Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: Establishment and characterization of GC (Gemcitabine and Cisplatin)-resistant bladder cancer cell lines and identification of resistance-related proteins. ( A ) GC-resistant T24-R and UC3-R cell lines were generated by gradually increasing GC concentrations. Created in BioRender ( https://BioRender.com ). ( B ) Dose-response curves and calculated half-maximal inhibitory concentration (IC50) values for cisplatin (upper panel) and gemcitabine (lower panel) in parental (T24, UC3) and GC-resistant (T24-R, UC3-R) cell lines. Data are presented as the mean ± SD from at least three independent experiments. ( C ) Apoptosis rates of parental and resistant cell lines after treatment with cisplatin, as determined by flow cytometry. Data are presented as the mean ± SD ( n ≥ 3). ( D ) Transcriptomic and proteomic analyses identified FN1 (Fibronectin), EEF1A2 (Eukaryotic Translation Elongation Factor 1 Alpha 2), MRC2 (Mannose Receptor C-Type 2), RAB6B (RAB6B, Member RAS Oncogene Family), THBS1 (Thrombospondin 1), DYSF (Dysferlin), TMOD1 (Tropomodulin 1), NES (Nestin), and APOE (Apolipoprotein E) as overexpressed in GC-resistant cells. ( E ) RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) and Western blot confirmed FN1 overexpression in T24-R and UC3-R. ( F ) FN1 staining was stronger in GC-resistant bladder cancer tissues (Chemotherapy Sensitive Group: n = 6; Chemotherapy Resistant Group: n = 6). For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, ** p < 0.01

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Generated, Concentration Assay, Flow Cytometry, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Staining

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: ITGB4 is critical for FN1-mediated chemotherapy resistance in bladder cancer cells. ( A ) Differential expression analysis revealed that ITGB4 (highlighted by the red box) was significantly overexpressed in T24-R cells compared to T24, suggesting a role in FN1-mediated resistance. ( B ) Structural modeling shows multiple binding sites between FN1 (blue ribbon) and ITGB4 (green ribbon). ( C ) The interaction between FN1 and ITGB4 had a binding score of −318.75 with a confidence score of 96%, indicating a stable interaction. ( D ) The Co-IP experiment confirmed that there is a mutual binding interaction between FN1 and ITGB4. ( E ) Adding rFN1 to resistant strains increased FAK (Y397) phosphorylation and inhibited apoptosis, whereas ITGB4 silencing reversed these effects, highlighting the dependency of FN1-mediated resistance on ITGB4 expression and activation. For panels ( A , C , E ), a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Quantitative Proteomics, Binding Assay, Co-Immunoprecipitation Assay, Phospho-proteomics, Expressing, Activation Assay

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 silencing enhances cisplatin-induced apoptosis in bladder cancer cells. ( A ) FN1 knockdown efficiency in T24-R and UC3-R cells was confirmed by Western blot analysis. ( B ) FN1 knockdown efficiency in T24-R and UC3-R cells was confirmed by RT-qPCR analysis. ( C ) Silencing FN1 significantly increased apoptosis in the resistant cell lines by TUNEL staining. ( D ) Silencing FN1 reduced the IC50 of cisplatin in the resistant cell lines by CCK-8 assay. ( E ) In resistant cells, FN1 knockdown induced the expression of pro-apoptotic mediators, including BAX and cleaved-caspase-3, but suppressed levels of the anti-apoptotic protein Bcl-2. ( F ) Cell apoptosis rates were quantified by flow cytometry. For all panels, a t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, ** p < 0.01

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, TUNEL Assay, Staining, CCK-8 Assay, Expressing, Flow Cytometry

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 silencing inhibits tumor growth in vivo , enhancing cisplatin sensitivity. ( A ) Representative images of tumors from each treatment group. ( B ) Tumor volume growth curves over time measured in the T24-R xenograft model. ( C ) Apoptosis in tumor tissues was detected by TUNEL staining. ( D ) Immunohistochemical analysis showed decreased FN1 expression in tumor sections from the FN1 knockdown group. For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. * p < 0.05, *** p < 0.001

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: In Vivo, TUNEL Assay, Staining, Immunohistochemical staining, Expressing, Knockdown

Journal: Oncology Research

Article Title: The FN1-ITGB4 Axis Drives Acquired Chemoresistance in Bladder Cancer by Activating FAK Signaling

doi: 10.32604/or.2025.072084

Figure Lengend Snippet: FN1 regulates FAK (Y397) phosphorylation and mediates cisplatin resistance in bladder cancer cells. ( A ) Silencing FN1 in resistant cell lines reduced the phosphorylation of FAK (Y397) as detected by Western blot. ( B ) T24 and UC3 parental and resistant cells were treated with a fixed dose of cisplatin along with a gradient of rFN1 for 48 h. Phosphorylation of FAK (Y397) was assessed by Western blot. Resistant cells (T24-R, UC3-R) showed sensitivity to rFN1 at lower concentrations under cisplatin stress. ( C ) Silencing FN1 in resistant cell lines reduced the phosphorylation of FAK (Y397) as detected by immunofluorescence. ( D ) rFN1 addition increased resistance index in resistant strains. ( E ) The addition of rFN1 reduced apoptosis in resistant strains. For all panels, a t -test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical significance was set at p < 0.05. ns, not significant, * p < 0.05, *** p < 0.001

Article Snippet: The resulting sample was subsequently analyzed using the Human FN1 Quantikine ELISA Kit (R&D Systems, DY1918, Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence

Journal: Molecular Cancer

Article Title: c-Rel drives pancreatic cancer metastasis through fibronectin-integrin signaling-induced isolation stress resistance and EMT

doi: 10.1186/s12943-025-02486-5

Figure Lengend Snippet: A Representative images of Masson's trichrome (p<0.0001) and Picosirius-red stained tumors and their quantification (p=0.0248) B Representative FN1-IHC images (p=0.0186). GCKP-1 reflects fibrillar FN1 deposition around cancer cells individually. GCKP-2 reflects the perinuclear FN1 signal intracellularly, as shown with arrowheads. C Western blot for FN1 in bulk tumors and isolated cancer cell lysates. The expression is normalized to HSP90 (tissues p=0.0002, cells n.s.) D Mouse serum FN1 ELISA results (One-way ANOVA for a linear trend p=0.0366) E Fibronectin adhesion assay. The number of cells attached to the FN1-coated wells was normalized to the seeding number (One-way ANOVA test for a linear trend p=0.0242) F Heatmap of the differentially expressed genes in the RNA-seq analysis of CCKP-CKP-GCKP cells. G Principal component analysis (PCA) plot of the cells used for the RNAseq analysis. H Venn diagrams illustrating the number of common genes up- or down-regulated in the given comparison pairs. I GSEA of the DEGs between the given comparison pairs. The red stars indicate enrichments related to ECM remodeling, whereas the blue stars indicate EMT-related pathways. Unless otherwise indicated, for all analyses, ordinary one-way ANOVA was used. ANOVA p values are given in the figure legends. Tukey's multiple comparison tests are shown in the figures

Article Snippet: The serum samples were diluted 1:8000 and analyzed with a Mouse Fibronectin ELISA Kit (Novus, NBP2-60517).

Techniques: Staining, Western Blot, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Adhesion Assay, RNA Sequencing, Comparison

Journal: Molecular Cancer

Article Title: c-Rel drives pancreatic cancer metastasis through fibronectin-integrin signaling-induced isolation stress resistance and EMT

doi: 10.1186/s12943-025-02486-5

Figure Lengend Snippet: A Colony-forming frequency was assessed by the ability of cells to form an organoid in Matrigel (one-way ANOVA, p=0.0015). Tukey's multiple comparisons test results are shown in the figure. B Representative microscopy images of spheroids formed under nonadherent conditions. The average spheroid diameter was quantified and analyzed (genotype factor p=0.0227). C Representative spheroid images and their diameter quantification. The spheroid medium was supplemented with either human plasma fibronectin (pFN1) or cellular fibronectin with EDB domain + type II domains 8−14 (EDB cFN1.3) (treatment factor p= n.s.) D Surface expression of selected proteins on spheroids as assessed by flow cytometry analysis (one-way ANOVA test for CD61 p=0.0244, CD133, EpCAM, CD44, CXCR4, Sca1 p= n.s.). Tukey's multiple comparisons test results are shown in the figure. E Representative macroscopic and microscopic images of the transplanted mouse lungs. The number of cell lines used per genotype are CKP=5, GCKP=5 and FNGCKP=4. Each cell line was injected two times to two individual mice. Nested one-way ANOVA was used for both analyses. Tukey's multiple comparisons test results are shown in the figure. Unless otherwise indicated, for all analyses, two-way ANOVA was used. ANOVA p values are given in the figure or their legends. Šídák's multiple comparison tests between genotypes are shown in the figures. F Schematic depicting the function of c-Rel in PDAC pathophysiology. c-Rel is an oncogenic factor involved in PDAC survival and metastasis. Increased c-Rel expression remodels the ECM by converting the collagen-rich stroma to a fibronectin-rich stroma. Cancer cells undergo epithelial-to-mesenchymal transition, increasing EMP by reducing the surface expression of E-cadherin and contractility via Src and Stat3 phosphorylation. Once isolation stress is induced under nonadherent conditions, c-Rel induces a niche enriched with fibronectin, coupled with increased Itgb3, Itga5 and Itgav expression. These changes are also supported by EMP, where c-Rel directly induces EMT-TFs such as Snai1, Snai2, Zeb2, and Tgfb . Increased tolerance to isolation stress ultimately drives metastasis. Created in BioRender. Kabacaoglu, D. (2025)

Article Snippet: The serum samples were diluted 1:8000 and analyzed with a Mouse Fibronectin ELISA Kit (Novus, NBP2-60517).

Techniques: Microscopy, Clinical Proteomics, Expressing, Flow Cytometry, Injection, Comparison, Phospho-proteomics, Isolation

Journal: Advanced Science

Article Title: Targeting Itga8 Mitigates Neurogenic Bladder Fibrosis Driven by Trem2⁺ Macrophage‐Derived Fn1 via FAK/RhoA/ROCK Signaling

doi: 10.1002/advs.202510631

Figure Lengend Snippet: Co‐culture experiments demonstrate that Fn1 derived from Trem2⁺ macrophages activates fibroblasts via Itga8. A) Schematic representation of the co‐culture experiment design. B) Flow cytometry confirms that IL‐17a induces the differentiation of BMDM into Trem2⁺ macrophages in vitro. C) Quantification of the percentage of Trem2 + macrophage in (B), n = 3 per group. D) Representative Western blot images of in vitro‐induced Trem2⁺ macrophages. E) ELISA analysis of Fn1 expression in the conditioned medium. F) Representative Western blot images of primary bladder fibroblasts treated with conditioned medium from IL‐17a‐induced BMDM. G,H) qRT‐PCR (G) and Western blot (H) analysis of Fn1 expression at the mRNA and protein levels following transfection with Fn1 siRNA. I) ELISA analysis of Fn1 expression in the culture supernatant following transfection with Fn1 siRNA. J) Representative Western blot images of fibroblasts treated with conditioned medium from IL‐17a‐induced BMDM transfected with either si‐NC or si‐Fn1. K) Representative images of the contraction assay in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with the conditioned medium. L) Quantification of cell contraction in (K), n = 4 per group. M) Representative Western blot images of fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with conditioned medium from IL‐17a‐induced BMDM. N) Representative images of the scratch assay in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with Fn1. O) Statistical analysis of the wound area over time in (N), n = 3 per group. P) Representative images of immunofluorescence staining in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with Fn1. Q) Quantification of α‐SMA/F‐actin in (P), n = 3 per group. R,S) qRT‐PCR (R) and Western blot (S) analysis of Trem2 expression at the mRNA and protein levels following transfection with OE‐Trem2. T) Flow cytometry confirms that IL‐17a induces the differentiation of BMDM into Trem2⁺ macrophages in vitro. U) Quantification of the percentage of Trem2 + macrophage in (T), n = 3 per group. V) Representative Western blot images of fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with conditioned medium from Trem2‐overexpressing BMDM. Mφ = Macrophage. Data represent mean ± SD. Unpaired two‐tailed t‐test was used for C, E, G, I, R, and U. One‐way ANOVA was used for L, and Q. Two‐way ANOVA was used for O. * P < 0.05, ** P < 0.01, *** p < 0.001. Figure was created in BioRender. Wang, J. (2025) https://BioRender.com/kp68v7m .

Article Snippet: The concentration of Fn1 in the supernatant of macrophage culture medium was determined using an ELISA kit (EK0350, Boster, Wuhan, China).

Techniques: Co-Culture Assay, Derivative Assay, Flow Cytometry, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Transfection, Contraction Assay, Transduction, Wound Healing Assay, Immunofluorescence, Staining, Two Tailed Test