Figure 4. Inhibition of Axl with BGB324 alters the immune landscape of PDA.

A) Tumor lysates from KIC mice treated with vehicle (Cntl), gemcitabine (Gem), BGB324 (BGB) or the combination (Combo) were analyzed for cytokine context by Milliplex assay. The levels of Ccl11, Il-7, Il-1β, and Il-6 are shown as mean ± SD (n = 2/group). B) Tumor lysates from KIC animals treated as above were probed for the level of active TBK1 and NF-κB by Western blotting for the indicated targets. C, D) The effect of Axl on TBK1 signaling was probed in AsPC-1 (C) and Panc-1 (D) cells by Western blot analysis. AsPC-1 cells were serum starved overnight and subsequently treated with increasing concentrations of BGB324 for 30 minutes. Panc-1 cells were serum starved and subsequently stimulated with control (0.02% DMSO in media), Gas6 (200 ng/ml), or Gas6 + BGB324 (2 μM) for 30 minutes. Tumor cell lysates were probed for phosphorylated TBK1 and downstream targets. E) Tumor tissue from KIC mice treated with BGB324 +/− gemcitabine were evaluated by immunofluorescence for macrophage (F4/80) and Arginase 1 (Arg1) and counterstained with DAPI. Images were analyzed using Elements software; quantification of % area fraction is shown. Data are displayed as mean ± SD and represent 5 images per tumor with 4 animals per group analyzed. **P < 0.01; ***P < 0.005; ****P < 0.001; by ANOVA with Tukey’s MCT. F) Flow cytometry of KPC-M09 subcutaneous tumors treated with vehicle (control) or BGB324 (50 mg/kg, PO, BID) for two weeks as described in the supplementary methods. BGB324 reduced monocytic MDSCs (CD11b⁺ Ly6G^- Ly6C⁺), PD-L1⁺ M-MDSCs, tumor associated macrophages (TAMs, CD11b⁺ Ly6G^- Ly6C^- F4/80⁺ CD11c⁺ MHCII⁺) and Arg1⁺ TAMs. *, P < 0.05; **, P < 0.01 by t-test.