conclusions: These findings support a post-germinal center origin for EBL, which arises in a background of chronic inflammation and persistent antigen stimulation.