Figure 3. VitE restores TADCs function by inhibiting SHP1 and boosts DCs-induced antitumor immunity.
A, Volcano plot of reverse phase protein array (RPPA) data of vehicle- or VitE (50μg/ml)-treated mouse BMDCs growing in tumor conditioned media (TCM) for 48 hours. Differentially expressed proteins are labeled, and the top hits are marked in red (up-regulated proteins) or blue (down-regulated proteins). The grey dashed line indicates P-value of 0.05.
B, Flow cytometric analysis of the intensity of pY564-SHP1 in spleen DCs and TADCs from EMT6 mammary tumor-bearing mice (n=6).
C, t-SNEs by CyTOF and quantification comparing the intensity of pY564-SHP1 in TADCs from the vehicle- or VitE-treated EMT6 tumors at day 15 post-implantation.
D, Flow cytometry analysis (top) and heat-map (bottom) depicting relative expressions of SHP1 and co-stimulatory molecules on siCtrl or siPTPN6 transfected human MoDCs.
E, The effective SHP1 knockdown in BMDCs by siPtpn6 versus siCtrl (left, top). CFSE-dilution analysis (left, bottom) and quantification (right panel) of OT-1 T cells co-cultured with OVA-pulsed BMDCs (ratio 10:1) transfected with siPtpn6 versus with siCtrl as a control (n=3).
F, Quantified proliferation of OT-1 T cell cocultured with OVA-loaded vehicle- or VitE-treated siCtrl- or siPtpn6-mouse BMDCs (n = 3).
G, Representative flow cytometric CFSE-dilution analysis of OT-1 T cells co-cultured with OVA-pulsed sgCtrl-DC2.4 or sgPtpn6-DC2.4 cells treated with vehicle or VitE (ratio 10:1) (n=3).
H, Quantification (absolute numbers) of OT-1 T cells co-cultured with OVA-pulsed sgCtrl-DC2.4 or sgPtpn6-DC2.4 cells treated with vehicle or VitE (ratio 10:1) (n=3) as in G.
I, Growth curves of B16-GMCSF melanoma cells alone (n=6), versus co-implanted with sgCtrl-DC2.4 cells (n=10) or sgPtpn6-DC2.4 cells (n=10) in C57BL/6 mice.
J, The quantification of tumor size (Day 20) in C57BL/6 mice transplanted with B16-GMCSF tumor cells alone, or co-implanted with sgCtrl- or sgPtpn6-DC2.4 cells and under the vehicle- or VitE- (50mg/kg, oral gavage, daily) treatment (n=4–5).
Mean ± s.e.m (B, C, E, F, H, I, J). Two-sided Student’s t-test (B, C). One-way analysis of variance (ANOVA) and Tukey’s test for multiple comparisons (E, F, H, I, J). The statistical significance is defined by P-value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.