Changes in intratumoral macrophages in responses to PLX3397 and PLX4032. a) C57BL/6 mice with SM1 tumors were treated with PLX3397 and PLX4032 for 5 days. Tissue immunofluorescence microscopy of tumor sections was used to assess prolonged effects of the drug on macrophages. Representative H&E (left) and immunofluorescence for macrophages stained with anti-F4/80-FITC (green, right), and nuclei stained with DAPI (blue, right). b) Effect of macrophages on SM1 cells. Bar-graph representation of bioluminescence activity of SM1 cells. SM1 cells were transduced with a lentivirus-firefly luciferase and co-cultured with and without intratuoral myeloid cells isolated from SM1 tumors (1:3 ratio) for 72 hours. Difference between SM1-luc and SM1-luc + TIMs: p = 0.4 c) SM1 cells co-cultured with myeloid cells (1:3 ratio) were treated with 1 μM PLX3397, 15 μM PLX4032, or in combination for 72 hours. Difference between vehicle and PLX3397: p = 0.83; PLX3397 and PLX4032: p = 0.0001; PLX4032 and combo: p = 0.39. d) SM1 cells expressing firefly luciferase were co-cultured with I-11.15 (1:3 ratio). Difference between SM1-luc and SM1-luc + I-11.15: p = 0.6. e) SM1-luc cells co-cultured with I-11.15 were treated with 1 μM PLX3397, 15 μM PLX4032, or in combination for 72 hours. Difference between vehicle and PLX3397: p = 0.05; PLX3397 and PLX4032: p = 0.0002; PLX4032 and combo: p = 0.7. f) Effect of growth factors on SM1 to PLX4032. SM1 cells were exposed to 15 μM PLX4032 with HGF or TNF-α (25 or 50 ng/mL). Cell viability assay (MTS) was performed after 72 hours. Difference between vehicle and PLX4032: p = 0.004; PLX4032 and PLX4032 + HGF (50 ng/mL): p = 0.06; PLX4032 and PLX4032 + TNF-α (50 ng/mL): p = 0.12.