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: RSC Advances

Article Title: Inhibitor-conjugated harmonic nanoparticles targeting fibroblast activation protein

doi: 10.1039/c9ra05299b

Figure Lengend Snippet: Fig. 3 Association of BFO-PEG-FAPi NPs to FAP through target- specific interactions. HrFAP (FAP, 100 ng) was incubated for 2 h at 37 C with BFO-PEG-FAPi NPs (100 mg mL1) or BFO-PEG NPs (100 mg mL1) in the presence of Human Fibroblast Activation Protein alpha/ FAP Alexa Fluor® 594-conjugated Antibody (anti-FAP) (dilution 1 : 50). The samples were centrifuged (5 min, 10 000 rpm) and the superna- tants were transferred into a flat bottom 96-well plate. Fluorescence was measured in a multi-well plate spectrophotometer (lex/lem ¼ 590/645 nm). HrFAP incubated with anti-FAP and BFO-PEG-FAPi NPs incubated with anti-FAP were used as controls. Results were analyzed using a Student's t-test. Fluorescence intensity of BFO-PEG-FAPi NPs incubated with FAP and anti-FAP was significantly lower than BFO- PEG NPs incubated with FAP and anti-FAP and the fluorescence intensity of the controls (all comparisons ***p < 0.001).

Article Snippet: Human Fibroblast Activation Protein alpha/FAP Alexa Fluor® 594- conjugated Antibody was purchased from R&D systems (Abingdon, UK).

Techniques: Incubation, Activation Assay, Fluorescence, Spectrophotometry

Journal: RSC Advances

Article Title: Inhibitor-conjugated harmonic nanoparticles targeting fibroblast activation protein

doi: 10.1039/c9ra05299b

Figure Lengend Snippet: Fig. 4 Association of BFO-PEG NPs and BFO-PEG-FAPi NPs to hrFAP quantified by multiphoton multispectral microscopy. HrFAP (100 ng) was incubated for 2 h at 37 C with BFO-PEG NPs (100 mg mL1) (A) or BFO- PEG-FAPi NPs (100 mg mL1) (B) in the presence of Human Fibroblast Activation Protein alpha/FAP Alexa Fluor® 594-conjugated Antibody (dilution 1 : 5). SH and fluorescence signals were epi-collected upon focused excitation at 840 nm. The ratio of the area of conjugates emitting in both SH and fluorescence channels to the area of SH emitting structures was averaged from four distinct regions of each sample (635 635 mm). Error bars represent standard deviation from n ¼ 4 calculations.

Article Snippet: Human Fibroblast Activation Protein alpha/FAP Alexa Fluor® 594- conjugated Antibody was purchased from R&D systems (Abingdon, UK).

Techniques: Microscopy, Incubation, Activation Assay, Standard Deviation