Journal: Genome Biology

Article Title: STHD: probabilistic cell typing of single spots in whole transcriptome spatial data with high definition

doi: 10.1186/s13059-025-03608-4

Figure Lengend Snippet: Spot-level predictions and benchmarking of a zoomed patch in a colon cancer VisiumHD sample. a Example of STHD results of a patch from the human colon cancer VisiumHD sample, revealing tumor-like epithelial cells and intercryptic immune population, visualized using the interactive STHDviewer. b Cell type posterior probabilities at spot level for the same patch, visualized using Squidpy. Top row, three epithelial cell types; Bottom row, two immune cell types. c A spatial plot of unsupervised Leiden clustering on spot gene expression. d Spatial plots of unsupervised Leiden clustering of gene expression aggregated by bins in size of 4 × 4 spots. Left, clustering bins within patch, right, global cluster group of bins. e Cell type proportion decomposed by RCTD on bins of size of 4 × 4 spots, demonstrating two tumor epithelial classes, enterocytes, and stem-like cells. f The H&E histopathology image in full resolution of the same crop. g Comparison to other cell typing and clustering methods adaptive to high-resolution spots. h The STHD predicted labels at spot-level and STHD-guided binning in bins of 4 × 4 spots and 8 × 8 spots, visualized using Squidpy using the same colon cancer cell type colormap. i Benchmarking with simulated high-resolution spatial data. Left, ground truth cell type labels; middle, STHD predicted cell type labels; right, receiver operating characteristic curve (ROC) curve for all cell types. j Benchmarking against other methods for spot annotation in repeated spatial transcriptomics simulations. k Expression dot plots for marker genes of normal epithelial cells, at spot level or STHD-guided bin level. Left to right: normal epithelial cell types in reference human colon cancer sample, normal epithelial cell types based on STHD predicted spots, normal epithelial cell types from STHD-guided bins of size 4 × 4 spots, normal epithelial cell types in STHD-guided bins of size 8 × 8 spots. The same cell type-specific gene markers from the original colon cancer study were used. ROC, Receiver operating characteristic. AUC, area under the curve

Article Snippet: Colon cancer VisiumHD data (patient P1, P3, P5) from Oliveira et al. [ ] include two colon cancer and one normal colon, available at https://www.10xgenomics.com/products/visium-hd-spatial-gene-expression/dataset-human-crc [ ].

Techniques: Gene Expression, Histopathology, Comparison, Expressing, Marker

Journal: Genome Biology

Article Title: STHD: probabilistic cell typing of single spots in whole transcriptome spatial data with high definition

doi: 10.1186/s13059-025-03608-4

Figure Lengend Snippet: STHD prediction facilitates segmentation of global structures and cell type-specific differential analyses across spatial regions. a Interactive spatial visualization by STHDviewer of predicted cell type labels for spots in the entire human colon cancer VisiumHD sample. b Annotation of regions of interest, separating epithelial regions and two T cell-infiltrated regions. c Cell type proportion bar plots at cell lineage and cell type level for epithelial regions and whole map. Upper: Proportions of major cell lineages including epithelial cells, fibroblasts, B cells, myeloid cells, and T cells. Bottom: Proportions of finer cell types of epithelial lineage. d Spot-level gene expression for epithelial-specific differential genes across regions. Top three genes by log fold change were shown. e Pathway enrichment of epithelial cell-specific genes differentially expressed in the Epi-Tumor-top region. f Macrophage-specific differential genes for the Epi-Tumor-inside region, where differential analyses were performed at bin level of size 4 × 4 spots with cell type stratification from STHD. DE, differential expression

Article Snippet: Colon cancer VisiumHD data (patient P1, P3, P5) from Oliveira et al. [ ] include two colon cancer and one normal colon, available at https://www.10xgenomics.com/products/visium-hd-spatial-gene-expression/dataset-human-crc [ ].

Techniques: Gene Expression, Quantitative Proteomics

Journal: Genome Biology

Article Title: STHD: probabilistic cell typing of single spots in whole transcriptome spatial data with high definition

doi: 10.1186/s13059-025-03608-4

Figure Lengend Snippet: STHD is scalable to other samples, revealing subcellular cell type identities and tissue structures. a STHD spot-level inference of a human colon cancer Visium HD sample showing tumor, colon tissue, and enriched lymphoid structure. b Left, spots (2 × 2 µm) labeled as germinal center B cells, with B cell marker expression aggregated per 8 × 8 µm bins. Right, spots (2 × 2 µm) labeled as T cells, and expression of TLS-specific T cell cytokine CXCL13 by 8 × 8 µm bins. Marker expression plots are illustrated by loupe browser. c Spot-level inference of human colon VisiumHD sample P5 (cancer) and P3 (normal adjacent tissue). d Immune cell-specific composition across all four human colon Visium HD samples. e STHD results for the mouse small intestine VisiumHD sample, with two zoomed-in regions highlighted. f Spots with immune cell identities in the lymph node-like region, showing T cell, Ms4a1 + B cell and Ighd + B cells. Top, STHD labels in 2 × 2 µm spots, Bottom, marker gene expression in 8 × 8 µm bins. g Spots of intestine cells forming layered structure, including intestine enterocyte cell, epithelial cell, goblet cell, Paneth cell, and fibroblasts. Top, STHD labels in 2 × 2 µm spots, Bottom, marker gene expression in 8 × 8 µm bins. GC, germinal center. CRC, colorectal cancer. NAT, normal adjacent tissue

Article Snippet: Colon cancer VisiumHD data (patient P1, P3, P5) from Oliveira et al. [ ] include two colon cancer and one normal colon, available at https://www.10xgenomics.com/products/visium-hd-spatial-gene-expression/dataset-human-crc [ ].

Techniques: Labeling, Marker, Expressing, Gene Expression

Journal: iScience

Article Title: Integrative spatial multi-omics reveal niche-specific inflammatory signaling and differentiation hierarchies in AML

doi: 10.1016/j.isci.2025.114289

Figure Lengend Snippet: Comparative spatial multi-omics analysis of acute myeloid leukemia patients’ bone marrow and extramedullary tissues (A) Schematic representation of the study workflow. Paired bone marrow (BM) samples (BM1 and BM2) and extramedullary (EM) samples (EM1, from skin; and EM2, from lymph node) from 2 newly diagnosed patients with acute myeloid leukemia (AML) (PT1 and PT2) were fixed in formalin and embedded in paraffin (FFPE) and then sectioned for use in Visium assays (v1 and v2), and Opal multiplex fluorescent immunohistochemistry (mfIHC). The Visium spatial transcriptomics (ST) results were validated using GeoMx digital spatial profiling (DSP) with tissue microarrays (TMAs) of samples from 3 newly diagnosed patients with AML (PT3, PT4, and PT5). An additional 4 AML bone marrow samples that performed the Visium gene and protein expression assay are used as a validational cohort (PT6, PT7, PT8, and PT9). Image created with BioRender ( https://biorender.com ). (B) Uniform manifold approximation and projection (UMAP) plot showing our reference map consisted of 79,029 cells collected from 9 healthy BM donors and 7 patients with AML with diploid cytogenetics to match the patient cytogenetic profiles, and included both newly generated scRNA data and previous works. This map consisted of 21 cell types, including T cells (CD4 + and CD8 + naive, effector, and memory T cells, T regulatory [Treg] cells, and unconventional T cells), other immune cells (Natural killer [NK] cells, B cells and plasma cells), hematopoietic progenitors (Hematopoietic stem cells [HSCs], common lymphoid progenitors [CLPs], granulocyte-monocyte progenitors [GMPs]), myeloid cells (megakaryocytes/platelets, monocytes, early and late erythroid cells, conventional and plasmacytoid dendritic cells) and leukemic (AML) cell populations. (C) Immunohistochemical staining of CD11c, MPO, and CD3e on BM1 sections that were used for histopathological annotation. The scale bar for the main tissue panels represents 1 mm. The scale bar for the zoomed-in panels, corresponding to the boxed regions, represents 100 μm. (D) Unsupervised clustering and pathology annotation for the projected spatial map of BM1, revealing 3 distinct regions with an adjusted rand index (ARI) of 0.46. (E) Spatial deconvolution of BM1 tissue, showing erythroid and AML cell populations, with CD11c immunohistochemistry (IHC) overlaid on an image of hematoxylin and eosin (H&E)-staining. The dotted red lines represent regions enriched for the erythroid cell population; dotted black lines, regions enriched for the AML cell population; and solid lines, regions that overlapped with other tissue sections. (F) Heatmap of Z score normalized canonical markers in pathology annotations, with matching unsupervised cluster distributions represented as a pie chart. HBB, HBD, HBA2, GATA1/2 are erythroid genes and S100A12, FCGR3A, CD14, MS4A7, and , CD33 are monocyte/leukemic genes. (G) Representative overlay of Visium H&E staining with Opal mfIHC and the generated spot-level data for CD33, CD71, CXCL12, CXCR4, CD68, and IL-6. Boxes illustrate magnified regions showing concordance between transcript-level (Visium) and protein-level (Opal) signals at the spot level. (H) Phenotype staining on near-adjacent tissue sections for markers of leukemic (CD33), monocytic (CD68), and erythroid (CD71) populations. DAPI was used as a nuclear counterstain. The spatial distribution of these markers corroborates ST-based spot deconvolution. Scale bars: 1 mm (whole-slide panels) and 100 μm (selected region panels). (I) Box and spatial plots of mfIHC staining intensities for phenotypic markers across ST-defined clusters in BM1, highlighting the enrichment of leukemic and monocytic populations in cluster 3 and that of erythroid populations in cluster 2 at BM1. Scale bars: 1 mm (whole-slide panels) and 100 μm (selected region panels). ns, not significant. ∗∗∗∗ p < 0.0001, Wilcoxon rank-sum test.

Article Snippet: Visium Spatial Transcriptomics Data , This paper , GEO: GSE279576.

Techniques: Biomarker Discovery, Multiplex Assay, Immunohistochemistry, Expressing, Generated, Clinical Proteomics, Immunohistochemical staining, Staining

Journal: iScience

Article Title: Integrative spatial multi-omics reveal niche-specific inflammatory signaling and differentiation hierarchies in AML

doi: 10.1016/j.isci.2025.114289

Figure Lengend Snippet: Spatial multi-omics profiling identifies leukemic infiltration and tissue composition in extramedullary acute myeloid leukemia samples (A) Unsupervised clustering of the extramedullary sample EM1 into 3 spatial clusters (left) compared against the pathology-based annotation (right; indicating a composition of leukemia, dermis, epidermis, and gland). The adjusted rand index (ARI; 0.51) reflects moderate agreement between the clusters and pathology annotations. (B) Spatial deconvolution scores obtained using the SpaCET algorithm show EM1’s malignant cell distribution overlaid on the hematoxylin and eosin (H&E) image. (C) Heatmap of canonical marker expression in EM1 regions, validating transcriptional segregation and matching pathologist-defined regions. Markers of leukemic populations and dermis regions show shared expression profiles. Unsupervised cluster overlap is represented as pie charts, with pathology annotation. (D) Phenotypic staining (Opal multiplex immunofluorescent) on near-adjacent sections validating the spatial distribution of CD33 (malignant cells), CD68, and CD71, which is consistent with the Visium malignant signature (spot-level) and CD68 and CD71 expression patterns. Scale bars: 1 mm (whole-slide panels) and 100 μm (selected region panels).

Article Snippet: Visium Spatial Transcriptomics Data , This paper , GEO: GSE279576.

Techniques: Biomarker Discovery, Marker, Expressing, Staining, Multiplex Assay

Journal: iScience

Article Title: Integrative spatial multi-omics reveal niche-specific inflammatory signaling and differentiation hierarchies in AML

doi: 10.1016/j.isci.2025.114289

Figure Lengend Snippet: Inflammatory microenvironment analysis reveals region-specific signatures in bone marrow and extramedullary tissues from patients with acute myeloid leukemia (A) Distribution of spatial inflammation classes in BM1 and EM1, based on composite inflammation scores from inflammation-related hallmark pathways (Inflammatory response, IL6/JAK/STAT3 signaling, TNF-α/NF-κB signaling, IFN-γ response, IFN-α response, Complement, IL2/STAT5 signaling). Classes were defined using Jenks' natural breaks optimization. (B) Mean activity comparison of individual inflammatory related pathways in spots with high-inflammatory activity revealed the highest activity of IFN-γ response in EM tissue. Complement pathway activity is higher in BM1 when compared with the EM1 inflammatory niche. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, Wilcoxon rank-sum test. (C) Boxplots of inflammation scores across the 3 clusters in BM1 (left) and EM1 (right). Each cluster displays significantly different levels of inflammatory activity; leukemia-enriched cluster 3 in BM1 and cluster 1 in EM1 have higher inflammation scores. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, Wilcoxon rank-sum test. (D) IL-6 staining (Opal multiplex fluorescent immunohistochemistry [mfIHC]) in whole-slide images (left) of BM1 (top) and EM1 (bottom) and corresponding magnified regions (center), aligned with Visium spot-level composite inflammation score (right). Scale bars: 1 mm (whole-slide panels) and 100 μm (selected region panels). (E) Scatterplots showing the correlation of IL-6 protein staining intensity (mfIHC IL-6) with the composite inflammation score in BM1 (left) and EM1 (right). IL-6 levels are higher in high-inflammation regions in both BM1 and EM1. (F) Dot plot showing the localization of T cell subtypes (exhausted, CD8 + dysfunction, senescence, regulatory T cells [Treg]) based on inflammation class in BM1 and EM1.

Article Snippet: Visium Spatial Transcriptomics Data , This paper , GEO: GSE279576.

Techniques: Activity Assay, Comparison, Staining, Multiplex Assay, Immunohistochemistry

Journal: iScience

Article Title: Integrative spatial multi-omics reveal niche-specific inflammatory signaling and differentiation hierarchies in AML

doi: 10.1016/j.isci.2025.114289

Figure Lengend Snippet: Chemokine signaling through the CXCL12-CXCR4 axis is linked to inflammatory niches and trans-differentiation in acute myeloid leukemia (A) Spatial and chord diagrams of the strength of interactions among acute myeloid leukemia (AML) cells, granulocyte-monocyte progenitors (GMP), and monocytes through the CXCL12-CXCR4 axis, as predicted by CellChat. (B) Boxplots of the expression levels of CXCL12 and CXCR4 in BM1, stratified by inflammation class (top), and corresponding spot-level expression maps (bottom) for the bone marrow sample BM1. Red spots indicate higher expression levels. (C and D) Whole-slide images of Opal multiplex fluorescent immunohistochemistry (mfIHC; left) for CXCR4 (turquoise) and CXCL12 (magenta) overlaid with DAPI (blue), alongside magnified Opal regions and Visium-based gene expression maps (right) in BM1 (C) and the extramedullary sample EM1 (D). Scale bars: 1 mm (whole-slide panels) and 100 μm (selected region panels). (E) Scatterplot shows the positive correlation of the PI3K/Akt/mTOR pathway score with the combined CXCL12-CXCR4 co-expression score (R = 0.50, p < 2.2e-16). Colors denote inflammation class. (F) Relationship between CXCR4 expression and inflammation score in EM1 (R = 0.19, p < 2.2e-16). Spatial maps show the distribution of CXCR4 expression. (G) Boxplots comparing CXCR4 protein signal intensity (mfIHC) across inflammation classes in BM1 (left) and EM1 (right). Spot-level images illustrate higher CXCR4 signal intensities in high-inflammation areas. (H) Sections 1 and 2 represent adjacent serial sections of the same EM1 biopsy embedded on a single Visium capture area. Visium ST visualization of PI3K/Akt/mTOR pathway (left) and trans -differentiation pathway (right) activity in these EM1 sections, revealing elevated pathway scores in high-inflammation and leukemic regions.

Article Snippet: Visium Spatial Transcriptomics Data , This paper , GEO: GSE279576.

Techniques: Expressing, Multiplex Assay, Immunohistochemistry, Gene Expression, Activity Assay

Journal: iScience

Article Title: Integrative spatial multi-omics reveal niche-specific inflammatory signaling and differentiation hierarchies in AML

doi: 10.1016/j.isci.2025.114289

Figure Lengend Snippet: Bone proximity analysis reveals the spatial distribution of acute myeloid leukemia cells in different differentiation states (A) Representative spatial map of SpatialTime calculated distances from trabeculae overlaid with hematoxylin and eosin (H&E) image. (B) Boxplots show deconvolution scores of primitive-like, granulocyte-monocyte progenitor (GMP)-like, and committed-like acute myeloid leukemia (AML) cells relative to their distance from bone in Visium data. ∗ p < 0.05, ∗∗∗∗ p < 0.0001, Wilcoxon rank-sum test. (C) GeoMx analysis of AML deconvolution in bone marrow regions from 3 patients with AML (PT3, PT4, PT5). D, distal (dark red); P, proximal (dark blue); B, bone (white). Stacked bar plots represent cell type deconvolution within distal and proximal regions. Scale bars: 250 μm. (D) Line graphs show proportions of primitive-like and GMP-like cells relative to distance from bone.

Article Snippet: Visium Spatial Transcriptomics Data , This paper , GEO: GSE279576.

Techniques: