B16 Tumor Growth Research Articles

Adenovirus Vector-Mediatedin VivoGene Transfer of OX40 Ligand to Tumor Cells Enhances Antitumor Immunity of Tumor-Bearing Hosts

OX40 ligand (OX40L), the ligand for OX40 on activated CD4+ T cells, has adjuvant properties for establishing effective T-cell immunity, a potent effector arm of the immune system against cancer. The hypothesis of this study is that in vivo genetic engineering of tumor cells to express OX40L will stimulate tumor-specific T cells by the OX40L-OX40 engagement, leading to an induction of systemic antitumor immunity. To investigate this hypothesis, s.c. established tumors of three different mouse cancer cells (B16 melanoma, H-2b; Lewis lung carcinoma, H-2b; and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of a recombinant adenovirus vector expressing mouse OX40L (AdOX40L). In all tumor models tested, treatment of tumor-bearing mice with AdOX40L induced a significant suppression of tumor growth along with survival advantages in the treated mice. The in vivo AdOX40L modification of tumors evoked tumor-specific cytotoxic T lymphocytes in the treated host correlated with in vivo priming of T helper 1 immune responses in a tumor-specific manner. Consistent with the finding, the antitumor effect provided by intratumoral injection of AdOX40L was completely abrogated in a CD4+ T cell-deficient or CD8+ T cell-deficient condition. In addition, ex vivo AdOX40L-transduced B16 cells also elicited B16-specific cytotoxic T lymphocyte responses, and significantly suppressed the B16 tumor growth in the immunization-challenge experiment. All of these results support the concept that genetic modification of tumor cells with a recombinant OX40L adenovirus vector may be of benefit in cancer immunotherapy protocols.

Inhibition of indoleamine 2,3-dioxygenase suppresses NK cell activity and accelerates tumor growth.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, is induced under various pathological conditions, including viral and bacterial infection, allograft rejection, cerebral ischemia, and tumor growth. We have previously reported that the expression of IDO mRNA was increased in some clinical cases of hepatocellular carcinoma in which the recurrence-free survival rate in these IDO-positive patients was significantly higher than that in patients without IDO mRNA induction in tumors. Additionally, IDO expressed in tumors was localized not to the tumor cells but instead to tumor-infiltrating cells by immunohistochemistry. In this study, in order to elucidate the mechanisms underlying anti-tumor effect of IDO, we investigated whether IDO inhibitor (1-methyl-dl-tryptophan, 1MT) affects the growth of subcutaneous B16 tumors in mice. Subsequently, the activity of natural killer (NK) cells was investigated under the conditions of inhibited IDO activity in vivo and in vitro. IDO mRNA expression of B16 cells, B16 subcutaneous tumor, sprenocytes of mice, and human NK cells were studied by reverse transcription-polymerase chain reaction. B16 subcutaneous tumor growth with or without IDO inhibition was observed and cytotoxic activity of NK cells were investigated under the conditions of inhibited IDO activity in vivo and in vitro. IDO mRNA was expressed in B16 subcutaneous tumor, splenocytes of tumor bearing mice, co-cultured splenocytes with B16, and human NK cells. On day 14, after injection of B16 melanoma cells, the sizes of tumors in IDO-inhibited mice were significantly larger than those in control mice. The cytotoxic activity of mice NK cells was reduced by IDO inhibition in vivo. In vitro inhibition of IDO, NK activity was reduced in dose-dependent manner of 1MT. In conclusion, these results indicated that IDO plays an important role in anti-tumor immunity by regulating cytotoxic activity of NK cells.

Specific peptide-mediated immunity against established melanoma tumors with dendritic cells requires IL-2 and fetal calf serum-free cell culture.

Melanoma, despite its aggressive growth characteristics, is an antigenic tumor expressing several characterized neo- and differentiation antigens. Dendritic cells (DC) when pulsed with defined peptides have been shown to effectively induce melanoma-specific T cell responses in humans and mice. These protect animals from challenge with melanoma, but so far have failed to induce significant tumor regressions. To study the efficacy of DC-based anti-tumor vaccinations, we set up a therapeutic model using C57BL/6J mice with established pulmonary and subcutaneous metastases induced by the B16-melanoma cell line B78-D14. Mice were vaccinated twice with 20,000 antigen-presenting cells, either bone marrow-derived DC or epidermal Langerhans cells (LC), which were loaded with the tyrosinase-related protein 2 (TRP2) peptide. Generally, DC cultured with fetal calf serum (FCS) induced a dominant unspecific response. This was not seen using LC cultured without serum; however, vaccination with TRP2-loaded FCS-free LC alone failed to influence the growth of established B16 tumors. A reproducible reduction of tumor size and weight was only obtained if LC vaccinations with TRP2 were followed by a 5-day treatment of mice with 200,000 IU IL-2 intraperitoneally twice/daily. Omitting the TRP2 peptide abolished the efficacy of this combined treatment, demonstrating the crucial role of priming a melanoma-specific T cell response. Microcytotoxic assays performed with spleen-derived T cells and melanoma as well as congenic fibroblast lines as targets confirmed the TRP2-dependent specificity of LC-induced immune responses. Thus, despite the fact that tumor-specific T cells were primed, an additional IL-2-dependent stimulus was needed to translate this immune response into a therapeutic effect against established tumors.

Pulsing of dendritic cells with cell lysates from either B16 melanoma or MCA-106 fibrosarcoma yields equally effective vaccines against B16 tumors in mice.

Dendritic cells (DC) pulsed in vitro with a variety of antigens have proved effective in producing specific antitumor effects in vivo. Experimental evidence from other laboratories has confirmed that shared antigens can be encountered in histologically distinct tumors. In our experiments, we set out to evaluate the immunotherapeutic potential of vaccines consisting of DC pulsed with MCA-106 fibrosarcoma or B16 melanoma cell lysates and to determine whether a cross-reactivity exists between the two tumors. DC were prepared from the bone marrow of C57BL/6 (B6) mice by culturing progenitor cells in murine granulocyte-macrophage colony-stimulating factor (GM-CSF). They were separated into three equal groups and were either pulsed with B16 melanoma cell lysates (BDC), pulsed with tumor extract from the syngeneic fibrosarcoma MCA-106 (MDC), or left unpulsed (UDC). DC were then used to immunize three groups of mice, with all mice receiving two weekly intravenous (IV) doses of 1 x 10(6) DC from their respective preparations on days -14 and -7. A fourth group of control mice were left untreated. On day 0, all mice were challenged with subcutaneous injections of 1 x 10(5) B16 and 1 x 10(5) MCA tumor cells, administered in the left and right thighs, respectively. After the inoculations, the mice were monitored closely with respect to tumor growth and survival. The MDC mice developed specific cellular immunity directed against not only MCA-106 tumor cells, but also against B16 melanoma, as measured through chromium-release assays of splenocyte preparations, while remaining ineffective at killing both L929 fibroblasts and CT26 tumor cells. By day 30 after tumor inoculations, control mice manifested the largest B16 tumor volumes at a mean of 2185 mm3, followed by the UDC, MDC, and BDC groups at 92 mm3 (P=0.00008), 3 mm3 (P=0.000002), and 2 mm3 (P=0.00004), respectively. The survival data mirrored this pattern, with control animals displaying the shortest mean survival time (37.1+/-4.0 days), followed by UDC (44.8+/-6.6), MDC (56.2 +/-14.7), and BDC (56.4+/-18.3) animals. No significant differences were noted between MCA-106 and B16 cell lysate-pulsed DC vaccines with respect to their abilities to inhibit B16 tumor growth and to prolong survival. These findings were confirmed using a B16 pulmonary metastasis model. Likewise, vaccination with interferon-gamma gene-modified MCA-106 tumor cells was shown to be effective at protecting against a subsequent subcutaneous B16 tumor challenge in 3 of 4 mice observed. These results demonstrate that immunization with antigen-pulsed DC confers cellular immunity, retards tumor growth, and prolongs the survival of tumor-challenged mice. The ability of MCA-106 cell lysate-pulsed DC vaccines to inhibit the growth of subcutaneous B16 tumors also suggests the presence of shared tumor-associated antigens between these two histologically distinct tumors.