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. 2022 May 23;10(5):e004258. doi: 10.1136/jitc-2021-004258

Figure 2.

Figure 2

SIK3 inhibits TC-mediated killing across solid tumors. (A) Luciferase-based cytotoxicity assay using different siRNA sequences targeting SIK3 or controls. Data were normalized to siCtrl. (B) Chromium release assay for the detection of TC-mediated cytotoxicity of PANC-1 cells after 6-hour coculture with PANC-TIL2. (C, D) Real-time live cell microscopy for the evaluation of tumor cell death using YOYO-1 dye. Seventy-two hours after siRNA transfection, MCF-7 (C) and SW480 (D) were cocultured either with survivin TCs or with TILs, respectively. Graph shows the area of YOYO-1+ dead cells/well (µm2/well). (E) M579-luciferase expressing melanoma cells were transfected with indicated siRNAs and cocultured with HLA-201+-matched melanoma TILs. TC-mediated cytotoxicity was assessed as in (A). (F) M579 cells were transfected with SIK3 ORF (Over) or EV. TC-mediated cytotoxicity was assessed as in (A). Data were normalized to siCtrl1. (G) siSIK3 or siCtrl PANC-1-luc cells were cocultured with EpCAM×CD3 (EpCAM BsAb) or control CD19×CD3 (Ctrl BsAb) bispecific antibodies in the presence of CD8+ TCs from healthy donors. TC-mediated cytotoxicity was measured as in (A). (H) PANC-1-luc cells were cocultured with PANC-TIL in the presence of increasing concentrations of the pan-SIK inhibitor HG-9-91-01. TC-mediated cytotoxicity was measured as in (A). (I) Luciferase-based cytotoxicity assay of siRNA-transfected PANC-1-luc cells challenged with PANC-TIL after knockdown of SIK family member proteins or controls. Cytotoxicity:viability ratio was calculated (see the Methods section). (J) shCtrl or shSIK3 M579 melanoma cells were coengrafted subcutaneously in NSG mice subjected to ACT of TIL209 or PBS (n=9). Mean±SEM tumor volume. Statistical difference was calculated using unpaired Mann-Whitney U test. Data information: (A, E, H) cumulative data of three independent experiments and (B–D, F, G, I, J) representative data of at least two independent experiments. Columns show mean±SEM. P values were calculated using two-tailed Student t-test. *P<0.05, **P<0.01. E:T, effector to target; EV, empty vector; shSIK3, SIK3-specific shRNA; SIK3, salt-inducible kinase 3; TC, T cell; TIL, tumor-infiltrating lymphocyte.