Sequential delivery of interferon-alpha gene and DCs to intracranial gliomas promotes an effective antitumor response.

Effective presentation of tumor antigens by dendritic cells (DCs) is considered to be essential for the induction of antitumor T-cell responses. Apoptotic and necrotic tumors have been noted to be a robust antigen source for DCs. Because glioma cells undergo apoptosis after transfection with the type I interferon (IFN) gene and type I IFNs promote the stimulatory activity of DCs, we hypothesized that transfection of glioma cells with type I IFN genes and provision of DCs would promote particularly effective antitumor activity by both facilitating apoptosis of glioma cells and the presentation of the glioma antigens, thereby inducing specific immune responses against glioma cells. We have previously reported the proof of this hypothesis in vitro and in a subcutaneous tumor model. Here we report an extension of this approach in intracranial (i.c.) gliomas using adenoviral IFN-alpha (Ad-IFN-alpha) vector. Mice bearing day-5 i.c. GL261 glioma received sequential intratumoral (i.t.) delivery of Ad-IFN-alpha and bone marrow-derived syngeneic DCs. This treatment prolonged survival in that nine of 17 animals survived long term (> 60 days versus 0 of 10 control animals). Specific CTL activity was demonstrated following this regimen in the cervical lymph nodes, and the therapeutic efficacy was dependent upon CD8+ cells. Furthermore, these animals were protected against subsequent re-challenge with GL261 gliomas. DCs injected i.t. survived in the tumor and migrated into cervical lymph node. In vitro migration assays revealed the ability of DCs to migrate toward the tumor, suggesting that i.t. injected DCs migrate through the glioma. Taken together, this combination of gene therapy and cellular immunotherapy may be an effective future strategy for treating human gliomas.

CD40 ligand immunotherapy in cancer: an efficient approach.

Cancer cells do not elicit a clinically sufficient anti-tumor immune response that results in tumor rejection. Recently, many investigators have been trying to enhance anti-tumor immunity and encouraging results have been reported. This review will discuss current anti-cancer immunotherapy; interleukin-2 therapy, tumor vaccine secreting Granulocyte macrophage-colony stimulating factor, dendritic cells fused with tumor cells, and CD40 ligand immunotherapy. Moreover, we introduce our two kinds of CD40 ligand immuno-genetherapy; (1) oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium (published in BLOOD 2000), (2) cancer vaccine transfected with CD40 ligand ex vivo for neuroblastoma (unpublished). Both approaches resulted in a high degree of protection against the tumor progression and they are simple and safe in the murine system.

Inhibitory effect of TNP-470 on hepatic metastasis of mouse neuroblastoma.

PURPOSE: TNP-470 is a strong inhibitor of angiogenesis. The present study was designed to determine whether the angiogenesis inhibitor TNP-470 inhibits metastasis of mouse neuroblastoma cells to the liver and thus increases survival. METHODS: A murine neuroblastoma cell line, C1300, and A/J mice were used in this study. First, to demonstrate the inhibitory effects of TNP-470 on angiogenesis, we quantified the area of angiogenesis on images made with SP-500 image analyzer (Olympus) 7 days after implanting a millipore chamber and compared the areas for the TNP-470-treated mice and control mice. Next, to determine the inhibitory effect of TNP-470 on metastasis of neuroblastoma cells to the liver, we made a murine hepatic metastasis model by implanting C1300 cells (1 x 10(6)) in the spleen of the mice and compared histologic findings, sizes, and weights of the livers of treated mice and control mice 14 days after the beginning of a 7-day infusion of TNP-470 (60 mg/kg). We also compared survival rates using the Kaplan-Meier method. RESULTS: When the angiogenesis inhibitor TNP-470 was infused into mice that received tumor cells, the area of angiogenesis in the TNP-470-treated mice was smaller than that in the control mice (52.5 +/- 6.3 SD vs 94.1 +/- 27.6 mm(2), P < 0.001). After the same treatment in other mice, no histologic evidence of metastasis was found, whereas control mice had countless tumor cell masses. Similarly, the weight of the liver was less in TNP-470-treated mice (0.8 +/- 0.1 g vs 4.5 +/- 0.3 g, P < 0.001). Survival was longer in the TNP-470-treated mice than in controls (80% of treated mice were alive more than 60 days after treatment, whereas all control mice died by Day 20). CONCLUSION: TNP-470 inhibits metastasis of mouse C1300 neuroblastoma cells to the liver, and thus increases survival. TNP-470 inhibits metastasis by inhibiting angiogenesis.

Organ-specific adhesion of neuroblastoma cells in vitro: correlation with their hepatic metastasis potential.

Neuroblastoma (NB) is the most common solid malignant tumor found in pediatric patients and the liver is one of the major sites of metastasis. To investigate the organ specificity of metastatic distribution, the adherence behavior of tumor cells was studied. The data presented are based on studies using a metastatic murine cell line C1300. In vivo, not only intrasplenic but also intravascular injection of C1300 NB cells consistently results in hepatic metastasis formation in syngeneic A/J mice. An in vitro assay was used in which C1300 NB cell attachment to cryostat sections of liver, spleen, brain, kidney and lung obtained from normal A/J mice was measured to compare organ-specific adhesion. A good correlation was found between their metastatic potential in the liver and the adhesion to the liver sections; C1300 NB cells adhered preferentially to liver cryostat sections. Enzyme assays indicated that cell surface glycoproteins were involved in cell adhesion. An adhesion assay with extracellular matrix proteins demonstrated that C1300 NB cells adhered preferentially to vitronectin and fibronectin, and the adherence was strongly inhibited by Arg-Gly-Asp (RGD)-containing peptides. Furthermore, adhesion of C1300 NB cells to liver cryostat sections could be blocked by the synthetic peptide GRGDS. This indicates that the interaction between RGD-containing matrix adhesion protein and cells has an important role for the specific adhesion of C1300 NB cells. The results suggested that tumor cell adhesion to liver cryostat sections could provide a useful tool in the study of host-tumor interactions in the metastasis of NB.

In vivo function of homing receptors participating in lymphocyte recirculation: transfer analysis in SCID mice.

In order to examine the in vivo function of the adhesion molecules implicated in lymphocyte homing, blocking effects of antibodies against various adhesion molecules on lymphocyte migration were tested in SCID mice into which BALB/c donor splenocytes had been transferred. It was proved that the transferred donor splenocytes migrated to peripheral lymph nodes (LNs) of SCID mice. T and B lymphocytes were distributed in the specialized compartments as seen in the LNs of normal mice. Migration of lymphocytes to the local LNs was accelerated by stimulation with ovalbumin and complete Freund's adjuvant. This experimental system with accelerated migration was applied to analyze the in vivo function of adhesion molecules, and the following findings were obtained. Combined use of antibodies against lymphocyte-function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) strongly inhibited the migration of T lymphocytes to the peripheral LNs. Antibodies against very late antigen 4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) led to diminished B lymphocyte migration and disturbed compartmentalization of T lymphocytes in the paracortex. Migration of both T and B lymphocytes to the LNs was completely inhibited by the antibody against L-selectin. These results indicate that L-selectin plays an essential role in migration of both T and B lymphocytes into peripheral LNs but LFA-1/ ICAM-1 and VLA-4/VCAM-1 play different roles in compartmentalization of T and B lymphocytes in the peripheral LNs. In contrast, these adhesion molecules were not involved in lymphocyte migration to the splenic white pulp, indicating that the mechanisms for lymphocyte homing to the white pulp are quite different from those to the peripheral LNs.