Journal: bioRxiv

Article Title: Collagen remodeling promotes a GPCR-mediated mechanosensory immune checkpoint in ADGRG1+ CD8+ T cells and serves as a spatial biomarker of response to immunotherapy

doi: 10.64898/2025.12.19.695209

Figure Lengend Snippet: (A) Volcano plot showing differentially expressed genes in ECM-high tumor samples from the Moffitt-IO HNSCC tissue microarray (TMA). (B) Representative images of low (n=33) and high (n=79) density LH2 staining in HNSCC cores. (C) Distribution of LH2 density scores in HNSCC cores; also represented across early and late stages. (D) Representative images of Picrosirius Red (PSR) stained cores visualized under bright field and polarized light. (E) Quantification of total collagen deposition in LH2-high (n=28) and LH2-low (n=24) primary tumor cores. (F) Quantification of collagen birefringence signals in LH2-high and LH2-low primary tumor cores. (G) Representative SHG ROI images of LH2 low and high tumor cores for analyzing collagen fiber features. (H-J) Analysis of fiber length, width and angular variance in LH2-low and high tumor cores by CT-FIRE. (K-M) Analysis of alignment, kurtosis and red pixel intensity in LH2-low and high tumor cores by CurveAlign. (N) Representative Orientation-J plots showing the distribution of collagen fiber orientations in LH2-low (cyan) and LH2-high (red) tumor cores (n = 5 per group). (O) Quantification of dominant fiber orientation (mode) frequency across all samples, illustrating the degree of collagen alignment in LH2-low and LH2-high groups. Statistical analysis was performed using two-tailed Mann–Whitney U test. Each dot in the scatter plot represents individual ROI/core, error bars are mean ± SEM. ns, not significant; *P < 0.05, **P < 0.01.

Article Snippet: For each marker, heat induced epitope retrieval (HIER) was performed (EDTA pH 9.0 buffer or citrate pH 6.0), followed by blocking and primary antibody incubation, LH2 (Proteintech, Rb Poly, 1:50, dye 570) at RT for 60 min followed by OPAL HRP polymer and one of the OPAL fluorophores during the final TSA step.

Techniques: Microarray, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Collagen remodeling promotes a GPCR-mediated mechanosensory immune checkpoint in ADGRG1+ CD8+ T cells and serves as a spatial biomarker of response to immunotherapy

doi: 10.64898/2025.12.19.695209

Figure Lengend Snippet: (A) Representative images of tumor cores stained for LH2 (orange), CD3 (magenta), Pan-cytokeratin (cyan) and DAPI (blue). (B) Cell types defined by multiplex immunofluorescence and segmentation; pie charts show distributions in LH2-low vs. LH2-high tumor cores. (C) Distribution of CD3+ T-cells across tumor (PCK+) and stromal areas (PCK-) of LH2 low (n=25) and LH2 high (n=31) cores. Statistical analysis was performed using two-tailed Mann–Whitney U-test ****P < 0.0001. (D) Schematic illustration of the Virtual Alignment of pathoLogy Image Series (VALIS) pipeline. (E) Gcross analysis of CD3+ to PCK+ proximity in LH2-high (n=13) and LH2-low (n=11) HNSCC patients. Lines represent group means with shaded areas indicating ±1 standard error. The dashed line denotes spatial independence. Higher curves (LH2 low group) suggest greater association between T-cells and tumor cells. (F) Resource selection analysis using the p’’ (rou-hat) rho-hat function showing the relative intensity of CD3+ cells as a function of local collagen density (within one cell diameter), which differs between LH2-high and LH2-low tumors. (G-H) Correlation of CD8+ and CD4+ T-cell infiltration with PLOD2 gene expression in HNSCC TCGA samples using EPIC deconvolution algorithm, tumor purity is depicted on the left. (I) Correlation of Treg cell infiltration with PLOD2 gene expression in HNSCC TCGA samples using X-CELL deconvolution algorithm. TIMER plots indicate computed Spearman’s rho and p-values.

Article Snippet: For each marker, heat induced epitope retrieval (HIER) was performed (EDTA pH 9.0 buffer or citrate pH 6.0), followed by blocking and primary antibody incubation, LH2 (Proteintech, Rb Poly, 1:50, dye 570) at RT for 60 min followed by OPAL HRP polymer and one of the OPAL fluorophores during the final TSA step.

Techniques: Staining, Multiplex Assay, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Selection, Gene Expression

Journal: bioRxiv

Article Title: Collagen remodeling promotes a GPCR-mediated mechanosensory immune checkpoint in ADGRG1+ CD8+ T cells and serves as a spatial biomarker of response to immunotherapy

doi: 10.64898/2025.12.19.695209

Figure Lengend Snippet: (A) Top panel , representative images of PSR stained sagittal sections of mouse tongue orthotopically transplanted with 4MOSC1 cells expressing inducible LH2, from mice maintained on regular chow (No Dox) or Doxycycline-containing chow (Dox). Bottom panel , ROIs depicting collagen architecture by multiplex IF imaging using Lunaphore COMET platform, H&E and PSR staining. (B) Representative SHG ROIs of mice tumors from No Dox and Dox conditions, with corresponding collagen fiber overlays (CT-FIRE), curvature maps (CurveAlign), and orientation-based color density overlays (OrientationJ). (C) Collagen fiber length analysis in mouse tumors under No Dox (n=5 mice) and Dox (n=5 mice) conditions, measured with CT-FIRE. Data points correspond to individual ROIs. Data points in the graph correspond to individual ROIs from the tumors. (D) Collagen fiber alignment analysis in mouse tumors under No Dox (n=5 mice) and Dox (n=5 mice) conditions, measured with CurveAlign. Data points in the graph correspond to individual ROIs from the tumors. (E) Mass spectrometry analysis of LH2-mediated DHLNL covalent cross-links in mice tumors from No Dox (n=3) and Dox (n=4) conditions. (F) AFM analysis of tumor stiffness in mouse tumors from No Dox (n = 3 mice) and Dox (n = 3 mice) conditions. Data point in the graph represents an individual indentation measurement. (G) Immune cell types represented as proportion of CD45+ cells infiltrating 4MOSC1 tumors from No Dox (n = 7 mice) and Dox (n = 7 mice) conditions. (H) Specific T-lymphocyte populations represented as proportion of CD3+ cells infiltrating 4MOSC1 tumors from No Dox (n = 7 mice) and Dox (n = 7 mice) conditions. (I) Mean fluorescence intensities of PD1 and CTLA-4 expressed on CD8+ T-cells infiltrating 4MOSC1 tumors from No Dox (n = 6 mice) and Dox (n = 6 mice) conditions. (J) Granzyme B, IFN-γ, and TNF-α expressing CD8⁺ T-cells are represented as a proportion of CD8⁺ T-cells infiltrating 4MOSC1 tumors under No Dox (n = 6 mice) and Dox (n = 6 mice) conditions. (K) Representative images from Lunaphore COMET multiplex staining of 4MOSC1 tumors (PCK+; cyan color) showing spatial distribution and proximity of T-cells (CD3+; red color) to collagen fibers (Pan-collagen+; white color) within the TME of control (No Dox) and LH2 (LH2+; yellow color) expressing Dox group. Nuclei are shown in blue. (L-M) Violin plots showing the predicted association of CD3+ and CD8+ cells with collagen within a 3-cell distance, as determined by Python-based analysis. (N) Representative images from Lunaphore COMET multiplex staining of 4MOSC1 tumors (PCK+; cyan color) showing spatial distribution and proximity of CD8+ T-cells (CD8+; yellow color) producing cytokines TNFα ( Tnf α+, red color) and IFN-γ ( Ifng +, blue) to collagen fibers (Pan-collagen+; white color) within the TME of control (No Dox) and LH2 (LH2+; yellow color) expressing Dox group. (O) Left, Violin plot showing abundance of Tnf α RNA transcript dots in CD8+ T-cells; Right, the predicted association of Tnf α + CD8+ cells with collagen within a 3-cell distance, as determined by Python-based analysis. (P) Left, Violin plot showing abundance of Ifng RNA transcript dots in CD8+ T-cells; Right, the predicted association of Ifng + CD8+ cells with collagen within a 3-cell distance, as determined by Python-based analysis. Statistical analyses were performed using unpaired two-tailed Mann–Whitney U-test (C, D, H, I, J, L, M, O and P), unpaired one-tailed Student’s t test (E). Bar graphs represent mean ± SEM. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: For each marker, heat induced epitope retrieval (HIER) was performed (EDTA pH 9.0 buffer or citrate pH 6.0), followed by blocking and primary antibody incubation, LH2 (Proteintech, Rb Poly, 1:50, dye 570) at RT for 60 min followed by OPAL HRP polymer and one of the OPAL fluorophores during the final TSA step.

Techniques: Staining, Expressing, Multiplex Assay, Imaging, Mass Spectrometry, Fluorescence, Control, Two Tailed Test, MANN-WHITNEY, One-tailed Test

Journal: bioRxiv

Article Title: Collagen remodeling promotes a GPCR-mediated mechanosensory immune checkpoint in ADGRG1+ CD8+ T cells and serves as a spatial biomarker of response to immunotherapy

doi: 10.64898/2025.12.19.695209

Figure Lengend Snippet: (A) HEK293T-cells transfected with plasmids to overexpress Adgrg1 and an SRE-luciferase reporter were treated with DHM, P19, Col3a1 or Col1a1. P19 agonist-peptide activated SRE-luciferase but DHM inhibited the P19 stimulus. Error bars are mean ± SEM of three technical replicates. Statistical analysis was performed using One-way ANOVA; *P < 0.05, **P < 0.01. (B) Python based spatial distances were calculated and graphed as violin plots to compare spatial association of CD8+ T-cells with collagen I and collagen III in the No Dox and Dox samples. Statistical analysis was performed using One-way ANOVA; **P < 0.01, ****P < 0.0001. (C) Representative contour plots showing ADGRG1 expression in CD8+ T-cells infiltrating tumors of mice from the No Dox and Dox cohorts analyzed by flow cytometry. Percentages shown are Mean±SD values from No Dox (n=9) and Dox (n=7) mice. (D) ADGRG1 expressing CD8⁺ T-cells are represented as a proportion of CD3⁺ cells infiltrating 4MOSC1 tumors under No Dox (n = 6 mice) and Dox (n = 6 mice) conditions (top); IFN-γ, TNF-α and Granzyme B expressing ADGRG1+ CD8⁺ T-cells in tumors from No Dox (n = 6 mice) and Dox (n = 6 mice) cohorts (bottom). Statistical analysis was performed using two-tailed Mann-Whitney U test; *P < 0.05, **P < 0.01. (E) UMAP of unbiased clustering of 26930 CD8+ T-cells to identify clusters representing cellular sub-states distinguished by different colors. (F) UMAP showing expression of Adgrg1 in CD8 T-cell sub states. (G) GSEA represented as dot plot displaying pathways enriched in exhausted T-cells from No Dox and Dox cohorts. (H) Bar graph showing absolute number of ADGRG1+ CD8+ T-cells in stromal and tumor compartments of LH2 low (n=5) and high (n=7) TMA cores. Statistical analysis was performed using two-tailed Mann-Whitney U test; *P < 0.05. (I) Representative images from Lunaphore COMET multiplex staining of TMA cores showing spatial distribution and proximity of ADGRG1+ CD8+ T-cells (CD3+: yellow; CD8+: red; ADGRG1: white) to collagen fibers (cyan color; overlaid using VALIS) within the TME of LH2 low and high cores.

Article Snippet: For each marker, heat induced epitope retrieval (HIER) was performed (EDTA pH 9.0 buffer or citrate pH 6.0), followed by blocking and primary antibody incubation, LH2 (Proteintech, Rb Poly, 1:50, dye 570) at RT for 60 min followed by OPAL HRP polymer and one of the OPAL fluorophores during the final TSA step.

Techniques: Transfection, Luciferase, Expressing, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Multiplex Assay, Staining