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: