Journal: Journal for Immunotherapy of Cancer

Article Title: SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation

doi: 10.1136/jitc-2020-001496

Figure Lengend Snippet: (A) Comparison of YES1, SRC, FYN and LYN mRNA expressions between non-malignant lung tissue and NSCLC (TCGA cohort). Mann-Whitney U test was used for the statistical analysis. (B, C) Kaplan-Meier survival curves showing that high YES1 and SFK levels (above the median) are associated with worse OS in NSCLC. The log-rank test was used for the statistical comparisons. (D, E) Quantification of FOXP3+CD4+ cells in NSCLC specimens from the CUN cohort. The percentage of Tregs (FOXP3+CD4+) in YES1-positive tumors in the upper quartile of the YES1 H-score is significantly higher than that found for the rest of the tumors (D) and in the lower quartile (E). (F, G) Quantification of FOXP3+CD4+ cells in patients with LUAD from CUN. The percentage of Treg cells in the YES1 upper quartile was compared with the rest of the quartiles (F) or the lower quartile (G). (H) Representative images of YES1 IHC and multiplex immunofluorescence for CD8+, CD4+ and FOXP3+CD4+ cells in patients with NSCLC. (I) Relative abundance of Treg cells in patients with LUAD from the TCGA database analyzed with CIBERSORT tool. Scale bar: 50 µm. (D–G, I) Data are expressed as median, and statistical comparisons were performed using the Mann-Whitney U test. *P<0.05, **P<0.01. CUN, University Clinic of Navarra; IHC, immunohistochemistry; LUAD, lung adenocarcinoma; ns, not significant; NSCLC, non-small cell lung cancer; OS, overall survival; SFK, SRC family kinase; SRC, Treg, regulatory T cell.

Article Snippet: For the detection of YES1 in the TMAs by immunohistochemistry, antigen retrieval was performed by heating the samples in citrate buffer (10 mM, pH 6), and the primary anti-YES1 antibody (Proteintech, 20 243–1-AP) was incubated at 1:100 dilution.

Techniques: Comparison, MANN-WHITNEY, Multiplex Assay, Immunofluorescence, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation

doi: 10.1136/jitc-2020-001496

Figure Lengend Snippet: (A) mRNA expression of YES1, SRC, FYN and LYN analyzed by RT-qPCR in a panel of 16 murine NSCLC cell lines. (B) Western blot analysis of YES1 expression in nine murine cell lines. (C) Effect of dasatinib on 393P and UN680 cell proliferation in vitro. (D) Western blot analysis showing pSFK protein inhibition by dasatinib at 10 hours post-treatment in vitro. (E) Subcutaneous tumor growth of 393 P cells injected in athymic nude mice treated with dasatinib (30 mg/kg) or vehicle. A two-way analysis of variance followed by a post hoc Bonferroni test was used. *P<0.05, ***P<0.001. NSCLC, non-small cell lung cancer; pSFK, phospho-SRC family kinase; RT-qPCR, real-time quantitative PCR; SFK, SRC family kinase.

Article Snippet: For the detection of YES1 in the TMAs by immunohistochemistry, antigen retrieval was performed by heating the samples in citrate buffer (10 mM, pH 6), and the primary anti-YES1 antibody (Proteintech, 20 243–1-AP) was incubated at 1:100 dilution.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, In Vitro, Inhibition, Injection, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation

doi: 10.1136/jitc-2020-001496

Figure Lengend Snippet: (A) Schematic representation of the experimental design followed for the depletion of CD8, CD4 and NK1.1 cell populations and the treatment with dasatinib+anti-PD-1 in vivo. (B) Tumor growth of 393P-inoculated cells in the presence of depleting antibodies and treatment with dasatinib+anti-PD-1 (n=6). (C) Treg cell proliferation 4 days after plating in the presence of IL-2, anti(α)CD3, αCD28 and dasatinib (2 and 10 nM) measured by flow cytometry. (D) IL-10 levels measured in Treg cells culture medium after 48 hours of exposure to dasatinib. (E) Protein expression of pLCK, LCK, YES1, pSTAT3, STAT3, pSTAT5 and STAT5 in Treg cells treated with dasatinib (10 nM) for 45 min. (F, G) In vitro experiment of TGF-β-dependent CD4+ T cell conversion into Tregs, in the presence of IL-2, αCD3, αCD28 and dasatinib (2, 10 and 20 nM), analyzed by flow cytometry. (H) Western blotting of pSTAT5, STAT5, pSMAD3 and SMAD3 in CD4+ T cells converted into Tregs after treatment with dasatinib (10 nM). Differences between groups were evaluated with a two-way (B) or a one-way analysis of variance test (C, D, F) followed by a post hoc Bonferroni test. **P<0.01, ***P<0.001, ****P<0.0001. IL, interleukin; LCK, lymphocyte-specific protein tyrosine kinase; PD-1, programmed cell death 1; pLCK, phospholymphocyte-specific protein tyrosine kinase; TGF-β, transforming growth factor beta; Treg, regulatory T cell.

Article Snippet: For the detection of YES1 in the TMAs by immunohistochemistry, antigen retrieval was performed by heating the samples in citrate buffer (10 mM, pH 6), and the primary anti-YES1 antibody (Proteintech, 20 243–1-AP) was incubated at 1:100 dilution.

Techniques: In Vivo, Flow Cytometry, Expressing, In Vitro, Western Blot

Journal: Reproduction, fertility, and development

Article Title: Role of Fyn kinase in oocyte developmental potential

doi: 10.1071/RD09311

Figure Lengend Snippet: Expression of c-Yes kinase in Fyn-null oocytes. Expression of c-Yes kinase in wild-type (WT) and Fyn-null oocytes was determined by western blot analysis of groups of 40 cumulus-free MII oocytes. The blots were probed with anti-c-Yes antibody and reprobed with anti-GAPDH. The upper panel shows a typical result. The average intensity of c-Yes and GAPDH bands was measured and the ratio of c-Yes/GAPDH was expressed graphically (lower panel). Values represent the mean ± s.d. from five groups of oocytes. Comparison of the from WT and Fyn-null oocytes by paired t-test indicated that c-Yes expression in Fyn-null oocytes was significantly higher than in controls (*).

Article Snippet: The primary antibodies used included mouse anti-phosphotyrosine (clone 4G10; Millipore, Temecula, CA, USA), rabbit anti-Fyn (Fyn3; 1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-c-Yes (1 : 1000; BD Transduction Laboratories, San Jose, CA, USA), mouse anti-GAPDH (1 : 2000; AbCam, Cambridge, MA, USA) and rat anti-α-tubulin (1 : 2000; AbCam).

Techniques: Expressing, Western Blot