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. Author manuscript; available in PMC: 2016 Mar 15.
Published in final edited form as: Clin Cancer Res. 2015 Jan 14;21(6):1438–1446. doi: 10.1158/1078-0432.CCR-14-1979

Figure 4. HIF-1α-triggered VEGFA-AKT/ERK axis promotes immune adaptation in human cancer.

Figure 4

Parental (P0) CaSki human cancer cells were pulsed with MART-1 peptide or irrelevant E7 peptide and subjected to selection with MART-1-specific human CTLs for 3 rounds to produce immune-resistant (P3) or control (N3) cells. (A) Top, P0, P3, or N3 CaSki cells were incubated with MART-1-specific human CTLs and the frequency of apoptotic tumor cells was determined by flow cytometry analysis of caspase-3 activation. Middle, Western blot analysis of HIF-1α expression in P0, P3 and N3 cells. Bottom, VEGFA concentration (pg/ml) in the supernatant of P0, P3 and N3 cells in culture. (B) Western blot analysis of AKT and ERK phosphorylation, as well as expression of anti-apoptotic proteins in P0, P3, and N3 cells. (C) Top, Western blot analysis of HIF-1α expression in P3 cells transfected with siRNA against HIF-1α or GFP. Bottom, VEGFA concentration (pg/ml) in the supernatant of HIF-1α or GFP siRNA-treated CaSki P3 cells. (D, E) Western blot analysis of AKT and ERK phosphorylation, as well as expression of anti-apoptotic proteins in HIF-1α or GFP siRNA and α-VEGFA mAb-treated CaSki P3 cells. (F) Control IgG or α-VEGFA mAb-treated CaSki P3 cells were pulsed with MART-1 peptide and mixed with MART-1-specific human CTLs. The frequency of apoptotic cells was determined by flow cytometry analysis of caspase-3 activation.