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: CD33 (clone PWS44) mouse mAb , CellMarque , Cat# 133M-14; RRID: AB_2861292.

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: CD33 (clone PWS44) mouse mAb , CellMarque , Cat# 133M-14; RRID: AB_2861292.

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

Journal: Cell Reports Medicine

Article Title: Identification of cells of leukemic stem cell origin with non-canonical regenerative properties

doi: 10.1016/j.xcrm.2024.101485

Figure Lengend Snippet: RECs demonstrate no stemness capacity and co-localize to CD34 + cells within leukemic tissue (A) Representative flow plot of hCD45 and CD33 expression in BM aspirates from PDXs 8 weeks post intra-femoral injection with RECs (n = 11, N = 3). (B) Bar graph of mean ± SD of CFU frequency (#colonies/cells seeded) of FACS-purified CD74 + /CD68 + cells and bulk AML patient MNCs normalized to average AML patient MNCs. (C) Bar graph of leukemic mutation VAF of FACS-purified CD74 + /CD68 + cells compared with control MNCs. (D) Whole H&E-stained tissue and representative images with and without spot overlays of BM tissue from AML patient 11 and BM donor 4. Scale bar, 100 μM. (E) Spots of CD74 + /CD68 + /CD34 + co-expression overlayed onto whole tissue section of AML BM11, and four representative images each from areas with and without CD74 + /CD68 + /CD34 + co-expression. Scale bars, 50 μM. (F) Xenograft BM sections of engrafted CB and AML, with hCD74 hCD68 hCD34 immunofluorescent labels by MIBI-TOF methodology. Examples of CD74 + /CD68 + cells and CD34 + are highlighted in white. (G) Bar graph of the mean +/- SD distance between CD74+/CD68+ and CD34 + cells in the AML vs. CB xenograft BM (∗∗∗∗p < 0.0001, Student’s t test). See also Figure S5 , Table S1 .

Article Snippet: PE mouse anti-human CD33 , BD Biosciences , Cat#347787; RRID: AB_400350.

Techniques: Expressing, Injection, Purification, Mutagenesis, Staining

Journal: Cell Reports Medicine

Article Title: Identification of cells of leukemic stem cell origin with non-canonical regenerative properties

doi: 10.1016/j.xcrm.2024.101485

Figure Lengend Snippet:

Article Snippet: PE mouse anti-human CD33 , BD Biosciences , Cat#347787; RRID: AB_400350.

Techniques: Recombinant, Selection, Software

Journal: bioRxiv

Article Title: Expansion of human hematopoietic stem cells by inhibiting translation

doi: 10.1101/2023.11.28.568925

Figure Lengend Snippet: A. Limiting dilution analysis (LDA) of human UCB CD34 + cells at day 0 or after 7 days of culture in CRCY+DMSO or CRCY with 4E1RCat (2 µM). Dose of cells injected is based on the number of CD34 + cells on day 0. Chimerism was measured as human CD45 + cells at 20 weeks in bone marrow with engraftment defined as ≥ 0.1% hCD45 + . Data are from 2 distinct LDA/transplant experiments. B. HSC frequency with 95% CI calculated with ELDA software. p = 0.001 for CRCY+4E1RCat compared to fresh cells. No significant difference for CRCY+DMSO compared to Day 0 cells. C-F. The percentage of human T cells (CD3 + cells), B cells (CD19 + cells), myeloid cells (CD33 + cells), and lineage negative (CD34 + CD38 - ) cells is shown for recipients injected with cells from ex vivo cultures corresponding to 5000, 500, 100, 25 CD34 + cells on day 0. G. Scheme for secondary transplantation from primary recipients that had received day 0 cells or cells cultured in CRCY+4E1RCat for 7 days. H. The percentage engraftment of human CD45 + cells in peripheral blood (16 weeks) in secondary recipients. I. The percentage engraftment of human CD45 + cells in the bone marrow (18 weeks) in secondary recipients. J-N. The percentage (log 10 scale) of human lineage negative cells (CD34 + CD38 - ), B cells (CD19 + ), myeloid cells (CD33 + ), megakaryocytes (CD41 + ) and erythroid cells (GlyA + ) in the bone marrow (18 weeks) of secondary recipients. Samples with 0% engraftment are shown at the baseline (with broken y-axes),

Article Snippet: After 18 weeks, bone marrow was collected and analyzed with PE anti-human CD45 antibody (CAT# 368510, Biolegend), APC/Cyanine7 anti-mouse CD45 antibody (CAT# 103116, Biolegend), APC anti-human CD3 antibody (CAT# 317318, Biolegend), BB515 Mouse anti-Human CD33 (CAT# 564588, BD Biosciences), BB700 mouse anti-Human CD19 (CAT# 566396, BD Biosciences), Brilliant Violet 421™ anti-human CD41 antibody (CAT# 303730, Biolegend), BUV395 Mouse anti-Human CD235a antibody (CAT# 563810, BD Biosciences), PE/Cyanine7 anti-human CD34 antibody (CAT# 343616, Biolegend), and Brilliant Violet 711™ anti-human CD38 antibody (CAT# 303528, Biolegend).

Techniques: Injection, Software, Ex Vivo, Transplantation Assay, Cell Culture

Journal: bioRxiv

Article Title: Expansion of human hematopoietic stem cells by inhibiting translation

doi: 10.1101/2023.11.28.568925

Figure Lengend Snippet: The percentage of human CD45 + cells, T cells (CD3 + ), B cells (CD19 + ), myeloid cells (CD33 + ), and CD34 + CD38 - cells in each primary recipient mouse at 20 weeks in bone marrow are represented by a heatmap. Red indicates percentage engraftment (human cells) is > 0.1%. Blue indicates human cell engraftment < 0.1%. White indicates percentage engraftment = 0.1%. Data show results from expansion/transplants using 2 distinct mixed donor UCB samples.

Article Snippet: After 18 weeks, bone marrow was collected and analyzed with PE anti-human CD45 antibody (CAT# 368510, Biolegend), APC/Cyanine7 anti-mouse CD45 antibody (CAT# 103116, Biolegend), APC anti-human CD3 antibody (CAT# 317318, Biolegend), BB515 Mouse anti-Human CD33 (CAT# 564588, BD Biosciences), BB700 mouse anti-Human CD19 (CAT# 566396, BD Biosciences), Brilliant Violet 421™ anti-human CD41 antibody (CAT# 303730, Biolegend), BUV395 Mouse anti-Human CD235a antibody (CAT# 563810, BD Biosciences), PE/Cyanine7 anti-human CD34 antibody (CAT# 343616, Biolegend), and Brilliant Violet 711™ anti-human CD38 antibody (CAT# 303528, Biolegend).

Techniques:

Journal: bioRxiv

Article Title: Expansion of human hematopoietic stem cells by inhibiting translation

doi: 10.1101/2023.11.28.568925

Figure Lengend Snippet: A-E. The percentage of human CD45 + cells, CD34 + CD38 - cells, myeloid cells (CD33 + ), T cells (CD3 + ), and B cells (CD19 + ) at day 29 post-transplant in bone marrow of mice used as donors for secondary transplant. Mice had received either uncultured (Day 0) CD34 + cells or cells cultured in CRCY+4E1RCat for 7 days. F. Table showing percent engraftment of each population in each mouse at 29 weeks.

Article Snippet: After 18 weeks, bone marrow was collected and analyzed with PE anti-human CD45 antibody (CAT# 368510, Biolegend), APC/Cyanine7 anti-mouse CD45 antibody (CAT# 103116, Biolegend), APC anti-human CD3 antibody (CAT# 317318, Biolegend), BB515 Mouse anti-Human CD33 (CAT# 564588, BD Biosciences), BB700 mouse anti-Human CD19 (CAT# 566396, BD Biosciences), Brilliant Violet 421™ anti-human CD41 antibody (CAT# 303730, Biolegend), BUV395 Mouse anti-Human CD235a antibody (CAT# 563810, BD Biosciences), PE/Cyanine7 anti-human CD34 antibody (CAT# 343616, Biolegend), and Brilliant Violet 711™ anti-human CD38 antibody (CAT# 303528, Biolegend).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Expansion of human hematopoietic stem cells by inhibiting translation

doi: 10.1101/2023.11.28.568925

Figure Lengend Snippet: Bone marrow harvested from primary recipients at 29 weeks was transplanted into conditioned secondary recipients as described in . The percentage of human CD45 + cells was measured in peripheral blood (PB) at 16 weeks. Bone marrow (BM) was harvested at 18 weeks and the percentage of human CD45 + cells T cells (CD3 + ), B cells (CD19 + ), myeloid cells (CD33 + ), megakaryocytic cells (CD41 + ), erythroid cells (GlyA + ) and CD34 + CD38 - cells was measured.

Article Snippet: After 18 weeks, bone marrow was collected and analyzed with PE anti-human CD45 antibody (CAT# 368510, Biolegend), APC/Cyanine7 anti-mouse CD45 antibody (CAT# 103116, Biolegend), APC anti-human CD3 antibody (CAT# 317318, Biolegend), BB515 Mouse anti-Human CD33 (CAT# 564588, BD Biosciences), BB700 mouse anti-Human CD19 (CAT# 566396, BD Biosciences), Brilliant Violet 421™ anti-human CD41 antibody (CAT# 303730, Biolegend), BUV395 Mouse anti-Human CD235a antibody (CAT# 563810, BD Biosciences), PE/Cyanine7 anti-human CD34 antibody (CAT# 343616, Biolegend), and Brilliant Violet 711™ anti-human CD38 antibody (CAT# 303528, Biolegend).

Techniques: