Journal: Nature Communications

Article Title: Targeted immunotherapy rescues pulmonary fibrosis by reducing activated fibroblasts and regulating alveolar cell profile

doi: 10.1038/s41467-025-59093-7

Figure Lengend Snippet: A Schematic diagram of fibrosis model establishment and treatment strategy in young or aged mice. B , C Young mouse BW changes ( B ) and survival curve ( C ) of control and BLM groups treated with IgG/ β LNP-FAPCAR 2.2, CD5/ β LNP-FAPCAR 2.2 (10 μg) and saline ( n = 10). The survival rate was analyzed using the log-rank (Mantel-Cox) test. D Lung coefficients (lung weight (mg)/BW (g)) ( n = 5,5,10,10,10,7). E tdTomato reporter gene expressed in T cells of spleen and lung at 24 h after the second CD5/ β LNP-FAPCAR 2.2 injection (Scar bar: 10 μm). F Flow cytometry plots for FAPCAR expression in CD3 + pulmonary T cells of BLM+Saline and CD5/ β LNP-FAPCAR 2.2 treatment groups and the ratio of FAPCAR expression to CD3 + pulmonary T cells ( n = 4). G Flow cytometry analysis of FAP + Pdgfra + fibroblasts in BLM+Saline and CD5/ β LNP-FAPCAR 2.2 treatment groups, ratio of FAP + Pdgfra + fibroblasts to all lung cells, and ratio of Pdgfra + fibroblasts (both FAP + and FAP — ) to all lung cells ( n = 4). H , I Representative micro-CT images and mean HU density ( H ), and lung air volume (mm 3 ) ( I ) computed according to CT results at postoperative 28 days ( n = 4,4,7,7,7,7). J Digital photos of lung tissue. ‘n’ means independent biological replications. The values are the means ± SDs. * P < 0.05. P -value determined by one-way ANOVA followed by Tukey’s multiple comparisons test ( D , F , H , I ) and two tailed unpaired Student’s t -test ( G ). (BW: body weight; HU: Hounsfield unit). Source data are provided as a Source Data file. Created in BioRender ( A , B ) .

Article Snippet: FVS 700 (live/death dye, #564997, BD Bioscience, CA, USA), CD3e (#562600, BD Bioscience), CD8a (#557654, BD Bioscience), F4/80 (#565411, BD Bioscience), CD86 (#558703, BD Bioscience), CD163 (#12-1631-82, Thermo Fisher), FAP (#FAB9727G, R&D system) and Pdgfra (#12-1401-81, Thermo Fisher) were used in experiments.

Techniques: Control, Saline, Injection, Flow Cytometry, Expressing, Micro-CT, Two Tailed Test

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: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: mRNA quantitative expression versus immunohistochemistry. (A) DCSIGN, CD163 M2, α‐smooth muscle actin (α‐SMA), fibroblast‐specific protein 1 (FSP1) and fibroblast activation protein (FAP) immunohistochemistry. Representative pictures of high and low protein expression levels of DCSIGN, CD163 M2 macrophage and α‐SMA, FSP1 and FAP cancer‐associated fibroblast (CAF) markers in human tumor samples. Arrows indicate positive cells. Original magnification, ×20. (B) Association between protein and mRNA expression levels. Statistical association between protein and mRNA expression levels of FSP1 and FAP, CAF markers. In addition, a clear trend toward statistical association is observed between protein and mRNA expression levels of DCSIGN and CD163, M2 macrophage markers and the α‐SMA, CAF marker. Bar charts show the average (±confidence interval) of mRNA expression levels of each gene in the low and high protein expression groups.

Article Snippet: Immunohistochemistry analysis and antibodies Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Immunohistochemistry, Activation Assay, Marker

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: Kaplan–Meier curves between expression levels of cancer‐associated fibroblast (CAF) and M2 macrophage markers (individually) and disease‐free survival (DFS) in colorectal cancer patients. Association between CD163, a M2 macrophage marker (A), and α‐smooth muscle actin (α‐SMA) (B), fibroblast‐specific protein 1 (FSP1) (C) and fibroblast activation protein (FAP) (D) CAF markers with DFS. “Low expression” refers to a low‐expression tertile for α‐SMA and low‐ and medium‐expression tertiles for CD163, FSP1 and FAP.

Article Snippet: Immunohistochemistry analysis and antibodies Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Marker, Activation Assay

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: Kaplan–Meier curves between expression levels of cancer‐associated fibroblast (CAF) and M2 macrophage markers (individually) and overall survival in colorectal cancer patients. Association of CD163, a M2 macrophage marker (A), and fibroblast‐specific protein 1 (FSP1) (C), a CAF marker, with overall survival. α‐Smooth muscle actin (α‐SMA) (B) and fibroblast activation protein (FAP) (D) expression levels, CAF markers, showed a trend towards an association with overall survival.

Article Snippet: Immunohistochemistry analysis and antibodies Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Marker, Activation Assay

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Increased perivascular fibrosis and pro-fibrotic cellular transition in intramyocardial blood vessels in myocardial infarction patients

doi: 10.1016/j.jmccpl.2024.100275

Figure Lengend Snippet: Gender-specific quantification of intramyocardial perivascular fibrosis, FAP expression and pro-fibrotic vascular cellular transition in MI patients. Shown are the quantifications of intramyocardial perivascular fibrosis (A), FAP expression (B), fibroblast-like transitioning of SMA + S100A4+ vascular smooth muscle cells (C) and CD31 + S100A4+ endothelial cells (D) in male (M) versus female (F) controls (n = 14), acute-phase (n = 24) and subacute-phase MI (n = 12) patients. Data are presented as median [IQR] in the graph. Each distinct point corresponds to the scores of individual patient: circles for controls, squares for acute-phase MI patients, triangles for the border zones and inverted triangles for the infarct cores of subacute-phase MI patients. A Mann-Whitney U test was used for statistical comparisons; Error bars represent IQR, ns indicated no statistic difference.

Article Snippet: For a CD31 + SMA + either S100A4 or FAP triple staining, the sections were then incubated with mouse-anti-human CD31 antibody (Dako; M0823, 1:200 dilution), mouse-anti-human SMA antibody (Dako; M0851, 1:200 dilution) and rabbit-anti-human S100A4 antibody (abcam; ab197896, 1:2000 dilution) or rabbit-anti-human FAP antibody (NBP2-66844; NOVUS, diluted 1:100) overnight at 4°C.

Techniques: Expressing, MANN-WHITNEY

Journal: Journal of Liver Cancer

Article Title: Isolation and characterization of cancer-associated fibroblasts in the tumor microenvironment of hepatocellular carcinoma

doi: 10.17998/jlc.2023.04.30

Figure Lengend Snippet: CAFs robustly express FAP and α-SMA, but do not express EpCAM, E-cadherin, and PECAM-1. (A) Single-stain flow cytometry was performed for SMA, FAP, AFP, and EpCAM in CAFs and non-tumor fibroblasts isolated from patients with HCC. (B) Similarly, single-stain flow cytometry was performed for the same markers in HCC cells. Graphs show the expression of each marker. (C) Expression of α-SMA, vimentin, and E-cadherin in CAFs, non-tumor fibroblasts, and hepatoma cell lines was analyzed using immunoblotting. (D) Expression of E-cadherin, PECAM-1, vimentin, and GAPDH in CAFs, non-tumor fibroblasts, and HUVECs was analyzed using immunoblotting. All means were statistically analyzed; * P <0.05, ** P <0.01, *** P <0.001. CAF, cancer-associated fibroblast; FAP, fibroblast activation protein-α; SMA, alpha-smooth muscle actin; EpCAM, epithelial cell adhesion molecule; MFI, mean fluorescence intensity; NF, non-tumor fibroblast; PECAM-1, platelet and endothelial cell adhesion molecule 1; HCC, hepatocellular carcinoma.

Article Snippet: The following antibodies were used: allophycocyanin-conjugated anti-human alpha-smooth muscle actin (α-SMA) (R&D system, Minneapolis, MN, USA), anti-human fibroblast activation protein (FAP) (R&D system), phycoerythrinconjugated anti-human alpha-fetoprotein (BD Biosciences, Franklin Lakes, NJ, USA), and anti-human epithelial cell adhesion molecule (EpCAM) (BD Biosciences).

Techniques: Staining, Flow Cytometry, Isolation, Expressing, Marker, Western Blot, Activation Assay, Fluorescence

Journal: Immunity, inflammation and disease

Article Title: Dendritic cells infected with recombinant adenoviral vector encoding mouse fibroblast activation protein-α and human livin α exert an antitumor effect against Lewis lung carcinoma in mice.

doi: 10.1002/iid3.1011

Figure Lengend Snippet: FIGURE 1 Identification of rAd‐infected DCs in vitro. (A–C) rAd‐FAP, rAd‐hlivin α, and rAd‐FAP/hlivin α were constructed using mouse FAP cDNA and human livin α cDNA, and then transduced into mouse bone marrow‐derived DCs, respectively. For evaluation of transduction efficacy, western blot was performed to measure protein expressions of FAP and livin α in cells, with GAPDH functioned as a loading control. ***p < .001, versus 1#; +++p < .001, versus 2#; ^^^p < .001, versus 3#. DCs, dendritic cells; FAP, fibroblast activation protein‐α; rAd‐FAP, recombinant adenoviral vector encoding mouse FAP cDNA; rAd‐hlivin α, recombinant adenoviral vector encoding human livin α cDNA.

Article Snippet: Following incubation with blocking buffer (P0231, Beyotime), Western blot was performed by incubating the membranes with primary antibodies against FAP (NB110‐85534, 88 kDa, Novus Biologicals), livin α (NB100‐56145, 33 kDa, Novus Biologicals), CD45 (ab10558, 147 kDa, Abcam), CD31 (ab281583, 82 kDa, Abcam), α‐SMA (ab5694, 42 kDa, Abcam), and internal control GAPDH (ab8245, 37 kDa, Abcam) at 4°C overnight.

Techniques: Infection, In Vitro, Construct, Derivative Assay, Transduction, Western Blot, Control, Activation Assay, Recombinant, Plasmid Preparation

Journal: Immunity, inflammation and disease

Article Title: Dendritic cells infected with recombinant adenoviral vector encoding mouse fibroblast activation protein-α and human livin α exert an antitumor effect against Lewis lung carcinoma in mice.

doi: 10.1002/iid3.1011

Figure Lengend Snippet: FIGURE 2 Identification of LLC tumor‐isolated CAFs and determination on expressions of FAP and livin α in LLC. (A–C) Western blot was performed to measure protein expressions of CD45, CD31, α‐SMA, and FAP in CAFs isolated from LLC‐bearing mice as well as the protein expression of livin α in mouse LLC tumors. GAPDH was used as the loading control. +++p < .001, versus lysate; ^^^p < .001, versus tumor; ##p < .001, versus Para. CAFs, cancer‐associated fibroblasts; LLC, Lewis lung carcinoma; Para, paracancerous tissues.

Article Snippet: Following incubation with blocking buffer (P0231, Beyotime), Western blot was performed by incubating the membranes with primary antibodies against FAP (NB110‐85534, 88 kDa, Novus Biologicals), livin α (NB100‐56145, 33 kDa, Novus Biologicals), CD45 (ab10558, 147 kDa, Abcam), CD31 (ab281583, 82 kDa, Abcam), α‐SMA (ab5694, 42 kDa, Abcam), and internal control GAPDH (ab8245, 37 kDa, Abcam) at 4°C overnight.

Techniques: Isolation, Western Blot, Expressing, Control

Journal: Immunity, inflammation and disease

Article Title: Dendritic cells infected with recombinant adenoviral vector encoding mouse fibroblast activation protein-α and human livin α exert an antitumor effect against Lewis lung carcinoma in mice.

doi: 10.1002/iid3.1011

Figure Lengend Snippet: FIGURE 3 The antitumor effect of rAd‐FAP/hlivin α‐transduced DCs on LLC in mice. (A and B) Mice were separately immunized with rAd‐EGFP‐transduced DCs, rAd‐FAP‐transduced DCs, rAd‐hlivin α‐transduced DCs, and rAd‐FAP/hlivin α‐transduced DCs (5 × 105 cells/ mouse) on Day 8 postinjection with LLC cells, and tumor volume (mm3) was calculated every 2 days for a total 24 days. (C) Mouse survival rate was observed for 80 days. (D) Splenic lymphocytes were obtained from mice on Day 7 after receiving injection with rAd‐EGFP‐ transduced DCs, rAd‐FAP‐transduced DCs, rAd‐hlivin α‐transduced DCs or rAd‐FAP/hlivin α‐transduced DCs. The cytotoxic effect of splenic lymphocytes on LLC‐derived CAFs was analyzed using Cytotox96 Non‐Radioactive Cytotoxicity Assay Kit. ***p < .001, versus 1#; +p < .05, +++p < .001, versus 2#; ^p < .05, ^^p < .01, ^^^p < .001, versus 3#. CAFs, cancer‐associated fibroblasts; DCs, dendritic cells; LLC, Lewis lung carcinoma; FAP, fibroblast activation protein‐α; rAd‐FAP, recombinant adenoviral vector encoding mouse FAP cDNA; rAd‐hlivin α, recombinant adenoviral vector encoding human livin α cDNA.

Article Snippet: Following incubation with blocking buffer (P0231, Beyotime), Western blot was performed by incubating the membranes with primary antibodies against FAP (NB110‐85534, 88 kDa, Novus Biologicals), livin α (NB100‐56145, 33 kDa, Novus Biologicals), CD45 (ab10558, 147 kDa, Abcam), CD31 (ab281583, 82 kDa, Abcam), α‐SMA (ab5694, 42 kDa, Abcam), and internal control GAPDH (ab8245, 37 kDa, Abcam) at 4°C overnight.

Techniques: Injection, Derivative Assay, Cytotoxicity Assay, Activation Assay, Recombinant, Plasmid Preparation