Journal: bioRxiv

Article Title: NicheSphere reveals Spp1⁺ macrophages as central hubs coordinating fibrotic remodeling in myeloproliferative neoplasms

doi: 10.64898/2026.03.16.711605

Figure Lengend Snippet: (A) Sankey plot showing top 30 differential LR interactions from OsteoCAR cells through ECM glycoproteins towards other co-localized cells. Colors represent LR expression per interaction: orange indicates stronger signaling in disease, blue indicates weaker signaling in disease. (B) Volcano plot for differential gene expression of OsteoCAR-Fibroblast multiplets cell. Genes with logFC > 1 and p-value < 0.05 shown in red; genes with p-value < 0.05 but low logFC in blue and others in green. (C) Representative images of Spp1 staining and quantification of mean DAB intensity in 4um femoral sections of TPO– or EV-induced murine BM fibrosis. Scale bar: 50um. P-value is based on an unpaired t-test.(*) = p<0.05. (D) Schematic representation of experimental timeline, where WT ckit+ donor cells are transduced with TPO-GFP or EV-GFP control lentiviral vectors, and transplanted into lethally-irradiated Spp1KO recipient mice (n=4-5/group). (E) Hemoglobin and red blood cell counts in TPO-driven fibrosis in WT and Spp1KO recipient mice at the time of sacrifice (82 days post-transplant). P-value is based on a one-way ANOVA.(*) = p<0.05,(**)= p<0.01, (***) = p<0.001, (****) = p<0.0001. (F) Blood monocytes quantified by flow cytometry in TPO-driven fibrosis in WT and Spp1KO recipient mice at the time of sacrifice (82 days post-transplant). (G) Spleen weight in TPO-driven fibrosis in WT and Spp1KO recipient mice at the time of sacrifice (82 days post-transplant). P-value is based on a one-way ANOVA.(*) = p<0.05,(**)= p<0.01, (***) = p<0.001, (****) = p<0.0001. (H) Image of spleens isolated from TPO-driven fibrosis in WT and Spp1KO recipient mice at sacrifice. (I) Bone marrow cellularity in TPO-driven fibrosis in WT and Spp1KO recipient mice at the time of sacrifice (82 days post-transplant). (J) Quantification of reticulin staining in 4um femoral sections in TPO-driven fibrosis in WT and Spp1KO recipient mice at the time of sacrifice (82 days post-transplant). (K) Representative imaging of reticulin staining in TPO-driven fibrosis in WT and Spp1KO recipient mice. Scale bar: 50um.

Article Snippet: Samples were diluted 1:50 and SPP1 concentration was quantified using the Human SPP1 Quantikine ELISA (R&D Systems, Human Osteopontin DOST00) according to the manufacturer’s instructions.

Techniques: Expressing, Gene Expression, Staining, Transduction, Control, Irradiation, Flow Cytometry, Isolation, Imaging

Journal: bioRxiv

Article Title: NicheSphere reveals Spp1⁺ macrophages as central hubs coordinating fibrotic remodeling in myeloproliferative neoplasms

doi: 10.64898/2026.03.16.711605

Figure Lengend Snippet: (A) Sankey plot showing top 25 LR pairs associated with Spp1+ macrophage-driven signaling via Spp1 as a ligand. (B) Heatmap of pathway score based on SPP1-associated intracellular signaling, highlighting differential transcriptional response between TPO vs. EV. Higher scores indicate stronger evidence of differential pathway regulation due to up-stream SPP1 signaling. (C) Sankey plot showing SPP1-associated intracellular signaling in Ly6C low infiltrating macrophages and resident-like macrophages, and pathways with enriched differential pathway score from panel B. Genes on the right represent genes downstream of SPP1. Here, only target genes that are ligands are shown. The color map reflects the log-transformed fold change (logFC) between TPO vs. EV. (D) Cytokines localized differential communication network showing cell types (nodes) colored by Niche with node size based on betweenness score. Edge colors represent normalized differential communication scores for cell type pairs. Red color in edges indicates increased signaling in disease, blue indicates decreased signaling in disease. (E) mRNA expression of Col1a1, αSMA and Spp1 in Gli1tdtomato MSCs stimulated with DMSO control, Tgf-β, IL-6, IL-1a, IL-1b and S100A8 for 48 hours, n=3/group, P-value is based on a one-way ANOVA.(*) = p<0.05,(**)= p<0.01, (***) = p<0.001, (****) = p<0.0001. (F) mRNA expression of Col1a1, Spp1 and IL-6 in Gli1tdtomato MSCs stimulated with DMSO control, Tgf-β and recombinant Spp1 for 48 hours, n=3/group, P-value is based on a one-way ANOVA.(*) = p<0.05,(**)= p<0.01, (***) = p<0.001, (****) = p<0.0001.

Article Snippet: Samples were diluted 1:50 and SPP1 concentration was quantified using the Human SPP1 Quantikine ELISA (R&D Systems, Human Osteopontin DOST00) according to the manufacturer’s instructions.

Techniques: Transformation Assay, Expressing, Control, Recombinant

Journal: bioRxiv

Article Title: NicheSphere reveals Spp1⁺ macrophages as central hubs coordinating fibrotic remodeling in myeloproliferative neoplasms

doi: 10.64898/2026.03.16.711605

Figure Lengend Snippet: A. Schematic representation of experimental timeline, where WT or Spp1KO ckit+ donor cells are transduced with TPO-GFP or EV-GFP control lentiviral vectors, and transplanted into lethally-irradiated WT recipient mice (n=5/group, experiment duration: 55 days after transplant). B. Hemoglobin and red blood cell counts in TPO-driven fibrosis in WT recipient mice with WT or Spp1KO BM at the time of sacrifice (55 days post-transplant). P-value is based on a one-way ANOVA.(*) = p<0.05,(**)= p<0.01, (***) = p<0.001, (****) = p<0.0001. C. Blood CD11b+ F4/80+ monocyte-derived macrophages quantified by flow cytometry in TPO-driven WT or Spp1KO fibrosis in WT recipient mice at the time of sacrifice (55 days post-transplant). D. Quantification of reticulin staining in 4um femoral sections in EV– or TPO-driven fibrosis in WT or Spp1KO-driven fibrosis at the time of sacrifice (55 days post-transplant). E. Representative imaging of reticulin staining in 4um femoral sections in TPO-driven WT or Spp1KO-driven fibrosis in WT recipient mice. Scale bar: 50um. F. ELISA analysis of IL-1b protein in plasma of WT or Spp1KO-driven TPO fibrosis. n=3-5/group. G. Representative imaging of IL-1b staining and DAB quantification in 4um femoral sections TPO-driven WT or Spp1KO-driven fibrosis in WT recipient mice. Scale bar: 50um. H. mRNA expression of Spp1, Tnfa and Il-6 in WT or Spp1KO FL-HoxB8 cells (undifferentiated) carrying either the EV or MPLW515L mutation. n=3/group, P-value is based on a one-way ANOVA.(*) = p<0.05,(**)= p<0.01, (***) = p<0.001, (****) = p<0.0001. I. Flow cytometry analysis of macrophage differentiation (CD11b+ F4/80+ cells) from FL-HoxB8 cells (WT vs. Spp1KO, MPLW515L vs. EV) over 5 days, followed by PE+ latex bead phagocytosis assay performed for 1 hour. n=3/group, P-value is based on a one-way ANOVA.(*) = p<0.05, (****) = p<0.0001.

Article Snippet: Samples were diluted 1:50 and SPP1 concentration was quantified using the Human SPP1 Quantikine ELISA (R&D Systems, Human Osteopontin DOST00) according to the manufacturer’s instructions.

Techniques: Transduction, Control, Irradiation, Derivative Assay, Flow Cytometry, Staining, Imaging, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Expressing, Mutagenesis, Phagocytosis Assay

Journal: bioRxiv

Article Title: NicheSphere reveals Spp1⁺ macrophages as central hubs coordinating fibrotic remodeling in myeloproliferative neoplasms

doi: 10.64898/2026.03.16.711605

Figure Lengend Snippet: (A) ELISA analysis of SPP1 protein in human blood plasma of MPN patients and healthy controls. n=6 healthy, 15 MPN. Statistical test performed is an unpaired t-test, p=0.2796. (B) Normalized SPP1 plasma protein abundance in MPN patients for distinct outcomes. Differences between groups were evaluated using a student t-test. (C) Kaplan–Meier survival curves of primary myelofibrosis patients stratified by quartiles of SPP1 concentration at baseline assessment. Differences between groups were evaluated using the log-rank test.

Article Snippet: Samples were diluted 1:50 and SPP1 concentration was quantified using the Human SPP1 Quantikine ELISA (R&D Systems, Human Osteopontin DOST00) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Quantitative Proteomics, Concentration Assay

Journal: bioRxiv

Article Title: NicheSphere reveals Spp1⁺ macrophages as central hubs coordinating fibrotic remodeling in myeloproliferative neoplasms

doi: 10.64898/2026.03.16.711605

Figure Lengend Snippet:

Article Snippet: Samples were diluted 1:50 and SPP1 concentration was quantified using the Human SPP1 Quantikine ELISA (R&D Systems, Human Osteopontin DOST00) according to the manufacturer’s instructions.

Techniques:

Journal: Nature Communications

Article Title: Perivascular mesenchymal cells instruct ST2+ reparative macrophages to promote endovascular injury-induced neointimal hyperplasia in mice

doi: 10.1038/s41467-026-68587-x

Figure Lengend Snippet: a The experimental design. BMDMs treated by IL-33 (20 ng/mL) for 24 h before additional 24 h fresh medium incubation to prepare CMs. b Representative images and quantification of EdU+ VSMCs co-cultured with the indicated CMs. Scale bar, 100 μm. c Analysis of the putative ligand-receptor communications between arterial macrophages and VSMCs based on the scRNA-seq data of D0 and D7 using CellChat. d Volcano plot depicting the upregulated and downregulated genes in macrophages at D0 and D7. e Violin plots showing Spp1 gene expression of the 7 immune cell populations based on scRNA-seq data. f (Left) Representative flow cytometry gating for quantifying OPN-expressing macrophages and neutrophils at D7. (Right) OPN+ cell percentages and OPN signal intensities in macrophages and neutrophils at D7. n = 6 mice per group. g Representative images of immunofluorescence staining of MAC2 and OPN using the artery sections from mice at D7. Scale bars, 50 μm. h mRNA levels of Spp1 in the artery from mice at D0 ( n = 4), D2 ( n = 8), D4 ( n = 8), D7 ( n = 8) and D14 (n = 4). i Plasma OPN protein levels in patients with or without in-stent restenosis (ISR, n = 25; Non-ISR, n = 25). j mRNA levels of Spp1 of the artery from Control ( n = 7) and Pb- Il33 KO ( n = 8) at D7. k mRNA levels of Spp1 in the artery from Control ( n = 10) and Mac- Il1rl1 KO ( n = 7) at D7. l mRNA levels of Spp1 in IL-33-treated (20 ng/mL) BMDMs for 24 h. m BMDMs electro-transfected with si Spp1 followed by IL-33 (20 ng/mL) treatment to prepare CMs. n mRNA levels of the cell proliferation-related genes in indicated CM-exposed VSMCs. o Representative H&E staining images and Intima/Media ratios and lumen diameter/aspect ratios in the artery sections at D28. Mice post-FAI were administrated with the hydrogel containing si Spp1 or si NC locally at the injured sites. n = 6 mice for each group. Scale bars, 50 μm. Data are all shown as the mean ± s.e.m. ** p < 0.01 or *** p < 0.001 by unpaired two-tailed Student’s t -test ( f , i , j , k , n , o ), one-way ( b , h ) or two-way ANOVA ( l ). Source data and statistic information are provided as Source data files. Figure 7a,c, m were created in BioRender. Shan, B. (2025) https://BioRender.com/x16x8wd .

Article Snippet: Plasma IL-33 and OPN protein levels were determined by a commercially available human IL33 ELISA Kit (#EK133, Multi sciences) and human OPN ELISA Kit (#EK1135, Multi Sciences) according to the manufacturer’s instructions.

Techniques: Incubation, Cell Culture, Gene Expression, Flow Cytometry, Expressing, Immunofluorescence, Staining, Clinical Proteomics, Control, Transfection, Two Tailed Test

Journal: Redox Biology

Article Title: Tumor-intrinsic redox programming drives an SPP1-CD44 axis of immune suppression in uveal melanoma

doi: 10.1016/j.redox.2026.104011

Figure Lengend Snippet: Intercellular communication networks between melanoma cells and T cells. (A, B) Number (A) and strength (B) of interactions among annotated tumor and T cell types. (C) Scatter plots illustrating the incoming and outgoing interaction strengths of the indicated cell types. (D) Heatmaps displaying representative outgoing (left) and incoming (right) signaling patterns among 14 cell types. (E) Hierarchical plot showing the inferred intracellular communication network of SPP1 signaling between annotated tumor and T cell types. (F) Violin plots comparing SPP1 expression level among five melanoma subtypes; ∗∗∗∗P < 0.0001. (G) Bar plot showing the major ligand-receptor pairs involved in SPP1 signaling. (H) Representative immunofluorescence images showing SPP1-CD44 ligand-receptor pairs in UM cohort (n = 7). HMB45 (green), SPP1 (yellow), CD8 (magenta), and CD44 (pink). Scale bar, 20 μm.

Article Snippet: A total of 5 × 10 5 cells were seeded into 6-well plates, and culture supernatants were collected after 48 h. The concentration of SPP1 in the supernatants was determined using a human SPP1 ELISA kit (Elabscience, E-EL-H1347) according to the manufacturer's instructions.

Techniques: Expressing, Immunofluorescence

Journal: Redox Biology

Article Title: Tumor-intrinsic redox programming drives an SPP1-CD44 axis of immune suppression in uveal melanoma

doi: 10.1016/j.redox.2026.104011

Figure Lengend Snippet: Redox-dependent SPP1 secretion drives immune suppression (A) ELISA measurement of SPP1 levels in conditioned medium from indicated UM cell lines. ∗P < 0.05, ∗∗∗∗P < 0.0001. (B) Immunoblot validation of SPP1 overexpression in UM cells. (C) ELISA analysis of secreted SPP1 following SPP1 overexpression. ∗∗∗∗P < 0.0001. (D) CCK-8 assay measuring proliferation of UM cells upon SPP1 overexpression. (E) Colony formation (top) and trans well assays (bottom) evaluating the clonogenic growth and invasive potential of UM cells upon SPP1 overexpression. (F) Schematic overview of the UM-CD8 + T cell coculture system. (G) Flow cytometry analysis of Ki-67 and IFN-γ expression levels in CD8 + T cells under different coculture conditions. ∗P < 0.05, ∗∗P < 0.01. (H) Proposed model of the SPP1-CD44 signaling axis in UM progression. (I) CCK-8 assay showing inhibitory effects of Mito-LND on MP46 cell viability. ∗∗P < 0.01. (J) OCR analysis assessing mitochondrial respiration in MP46 cells following Mito-LND treatment. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (K) ROS levels in UM cells after Mito-LND treatment. Scale bar, 200 μm. ∗P < 0.05. (L) Immunoblot analysis of SPP1 expression under oxidative stress induced by Tert -Butyl hydroperoxide solution (TBHP) or Mito-LND. (M) ELISA quantification of secreted SPP1 levels after Mito-LND treatment. ∗∗P < 0.01. (N) Immunoblot analysis of ER stress markers CHOP in UM cells treated with Mito-LND.

Article Snippet: A total of 5 × 10 5 cells were seeded into 6-well plates, and culture supernatants were collected after 48 h. The concentration of SPP1 in the supernatants was determined using a human SPP1 ELISA kit (Elabscience, E-EL-H1347) according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Biomarker Discovery, Over Expression, CCK-8 Assay, Flow Cytometry, Expressing