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: 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

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Assessment of the binding properties of FAPI TMs to FAP-positive target cells. The surface expression of FAP on HT1080 hFAP (a) and MRC-5 (b) cells was assessed by flow cytometry using commercial anti-hFAP mAb and binding was detected using goat anti-mouse IgG Pacific Blue. Data are shown for one representative experiment out of three independent experiments with similar results ( n = 3). The number of hFAP surface antigens per cell was determined for the HT1080 hFAP and MRC-5 cell lines using QIFIKIT ( n = 1) (c). The FAP-specific binding of 64 Cu-radiolabeled FAPI TMs to HT1080 hFAP cells was evaluated in vitro after 1 h of incubation at 37 °C ( n = 1) (d). The binding of NODA-GA-FAPI-PEG 4/12/24 -E5B9 TMs and NODA-GA-FAPI-PEG 4 to HT1080 hFAP was investigated using flow cytometry. The target cells were incubated with 200 nM of either NODA-GA-FAPI-PEG 4 (black), NODA-GA-FAPI-PEG 4 -E5B9 (red), NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green), and binding was detected using anti-La mAb 5B9 (e). The data are shown for one representative experiment out of three independent experiments with similar results ( n = 3). The binding affinity of NODA-GA-FAPI-PEG 12 -E5B9 (blue) and NODA-GA-FAPI-PEG 24 -E5B9 (green) TMs to HT1080 hFAP (f, g) and MRC-5 (h) cell lines were further studied by flow cytometry using anti-La mAb 5B9 and the goat anti-mouse IgG Pacific Blue. Titration curves are plotted as the mean fluorescence intensity (MFI) ± SD from three independent experiments ( n = 3). K D : equilibrium dissociation constant; n.a.: not applicable.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Binding Assay, Expressing, Flow Cytometry, In Vitro, Incubation, Titration, Fluorescence

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Evaluation of cytotoxicity and immunostimulatory cytokine production by UniCAR T-cells redirected by FAPI TMs. UniCAR T-cells were cultivated together with HT1080 hFAP or MRC-5 for 16 h in the absence (dark gray) or presence of NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green) TM. Target cell lysis was measured using luciferase-based assays. UniCAR T-cells were incubated with target cells at varying E:T ratios in the absence or presence of NODA-GA-FAPI-PEG 12 -E5B9 or NODA-GA-FAPI-PEG 24 -E5B9 TM (a, b). Titration curves were obtained for UniCAR T-cells co-cultured with target cells at an E:T ratio of 5:1. The dotted lines indicate the level of target cell lysis in the absence of FAPI TM (c, d). The statistical significance of the differences between the dose‒response curves was determined by nonlinear regression analysis using the extra sum-of-squares F-test in GraphPad Prism. Cytokines released by UniCAR T-cells cultivated with target cells and FAPI-TMs were quantified by ELISA (e, f). All results are plotted as the mean ± SD from three independent donors and experiments ( n = 3), each performed with three technical replicates. EC 50 : half-maximal effective concentration.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Lysis, Luciferase, Incubation, Titration, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Assessment of in vivo killing of UniCAR T-cells redirected by NODA-GA-FAPI-PEG 12/24 -E5B9 TMs. Female NXG mice were injected with either HT1080 hFAP cells (black), HT1080 hFAP and UniCAR T-cells (gray), or HT1080 hFAP and UniCAR T-cells at an E:T ratio of 1:1 in combination with 1 nmol of either NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green). The bioluminescence signal of HT1080 hFAP cells across all groups is shown for days 0 (1 h post-injection), 1, 2, 3 and 6 d post-injection (a). Quantitative analysis of the bioluminescence was conducted, and the results were plotted as mean ± SD from five mice per group ( n = 5) (b). Statistical significance was assessed using two-way ANOVA with Greenhouse–Geisser correction and with Dunett ´ s multiple comparison test with respect to the control group injected with only HT1080 hFAP cells. Stars indicate statistical significance; p -values close to the cutoff (0.05) are shown on the graph.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: In Vivo, Injection, Comparison, Control

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Binding characteristics and pharmacokinetics of 64 Cu-radiolabeled FAPI TMs with different PEG spacer lengths. Maximum intensity projection (MIP) of NRMI nude mice bearing HT1080 (left flank, dashed white circles) and HT1080 hFAP tumor (right flank, white circles) after i.v. injection of 64 Cu-radiolabeled FAPI TMs ( a ). Time–activity curves (TAC, n = 2–3 mice per group) were analyzed for HT1080 hFAP tumor (b) and kidney (c) to determine tumor-to-kidney ratios over time (d). *: free urine.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Binding Assay, Drug discovery, Injection, Activity Assay