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 Assay, Transformation Assay, Western Blot, Inhibition, Activation Assay

Journal: Molecular Cancer

Article Title: CREB3L1 promotes tumor growth and metastasis of anaplastic thyroid carcinoma by remodeling the tumor microenvironment

doi: 10.1186/s12943-022-01658-x

Figure Lengend Snippet: CREB3L1 is associated with ECM signaling in thyroid cancer. A GSEA determined the enrichment differences of biological processes depending on the CREB3L1 expression. B-C The evaluated activity scores of the extracellular matrix (ECM) and collagen signaling in NT, PTC and ATC samples. D-E The Pearson correlation analysis of CREB3L1 and ECM or collagen signaling. F Immunofluorescence staining of CREB3L1 and fibroblast marker FAP in thyroid cancer samples. G RT - PCR was used to detect the expression of collagen signals after CREB3L1 knockdown. H The expression of COL5A1 was detected, after CREB3L1 knockdown or overexpression in thyroid cancer cell lines. I-J After CREB3L1 knockdown, Masson trichrome staining and IHC staining were used to detect changes in collagen fibril abundance and the expression of α - SMA after CREB3L1 knockdown, respectively. K Immunofluorescence staining of COL5A1 in ATC cell-derived xenografts after CREB3L1 knockdown. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 NC versus CREB3L1 - KD

Article Snippet: To analyze the α - SMA - , FAP - , or PDGFRα - positive CAFs, found in the 8505C - derived sphere or co - culture with 8505C, the samples were digested and resuspended in PBS, containing 1.5% BSA and incubated with the primary antibodies anti - α - SMA (Cat#14,395–1 - AP, Proteintech), anti - FAP (FAB3715A, R&D Systems, Minnesota, USA), and anti - PDGFRα (#567,950, BD biosciences), respectively.

Techniques: Expressing, Activity Assay, Immunofluorescence, Staining, Marker, Reverse Transcription Polymerase Chain Reaction, Knockdown, Over Expression, Immunohistochemistry, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Radiation-induced FAP + fibroblasts are involved in keloid recurrence after radiotherapy

doi: 10.3389/fcell.2022.957363

Figure Lengend Snippet: Irradiation (IR) promoted FAP + KF proliferation and inhibited senescence, accompanied by high energy production. In vitro , primary KFs with or without IR treatment were analyzed by anti-hFAP and anti-hVimentin antibody staining (A) . Representative images of SA-β-gal staining (B) ; senescent cells are stained green, scale bar = 100 μm. The percentage of senescent cells in FAP- versus FAP + KFs at 72 h after IR (C) . Data are representative of three independent samples and are presented as the mean ± SD; n = 6; ****, p < 0.001. IR promoted FAP + KF proliferation (D) and increased intracellular ATP production (E) . Data from three experiments with essentially the same results were combined and are presented as the mean ± SD; n = 3; *, p < 0.05; **, p < 0.01; ****, p < 0.001.

Article Snippet: The cells were analyzed by staining with an AF488-conjugated anti hFAP antibody (R&D Systems, FAB3715G-100) and a PE-conjugated anti-hVimentin antibody (BD, 562337).

Techniques: Irradiation, In Vitro, Staining

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: 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: 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: 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