Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) Human FGF-21 is cleaved by FAP. Recombinant human FGF-21 was digested by recombinant human FAP and visualized by Coomassie staining of SDS-Page gel. (B) Time course of FGF-21 digestion by FAP quantified by LC/MS extracted ion integration of peaks corresponding to intact (1–181) and cleaved (1–171) forms of FGF-21 (n = 3 per time point per group). Values are mean ± SEM with one phase decay curve fit superimposed. (C) FAP cleavage of FGF-21 is prevented by ARI-3099. ARI-3099 was pre-incubated with recombinant FAP for 30 minutes prior to addition of FGF-21. Reaction products were visualized by Coomassie staining of SDS-Page gel. (D) Recombinant PREP does not cleave FGF-21. Recombinant human PREP was added to recombinant FGF-21 and visualized by Coomassie staining of SDS-Page gel.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Recombinant, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) FAP cleaves human FGF-21 in mouse, monkey and human plasma. Recombinant FGF-21 was added to plasma to a final concentration of 1 μM in the presence or absence of 16 μM ARI-3099 followed by assessment of intact FGF-21 by sandwich ELISA (n = 3 per group). Values are mean ± SEM. *P < .05 ***P < .001 by ANOVA . (B) FAP activity of mouse, monkey and human plasma as assessed by the FAP-specific fluorescent substrate ARI-3144.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Clinical Proteomics, Recombinant, Concentration Assay, Sandwich ELISA, Activity Assay

Journal: Genes & Diseases

Article Title: Cancer-associated fibroblasts derived fibronectin extra domain A promotes sorafenib resistance in hepatocellular carcinoma cells by activating SHMT1

doi: 10.1016/j.gendis.2024.101330

Figure Lengend Snippet: CAFs promote sorafenib resistance by activating NF-κB in HCC cells. (A) Immunofluorescence analysis was performed to assess the expression of FAP and α-SMA on primary CAFs. Scale bars, 50 μm. (B) Co-culture with CAFs significantly reduced apoptosis of HepG2 and Huh7 upon sorafenib treatment. (blue bar: sorafenib-treated tumor cells cultured alone; red bar: sorafenib-treated tumor cells co-cultured with CAFs). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (C) Enrichment score of the “I-κB kinase/NF-κB signaling” in sorafenib-resistant group versus sorafenib-sensitive group after sorafenib treatment, analyzed by Gene Set Enrichment Analysis based on the RNA-seq data (after performing log 2 transformation, normalization, and mean value calculation) obtained from the GEO database (GSE182593). (D) Western blot analyses of the protein level of P-p65 in the indicated HCC cells with different treatments. Results are representative of three experiments. (E) Inhibition of the NF-κB signaling pathway in HCC cells induced apoptosis significantly upon sorafenib treatment (blue bar: sorafenib-treated group; red bar: sorafenib and BAY11-7082-treated (100 μM) group). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CAFs, cancer-associated fibroblasts; NF-κB, nuclear factor kappa B; HCC, hepatocellular carcinoma; FAP, fibroblast activation protein; α-SMA, alpha-smooth muscle actin.

Article Snippet: The cells on slides were fixed with 4% paraformaldehyde for 10 min and permeabilized in phosphate buffer saline for 20 min. Then, cells were blocked with goat serum at room temperature for 60 min and incubated with primary antibodies against FAP (fibroblast activation protein; R&D system, #FAB3715A, RRID: AB_2884010) (1:200) and α-SMA (alpha-smooth muscle actin; R&D system, #MAB1420, RRID: AB_262054) (1:200) for 2 h. Then, the cells on slides were reheated and incubated with the corresponding secondary antibody at 37 °C in the dark for 2 h. Nuclei were counter-stained with DAPI.

Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Transformation Assay, Western Blot, Inhibition, Activation Assay

Journal: Frontiers in Oncology

Article Title: Functional roles of FAP-α in metabolism, migration and invasion of human cancer cells

doi: 10.3389/fonc.2023.1068405

Figure Lengend Snippet: (A) Representative immunoblots of FAP-α with GAPDH used as loading control in 231 WT and 231-FAP cells cultured with (+) or without (–) FBS. -1d: 1 day without FBS. (B) Quantitative analysis of the immunoblots represented as band intensity ratio of FAP-α to GAPDH demonstrating significant increase of FAP-α in 231-FAP cells compared to 231 WT cells. Values represent Mean ± SEM (n = 2-3). n.d., non-detectable.

Article Snippet: After blocking in 5% milk-TBST (TBS Tween) or 5% BSA-TBST, the membrane was separately probed with antibodies against FAP-α (R&D Systems Inc AF3715.

Techniques: Western Blot, Control, Cell Culture

Journal: Cell Death & Disease

Article Title: Colorectal cancer cell intrinsic fibroblast activation protein alpha binds to Enolase1 and activates NF-κB pathway to promote metastasis

doi: 10.1038/s41419-021-03823-4

Figure Lengend Snippet: A Immunofluorescence analysis of FAP location in HCT116-FAP and HCT8-FAP cells. Error bar = 30 μm. B ELISA assay detects the concentration of FAP in media of HCT116-FAP, HCT8-FAP, HCT116-Ctrl, and HCT8-Ctrl cells. Ctrl: control; FAP: FAP overexpression. C Migration assays of HCT116 and HCT8 cells treated with control medium and FAP-overexpressed medium. Error bar = 100 μm. * p < 0.05, *** p < 0.001. The CM was added into the upper chamber. CM: conditioned medium. D Invasion assays of HCT116 and HCT8 cells treated with control medium and FAP-overexpressed medium. Error bar = 100 μm. * p < 0.05, *** p < 0.001. The CM was added into the upper chamber. E Migration assays of HCT116 and HCT8 cells treated with different concentration of recombinant FAP. Error bar = 100 μm. ** p < 0.01, *** p < 0.001. F Invasion assays of HCT116 and HCT8 cells treated with different concentration of recombinant FAP. Error bar = 100 μm. ** p < 0.01, *** p < 0.001.

Article Snippet: FAP concentration was detected using FAP DuoSet ELISA kit (R&D Systems, DY3715) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control, Over Expression, Migration, Recombinant

Journal: Current Issues in Molecular Biology

Article Title: Oncological Safety of High Hydrostatic Pressure Treatment: Effects on Cancer-Associated Fibroblast-like Transdifferentiation of Adipose Stromal Cells

doi: 10.3390/cimb48010091

Figure Lengend Snippet: Protein expression analysis of cancer-associated fibroblast (CAF) markers in stimulated human adipose stromal cells (hASCs). HASCs were incubated for 96 h with tumor-conditioned medium (TCM), 0 MPa-CM, or 300 MPa-CM derived from HNSCC16 or UTSCC14 cells, TGF-β1 (10 ng/mL), or serum-free control medium, respectively. ( A ) α-SMA protein expression was determined by Western blot analysis. Representative Western blot membrane with protein bands of α-SMA following incubation with primary and secondary antibody is shown. Precision Plus Protein™ All Blue prestained standard served as molecular weight marker. Densitometric quantification of α-SMA protein expression was evaluated and total protein normalization was performed using Bio-Rad Stain-Free™ technology. Data represents n = 4 independent donors (mean ± SD). Results are presented as relative protein expression normalized to the serum-free control (set to 1; illustrated as dashed lines). ( B ) FAP levels were determined by ELISA in hASCs from six independent donors after 96 h of stimulation with the respective conditioned media. Results are presented as relative protein expression normalized to the serum-free control (set to 1; illustrated as dashed lines). Statistical significance was assessed using predefined post hoc comparisons following one-way ANOVA or Kruskal–Wallis testing. Differences were evaluated relative to the control group or between specific treatment conditions as indicated in the figures. Statistically significant differences are indicated by asterisks (* p < 0.05, ** p < 0.01).

Article Snippet: Determination of FAP concentration was performed on cell lysates using the DuoSet Human FAP enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Derivative Assay, Control, Western Blot, Membrane, Molecular Weight, Marker, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip

doi: 10.1016/j.xcrm.2024.101549

Figure Lengend Snippet: Lung tumor-on-chip (ToC) platforms for personalized immunotherapy response profiling (A) Workflow for lung ToC generation and analysis. Cancer cells, T cells, and fibroblasts are isolated from the tumor and co-cultured embedded in a biomimetic collagen gel within microfluidics devices. The microfluidics setup allows us to perfuse the immunotherapy drugs into the ToC, which is live imaged by video microscopy. Automated advanced methods of image analysis are used to measure the anti-cancer cytotoxic activity and the kinematics of immune cells. (B) Representative confocal images of the reconstituted 3D lung tumor microenvironment. Autologous cancer cells (IGR-Heu) and CD8 + CTLs (H5B) are labeled in red and blue (Cell Trace), respectively. CAFs (heterologous) are labeled in green. a: top view. b: lateral view. c: magnified view. (C) Patients’ clinical data. N/D, not determined. (D) Representative immunostaining of human lung adenocarcinoma. Top: co-immunostaining of pancytokeratin (brown), highlighting tumor cells, and FAP (red), highlighting CAFs, with a magnified view on the right, used for manual counting. Bottom: CD8 immunostaining before (left) and after (right) supervised automated quantification using QuPath software (red, CD8 + T lymphocytes; blue, CD8 − lymphocytes and tumor cells). (E) Density of tumor cells, FAP + CAFs, and CD8 + T cells for all patients and cell ratio calculation.

Article Snippet: The used antibodies were: CD29 AF700 (Biolegend, #303020) and FAP (R&D Systems, #MAB3715-500), conjugated to APC using the Zenon Mouse IgG1 Labeling Kit (Invitrogen, #Z25051).

Techniques: Isolation, Cell Culture, Microscopy, Activity Assay, Labeling, Immunostaining, Software

Journal: Cell Reports Medicine

Article Title: Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip

doi: 10.1016/j.xcrm.2024.101549

Figure Lengend Snippet:

Article Snippet: The used antibodies were: CD29 AF700 (Biolegend, #303020) and FAP (R&D Systems, #MAB3715-500), conjugated to APC using the Zenon Mouse IgG1 Labeling Kit (Invitrogen, #Z25051).

Techniques: Control, Labeling, Purification, Recombinant, Activation Assay, Cell Isolation, Red Blood Cell Lysis, Software, Flow Cytometry, Polymer

Journal: Frontiers in Drug Discovery

Article Title: Mimicking the immunosuppressive impact of fibroblasts in a 3D multicellular spheroid model

doi: 10.3389/fddsv.2024.1427407

Figure Lengend Snippet: FIGURE 5 Increase in FAP surface expression in spheroids compared to monolayer culture. For flow cytometry, cells were stained with two dilutions of PE- conjugated FAP antibody and SYTOX™Red dead cell stain. Measurements with PBS/1% BSA served as a negative control. (A) Surface antibody binding in 2D and 3D mono-culture of MDA-MB-231 fluc GFP cells, mono-culture of WS1 cells, and co-culture of both cell lines after 72 h. Only the viable cells were examined for the event analysis. The cells were separated into GFP-positive (GFP+) and GFP-negative (GFP-) populations. (B) The % fluorescence signal of the median of viable GFP- cells was calculated by normalizing to the respective negative controls. Statistical significance was calculated using a Kruskal–Wallis one-way ANOVA in GraphPad Prism v9.1.2; *p < 0.05; ***p < 0.0005. (mean ± SEM, n = 3; n = 2 2D mono-culture WS1 1:50 AB).

Article Snippet: Thereafter, cells were washed twice with cold 1x DPBS supplemented with 1% BSA and stained with a PE-labeled antihuman FAPα antibody (Cat. No. FAB3715P, R&D Systems) or with a TAMRA-dextran-oncoFAP construct for 30 min on ice in the dark.

Techniques: Expressing, Cytometry, Staining, Negative Control, Binding Assay, Co-Culture Assay

Journal: Frontiers in Drug Discovery

Article Title: Mimicking the immunosuppressive impact of fibroblasts in a 3D multicellular spheroid model

doi: 10.3389/fddsv.2024.1427407

Figure Lengend Snippet: FIGURE 6 Strong IFNγ release after simultaneous FAP and CD3 binding through oncoFAP-bearing construct. (A) Labeling of alkyne-dextran-cadaverine with NHS-TAMRA and subsequent attachment of multiple oncoFAP moieties via CuAAC. Created with BioRender.com. (B) The % fluorescence signal was calculated by normalizing the median of viable GFP- cells stained with TAMRA-dextran-oncoFAP to the median viable GFP- unstained cells measured by flow cytometry. The graph was created in GraphPad Prism v9.1.2. (mean ± SEM, n = 2). (C) mTG mediated conjugation of α-CD3-scFv-Fc protein and alkyne-dex-cadaverine. Subsequently, oncoFAP was introduced via CuAAC. Created with BioRender.com. (D) The α-CD3-scFv-Fc-dextran-oncoFAP construct (abbreviated as construct) was added together with naïve T cells on day 2. As control α-CD3-scFv-Fc (abbreviated as CD3) alone was tested. IFNγ concentration [pg/mL] was measured in triple-culture after 48 h of treatment. The dotted line represents the lowest standard. Concentrations were calculated with GraphPad Prism v9.1.2 and statistical significance was calculated using a Kruskal–Wallis one-way ANOVA in GraphPad Prism v9.1.2; ***p < 0.001. (mean ± SEM; n = 3 with 8 different T cell donors for α-CD3-scFv-Fc-Dex-oncoFAP, n = 1 with 3 different T cell donors for α-CD3-scFv-Fc).

Article Snippet: Thereafter, cells were washed twice with cold 1x DPBS supplemented with 1% BSA and stained with a PE-labeled antihuman FAPα antibody (Cat. No. FAB3715P, R&D Systems) or with a TAMRA-dextran-oncoFAP construct for 30 min on ice in the dark.

Techniques: Binding Assay, Construct, Labeling, Staining, Cytometry, Conjugation Assay, Control, Concentration Assay