Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: Mice bearing Cer2-OVA MMTV-PyMT lung metastases were treated with vehicle (0.5% HPMC) or 75 mg/kg VS-4718 p.o. for 2 weeks starting 4 weeks post-implantation. ( A & B ) Representative images and quantification of collagen VIIIα1 and laminin 5α in metastatic versus non-metastatic lung regions at 6 weeks (H-score; each dot represents one lesion; n = 5 mice/group). Kruskal-Wallis test and Dunn’s multiple comparison. ( C & D ) Collagen VIIIα1 and laminin 511 suppress CD8⁺ T cell activation in vitro . CD8⁺ T cells were plated on 1 μg/mL ECM components, stimulated with CD3/CD28 + IL-2 for 48 h, and assessed by flow cytometry for CD25, granzyme B, IFNγ, and TNFα expression (n = 3-5). ( E ) ECM-mediated inhibition of CD8⁺ T cell migration. Transwell inserts coated with laminin 511, or collagen VIIIα were used to assess migration toward serum-rich media for 5 h (n = 3-4). ( F ) ECM-dependent CD8⁺ T cell adhesion. Adhesion to laminin 511, or collagen VIIIα-coated plates was quantified after 90 min and normalized to uncoated plates (n = 3-4). (G & H) Kaplan-Meier curves showing progression-free survival (PFS) of patients with invasive breast carcinoma stratified by (G) COL8A1 and (H) LAMA5 expression levels. Patients were divided into high and low expression groups based on the median RNA-seq expression. Point-wise z-test using Greenwood’s standard error. Data source = Breast invasive carcinoma TCGA. ( I-K ) Spearman’s Correlation of Metastatic burden signature correlated to FAK-dependent ECM score (I ), COL8A1 ( J ) and LAMA5 ( K ) expression in triple negative breast cancer patients of the SCAN-B dataset . Spearman’s correlation of cytotoxic CD8 + T cells with COL8A1 ( L ) and LAMA5 ( M ) expression in triple negative breast cancer patients of the SCAN-B dataset. ( L & M ). Log 2 mRNA abundance of COL8A1 and LAMA5 in triple negative breast cancer patients with complete response (pCR) or residual disease (RD) in biopsies taken before treatment with either Palclitaxel and Pembrolizumab ( O ) or Palclitaxel, ABT888 and Carboplatin ( N ) in ISPY-2 trial.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Comparison, Activation Assay, In Vitro, Flow Cytometry, Expressing, Inhibition, Migration, RNA Sequencing

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: Mice carrying Cer2-OVA MMTV-PyMT metastatic lesions or without Cer2-OVA MMTV-PyMT injection were treated for two weeks with either vehicle control (0.5% HPMC) or VS-4718 (75 mg/kg). Quantification (H-score) of concentration of (A) Collagen VIIIα1 and (B) Laminin 5α in metastatic lesions and tumor adjacent areas at 5 weeks post implantation of Cer2-OVA MMTV-PyMT cells. Each dot represents a metastatic lesion. n = 5 mice/group using whole lung slide scans. One-way ANOVA for normal distributed with Tukey’s multiple comparison test. Mean ± SEM; ** = p < 0.01.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Injection, Control, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: (A) Representative images of collagen VIIIα1 in metastatic lesions. Scalebar = 50 μm. (B) Quantification (H-score) of collagen VIIIα1 in metastatic lesions and tumor adjacent areas. (C) Representative images of laminin 5 α in metastatic lesions and tumor adjacent areas. Scalebar = 50 μm. (D) Quantification (H-score) of laminin 5 α in metastatic lesions and tumor adjacent areas. Dots represent metastatic lesions. n = 5 mice/group using whole lung slide scans. Kruskal-Wallis test and Dunn’s multiple comparison for not normal distributed. Mean ± SEM; **** = p < 0.0001.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Comparison

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: (A) Mean migration velocity and (B) directional change rate/ average turning frequency of activated CD8⁺ T cells migrating on laminin 511 or collagen VIIIα1 (1 µg/cm²). T cells were CellTracker™ Deep Red-labeled and imaged for 6 h at 5-min intervals; tracks with >11 spots were analyzed using TrackMate . Laminin-511 reduced velocity and increased turning frequency, whereas collagen VIIIα1 had no significant effect. n = 3 independent experiments with 3-5 field of views per group. Unpaired t-tests. Mean ± SEM; **** = p < 0.0001.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Migration, Labeling

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: FAK inhibition reduces laminin-α5 and collagen VIIIα1 within metastatic lesions, weakening basement-membrane-derived physical and inhibitory barriers, thereby improving CD8⁺ T-cell infiltration, migration, tumor-cell engagement, and cytotoxic activity to promote metastatic regression.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Inhibition, Membrane, Derivative Assay, Migration, Activity Assay