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 cell

Article Title: Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer.

doi: 10.1016/j.ccell.2018.01.011

Figure Lengend Snippet: Figure 7. Schematic Representation of CAF Heterogeneity and T Lymphocyte Content in BC (A) CAF heterogeneity in BC was addressed by analyzing six CAF markers (CD29, FSP1, FAP, aSMA, PDGFRb, and CAV1) concomitantly. We identify four CAF subsets (CAF-S1 to CAF-S4) that accumulate differently in juxta-tumors compared with tumors and in BC subtypes. TNBC could be divided in two sub- groups according to their enrichment in either CAF-S1 or CAF-S4. (B) CAF-S1-enriched TNBC exhibit high content in FOXP3+ T cells and low infiltration of CD8+ T cells compared with CAF-S4-enriched tumors. (C) CAF-S1 is associated with an immunosuppressive environment by acting at complementary levels: CAF-S1 attract CD4+CD25+ T cells mainly through the release of CXCL12, retain them through OX40L, PD-L2, and JAM2, promote their survival and stimulate their differentiation into CD25HighFOXP3High, enriched in regulatory T cells, through B7H3, CD73, and DPP4. Finally, CAF-S1 enhance Treg-mediated inhibition of T effector proliferation, in contrast to CAF-S4. In summary, we demonstrate that half of TNBC accumulate a specific CAF subset that promotes an immunosuppressive microenvironment.

Article Snippet: Isotype control antibodies for each CAFmarker usedwere: iso-anti-CD29 (BioLegend, #400144), iso-FAP (primary antibody, R&DSystems, #MAB002), iso-anti-PDGFRb (BioLegend, #400114), iso-anti-CAV1 (Santa Cruz, #2857), iso-anti-SMA (R&D Systems, #IC003A) and iso-anti-FSP1 (primary antibody, Abcam, #ab27472).

Techniques: Inhibition