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: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: (A) Kaplan-Meier analysis showing that neutrophil density negatively correlates with patient outcome, n=83 patients. (B) CD66b + TAN were analyzed by immunofluorescence and quantified in tumor, tumor margin and stroma of HNSCC patients (mean ± SD, n=83, circles = males, open circles = females). (C) Schematic overview of the in vitro system for tumor-stroma communication, generated using BioRender. (D) SNs from tumor (FaDu, PCI13), MSC-primed tumor, MSCs and tumor-primed MSCs were analysed for released CXCL8 by ELISA, mean + SD, n=3. (E) The impact of these SN on PMN chemotaxis was analyzed using transwell assays, mean + SD, n=3-8. (F) Luminex screening was performed on SNs from FaDu cells and seven patient-derived MSC lines, analyzed both in their naïve state and after FaDu-priming. CXCL8 was measured using ELISA. Data are depicted in pg/mL. Statistical analysis was performed with Kruskal-Wallis (B) and ordinary one-way ANOVA (D, E). P -values are indicated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Immunofluorescence, In Vitro, Generated, Enzyme-linked Immunosorbent Assay, Chemotaxis Assay, Luminex, Derivative Assay

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: (A) MSCs were stimulated for 24 h with FaDu tumor-conditioned SNs. Tumor-derived cytokines (columns) were quantified by Luminex, and MSC-derived CXCL8 and G-CSF (rows) by ELISA. Data are displayed as a correlation matrix. (B) MSCs were treated with FaDu tumor SNs for 24 h. Tumor-derived factors were analyzed by Luminex; MSC-derived CXCL8 and G-CSF were quantified by ELISA. Tumor-derived IL-1α correlates with MSC-derived CXCL8 and G-CSF. (C) MSCs were treated with recombinant IL-1α for 24 h. Release of CXCL8 and G-CSF was analyzed by ELISA. (D) IL-1α was measured in control (non-sense, NS) and IL-1α overexpressing FaDu cells (IL-1α-OE) using Luminex. (E) MSCs were treated with SNs from non-sense and IL-1α-OE cells. MSC-derived CXCL8 and G-CSF were quantified by ELISA. (F) IL-1α release was determined in the SN of viable and necrotic FaDu and A431 cells. MSCs were treated with the SN of viable and necrotic FaDu (G) or A431 (H) cells for 24 h. MSC-derived CXCL8 and G-CSF were quantified by ELISA. Statistical significance was assessed after log-transformation using an ordinary one-way ANOVA with Tukey’s multiple comparisons test (C), while paired t -tests were applied for panels D-H. Data are shown as mean ± SD. In panel C, significance levels are indicated as # or * (p ≤ 0.05), ## or ** (p ≤ 0.01), ### or *** (p ≤ 0.001), and #### or **** (p ≤ 0.0001); all other p values are shown numerically. Symbols (BioRender) are included to show the origin of the analyzed SNs.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Derivative Assay, Luminex, Enzyme-linked Immunosorbent Assay, Recombinant, Control, Transformation Assay

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: (A,B) Tumor-derived cytokines were quantified by Luminex analysis. (A) Data are displayed as Pearsońs correlation matrix. Significant correlations among tumor-derived factors are indicated by black asterisks (B). Quantification of tumor-derived mediators known to be involved in tumor-stroma communication. (C) MSCs were treated with FaDu-CXCL8KO tumor conditioned medium in the presence of 10 µg/mL IL-1α neutralizing antibody or isotype control. Released CXCL8 and G-CSF were quantified by ELISA. (D) Quantification of tumor-derived mediators of tumor-stroma communication in non-sense and IL-1α overexpressing (IL-1α-OE) cells was performed by Luminex. (E) Quantification of tumor-derived factors implicated in neutrophil recruitment and survival in SN from non-sense and IL-1α-OE cells (Luminex and CXCL8-ELISA). (F) LDH assay quantifying cytotoxicity in SN of viable and necrotic FaDu and A431 tumor cells. Statistical analysis was performed using paired t -tests (C,F) and unpaired t -tests (D). Data are presented as mean ± SEM (C) and mean ± SD (D,E), p values are indicated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Derivative Assay, Luminex, Control, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: MSCs were treated with recombinant IL-1α for 6 and 24 h. Gene expression was analyzed by Bulk RNAseq. (A) Heatmap depicts functional DEGs linked to the biology and function of CAFs. (B) Enriched hallmark pathways are shown for untreated (ctrl) and 24 h IL-1α-treated MSCs. C) Pathway analysis (STRING v12.0) highlights local network cluster: IL-1 receptor activity and IL-1 family (yellow), chemokine receptors bind chemokines (green), extracellular matrix remodeling (pink), JAK-STAT (blue) and NFκB (red) signaling pathways. (D) Release of cytokines, chemokines and growth factors was analyzed by Luminex. Heatmap depicts proteins functioning in PMN survival, activation and chemotaxis. (E) Surface markers were analyzed by flow cytometry in different MSCs. (F) Inhibition of the Jak/Stat pathway by AZD1480 (JAK2i) or NFκB by Activation Inhibitor III (NFκBi) inhibit the release of CXCL8, G-CSF and IL6 in different MSCs. Red dots in E and F indicate the MSC cell line that was used for A-D. Statistical analysis was performed with paired t-tests. P -values are depicted.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Recombinant, Gene Expression, Functional Assay, Activity Assay, Protein-Protein interactions, Luminex, Activation Assay, Chemotaxis Assay, Flow Cytometry, Inhibition

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: MSCs were treated with recombinant IL-1α for 6 and 24 h. Gene expression was analyzed by Bulk RNAseq. Volcano plots are shown for untreated vs. 6 h (A), and untreated vs. 24 h IL-1α treatment (B). (C) Enriched hallmark pathways are shown for untreated vs. 6 h. (D) Venn diagram showing the number of upregulated DEGs in two MSC cell lines after 6 h of IL-1α treatment. (E) MSC cells were treated for 6 h and 24 h with IL-1α and were analyzed by RNAscope for the expression of CXCL8 (red) and CSF3 (green) with consecutive COX2 immunostaining (white). (F) FaDu spheroids were cultured in the presence or absence of SNs derived from MSCs or IL-1α-stimulated MSCs. Relative tumor spheroid area was quantified for two independent experiments. Statistical analysis was performed using Mann-Whitney test. Data depict mean ± SD. P -values are indicated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Recombinant, Gene Expression, RNAscope, Expressing, Immunostaining, Cell Culture, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: GSEA was performed on genes differentially expressed in MSCs after 24 h IL-1α treatment versus untreated controls. (A) Enrichment analysis of the IL-1α signature across eight CAF clusters . Clusters 3, 4, and 6 are associated with ECM remodeling, EMT, antigen presentation, and worse overall survival. (B) Enrichment analysis across five CAF clusters . HNCAF-0 (iCAF), HNCAF-3 (iCAF-like/ECM remodeling), and HNCAF-1 (iCAF, immunosuppressive) show high similarity to the IL-1α program; HNCAF-1 is linked to poor prognosis, whereas HNCAF-0/3 have been associated with favorable responses to anti-PD-1 immunotherapy. (C) The heatmap shows log2 fold changes of published CAF cluster marker genes in IL-1α-treated MSCs relative to control. The CF1/CXCL8 cluster represents an iCAF cluster and the most progressed fibroblast state.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Immunopeptidomics, Marker, Control

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: PMNs were treated with recombinant IL-1α and SNs from non-sense (NS) or IL-1α-overexpressing (OE) FaDu cells, as well as from MSCs preconditioned with these tumor cell SNs. A) PMN survival was analyzed by Annexin V/7-AAD staining (dotted line indicates the mean of untreated PMNs and (B) in the presence of neutralizing antibodies against G-CSF, GM-CSF, or both. (C,D) Activation was assessed using flow cytometry, determining changes in cell size (C) and granularity (D). (E) CCL4 release was quantified by ELISA. (F) Surface activation marker CD11b and CD54 were analyzed by flow cytometry. (G) ROS productionwas measured using 123-DiRhodamine. (H) Expression of marker genes was assessed by qRT-PCR. Statistical analysis was performed with paired t-tests (A,B), ordinary one-way ANOVA with Tukey’s multiple comparisons test (C) or Wilcoxon signed rank test (E-H). Data are plotted as mean + SD (E, G, H) or mean ± SD (A,B,F). P -values are stated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Recombinant, Staining, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Marker, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: PMNs were treated with SN from non-sense (NS) or IL-1α-overexpressing (OE) FaDu cells, as well as from MSCs preconditioned with these tumor cell SN. (A) PMN migration toward tumor– and MSC-derived factors was analyzed by trans-well assays (dotted line indicates the mean of untreated PMNs) and (B) in the presence of neutralizing antibodies against CXCL8. (C) PMN migration was assessed by transwell assays toward SN collected from IL-1α–treated or untreated wild-type (WT) and CXCL8-knockout (KO) MSCs. (D) These SN were analyzed for released chemoattractants using Luminex. (E) Representative images of PMN recruitment into mixed NS FaDu-MSC or IL-1α OE-FaDu-MSC spheroids (tumor spheroids, blue; MSC spheroids, orange; PMNs, gray). (F) Quantification of PMN recruitment into tumor spheroids (NS or IL-1α-OE FaDu only), MSC spheroids (untreated vs. IL-1α-treated), and mixed tumor-MSC spheroids was analyzed by confocal microscopy (n = 6–10). (G) Representative images of FaDu cells (blue), MSCs (orange) and neutrophils (gray) 24 h post-injection into the perivitelline space of zebrafish larvae. Upper row: FaDu NS control cells; lower row: IL-1α-OE FaDu cells. Left panel: FaDu cells only; middle panel: FaDu cells + MSC1; right panel: FaDu cells + MSC2. (H) Quantification of infiltrated neutrophils per graft at 24 h post-injection. Statistical analysis was performed using paired t -tests (A-C), Mann–Whitney tests (F), or Wilcoxon signed-rank tests (H). Data are presented as mean ± SD (A-C), median (F), and mean ± SEM from three independent experiments (H). P values are indicated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Migration, Derivative Assay, Knock-Out, Luminex, Confocal Microscopy, Injection, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: (A) Representative tumor section illustrating tumor-stroma segmentation, RNAscope detection of IL1A (pink), CXCL8 (red), and CSF3 (yellow), and zonal analysis of tumor-stroma communication. Shown is also an example of an IL1A + niche and an IL1A − niche with corresponding RNA probe signals. (B,C) Correlation of tumor IL1A expression with stromal CXCL8 + CSF3 + double-positive cells across the total cohort (B; n = 21; black circles = males, open circles = females) and in male patients only (C; n = 16). (D,E) Frequency of CXCL8 + CSF3 + double-positive stromal cells stratified by IL1A -negative vs. IL1A-positive tumors (D) and by TSR-low vs. TSR-high tumors (E). (F) Total TAN density and frequency in patients with low vs. high stromal CXCL8 + CSF3 + cells. (G) Spearman correlation analysis of an IL-1α-induced MSC/iCAF gene signature (SIG) with published TAN signatures across TCGA HNSCC tumor samples (left, n = 515) and matched tumor-adjacent normal tissue (right, n = 44). The MSC/iCAF signature (MSC-SIG) was defined by the top 100 genes upregulated in MSCs following 24 h IL-1α stimulation. TAN signatures from four independent studies – represent immature, inflammatory, and activated neutrophil populations. Individual cytokine and chemokine expression ( IL1A, IL1B, CSF2, CSF3, CXCL8 ) shows concordant associations with multiple TAN subset signatures. Statistical analysis was performed using Spearman correlation (B,C,G) and Mann–Whitney test (D-F). Data are presented scatter plots (B,C), or mean ± SEM (E–J). P values are indicated (A-D). In (G) p values are indicated as follow: * p value < 0.05, ** p value < 0.01, *** p value <0.001.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: RNAscope, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet:

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: