Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors

Abstract

Recent studies have shown that the brain is not a barrier to successful active immunotherapy that uses gene-modified autologous tumor cell vaccines. In this study, we compared the efficacy of two types of vaccines for the treatment of tumors within the central nervous system (CNS): dendritic cell (DC)-based vaccines pulsed with either tumor extract or tumor RNA, and cytokine gene-modified tumor vaccines. Using the B16/F10 murine melanoma (B16) as a model for CNS tumor, we show that vaccination with bone marrow-generated DCs, pulsed with either B16 cell extract or B16 total RNA, can induce specific cytotoxic T lymphocytes against B16 tumor cells. Both types of DC vaccines were able to protect animals from tumors located in the CNS. DC-based vaccines also led to prolonged survival in mice with tumors placed before the initiation of vaccine therapy. The DC-based vaccines were at least as effective, if not more so, as vaccines containing B16 tumor cells in which the granulocytic macrophage colony-stimulating factor gene had been modified. These data support the use of DC-based vaccines for the treatment of patients with CNS tumors.

Figure 1

Induction of specific lytic activity against tumor cells by immunization with DC pulsed with tumor extracts or with tumor-derived total RNA. Triplicate C57BL/6 mice were immunized three times with either DC pulsed with (open circles) B16 tumor extract or (filled squares) SMA 560 tumor extract (A and B, respectively), or with DC pulsed with (open circles) B16 tumor RNA or (filled squares) SMA 560 RNA (C and D, respectively) or with PBS (filled triangle). 7 d later, splenocytes were isolated and restimulated for 5 d with irradiated B16 cells (A and C) or SMA 560 cells (B and D). Cytotoxic activity was then measured by chromium release assay using the targets indicated in each panel. *(P <0.05) indicate points of significant differences in lysis when B16-immunized animals are compared to SMA 560–immunized animals. Error bars indicate 1 SD of the mean.

Figure 2

Vaccination with DCs pulsed with tumor extract or tumor RNA protects against CNS challenge with B16 tumor and is equipotent to vaccination with GM-CSF–producing B16 cells. Vaccination of C57BL/6 mice was performed a total of three times before intracranial tumor challenge with B16 cells as described in Materials and Methods. Mice were evaluated daily until death. Data are representative of two experiments performed with similar results. The results are divided in two panels for clarity. The results for the PBS-immunized group (closed squares) and the GM-CSF modified B16 cell immunized group (open squares) are represented in both A and B for ease of comparison. Median days of survival, range, number of animals, and significance compared to PBS-immunized animals based on log-rank analysis for each group are as follows: closed squares, PBS: 21, 13–41, n = 10; X, DC pulsed with B16 tumor extract: >80, 17–>80, n = 13, P = 0.0006; closed diamonds, DCs pulsed with SMA 560 extract; 24, 19–30, n = 11, P = 0.50; closed triangles, B16 extract: 21, 14–21, n = 5, P = 0.45; open squares, GM-CSF– modified B16 cells: 36, 30–>80, n = 6, P = 0.022; open triangles, DC pulsed with B16 RNA: 31, 19–>80, n = 10, P = 0.0001; open diamonds, DC pulsed with SMA 560 RNA: 21, 15–31, n = 9, P = 0.40; open circles, unmodified B16 cells: 23, 18–45, n = 5, P = 0.39.

Figure 3

Treatment of established CNS B16 tumors with DCs pulsed with tumor extract prolongs survival. Intracranial tumor challenge with B16 cells was performed first and then, starting 4 d later, C57BL/6 mice were treated with a total of three immunizations as described in Materials and Methods. Mice were evaluated daily until death. Median days of survival, range, number of animals, and significance compared to PBS-immunized animals based on log-rank analysis for each group are as follows: filled squares, PBS: 16, 15–18, n = 6; X, DC pulsed with B16 tumor extract: 26, 14– >80, n = 7, P = 0.037; (filled diamonds), DC pulsed with SMA 560 extract: 16, 15–26, n = 7, P = 0.57.

Figure 4

CNS B16 tumors from animals vaccinated with DC pulsed with B16 tumor extract demonstrate large areas of necrosis, hemorrhage, and inflammatory infiltrate (b). No such changes are seen in CNS B16 tumors from animals vaccinated with DC pulsed with control tumor extract (a) or PBS (not shown).

Figure 4

CNS B16 tumors from animals vaccinated with DC pulsed with B16 tumor extract demonstrate large areas of necrosis, hemorrhage, and inflammatory infiltrate (b). No such changes are seen in CNS B16 tumors from animals vaccinated with DC pulsed with control tumor extract (a) or PBS (not shown).