Mouse Bone Marrow-derived DCs Activation and Phagocytosis Assays

Mouse bone marrow-derived DCs (BMDCs) were extracted from the femurs of male C57BRL/6 mice (eight to twelve weeks old) and cultured in RPMI 1640 complete culture medium containing GM-CSF (20 ng/ml, Biolegend) for 7 days. The suspended BMDCs were half-changed on the 3^rd and 5^th day, respectively. To determine the phagocytosic ability and activation status of BMDCs after direct treatment with BI2536, BMDCs were incubated with a series of concentrations of BI2536 for 24 h. Then, LLC cells were stained with CFSE (Biolegend, 5 mM, 1:1000) and co-cultured with BMDCs at a ratio of 1:1 in the normal culture medium for another 24 h. To determine the phagocytosis status of BI2536-treated cancer cells, LLC cells were labeled with CFSE (Biolegend, 5 μM) and treated with BI2536 for 24 h. Then the treated LLC cells and the induced DCs were co-cultured at a ratio of 1:1 for another 24 h, as indicated in Fig ​Fig4A.4A. DCs were labeled by CD11c (117309) fluorescence-labeled Ab, and the fluorescence data of CD80 (104708), CD86 (105012) and MHCII (107630) were used as indicators of the DC maturation. Tumor cell phagocytosis was quantitatively determined by detecting CFSE (tumor)/CD11c (DC) double-positive signals. For immunofluorescence staining, tumor cells were incubated with CFSE for 5 minutes and co-cultured with DiI (C1036, Beyotime, 10 µM)-stained DC cells for 0-2 h at a 1:1 ratio. The cells were fixed in 4% PFA for 20 min and then stained with DAPI, and the fluorescent images were acquired on the Olympus FVMPE-RS platform after a series of washes.

BI2536-treated tumor cells were phagocytosed and could promote BMDC activation. A. Schematic diagram of co-culture system illustrating the effect of BI2536-treated tumor cells on BMDC function. B. Representative immunofluorescence images showing nucleus (blue, DAPI), BMDCs (red, DiI) and BI2536-treated LLC cells (green, CFSE) 0, 1 and 2 h after co-culture (confocal imaging, objective: 20×). The arrow indicates the surrounding BMDCs ready to engulf tumor cells. Scale bars, 20 µm. C. The phagocytosis of CD11c⁺ DCs after co-culture with BI2536-treated LLC cells (CFSE), as assessed by flow cytometry. D-F. The expression of CD80, CD86 and MHCII in BMDC after incubation with treated tumor cells. G-H. LLC cells treated with BI2536 were inoculated subcutaneously into mice and the activation of DCs in inguinal lymph nodes was flow cytometrically detected at different time points after vaccination. The MFI of CD80 and CD86 on CD11c⁺ cells are listed.