Fig 2: The induction of long-lasting anti-tumor effects by PLGA/PEI-HMGB1-GPC3 vaccineMice that received PLGA/PEI-HMGB1-GPC3 vaccine were rechallenged with Hepa1-6-hGPC3 cells on the left flank accompanied by naive mice as the control group.(A) Growth curve of individual mice over time in 70 days.(B) The percentage survival of the control group and PLGA/PEI-HMGB1-GPC3 immunized group.(C) Representative images of flow cytometry and statistical analysis of CD8+T cell cells labeled by CD44 and CD62L in the splenocytes from various vaccine immunization groups.(D–F) The frequencies of Naive: CD44-/CD62Lhigh, Central memory: CD44+/CD62Lhigh, Effector memory: CD44+/CD62Llow CD8+CD11C+ or CD103+CD11C+ in and TILs were analyzed statistically. Data are from one representative experiment of three performed and presented as the mean ± SD, **p < 0.01.

Fig 3: The expression of PLGA/PEI-GPC3 and PLGA/PEI-HMGB1 vaccine(A and B) The protein expression of HMGB1 or GPC3 was evaluated by flow cytometry in pHMGB1-transfected or pGPC3-transfected 293T cells.(C and D) Statistical analysis of the proportions of the positive cells in A and B.(E and F) The mice were intramuscularly administered PLGA/PEI-GPC3 and PLGA/PEI-HMGB1 vaccines. The injected muscular tissues were collected at 72 h after immunization. In vivo protein expression of HMGB1 and GPC3 was determined by western blot assay in muscular tissues. Data are from one representative experiment of three performed and presented as the mean ± SD, ****p < 0.001.

Fig 4: Anti-tumor effect of PLGA/PEI-HMGB1/GPC3 vaccine in the subcutaneous Hepa1-6-hGPC3 tumor model(A) The frequency of hGPC3-positive cells was assessed by flow cytometry in Hepa1-6 cells infected with hGPC3-lentivirus. C57BL/6 mice were s.c. implanted with 5×106 Hepa1-6-hGPC3 cells on the right flank. One week following inoculation, the mice were randomly sub-grouped and respectively vaccinated with PLGA/PEI-Vector, PLGA/PEI-HMGB1, PLGA/PEI-GPC3, or PLGA/PEI-HMGB1/GPC3 vaccines.(B) Tumor growth was recorded once a week from day 7 to day 42.(C) Tumor weights.(D) Tumor inhibition rate.(E and F) Immune cell subsets, including CD3+T, CD8+T, CD4+T, DCs, NK, Mf, and MDSCs, were quantified by flow cytometry in splenocytes and TILs.(G and H) The frequency of CD8+CD11C+ or CD103+CD11C+ in the splenocytes and TILs was detected by flow cytometry. Data are from one representative experiment of three performed and presented as the mean ± SD, **p < 0.01; ***p < 0.001.

Fig 5: CTL effect induced by PLGA/PEI-HMGB1/GPC3 co-immunizationThe lymphocytes obtained from the spleens were restimulated with GPC3 protein (10 µg/mL) ex vivo.(A and B) The proliferation ability of CD8+T was assessed by EdU assay.(C and D) The frequencies of IFN-?+CD8+T, IL-2+CD8+T, or TNF-a+CD8+T cells were analyzed by flow cytometry in GPC3 protein stimulated lymphocytes following incubation with 500 ng/mL Ionomycin, 50 ng/mL PMA, and 1× Brefeldin A for 5h.(E) The number of IFN-? secreting CD8+T cells was assessed by ELISPOT assay.(F) A co-culture experiment evaluated the antigen clearance effect. Data are from one representative experiment of three performed and presented as the mean ± SD, **p < 0.01.