Dendritic cell-directed vaccination with a lentivector encoding PSCA for prostate cancer in mice

Abstract

Many studies have demonstrated that prostate stem cell antigen (PSCA) is an attractive target for immunotherapy based on its overexpression in prostate tumor tissue, especially in some metastatic tissues. In this study, we evaluated dendritic cell (DC)-directed lentiviral vector (DCLV) encoding murine PSCA (DCLV-PSCA) as a novel tumor vaccine for prostate cancer in mouse models. We showed that DCLV-PSCA could preferentially deliver the PSCA antigen gene to DC-SIGN-expressing 293T cells and bone marrow-derived DCs (BMDCs). Direct immunization with the DCLV-PSCA in male C57BL/6 mice elicited robust PSCA-responsive CD8(+) and CD4(+) T cells in vivo. In a transgenic adenocarcinoma mouse prostate cell line (TRAMP-C1) synergetic tumor model, we further demonstrated that DCLV-PSCA-vaccinated mice could be protected from lethal tumor challenge in a prophylactic model, whereas slower tumor growth was observed in a therapeutic model. This DCLV-PSCA vaccine also showed efficacy in inhibiting tumor metastases using a PSCA-expressing B16-F10 model. Taken together, these data suggest that DCLV is a potent vaccine carrier for PSCA in delivering anti-prostate cancer immunity.

Figure 1. Targeted transduction and delivery of PSCA antigen gene into dendritic cells (DCs) by DCLV-PSCA.

(A) 293T cells were transfected transiently with plasmids FUW-Null (mock control, blue line) or FUW-PSCA (red line). Two days later, cells were collected and stained for PSCA expression analyzed by flow cytometry. 293T cells stained with the isotype antibody were included as a control (grey shade area). (B) 293T cells were transfected transiently with plasmids FUW-PSCA, SVGmu, and other necessary lentiviral packaging plasmids to produce DCLV-PSCA vectors. Fresh virus supernatant was used to transduce 293T cells (blue line) or 293T.hDC-SIGN cells (red line) with MOI = 10. PSCA expression was analyzed by flow cytometry 3 days post-transduction. (C) Bone marrow-derived DCs were transduced with a mock vector DC-LV-Null or DC-LV-PSCA vector. Five days later, CD11c and PSCA expression were assessed by flow cytometric analysis. All experiments were repeated three times and the representative data is shown.

Figure 2. PSCA-specific T cell response after a single dose of in vivo immunization with DCLV-PSCA.

(A) Male C57BL/6 mice were immunized with 6×10⁷ TU of DCLV-PSCA through different administration routes: intraperitoneal space (i.p.), subcutaneous area (s.c.), intramuscular area (i.m.), footpad (f.p.), or intradermal (the base of tail, i.d.). One immunization group was included as a negative control. Two weeks after immunization, splenocytes from mice were harvested and analyzed for the presence of PSCA-specific CD8⁺ T cells by restimulating splenocytes with a PSCA peptide (PSCA_83-91), followed by intracellular staining for IFN-γ and surface staining for CD8. Percentage of IFN-γ-secreting CD8⁺ T cells is indicated. (B) Statistical comparison of immunization elicited by administration of DCLV-PSCA among different administration routes. (C) Male C57BL/6 mice were immunized with different doses of DCLV-PSCA vectors (0, 2, 10, 40 and 80 million TU) at the base of tail. Two weeks post-vaccination, PSCA-specific CD8⁺ T cells from the spleen were analyzed by restimulating with the peptide PSCA_83-91, followed by intracellular staining for IFN-γ. (D) Production of PSCA-specific IFN-γ-secreting cells from both spleen (SP) and inguinal lymph node (LN) was evaluated by restimulation with the PSCA_83-91 peptide, followed by ELISPOT analysis for IFN-γ. (E) Production of PSCA-specific IL-2 from splenocytes (with CD8⁺ T cells depleted) was measured by restimulation with 293T cell lysate transfected to express PSCA, followed by the ELISPOT analysis for IL-2. (**: P<0.01; *: P<0.05; One-way ANOVA followed by Bonferroni's multiple comparison test. Error bars represent SD.) All experiments were repeated three times and the representative data is shown.

Figure 3. Prophylactic and therapeutic anti-TRAMP-C1 prostate cancer immunity elicited by in vivo immunization with DCLV-PSCA.

(A, B) Male C57BL/6 mice were immunized with 8×10⁷ TU of DCLV-PSCA, mock vector DC-LV-Null, or PBS control at the base of tail. Ten days post-immunization, these mice were challenged subcutaneously with 5×10⁵ of TRAMP-C1 tumor cells. Tumor growth curves were monitored with a fine caliper, and tumor volume was calculated based on the largest perpendicular diameters (mm³), according to the formula V = ab²π/6, where a and b are the largest perpendicular diameters. Representative Kaplan Meyer survival curve for prophylactic tumor challenge (n = 12). (C, D) Male C57BL/6 mice were implanted with 5×10⁵ TRAMP-C1 tumor cells subcutaneously, and 18 days later, these tumor-bearing mice were treated with 8×10⁷ TU of DCLV-PSCA (n = 12) or DCLV-Null (n = 12) at the base of tail. Tumor volume was monitored and calculated as previously described. Representative Kaplan Meyer survival curve for therapeutic tumor challenge. (***: P<0.001; Log-rank (Mantel-Cox) test. Error bars represent SEM.) All experiments were repeated twice and the representative data is shown.

Figure 4. CD8⁺/CD4⁺ T cell-dependent immune protection against TRAMP-C1 tumors induced by DC-LV-PSCA immunization.

(A) Infiltration of T cells into tumor tissues. TRAMP-C1 tumors from tumor-bearing mice were excised 3 weeks post-immunization, paraffin-embedded, and stained for immunofluorescence-conjugated CD3, CD4 and CD8 antibody (green color as indicated by white arrows) together with nuclear staining (red color). Representative images showing CD4⁺ and CD8⁺ T cells infiltrated to tumor tissues from DCLV-PSCA-immunized mice as compared to those of DCLV-Null-immunized mice. (B) Four groups of male C57BL/6 mice (n = 8 for each group) were transplanted with 5×10⁵ TRAMP-C1 cells subcutaneously at day 0. Fourteen days later, 3 groups were immunized with DCLV-PSCA, while the other group was immunized with mock vector DCLV-Null. Two groups of mice from the DCLV-PSCA-immunized groups were subjected to CD4⁺ or CD8⁺ T cell depletion by injecting CD4- or CD8-depletion antibody intraperitoneally. (C) Tumor volume for each group of mice was monitored. Error bars represent SEM. All experiments were repeated twice and the representative data is shown.

Figure 5. The ability of DCLV-PSCA immunization to suppress lung metastases.

(A) Male C57BL/6 mice were immunized with DCLV-PSCA or DCLV-Null as a mock control. Ten days later, mice were challenged with 0.2 million B10-F10-PSCA cells by intravenous injection through tail vein. Two weeks later, mice were sacrificed, and macroscopic views of the lungs were shown. (B) Microscopic H&E staining (20×) of lung tissue samples from mice immunized with DCLV-PSCA or DCLV-Null. (C) Statistical quantification of melanoma lung metastases (number of black nodules on the lungs) of immunized mice; similar immunization, but with the original B16-F10 melanoma metastases included as a control. (**: P<0.01 and n/s: not statistically significant; One-way ANOVA followed by Bonferroni's multiple comparison test. Error bars represent SD, n = 4). All experiments were repeated twice and the representative data is shown.