e14210 Background: The purpose of the study was to determine the effect of a strain No. 228 of a new rotavirus group of the Reoviridae family on the growth of transplantable В16/F10 melanoma in mice and survival of animals. Methods: The study was performed using a strain No.228 of a new group of rotaviruses (working title «group K», RVK) ( http://jbks.ru/archive/issue-10/article-6 ). RVK is a live attenuated and apathogenic strain growing on the PEKC culture (pig embryo kidney cells) with 5·109 viral particles/mL. The dynamics of melanoma growth and survival of animals receiving RVK were studied on 25 male mice divided equally into 5 groups. RVK was administered intramuscularly and Per os once a week, respectively 0.3 mL and 25 µL to groups 1-2 21 days prior to the tumor inoculation, a total of 4 injections (“vaccination” regimen); to groups 3-4 – 7 days after tumor inoculation with the appearance of tumor nodes, a total of 3 injections (“treatment” regimen); group 5 – controls receiving saline. Groups 1 and 3 received live RVK, groups 2 and 4 - RVK inactivated by UV radiation. Results: Survival in both groups of animals with “vaccination” regimen was improved significantly, compared to controls (group 1 – 25.8±2.2 days after melanoma transplantation; group 2 – 25.4±1.1 days; controls – 14.6±3.08 days, p < 0.05). In groups with “treatment” regimen, survival was 19.2±3.08 days in group 3 and 21.6±3.52 days in group 4, the latter significantly higher than in controls (p < 0.05). Introduction of a live virus in the “vaccination” regimen resulted in significantly better survival compared to the “treatment” regimen (group 1 vs. group 3), while inactivated virus did not caused such a difference (groups 2 and 4). Conclusions: Improved survival of mice with B16/F10 melanoma after administration of RVK demonstrated its ability to inhibit tumor growth and confirmed our previously published data obtained on Guerin's carcinoma in rats. Administration of a live virus prior to the tumor inoculation cause stronger effect than after inoculation. Since inactivation of the virus does not eliminate its effect, apparently, it is associated with immunostimulating action, rather than with oncolytic one.

The antitumor activity of the antifibrinolytic drug ε-aminocaproic acid (ACC) in the therapeutic form (5% ACC solution in physiological solution instead of drinking water) was studied in the ascitic and solid Ehrlich tumor. ACC did not affect the growth of ascitic, as well as a solid Ehrlich tumor, when the drug was administered 72 hours after the tumor inoculation. At the same time, when it was administered 7 days after tumor inoculation, the drug had a statistically significant antitumor effect on the tumor nodes formed (8-10 mm in diameter). ACC antitumor effect allows to assume that it influences tumor stroma rather than the parenchyma. This effect is not fully understood and needs further study.

An alternate thermal physical treatment was developed to destroy tumor tissue using liquid nitrogen cooling and RF heating treatment in our pervious study. Our pervious reports had shown that anti-tumor immunity was induced by the alternate treatment. Myeloid derived suppressor cells (MDSCs) are a subset of heterogeneous, bone marrow derived hematopoietic cells that accumulate in the spleen, bone marrow, blood and tumor sites of tumor-bearing mice and cancer patients. MDSCs are one of the key suppressor cells that regulate anti-tumor immune responses in tumor-bearing hosts. MDSCs have been shown to inhibit the function of various types of cells mediating anti-tumor immunity, such as T cells, B cells, NK cells and dendritic cells. MDSCs are recruited specifically to the tumors and contribute indirectly to angiogenesis, growth and metastasis. MDSCs also exert resistance to cancer therapies, such as anti-VEGF strategies and cancer immunotherapy. Given the role of MDSCs in tumor invasion and metastasis and anti-tumor immune responses, therapeutics targeting MDSCs might offer a new strategy for cancer treatment. In this study, the therapeutic effect and MDSCs changes after the alternate cooling and heating treatment was studied using the 4T1 murine mammary carcinoma, a common animal model of human metastatic breast cancer. Due to its highly invasive and poorly immunogenic characters, the 4T1 tumor could cause death even after the primary tumor was surgically removed. The treatment was carried out when micro-metastases were well established. Comparisons were made with the results from the surgery and hyperthermia groups, respectively. The results showed that MDSCs in blood increased rapidly with time after tumor inoculation, and in 66 days, all the mice died in the control group. The statistical results indicated a significant increase in circulating MDSC numbers at different tumor growth stages. In the surgical resection group, MDSCs in blood did not decrease, but increased rapidly to a level much higher that of the control group in 39 day after tumor inoculation. In the hyperthermia group, MDSCs in blood increased rapidly with time after tumor inoculation, and in 39 day, MDSCs was up to 3 times higher than that of the control group. Mice died in 45 day after initial tumor inoculation. But in the alternate treatment group, the number of MDSCs decreased rapidly and recovered to the normal healthy level in 11 days after the treatment. No metastatic tumor could be observed in these mice, and they were in good physiological conditions as observed in the following 3 month. In conclusion, the alternate treatment was found extremely effective against MDSCs in the very aggressive and highly metastatic mouse mammary carcinoma. The good prognosis was expected in relation to the significant decrease in MDSCs and thus the relief of the immune suppression, induced by the alternate cooling and heating treatment. It could be further developed as a novel therapeutic method against metastatic tumor. On the other hand, combining the alternate treatment with other strategies, such as anti-VEGF and cancer immunotherapy, the best therapeutic effect would be achieved through synergy.

Abstract Abstract 2941 Current approaches for vaccination and immunotherapy are often capable of eliciting strong T-cell responses against tumor antigens. However, negative regulatory mechanisms within the tumor microenvironment inhibit antitumor T-cell function, leading to evasion from immune attack. One inhibitory mechanism is upregulation of programmed death-ligand 1 (PD-L1/B7H1) expressed on tumor or stromal cells, which binds to programmed death-1 (PD-1) on activated T cells. PD-1 and B7H1 engagement results in diminished antitumor T-cell responses and correlates with poor outcome in murine and human cancers. To cure mice with large tumor burdens by administering tumor vaccines, we hypothesized that it is necessary not only to enhance the immunogenicity of the vaccines, but also to target the suppressive tumor microenvironment and break immune suppression on effector cells. Therefore, we explored combinational treatments in a myeloma setting by using vaccination and anti-B7H1 blocking mAbs to enhance clinical efficacy of cancer vaccines. To establish mouse tumor models for evaluation of tumor-associated B7H1 in the inhibition of T-cell immunity, first, we examined the expression of B7H1 in four murine myeloma cell lines that originated from Balb/c mice. Results showed that all the myeloma cells expressed a negative T-cell costimulatory molecule B7H1, and blocking surface B7H1 by using a specific mAb (M5H1) enabled tumor-specific CTLs from vaccinated mice to be more efficient at lysing the tumor cells. Thus these results suggest that B7H1 on tumor cells and PD-1 on T cells may form a B7H1/PD-1 molecular shield to prevent lysis by T cells and actively inhibit the cytolytic function of tumor-specific CTLs by expressing B7H1. Next, we evaluated the therapeutic efficacy of DKK1-DNA vaccine in mice with established myeloma. In these experiments, DKK1-DNA vaccine plus CpG was used as a standard vaccine. Anti-B7H1 (M5H1) mAb was used to block negative T-cell signaling in mice receiving DKK1-DNA vaccination. Balb/c mice were first inoculated subcutaneously with the myeloma cells (1 million cells per mouse). Ten days after tumor inoculation, mice were immunized with three injections of the vaccines on days 10, 14, and 17 after tumor inoculation. Mice were also injected intraperitoneally with B7H1 (200 μg per mouse per treatment) on days 1, 4, 7, and 10 following the first vaccination. Mice receiving injections of PBS, isotype IgG (200 μg per mouse per treatment), or CpG- (50 μg per mouse per treatment) alone were used as controls. Results show that mice receiving DKK1-DNA vaccine (plus CpG) together with B7H1 induced more robust tumor growth inhibition (P < 0.01, compared with PBS control mice). The survival of DKK1-CpG vaccine- and vaccine plus B7H1 antibody-treated mice were 60% and 80%, respectively. Thus, our data showed that B7H1 blocking mAbs can further enhance the therapeutic efficacy of the vaccine. Finally, we investigated the mechanism of therapeutic efficacy of vaccine enhanced by anti-B7H1 mAbs. Our results showed that the percentages of IL-10-secreting or Foxp3+ Tregs in the spleens and tumors are significantly decreased in mice vaccinated with DKK1 DNA plus CpG in combination with anti-B7H1 antibodies (P < 0.01, compared with mice receiving DKK1 DNA alone or vaccine plus CpG). These results indicate that myeloma cells are able to induce or recruit regulatory T cells, and the beneficial effects of anti-B7H1 mAbs were derived from their ability to reduce the numbers of Tregs in tumor-bearing mice. Taken together, these results support the concept that B7H1/PD-1 forms a molecular shield to prevent destruction by CTLs and implicate new approaches for immunotherapy of human cancers by B7H1 blocking mAbs. Disclosures: No relevant conflicts of interest to declare.

The methanol extract of Diospyros peregrina (Ebenaceae) bark (MEDP) were evaluated for antitumor activity against Ehrlich ascites carcinoma (EAC)-bearing swiss albino mice. The extract was administered at the doses of 200 and 400 mg/kg body weight per day for 14 days after 24 h of tumor inoculation. After the last dose and 18 h fasting, the mice were sacrificed. The present study deals with the effect of MEDP on the growth of transplantable murine tumor, life span of EAC-bearing hosts and hematological profile  MEDP caused significant (P < 0.01) decrease in tumor volume, packed cell volume, and viable cell count; and it prolonged the life span of EAC-tumor bearing mice. Hematological profile converted to more or less normal levels in extract-treated mice. The results indicate that MEDP exhibited significant antitumor activity in EAC-bearing mice. Keywords: Diospyros peregrina; Ehrlich ascites carcinoma; Antitumor. © 2011 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi:10.3329/jsr.v3i2.6787                J. Sci. Res. 3 (2), 413-419 (2011)

PURPOSE: To develop a model to evaluate the effects of focal pulsed ultrasound (US) waves as a source of heat for treatment of murine subcutaneous implanted Walker tumor. METHODS: An experimental, controlled, comparative study was conducted. Twenty male Wistar rats (160-300 g) randomized in 2 equal groups (G-1: Control and G-2: Hyperthermia) were inoculated with Walker-256 carcinosarcoma tumor. After 5 days G-2 rats were submitted to 45ºC hyperthermia. Heat was delivered directly to the tumor by an ultrasound (US) equipment (3 MHz frequency, 1,5W/cm³). Tumor temperature reached 45º C in 3 minutes and was maintained at this level for 5 minutes. Tumor volume was measured on days 5, 8, 11, 14 e 17 post inoculation in both groups. Unpaired t-test was used for comparison. P<0.05 was considered significant. RESULTS: Tumor volume was significantly greater in day 5 and decreased in days 11, 14 and 17 in treated rats. Rats treated with hyperthermia survived longer than control animals. On the 29th day following tumor inoculation, 40% of control rats and 77.78% of hyperthermia-treated rats remained alive. CONCLUSION: The proposed model is quite simple and may be used in less sophisticated laboratory settings for studying the effects of focal hyperthermia in the treatment of malignant implanted tumours or in survival studies.

Abstract The derivation of both myeloid and lymphoid cells from human embryonic stem cells (hESCs) clearly establish hESCs as an important model system to study human hematopoietic ontogeny. However, the potential for clinical applications of hESC-derived hematopoietic cells still remain poorly characterized. Here we have analyzed the efficacy of hESC-derived natural killer (NK) cells in a model of anti-tumor immunotherapy. hESC-derived NK cells were compared to NK cells derived from human umbilical cord blood (UCB) for ability to clear both established human tumors and metastatic disease in an in vivo model. Using a two-step differentiation process, we have demonstrated effective derivation of NK cells from hESCs. The hESC-derived NK cells express activating and inhibitory receptors similar to NK cells derived from UCB. These receptors include C-type lectin-like receptors, natural cytotoxicity receptors, CD16 and diverse killer-cell Ig-like receptors. More importantly, the hESC-derived NK cells also demonstrate cytokine production and potent direct cytolytic activity against multiple types of tumors, including leukemia, lymphoma, glioma, testicular cancer and breast cancer cells lines. This in vitro cytolytic activity is similar to what found for UCB-derived NK cells cultured in identical conditions. To advance these studies to a more relevant pre-clinical model, we have now investigated the in vivo activity of hESC-derived NK cells in a xenogeneic mouse model. Here, K562 erythroleukemia cells stably expressing firefly luciferase (luc) were injected subcutaneously into sub-lethally irradiated NOD/SCID mice. The luc+ K562 cells allows serial bioluminescent imaging to follow growth of the tumor cells non-invasively over a prolonged time course, as well as sensitive detection of micro-metastasis. Three days after tumor-inoculation, mice received one of three treatment courses: NK cells derived from hESCs, NK cells derived from UCB, or no cells. Each group received ip injections of IL-15 every 2–3 days for the first 7 days after treatment, then IL-2 every 2–3 days for an additional 2 weeks. In this model, mice that received cytokine treatment but did not receive NK cells (n=11) consistently developed large tumors within three weeks. Remarkably, all mice treated with hESC-derived NK cells demonstrated complete clearance of the primary tumor two weeks after tumor inoculation (n=8). In contrast, mice treated with UCB-derived NK cells had significantly less anti-tumor activity in vivo, with only 50% tumor-free animals treated with UCB-derived NK cells (n=8). Some mice treated with hESC-derived NK cells were monitored up to 8 weeks with no evidence of tumor development. Furthermore, liver, lungs, spleen and kidneys were harvested at the time of sacrifice and analyzed for presence of micro-metastasis by detection of luc. In animals receiving cytokines alone, 50% of the organs analyzed displayed metastatic presence of luc+ cells. However, there was a significant reduction of metastases in UCB-NK-treated (9%) and hESC-NK-treated (4%) animals. These results suggest that hESC-derived NK cells are capable of clearing human tumor cells in vivo more effectively than UCB-derived NK cells. Current studies are underway to investigate in vivo activity of hESC-derived NK cells in other tumor models, and to evaluate specific mechanisms that might regulate improved in vivo activity of hESC- compared to UCB-derived NK cells, focusing on in vivo cell migration, cell survival and proliferation.