Research ArticleIMMUNOLOGY

Engineering autologous tumor cell vaccine to locally mobilize antitumor immunity in tumor surgical bed

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Science Advances  19 Jun 2020:
Vol. 6, no. 25, eaba4024
DOI: 10.1126/sciadv.aba4024
  • Fig. 1 Scheme illustrating the formation and function of PC-Cell@gel.

    (A) On-demand gelation in residual tumor areas was activated via two steps. First, FK-PBA was locally injected into tumor surgical bed, enabling sialic acid–targeted accumulation of FK-PBA in residual tumors. (B) Then, PC-Cell was prepared by coating oxidized autologous tumor cells with PEI-Ce6. PC-Cell dispersed in 1 mM Na2CO3 solution was sequentially injected to activate the gelation of FK-PBA. (C) Upon NIR laser irradiation, engineered hydrogel vaccine matured DCs and mobilized neoepitope-specific CTLs in surgical bed. The synergetic effects of ATVs and PDT highly efficiently inhibit relapse of postoperative tumors.

  • Fig. 2 Preparation and characterization of PC-Cell@gel.

    (A) Scanning electron microscopy (SEM) images of oxidized tumor cells and PC-Cell. Scale bars, 5 μm. (B) Loading and encapsulation efficacy of PEI-Ce6 on tumor cells. (C) ζ-Potential of PC-Cell as a function of PEI-Ce6 incubation time. (D) SDS-PAGE electrophoresis of proteins in B16-F10–derived PC-Cell complexes. (E) Western blot assay and semiquantification of intracellular Toll-like receptor 3 (TLR-3), nuclear factor κB p65 (NF-κB p65), β-actin, and high-mobility group box 1 (HMGB-1) proteins. (F) SEM image of FK-PBA hydrogel. Scale bar, 10 μm. (G) Frequency-dependent rheological properties of FK-PBA hydrogel at 37°C (peptide concentration, 0.5 weight %; strain, 1%). Inner photographs are FK-PBA solution and formed hydrogel. (H) Three-dimensional (3D) construction image of FK-PBA hydrogel encapsulating PC-Cell (left) and 2D images of PC-Cell@gel (right). The cell membrane was prelabeled with wheat germ agglutinin (WGA)–fluorescein isothiocyanate (FITC). Scale bars, 500 μm. DAPI, 4′,6-diamidino-2-phenylindole. (I) ROS generation following increased Ce6 concentration (5, 10, 15, and 20 μg/ml) and laser power density (100, 200, 300, 400, and 500 mW/cm2). Data are shown as means ± SD (n = 3). a.u., arbitrary units.

  • Fig. 3 PC-Cell@gel induced PDT and DC maturation in vitro.

    (A) Scheme illustrating the gelation of FK-PBA in vitro. Seeded B16-OVA cells were stained by Hoechst, fixed, and sequentially treated with FK-PBA and PC-Cell in 1 mM Na2CO3 solution. (B) 3D construction of PC-Cell@gel on the surface of B16-OVA cells (right). Scale bars, 200 μm. Side view of the 3D structures (left). Scale bars, 50 μm. (C) PC-Cell@gel induced ROS production in B16-OVA cells. Scale bars, 20 μm. (D) Laser power density–dependent phototoxicity of PC-Cell@gel. (E) Cell@gel promoted BMDC maturation following increased vaccine cell concentrations. (F) Percentage of CD11c+CD80+ BMDCs and (G) CD11c+CD86+ BMDCs after incubated with PC-Cell@gel or laser-pretreated PC-Cell@gel. LPS, lipopolysaccharide. (H) Mice with partially resected CT26-GFP tumors were first locally treated with FK-PBA-Cy5.5 or FK-Cy5.5 at 5 mg/ml, respectively. Four hours later, 1 mM Na2CO3 solution was sequentially injected. Tissues in surgical bed were collected and sliced for CLSM imaging at 24 hours. Scale bars, 200 μm. Data are means ± SD (n = 3). **P < 0.01, ***P < 0.001.

  • Fig. 4 P-ATV elicited neoepitope-specific CTLs to inhibit relapse of postoperative B16-OVA and B16-F10-Luc tumors.

    (A) Treatment schedule of antirelapse study in B16-OVA and B16-F10-Luc mouse tumor model. (B) Growth kinetics of recurrent B16-OVA tumors in C57BL/6 mice model (n = 4). (C) Body weight of mice during the antirelapse study (n = 4). (D) Fluorescent staining of CD8+ T cells in tumor slices collected from control and treated groups. (E) Lymphocytes isolated from lymph nodes of vaccinated mice were determined for the presence of matured DC (CD11c+CD80+CD86+). (F) Frequency of OVA-specific CD8+ T cells in peripheral blood 3 days after the second vaccination. (G) Bioluminescence images of B16-F10-Luc tumor–bearing mice collected on days −1, 0, 6, and 15 of the antirelapse study (n = 3). (H) Quantification of BLI in tumor surgical bed at desired time points after different treatments (n = 3). (I) Growth curves of relapsed B16-F10-Luc tumors in C57BL/6 mice (n = 6). (J) Survival kinetics of postoperative B16-F10-Luc tumor–bearing mice in all groups (n = 6). (K) Bioluminescence images (left) and BLI quantification (right) of lungs collected from metastatic B16-F10-Luc mouse model after various treatments (n = 3). Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 P-ATV promoted antitumor immunity in postoperative CT26 tumor model.

    (A) Fluorescent imaging of PC-Cell– and PC-Cell@gel–treated mice by subcutaneous injection within 28 days. (B) Time course quantification of average fluorescence intensity at injection sites (n = 3). (C) Vaccination schedule for immunotherapy. (D) Secretion levels of TNF-α and (E) IFN-γ in serum on days 3, 6, and 9 after treatments, respectively. (F) Frequency of CD8+ T cells in tumor surgical bed on days 4, 8, and 12 in control and treated groups. (G) Frequency of CD4+ and CD8+ T cells in lymphocytes harvested from tumor surgical bed in control and treated groups on day 8. (H) Frequency of Tregs in tumor surgical bed with different treatments on day 8. (I) Weight-normalized number of CD8+ T cells and (J) IFN-γ+ CD8+ T cells in residue tumors of control and treated groups. (K) CD8+ T cell–to–Treg ratio in tumor surgical bed. Data are means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Inhibiting the relapse of postoperative tumor by PC-Cell@gel.

    (A) Treatment schedule in CT26 recurrent mouse tumor model. (B) Average growth kinetics of recurrent tumors after treated by different suspensions (n = 5). (C) Survival percentage of CT26 recurrent tumor-bearing mice in control and treated groups (n = 6). (D) Body weight of mice in all groups (n = 5). (E) Serological and physiological-biochemical analysis of vaccinated mice was performed at the end of study (n = 5). 1#, PBS; 2#, PC-Cell@gel + Laser. (F) Histological assessment of recurrent tumors. Scale bars, 100 μm. (G) TUNEL staining of tumor sections in all groups. Scale bars, 100 μm. Data are means ± SD. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test and log-rank (Mantel-Cox) test (ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001).

Supplementary Materials

  • Supplementary Materials

    Engineering autologous tumor cell vaccine to locally mobilize antitumor immunity in tumor surgical bed

    Lei Fang, Zitong Zhao, Jue Wang, Pengcheng Zhang, Yaping Ding, Yanyan Jiang, Dangge Wang, Yaping Li

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