Journal: STAR Protocols

Article Title: Protocol for isolating stromal cells from lymphoid tissue for performing scRNA-seq

doi: 10.1016/j.xpro.2026.104501

Figure Lengend Snippet: Composition of cells recovered after each digestion step Collected cells were analyzed after each digestion step to determine cell phenotypes, numbers and viability. When performing transcriptomics analysis all digestion fractions will be combined. (A) Percentages of immune cells (CD45 + cells) and stromal cells (CD45 - cells) extracted from each digestion step analyzed via flow cytometry. (B) Representative viability images of each digestion step acquired on automated cell counter (LUNA 7-FX™) where red indicates dead cells and green live cells. (C) Immunofluorescence images of cells stained after each digestion step for CD45 (yellow) and Nuclei (blue). Representative Cytospin slides. Red arrows showing examples of CD45 - cells. Scale bars indicate 100 μm for main images and 10 μm for zoom. (D) After each digestion step the (i) percentage of live cells, (ii) number of live cells after each digestion measured by automated cell counting and (iii) percentage of immune (gray) and stromal cells (red) after each digestion fraction. (E) Combined cells from all digestions showing percentage and number of cells from each lymph node. (F) Representative gating strategy for lymph node cells showing stromal cell percentages. (G–L) Number of (G) immune cells, (H) stromal cells, (I) fibroblastic reticular cells (FRC), (J) lymphatic endothelial cells (LEC), (K) blood endothelial cells (BEC) and (L) double negative cells (DNC).

Article Snippet: CD45 antibody, anti-mouse, Biotin (Dilutions in 1:50) , Miltenyi Biotec , Cat# 130-124-209, RRID: AB_2819580.

Techniques: Transcriptomics, Flow Cytometry, Immunofluorescence, Staining, Cell Counting

Journal: STAR Protocols

Article Title: Protocol for isolating stromal cells from lymphoid tissue for performing scRNA-seq

doi: 10.1016/j.xpro.2026.104501

Figure Lengend Snippet: Cell selection using automated magnetic cell sorting (A) Cells were stained with CD45-biotin and CD31-biotin and sorted using autoMACS® Pro Separator. (B) Number of cells before staining for autoMACS® separation (step 11), and number of cells recovered from positive selection (CD45 + and CD31 + cells) and negative selection (CD45 - and CD31 - cells) in step 23. Each dot represents combined numbers from inguinal, axillary and brachial lymph nodes from 6 mice. Colors indicate biological replicates. (C) Percentages of cells after separation compared to the pre-staining cell count performed in step 11. (D) Purity check of separated cells using flow cytometry and staining for CD45 and CD31. (E) Overlay plots of positive selection (blue) and negative selection (red). (F) Percentage of CD45 + , CD45 - and CD31 + cells recovered (∗∗∗∗ p value < 0.0001, ∗ p value < 0.05). (G) Final viability check of positive and negative selected cells acquired just before performing scRNA-sequencing analysis.

Article Snippet: CD45 antibody, anti-mouse, Biotin (Dilutions in 1:50) , Miltenyi Biotec , Cat# 130-124-209, RRID: AB_2819580.

Techniques: Selection, FACS, Staining, Cell Characterization, Flow Cytometry, Sequencing

Journal: Molecular Therapy Oncology

Article Title: Oncolytic virotherapy mobilizes tumor-resident, granzyme B-producing bystander CD4 + T cells to inhibit systemic microbial infection

doi: 10.1016/j.omton.2026.201187

Figure Lengend Snippet: OV-BYTE strategy boosts systemic pathogen-specific CD4 + T MEM cell responses (A) Schematic of the experimental design. Congenic CD45.1 + SM CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LCMV Armstrong and engrafted with MC38 cells on day 60 post infection. On days 7–12 after tumor engraftment, recipients were administered PBS, NDV-WT, or NDV-GP daily via the intratumoral route. On day 15 post-tumor engraftment, SM CD4 + T cells in various organs were analyzed. (B and C) Frequency (B) and number (C) of SM CD4 + cells from the indicated organs of groups administrated PBS (indicated by gray dots), NDV-WT (indicated by blue dots), or NDV-GP (indicated by red dots). Quantification of SM CD4 + cells from 100 μL of peripheral blood. (D) Flow cytometry analysis of SM CD4 + T cells on day 15 post-tumor engraftment from the PBS-, NDV-WT-, or NDV-GP-treated group. Numbers adjacent to the outlined areas indicates percentages of SM CD4 + cells among total CD4 + T cells. (E) Schematic of the experimental design. Naive C57BL/6 mice were infected with LCMV Armstrong and engrafted with MC38 cells on day 60 post infection. On days 7–12 after tumor engraftment, recipients were administered PBS, NDV-WT, or NDV-GP daily via the intratumoral route. On day 15 post-tumor engraftment, endogenous LCMV GP 66-77 tetramer + CD4 + T cells in various organs were analyzed. (F and G) Frequency (F) and number (G) of endogenous LCMV GP 66-77 tetramer + CD4 + T cells from the indicated organs of groups administrated PBS (indicated by gray dots), NDV-WT (indicated by blue dots), or NDV-GP (indicated by red dots). (H) Schematic of the experimental design. Congenic CD45.1 + OT-II CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LM-OVA and engrafted with MC38 cells on day 60 post infection. On days 7–12 after tumor engraftment, recipients were administered PBS, Ad5-WT, or Ad5-OVA daily via the intratumoral route. On day 15 post-tumor engraftment, OT-II CD4 + T cells in various organs were analyzed. (I and J) Frequency (I) and number (J) of OT-II CD4 + cells in the indicated organs of groups administrated PBS (indicated by gray dots), Ad5-WT (indicated by blue dots), or Ad5-OVA (indicated by red dots). All data are representative of at least two independent experiments with at least four mice per group. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by one-way ANOVA with Turkey’s test (B, C, F, G, I, J). Center values and error bars (B, C, F, G, I, J) indicate mean and SEM.

Article Snippet: C57BL/6, OT-II, and CD45.1 + (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from Jackson Laboratories.

Techniques: Infection, Flow Cytometry

Journal: Molecular Therapy Oncology

Article Title: Oncolytic virotherapy mobilizes tumor-resident, granzyme B-producing bystander CD4 + T cells to inhibit systemic microbial infection

doi: 10.1016/j.omton.2026.201187

Figure Lengend Snippet: OV-BYTE-educated systemic TCF-1 lo CD39 hi CD4 + T cells are imprinted with enhanced effector functionality (A) Venn diagram showing overlap of up-regulated genes in NDV-GP-educated SM CD4 + T cells across different organs. (B) GSEA results showing the normalized enrichment scores (NES) for selected biological process gene sets based on the overlap of 299 DEGs across samples in (A). (C) Heatmap showing selected genes in tumor, spleen, and lung from the indicated groups. (D) Flow cytometry analysis of Ki67 + cells and IFN-γ + TNF-ɑ + cells in indicated SM CD4 + T cells from spleens of indicated groups on day 15 post-tumor engraftment. Numbers adjacent to the outlined areas indicate percentages of Ki67 + cells and IFN-γ + TNF-ɑ + cells among the indicated SM CD4 + T cells. (E and F) Frequency of Ki67 + cells (E) and IFN-γ + TNF-ɑ + cells (F) in PBS-treated TCF1 hi CD39 lo (indicated by gray dots), NDV-WT-treated TCF1 hi CD39 lo (indicated by blue dots), NDV-GP-treated TCF1 hi CD39 lo (indicated by red dots), or NDV-GP-treated TCF1 lo CD39 hi (indicated by purple dots) SM CD4 + T cells from spleens. (G) Flow cytometry analysis of GzmB expression in indicated SM CD4 + T cell populations in spleens. Percentages of GzmB + cells in the indicated SM CD4 + T populations are shown at the top of the flow charts. (H) Frequency of GzmB + cells in PBS-treated TCF1 hi CD39 lo (indicated by gray dots), NDV-WT-treated TCF1 hi CD39 lo (indicated by blue dots), NDV-GP-treated TCF1 hi CD39 lo (indicated by red dots), and NDV-GP-treated TCF1 lo CD39 hi (indicated by purple dots) SM CD4 + T cells in spleens. (I) Schematic of the experimental design. Naive C57BL/6 mice and SM CD4 + T cell chimera C57BL/6 mice were infected with LCMV Armstrong and engrafted with MC38 cells on day 60 post infection. On day 10 after tumor engraftment, splenic Ly108 hi CD39 lo or Ly108 lo CD39 hi SM cells from SM CD4 + T cells chimera recipients were isolated, CTV labeled, and transferred into the other LCMV Armstrong memory cohort via the intravenous route. On days 11–13 after tumor engraftment, recipients were administered NDV-GP daily via the intratumoral route. On day 15 post-tumor engraftment, transferred CTV-labelled SM CD4 + T cells in the spleen were analyzed. (J) Flow cytometry analysis of proliferating cells in the indicated SM CD4 + T cell populations in spleens. Numbers adjacent to the outlined areas indicate percentages of proliferating cells among the indicated SM CD4 + T cells. (K) Frequency of proliferating cells among transferred NDV-GP-treated TCF-1 hi CD39 lo (indicated by red dots) and TCF-1 lo CD39 hi (indicated by purple dots) SM CD4 + T cells from spleens. (L) Schematic of the experimental design. Congenic CD45.1 + SM CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LCMV Armstrong and engrafted with MC38 cells on day 60 post infection. On days 7–12 after tumor engraftment, recipients were administered NDV-GP daily via the intratumoral route. On day 15 post-tumor engraftment, Ly108 hi CD39 lo or Ly108 lo CD39 hi SM cells in spleens were sorted, and naive SM cells were isolated as a control. Then, Ly108 hi CD39 lo , Ly108 lo CD39 hi , or naive SM cells were in vitro co-cultured with violet hi -labelled LCMV GP 61-80 peptide-coated splenocytes and violet lo -labelled uncoated splenocytes. After 5 h, violet-labeled splenocytes were analyzed. (M) Flow cytometry analysis of violet hi -labelled LCMV GP 61-80 peptide-coated cells and violet lo -labeled uncoated control cells among live target cells in the presence of naive, TCF-1 hi CD39 lo , or TCF-1 lo CD39 hi SM cells. Numbers adjacent to the red outlined areas indicate percentages of LCMV GP 61-80 peptide-coated cells, and numbers adjacent to the blue outlines indicate percentages of uncoated control cells. (N) Ex vivo killing efficacy of TCF-1 hi CD39 lo (indicated by red dots) and TCF-1 lo CD39 hi SM cells (indicated by purple dots). All data are representative of at least two independent experiments with at least three mice per group. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001 by one-way ANOVA with Turkey’s test (E, F, H) and two-tailed unpaired Student’s t test (K, N). Center values and error bars (E, F, H, K, N) indicate mean and SEM.

Article Snippet: C57BL/6, OT-II, and CD45.1 + (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from Jackson Laboratories.

Techniques: Flow Cytometry, Expressing, Infection, Isolation, Labeling, Control, In Vitro, Cell Culture, Ex Vivo, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: Oncolytic virotherapy mobilizes tumor-resident, granzyme B-producing bystander CD4 + T cells to inhibit systemic microbial infection

doi: 10.1016/j.omton.2026.201187

Figure Lengend Snippet: Systemic TCF-1 lo CD39 hi CD4 + T cells are more prone to T H 1 differentiation upon OV-BYTE treatment (A) Bubble chart showing CD4 + T cell lineage-associated key genes in Tcf7 lo Entpd1 hi clusters and Tcf7 hi Entpd1 lo clusters in tumor, spleen, and lung. (B) Schematic diagram of the experimental design. Congenic CD45.1 + CXCR5-GFP SM CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LCMV Armstrong and engrafted with MC38 cells on day 60 post infection. On days 7–12 after tumor engraftment, recipients were administered PBS, NDV-WT, or NDV-GP daily via the intratumoral route. On day 15 post-tumor engraftment, CXCR5-GFP SM CD4 + T cells from tumor, spleen, and lung were analyzed. (C) Flow cytometry analysis of CXCR5/Ly6C expressions in the indicated SM CD4 + T cell populations in the indicated organs. (D) Frequency of CXCR5/Ly6C expressions in the indicated SM CD4 + T cell populations from tumor, spleen, and lung. (E) Flow cytometry analysis of T-bet expression levels in the indicated SM CD4 + T cell populations from tumor, spleen, and lung. Numbers in the flow charts indicate T-bet MFI of PBS-treated TCF-1 hi CD39 lo (gray), NDV-WT-treated TCF-1 hi CD39 lo (blue), NDV-GP-treated TCF-1 hi CD39 lo (red), and NDV-GP-treated TCF-1 lo CD39 hi (purple) SM CD4 + T cells. (F) Comparison of T-bet expression levels among the indicated SM CD4 + T cell populations from tumor, spleen, and lung. All data are representative of at least two independent experiments with at least four mice per group. Not significant (ns), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by one-way ANOVA with Turkey’s test (D, F). Center values and error bars (D, F) indicate mean and SEM.

Article Snippet: C57BL/6, OT-II, and CD45.1 + (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from Jackson Laboratories.

Techniques: Infection, Flow Cytometry, Expressing, Comparison

Journal: Molecular Therapy Oncology

Article Title: Oncolytic virotherapy mobilizes tumor-resident, granzyme B-producing bystander CD4 + T cells to inhibit systemic microbial infection

doi: 10.1016/j.omton.2026.201187

Figure Lengend Snippet: OV-BYTE-educated systemic TCF-1 lo CD39 hi CD4 + T cells epigenetically resemble tumor-resident CD4 + T BYS cells (A) Schematic of the experimental design for (B–F). Congenic CD45.1 + SM CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LCMV Armstrong and engrafted with MC38 cells on day 60 post infection. On days 7–12 after tumor engraftment, recipients were administered PBS, NDV-WT, or NDV-GP daily via intratumoral route. On day 15 post-tumor engraftment, PBS-treated Ly108 hi CD39 lo , NDV-WT-treated Ly108 hi CD39 lo , NDV-GP-treated Ly108 hi CD39 lo , and NDV-GP-treated Ly108 lo CD39 hi SM CD4 + T cells from tumors and spleens were isolated and subjected to ATAC-seq. (B) PCA analysis of peak accessibility in sorted cells from (A). The plot shows the first two principal components (PC1 and PC2), which explain 71.3% and 14.4% of the total variance, respectively. Each group includes two biological replicates from two independent experiments. (C) UpSet plot illustrating uniquely open DARs in each group, as well as the overlap of open DARs between each pair of groups. (D) Venn diagram showing the overlap of open DARs among the indicated groups. The numbers and percentages above each section represent the overlap of DARs for the respective groups. (E) Venn diagram showing the overlap of open DARs among the indicated groups. These open DARs correspond to the top 60 up-regulated DEGs in the tumor TCF-1 lo CD39 hi subset. The numbers above each section represent the overlap of DARs for the respective groups. (F) ATAC-seq signal profiles of the indicated gene loci. Differential peaks are highlighted in red frames.

Article Snippet: C57BL/6, OT-II, and CD45.1 + (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from Jackson Laboratories.

Techniques: Infection, Isolation

Journal: Molecular Therapy Oncology

Article Title: Oncolytic virotherapy mobilizes tumor-resident, granzyme B-producing bystander CD4 + T cells to inhibit systemic microbial infection

doi: 10.1016/j.omton.2026.201187

Figure Lengend Snippet: Tumor-resident CD4 + T BYS cells serve as the reservoir of OV-BYTE-induced systemic TCF-1 lo CD39 hi CD4 + T cells (A) Schematic of the experimental design for (B–H). Congenic CD45.1 + CD45.2 - SM CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LCMV Armstrong. In another cohort, naive C57BL/6 recipients (CD45.2 + ) receiving congenic CD45.1 + CD45.2 + SM CD4 + T cells were then infected with LCMV Armstrong. On day 60 post-infection, recipients that received CD45.1 + CD45.2 - SM CD4 + T cells were engrafted with MC38 cells. On day 10 post-tumor growth, tumor masses were excised and transplanted into recipients that received CD45.1 + CD45.2 + SM CD4 + T cells. The transplanted tumors were then subjected to intratumoral injections of PBS, NDV-WT, or NDV-GP, and flow cytometry analysis was performed at the indicated time point. (B) Flow cytometry analysis of CD45.1 + CD45.2 - and CD45.1 + CD45.2 + SM CD4 + T cells in total SM CD4 + T cells from the indicated organs of PBS-, NDV-WT-, or NDV-GP-treated group. Numbers adjacent to the red outlined areas indicate the percentages of CD45.1 + CD45.2 - SM CD4 + T cells (donor), and numbers adjacent to the blue ones indicate the percentage of CD45.1 + CD45.2 + SM CD4 + T cells (host). (C and D) Frequency (C) and number (D) of CD45.1 + CD45.2 - SM CD4 + T cells (donor) from the indicated organs of group treated with PBS (indicated by gray dots), NDV-WT (indicated by blue dots), or NDV-GP (indicated by red dots). Quantification of SM CD4 + cells from 100 μL of peripheral blood. (E and F) Frequency of TCF-1 hi CD39 lo (E) and TCF-1 lo CD39 hi (F) cells among host-derived PBS-treated (indicated by gray dots), NDV-WT-treated (indicated by blue dots), NDV-GP-treated (indicated by red dots) SM CD4 + T cells, and donor-derived NDV-GP-treated (indicated by purple dots) SM CD4 + T cells in the indicated organs. (G) Frequency of circulating cells (intravenous [i.v.] CD4 staining positive) of host-derived PBS-treated (indicated by gray dots), NDV-WT-treated (indicated by blue dots), NDV-GP-treated (indicated by red dots) TCF-1 hi CD39 lo SM CD4 + T cells, and NDV-GP-treated TCF-1 lo CD39 hi (indicated by purple dots) SM CD4 + T cells, together with donor-derived NDV-GP-treated TCF-1 hi CD39 lo (indicated by red cubes) and TCF-1 lo CD39 hi (indicated by purple cubes) SM CD4 + T cells in the indicated organs. (H) Comparison of GzmB expression levels in host-derived PBS-treated (indicated by gray dots), NDV-WT-treated (indicated by blue dots), NDV-GP-treated (indicated by red dots) TCF-1 hi CD39 lo SM CD4 + T cells, and NDV-GP-treated TCF-1 lo CD39 hi (indicated by purple dots) SM CD4 + T cells, together with donor-derived NDV-GP-treated TCF-1 hi CD39 lo (indicated by red cubes) and TCF-1 lo CD39 hi (indicated by purple cubes) SM CD4 + T cells in the indicated organs. All data are representative of at least two independent experiments with at least four mice per group. Not significant (ns), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by one-way ANOVA with Turkey’s test (C–H). Center values and error bars (C–H) indicate mean and SEM.

Article Snippet: C57BL/6, OT-II, and CD45.1 + (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from Jackson Laboratories.

Techniques: Infection, Flow Cytometry, Derivative Assay, Staining, Comparison, Expressing

Journal: Molecular Therapy Oncology

Article Title: Oncolytic virotherapy mobilizes tumor-resident, granzyme B-producing bystander CD4 + T cells to inhibit systemic microbial infection

doi: 10.1016/j.omton.2026.201187

Figure Lengend Snippet: Turnover of OV-BYTE-educated Ly108 hi CD39 lo and Ly108 lo CD39 hi CD4 + T cells (A) Schematic of the experimental design for (B–I). Congenic CD45.1 + SM CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LCMV Armstrong. In another cohort, naive C57BL/6 recipients (CD45.2 + ) were simultaneously infected with LCMV Armstrong without SM CD4 + T cell transfer. On day 60 post-infection, both cohorts were engrafted with MC38 cells. On days 7–12 post-tumor engraftment, the cohort that received SM CD4 + T cells was administered NDV-GP daily via the intratumoral route. Then, Ly108 hi CD39 lo and Ly108 lo CD39 hi SM CD4 + T cells in tumor and spleen were isolated on day 15 post-tumor engraftment and subsequently transferred into MC38 tumor-bearing mice (no SM CD4 + T cells transfer) via intratumoral injection and intravenous injection, respectively. These recipients were then administered NDV-GP intratumorally for five consecutive days, and flow cytometry analysis was thereafter. (B and C) Frequency (B) and number (C) of transferred tumor-derived Ly108 hi CD39 lo (indicated by blue dots) and Ly108 lo CD39 hi (indicated by red dots) SM CD4 + T cells in the indicated organs. Quantification of SM CD4 + cells from 100 μL of peripheral blood. (D) Flow cytometry analysis of TCF-1 hi CD39 lo and TCF-1 lo CD39 hi cells in transferred tumor-derived Ly108 hi CD39 lo or Ly108 lo CD39 hi SM CD4 + T cells (red contour lines) in the indicated organs. TCF-1/CD39 expression of tumor-resident polyclonal CD4 + T cells (gray contour lines) is shown as an internal control in the bottom-left plot. Numbers adjacent to the blue outlined areas indicate the percentages of TCF-1 hi CD39 lo cells in the indicated transferred SM CD4 + T cells, and numbers adjacent to the red ones indicate the percentage of TCF-1 lo CD39 hi cells in the indicated transferred SM CD4 + T cells. (E and F) Frequency of TCF-1 lo CD39 hi (E) and TCF-1 hi CD39 lo (F) cells in transferred tumor-derived Ly108 hi CD39 lo (indicated by blue dots) and Ly108 lo CD39 hi (indicated by red dots) SM CD4 + T cells in the indicated organs. (G and H) Frequency (G) and number (H) of transferred spleen-derived Ly108 hi CD39 lo (indicated by blue dots) and Ly108 lo CD39 hi (indicated by red dots) SM CD4 + T cells in the indicated organs. Quantification of SM CD4 + cells from 100 μL of peripheral blood. (I) Frequency of TCF-1 hi CD39 lo and TCF-1 lo CD39 hi cells in transferred spleen-derived Ly108 lo CD39 hi SM CD4 + T cells. All data are representative of at least two independent experiments with at least four mice per group. Not significant (ns), ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by two-tailed unpaired Student’s t test (B, C, E–H). Center values and error bars (B, C, E–H) indicate mean and SEM.

Article Snippet: C57BL/6, OT-II, and CD45.1 + (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from Jackson Laboratories.

Techniques: Infection, Isolation, Injection, Flow Cytometry, Derivative Assay, Expressing, Control, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: Oncolytic virotherapy mobilizes tumor-resident, granzyme B-producing bystander CD4 + T cells to inhibit systemic microbial infection

doi: 10.1016/j.omton.2026.201187

Figure Lengend Snippet: Dual protection against tumor and pathogen infection by the OV-BYTE strategy (A) Schematic of the experimental design for (B–D). C57BL/6 mice were infected with LCMV Armstrong and engrafted with MC38 cells on day 60 post-infection. On days 7–12 after tumor engraftment, recipients were daily administered PBS, NDV-WT, or NDV-GP daily via the intratumoral route. On day 15 after tumor engraftment, recipients were infected with either LM-GP 61-80 or IAV-GP 61-80 at an LD 50 dose. (B) Tumor growth curve of MC38 tumor-bearing mice intratumorally treated with PBS, NDV-WT, or NDV-GP as described in (A). (C and D) Survival curves of LM-GP 61-80 (C) and IAV-GP 61-80 (D) infection in MC38-engrafted mice treated with PBS, NDV-WT, or NDV-GP as described in (A). (E) Schematic of the experimental design for (F–H). C57BL/6 mice were infected with LCMV Armstrong and engrafted with B16F10 cells on day 60 post-infection. On days 7–12 after tumor engraftment, recipients were administered PBS, Ad5-WT, or Ad5-GP daily via the intratumoral route. On day 15 after tumor engraftment, recipients were infected with either LM-GP 61-80 or IAV-GP 61-80 at an LD 50 dose. (F) Tumor growth curve of B16F10 tumor-bearing mice intratumorally treated with PBS, Ad5-WT, or Ad5-GP as described in (E). (G and H) Survival curves of LM-GP 61-80 (G) and IAV-GP 61-80 (H) infection in B16F10-engrafted mice treated with PBS, Ad5-WT, or Ad5-GP as described in (E). (I) Schematic of the experimental design. Congenic CD45.1 + SM CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LCMV Armstrong. On day 60 post-infection, these recipients were engrafted with MC38 cells. On days 7–12, these recipients were administered NDV-GP daily via the intratumoral route. Then, Ly108 hi CD39 lo and Ly108 lo CD39 hi SM CD4 + T cells in the spleens were isolated on day 15 post-tumor engraftment and subsequently transferred into MC38 tumor-bearing mice (no LCMV Armstrong infection) via intravenous injection, along with MC38 tumor-bearing mice receiving no cell transfer as control. One day later, all recipients were infected with LM-GP 61-80 at an LD 50 dose. (J) Survival curve of LM-GP 61-80 infection in groups described in (I). (K) Schematic of the experimental design. WT and Gzmb KO mice were infected with LCMV Armstrong. On day 60 post-infection, splenic LCMV Armstrong-activated CD4 + T MEM cells were harvested and adoptively transferred into another cohort of naive C57BL/6 mice. These recipients, along with control C57BL/6 mice with no CD4 + T MEM cell transfer, were then engrafted with MC38 tumor cells, intratumorally administrated NDV-WT or NDV-GP, and infected with LM-GP 61-80 at the indicated time points. (L) Survival curve of LM-GP 61-80 infection in groups described in (I). All data are representative of at least two independent experiments with at least eight mice per group. Not significant (ns), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by two-way ANOVA (B, F) and log rank (Mantel-Cox) test (C, D, G, H, J, L). Center values and error bars (B, F) indicate mean and SEM.

Article Snippet: C57BL/6, OT-II, and CD45.1 + (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from Jackson Laboratories.

Techniques: Infection, Isolation, Injection, Control

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS reprograms the lineage commitment of MSCs after GC treatment and inhibits the generation of primary senescent adipocytes. ( A ) Schematic illustration of the in vitro investigation of SCS targeting the prostaglandin/PPARγ/INK positive feedback loop in MPS-induced primary senescent adipocytes. ( B ) Representative flow cytometry plot showing p16 + senescent cells in adipocytes derived from bone marrow after 14 days of in vivo MPS induction and subsequently treated with SCS in vitro . ( C ) qPCR analysis of 12 senescence-associated markers in primary senescent adipocytes after in vitro SCS treatment. n = 3 biological replicates. ( D ) ELISA analysis of IL-1β levels in adipocyte supernatant following in vitro SCS treatment. n = 6 biological replicates. ( E ) ELISA analysis of secreted prostaglandins PGD2 and PGE2 in adipocytes under different treatment conditions. D-PBS: bone marrow adipocytes isolated from mice treated in vivo with the solvent control DMSO, followed by in vitro treatment with PBS; M-PBS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with PBS. M-SCS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with SCS. ( F ) Western blot analysis of intracellular COX-2 protein levels in adipocytes across the three treatment conditions. ( G ) Schematic illustration of competitive osteogenic–adipogenic differentiation of CD45 − Ter119 − CD31 − LepR + MSCs after 7 days of in vivo SCS and MPS co-treatment. ( H ) qPCR analysis of pan-adipocyte markers ( Fabp4 , Adipoq , Plin1 , Cd36 , and Lep ) in CD45 − Ter119 − CD31 − LepR + MSCs after 14 days of in vitro competitive lineage differentiation. n = 3 biological replicates. ( I and J ) Representative immunofluorescence images (I) and quantification (J) of perilipin + adipocytes and osteopontin + mature osteoblasts derived from lineage-committed MSCs. n = 6 biological replicates. (Scale bars, 30 μm, 15 μm and 15 μm). ( K ) Western blot analysis of adipogenesis-related markers C/EBPα, PPARγ, and C/EBPβ in the lineage-mixed cells after in vitro competitive differentiation of CD45 − Ter119 − CD31 − LepR + MSCs. ( L ) qPCR analysis of lipogenesis-related markers Fasn , Scd1 , Srebf1 , Acaca , and Acacb . n = 3 biological replicates. ( M and N ) Representative H&E staining images (M) of the femurs at day 14 following SCS and MPS co-treatment. Yellow arrows indicate bone marrow adipocytes. Magnified images show hypertrophic adipocyte morphology, with quantification of adipocyte diameter (N). n = 19 biological replicates. (Scale bars, 200 μm, 50 μm and 20 μm). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( C, D, H, J, L and N ), or one-way ANOVA with Tukey's post hoc test ( E ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: In Vitro, Flow Cytometry, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Solvent, Control, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Gene expression profiles of bone marrow-derived LepR + MSCs after 7-day in vivo co-treatment with SCS and MPS. ( A ) Heatmap showing DEGs in CD45 − Ter119 − CD31 − LepR + MSCs sorted from bone marrow at day 7 post-treatment with SCS versus PBS ( P < 0.05, |log fold change| > 1.5). n = 3 biological replicates. ( B ) Representative GO biological process enrichment analysis of downregulated DEGs. ( C ) Top 20 enriched KEGG pathways of downregulated DEGs in SCS versus PBS. ( D ) GSEA plots of biological processes positively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25). ( E ) Representative downregulated DEGs associated with adipogenesis and lipogenesis identified through KEGG pathway analysis. n = 3 biological replicates. ( F ) Top 20 enriched KEGG pathways of upregulated DEGs in SCS versus PBS. ( G ) Representative GO biological process enrichment analysis of upregulated DEGs. ( H ) Representative upregulated DEGs identified through biological process enrichment analysis. n = 3 biological replicates. ( I and J ) GSEA plots of KEGG pathways negatively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Gene Expression, Derivative Assay, In Vivo

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: P300/CBP inhibition with inobrodib in combination with gilteritinib and venetoclax targets leukemia stem cells in epigenetic mutant AML

doi: 10.1126/sciadv.aec9305

Figure Lengend Snippet: ( A ) In vivo CCS1477 treatment schematic for survival analyses. CD45.2 + D/F or T/F AML cells were transplanted into sublethally irradiated CD45.1 + WT recipient mice. Treatment was initiated 14 days posttransplant. CCS1477 (20 mg/kg) or vehicle was dosed by mouth (PO) once daily (QD). ( B ) Average ± SEM percent c-Kit + cells in the CD45.2 + peripheral blood (PB) of CCS1477- and vehicle-treated D/F ( n = 7 per treatment) and T/F ( n = 11 per treatment) transplanted AML mice over 2 weeks. Week 0 time point was immediately before the first treatment, followed by weeks 1 and 2 on treatment. Significant differences were evaluated by two-way analysis of variance (ANOVA) Šídák’s multiple comparisons test. Average ± SEM ( C ) spleen weight, and numbers of ( D ) CD45.2 + and ( E ) CD45.2 + LSK (Lin − Sca1 + Kit + ) bone marrow cells of D/F ( n = 8 per treatment) or T/F ( n = 8 per treatment) AML mice after 2 weeks of CCS1477 or vehicle. Significant differences were evaluated by unpaired t test. ( F ) Kaplan-Meier survival analysis of CCS1477-treated and vehicle-treated D/F AML (CCS1477, n = 12; vehicle, n = 14) and T/F AML (CCS1477, n = 9; vehicle, n = 9) transplant recipient mice. Overall survival (OS) of moribund D/F AML mice was 94 and 73 days for CCS1477 and vehicle, respectively. OS of moribund T/F AML mice was 51 and 39 days for CCS1477 and vehicle, respectively. Significance determined by the log-rank (Mantel-Cox) test. ( G ) Average ± SEM spleen weight at mouse moribundity of CCS1477- or vehicle-treated D/F ( n = 3 per treatment) or T/F ( n = 3 per treatment) AML mice. Significant differences were evaluated by unpaired t test. Individual data points represent biological replicates. For all panels, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For CCS1477 monotherapy in vivo studies, CD45.1 + C57BL6/J mice (Charles River Laboratories), 7 to 8 weeks of age, were irradiated with 500 rads before transplantation with either 10 million CD45.2 + D/F AML cells or 2.5 million CD45.2 + T/F AML cells via retro-orbital injection.

Techniques: In Vivo, Irradiation