B16 Melanoma Tumor Cells Research Articles

Deficiency of plasminogen activator inhibitor‐2 results in accelerated tumor growth

BackgroundUpregulation of the plasminogen activation system, including urokinase plasminogen activator (uPA), has been observed in many malignancies, suggesting that co‐opting the PA system is a common method by which tumor cells accomplish extracellular matrix proteolysis. PAI‐2, a serine protease inhibitor, produced from the SERPINB2 gene, inhibits circulating and extracellular matrix‐tethered uPA. Decreased SERPINB2 expression has been associated with increased tumor invasiveness and metastasis for several types of cancer. PAI‐2 deficiency has not been reported in humans and PAI‐2‐deficient (SerpinB2−/−) mice exhibit no apparent abnormalities. ObjectivesWe investigated the role of PAI‐2 deficiency on tumor growth and metastasis. MethodsTo explore the long‐term impact of PAI‐2 deficiency, a cohort of SerpinB2−/− mice were aged to >18 months, with spontaneous malignancies observed in 4/9 animals, all of apparently vascular origin. To further investigate the role of PAI‐2 deficiency in malignancy, SerpinB2−/− and wild‐type control mice were injected with either B16 melanoma or Lewis lung carcinoma tumor cells, with markedly accelerated tumor growth observed in SerpinB2−/− mice for both cell lines. To determine the relative contributions of PAI‐2 from hematopoietic or nonhematopoietically derived sources, bone marrow transplants between wild‐type C57BL/6J and SerpinB2−/− mice were performed. Results and ConclusionsOur results suggest that PAI‐2 deficiency increases susceptibility to spontaneous tumorigenesis in the mouse, and demonstrate that SerpinB2 expression derived from a nonhematopoietic compartment is a key host factor in the regulation of tumor growth in both the B16 melanoma and Lewis lung carcinoma models.

Abstract PR02: Melanoma evolves complete immunotherapy resistance through acquisition of a hypermetabolic phenotype

Abstract Advances in our understanding of tumor immune biology and development of cancer immunotherapies have led to improved outcomes for patients who suffer from aggressive cancers such as melanoma. Despite the clinical success of immune checkpoint blockade, a majority of patients still fail to respond, and the underlying mechanisms that drive resistance remain unclear. To understand why a subset of tumors fail to respond to immunotherapy, we established a novel murine model of melanoma that is fully resistant to immune checkpoint blockade. By in vivo passaging nonresponding B16 melanoma tumor cells, we selected for a resistant variant that fails to respond to the combination of CTLA-4, PD-1, and PD-L1 blockade. In comparing gene expression of parental versus resistant tumor cells and analyzing the corresponding immune infiltrate, we determined the adaptations associated with resistance to therapy. We found that evasion of immunotherapy was associated with a “hypermetabolic” phenotype, characterized by an upregulation of glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways to establish a hypoxic, metabolically hostile microenvironment. Enforced expression of two key genes associated with these pathways in parental tumor cells was sufficient to mediate resistance to triple checkpoint blockade. Flow cytometry assays determined that T cells infiltrating resistant tumors had diminished glycolytic capacity and effector function, indicating a metabolic disadvantage. Consistent with our findings, melanoma patients who failed dual checkpoint blockade exhibited similar metabolic alterations as seen in our resistant variant. Using a novel MRI-based imaging approach, we observed distinct metabolic changes that stratified responding versus nonresponding tumors in live mice. Applying this method to patients could provide insight into predicting response rates to checkpoint modulation. Overall, our data indicate that resistant melanoma tumor cells acquire a “hypermetabolic” phenotype to establish a hostile microenvironment that is capable of inhibiting the antitumor immune response. This abstract is also being presented as Poster A24. Citation Format: Ashvin R. Jaiswal, Arthur J. Liu, Shivanand Pudakalakatti, Prasanta Dutta, Priyamvada Jayaprakash, Todd Bartkowiak, Casey Ager, Zhiqiang Wang, Alex Reuben, Zachary Cooper, Cristina Ivan, Zhenlin Ju, Felix Nwajei, Jing Wang, Michael A. Davies, R. Eric Davis, Jennifer A. Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Melanoma evolves complete immunotherapy resistance through acquisition of a hypermetabolic phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR02.

Abstract 2403: Targeting 4-1BB in tumors enhances anti-tumor immune responses

Abstract 4-1BB (CD137) is a co-stimulatory receptor expressed on activated T cells and other immune cells and is associated with enhanced T cell proliferation, survival, and increased cytokine production upon engagement with its ligand (4-1BBL). We have previously shown that addition of agonistic 4-1BB antibody to tumor fragments increased tumor infiltrating lymphocyte (TIL) expansion in human melanoma and pancreatic tumors. Furthermore, agonistic 4-1BB antibody led to increased expression of maturation markers in resident dendritic cells (DCs) present within tumor fragments. While the role of 4-1BB in T cell biology is well described, little is known about 4-1BB signaling in tumor-resident DCs. We hypothesized that 4-1BB agonism in tumor-resident DCs would promote anti-tumor T cell immune responses. To evaluate the role of 4-1BB signaling on dendritic cells, murine bone marrow derived-DCs were cultured with agonistic 4-1BB antibody and loaded with OVA protein. Treatment with agonistic 4-1BB in vitro led to increased OVA-FITC uptake by DCs (32.6% compared to 15.9% in DCs treated with control antibody). DCs treated with agonistic 4-1BB antibody demonstrated increased T cell stimulatory capacity upon co-culture with OVA-specific OT-I and OT-II T cells. We next compared systemic and intralesional (IL) treatment with agonistic 4-1BB antibody. For the systemic model, mice were injected subcutaneously (SC) with B16 melanoma or Panc02 pancreatic tumor cells and treated by intraperitoneal injection of control or anti-4-1BB antibodies. Mice treated systemically with agonistic 4-1BB antibody exhibited a minimal reduction in tumor growth. In contrast, IL injection of agonistic 4-1BB antibody led to dramatic decreases in tumor growth. Next, we wanted to determine whether IL treatment with agonistic 4-1BB antibody would improve DC stimulatory capacity. Mice bearing B16 or Panc02 tumors were treated IL with normal control or anti-4-1BB antibody. After 7 days, tumor-resident CD11b+ DCs exhibited enhanced in vivo antigen uptake. In addition, an increased number of CD8+ T cells was measured within tumors treated with agonistic 4-1BB antibody (28.1% compared to 20% in NrIgG-treated Panc02OVA tumors and 19.45% compared to 7.3% in NrIgG treated B16-OVA tumors; p=0.0059). Together, these studies demonstrate that agonistic 4-1BB can enhance the ability of DCs to stimulate T cells and IL administration of agonistic 4-1BB antibody can significantly reduce tumor growth. This suggests that IL treatment of agonistic 4-1BB may yield greater efficacy than systemic therapy in patients with melanoma or pancreatic cancer. Citation Format: Amy Mackay Weber, Patrick Innamarato, Jennifer Morse, Scott Kidd, Sarah Asby, Amod Sarnaik, Shari Pilon-Thomas. Targeting 4-1BB in tumors enhances anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2403.

Wheel running predicts resilience to tumors in old mice.

ABSTRACTAging intervention studies are hampered by the lack of predictive measures for determination of individuals at risk of age-associated chronic disease. Assessment of physical resilience could be informative in this regard, especially for age-related diseases such as cancer. Voluntary wheel running is a mildly stressful physical activity that is easily quantifiable in the mouse but has not been studied as a predictor of resistance to tumor invasiveness with increasing age. Male C57BL/6 mice in cohorts of 4, 12, 20, and 28 months of age were allowed access to a slanted in-cage running wheel for 3 days. Three months later, mice were injected subcutaneously with B16 melanoma tumor cells and followed for two weeks before harvesting. No relation was observed between running distance and tumor burden in the 4-month age group. The 12-month age group showed a trend, and the 20- and 28-month age groups showed a negative correlation (P < 0.05) between running distance and tumor burden. Mice in the 20-month age group that ran longer distances had lower tumor invasive scores compared to mice in the same age group that ran shorter distances. In conclusion, short term exercise capability could be a marker for resilience to cancer, and possibly other age-related disease conditions, in mice.

Abstract A02: Gut microbiome controls growth of liver tumors

Abstract Aim: The gut microbiome can modify tumor immunity and has been suggested to be involved in the development and growth of liver cancer as well as metastasis in the liver. However, it remains unknown how the gut microbiome controls hepatic immune responses. This study was designed to exam the effect of the gut microbiome on liver antitumor immunity, and to study potential mechanism. Experimental Procedure: An antibiotic cocktail containing 0.5g/L vancomycin, 0.5 g/L neomycin and 0.6 g/L primaxin in drinking water was given to reduce mouse gut microbiota. Control mice were kept on regular water. EL4 thymoma cells were injected s.c. to induces spontaneous liver metastasis. B16 melanoma and CT26 colon cancer cells were injected intrasplenically to form liver metastasis. Lung metastasis was induced by tail injection of tumor cells. Spontaneous hepatocellular carcinomas were studied in TRE-MYC mice. Gut bacteria and metabolic studies were performed. Results: Antibiotic cocktail efficiently depleted gut bacteria. Removing gut commensal bacteria did not affect the growth of primary s.c. EL4 tumors, but impaired formation of liver metastasis in different models. The inhibitory effect on liver metastasis by removing gut microbiome was found after intrasplenic injection of tumor cells to form liver metastasis using both B16 melanoma and CT26 colon cancer tumor cells as well as in TRE-MYC mice. Interestingly, formation of lung metastasis caused by tail vein injection of B16 cells was not impaired by antibiotics treatment, suggesting a liver specific effect. The inhibition of liver metastasis by antibiotic treatment was absent in Rag1 knockout mice, suggesting that the observed mechanism is mediated by the adaptive immune system. A detailed mechanism how the gut microbiome causes metabolic liver changes and thereby growth of liver tumors will be presented. Conclusion: Our results suggest that the gut microbiome affects the liver immune microenvironment and modulates antitumor immunity Citation Format: Chi Ma, Miaojun Han, Bernd Heinrich, Qiong Fu, Qianfei Zhang, Xin W. Wang, Giorgio Trinchieri, Tim Greten. Gut microbiome controls growth of liver tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A02.

Abstract 3057: Gut microbiome controls liver metastasis

Abstract Aim: The gut microbiome can modify tumor immunity and has been suggested to be involved in the development of liver cancer. However, it remains unknown how the gut microbiome controls hepatic immune responses. This study was designed to exam the effect of the gut microbiome on liver antitumor immunity, and to study potential mechanism. Experimental procedure: Antibiotic cocktail in drinking water was given to remove mouse gut microbiota. Control mice were kept on regular water. EL4 thymoma cells were injected s.c. to induce spontaneous liver metastasis. B16 melanoma and CT26 colon cancer cells were injected intrasplenically to form liver metastasis. Lung metastasis was induced by tail injection of tumor cells. Results: Antibiotic cocktail efficiently depleted gut bacteria. Removing gut commensal bacteria did not affect the growth of primary s.c. tumors, but impaired formation of liver metastasis in different models. The inhibitory effect on liver metastasis by removing gut microbiome was found after intrasplenic injection of tumor cells to form liver metastasis using both B16 melanoma and CT26 colon cancer tumor cells. Interestingly, formation of lung metastasis caused by tail vein injection of B16 cells was not impaired by antibiotics treatment, suggesting a liver specific effect. The inhibition of liver metastasis by antibiotic treatment was absent in Rag1 knockout mice, suggesting that the mechanism is mediated by the adaptive immune system. Conclusion: Our results suggest that the gut microbiome affects the liver immune microenvironment and modulates antitumor immunity. Citation Format: Chi Ma, Qiong Fu, Tim Greten. Gut microbiome controls liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3057. doi:10.1158/1538-7445.AM2017-3057

BAP31, a promising target for the immunotherapy of malignant melanomas.

PurposeMalignant melanoma’s (MM) incidence is rising faster than that of any other cancer in the US and the overall survival at 5 years is less than 10%. B cell associated protein 31 (BAP31) is overexpressed in most MMs and might be a promising target for immunotherapy of this disease.Experimental designFirstly, we investigated the expression profiles of human BAP31 (hBAP31) and mouse BAP31 (mBAP31) in human and mouse normal tissues, respectively. The expression level of hBAP31 in human MMs and mBAP31 in B16 melanoma cells was also analyzed. Then we constructed novel mBAP31 DNA vaccines and tested there ability to stimulate mBAP31-specific immune responses and antitumor immunity in B16 melanoma-bearing mice.ResultsFor the first time, we found that protein expression of hBAP31 were dramatically upregulated in human MMs when compared with human normal tissues. Predominant protein expression of mBAP31 was found in mouse B16 melanoma cells but not in mouse important organs. When mice were immunized with mBAP31 DNA vaccines, strong cellular response to mBAP31 was observed in the vaccinated mice. CTLs isolated from immunized mice could effectively kill mBAP31-positive target mouse B16 melanoma tumor cells in vitro and vaccination with mBAP31 DNA vaccines had potent anti-tumor activity in therapeutic model using B16 melanoma cells.ConclusionsThese are the first data supporting a vaccine targeting BAP31 that is capable of inducing effective immunity against BAP31-expressing MMs and will be applicable to human MMs and hBAP31 DNA vaccine warrants investigation in human clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0153-6) contains supplementary material, which is available to authorized users.

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia. Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune “escape,” including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia. Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune "escape," including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

USP18 is crucial for IFN-γ-mediated inhibition of B16 melanoma tumorigenesis and antitumor immunity.

BackgroundInterferon (IFN)-γ-mediated immune response plays an important role in tumor immunosurveillance. However, the regulation of IFN-γ-mediated tumorigenesis and immune response remains elusive. USP18, an interferon stimulating response element, regulates IFN-α-mediated signaling in anti-viral immune response, but its role in IFN-γ-mediated tumorigenesis and anti-tumor immune response is unknown.MethodIn this study, USP18 in tumorigenesis and anti-tumor immune response was comprehensively appraised in vivo by overexpression or downregulation its expression in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice.ResultsEctopic expression or downregulation of USP18 in B16 melanoma tumor cells inhibited or promoted tumorigenesis, respectively, in immunocompetent mice. USP18 expression in B16 melanoma tumor cells regulated IFN-γ-mediated immunoediting, including upregulating MHC class-I expression, reducing tumor cell-mediated inhibition of T cell proliferation and activation, and suppressing PD-1 expression in CD4+ and CD8+ T cells in tumor-bearing mice. USP18 expression in B16 melanoma tumor cells also enhanced CTL activity during adoptive immunotherapy by prolonging the persistence and enhancing the activity of adoptively transferred CTLs and by reducing CTL exhaustion in the tumor microenvironment. Mechanistic studies demonstrated that USP18 suppressed tumor cell-mediated immune inhibition by activating T cells, inhibiting T-cell exhaustion, and reducing dendritic cell tolerance, thus sensitizing tumor cells to immunosurveillance and immunotherapy.ConclusionThese findings suggest that stimulating USP18 is a feasible approach to induce B16 melanoma specific immune response.

B16 melanoma cells increase B-1 cell survival, IL-10 production and radioresistance in vitro

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. The role of both cell populations in cancer progression is still controversial. Previous studies have indicated that direct contact between B-1 cells and B16 melanoma tumor cells (B16) increases the metastatic potential of the tumor cells. However, cellular changes that are induced in B-1 cells during the interaction between these two cell types have not been evaluated. In the present study, it is hypothesized that B-1 cells are modified after their interaction with tumor cells, leading to both increased cell viability and rate of proliferation. Additionally, soluble factors that were secreted by B16 cells were sufficient to augment B-1 cell viability and to modify the production of IL-10 by B-1 cells. Impressively, after direct or indirect contact with the B16 cells, B-1 cells became resistant to radiation-induced cell death. Thus, future studies that assess the importance of concomitant immunity and other conventional therapies in cancer treatment are needed.

Enhanced memory development in mice by aptamer targeted mTORC1 inhibition in CD8+ T cells (110.3)

Abstract Recent studies have highlighted the importance of immunological memory in mediating protective immunity in chronic infectious diseases and cancer. Inhibition of intracellular mediators of T cell differentiation with pharmacological agents was shown to promote memory differentiation in mice. However, given their often pleiotropic effects, non-targeted administration of pharmacological agents could compromise their immune promoting properties. We have shown that a CD8+ T cell targeted 4-1BB aptamer -raptor siRNA conjugate downregulated mTORC1, while preserving mTORC2, activity in 60% of adoptively transferred OVA-primed OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced proliferative recall response. However, the rapamycin-generated memory cells were defective in their in vivo cytotoxic effector functions which correlated with reduced perforin expression. Likewise, rapamycin or aptamer-siRNA conjugate treatment enhanced the in vivo differentiation of gp100-primed Pmel-1 cells into memory cells. Consistent with a cytotoxic defect in the rapamycin-generated memory cells, the rapamycin-treated mice were not able to inhibit a challenge with gp100-expressing B16 melanoma tumor cells. These observations support the premise that in vivo cell-targeted inhibition of mTOR, and conceivably other mediators of T cell differentiation, will circumvent undesirable effects of non-targeted inhibition by pharmacological agents.

Abstract LB-107: Aptitude for running in mice is age and strain-dependent but not predictive of tumor response

Abstract Quality of life is a major consideration in attempting to design intervention strategies for delaying aging and age-related diseases. Preclinical studies depend on the use of reliable animal models, and the mouse has been used extensively in this regard. Quality of life can be evaluated in mice by measuring the functional status of various organ and physiological systems that are important in the health/physiological performance of an animal and that are shown to change with age in mice and/or humans. These physiological parameters would then be predicted to give a read-out of the animal's functional status relevant to human aging and quality of life. We conducted various assays of physiological function over the lifespan of male C57Bl/6 and BC6 F1 mice at 4, 12, 20, and 28 months of age. We report here on the correlation of running activity with tumor response. The inclination and capacity of mice to engage in voluntary spontaneous activity is a viable measure of their general physical aptitude. Response to the B16 melanoma tumor cell line is a measure of the host microenvironment to enhance or suppress tumor progression. We used in-cage running wheels connected to computers with monitoring software to assess the speed, distance and time the mice engaged in running behavior. Two months later, 1x105 B16 melanoma tumor cells were injected subcutaneously into the left inguinal space and repeated in the right axillary space. At 14 days post-injection, mice were terminated and tumors weighed and measured for size. Cohort numbers were 11-12 mice per age group and genotype. Four-month-old CB6 F1 mice ran further and had less tumor volume than age-matched B6 mice (16 Km vs. 11 Km and 360 mm vs. 800 mm, respectively). Total distance ran significantly decreased in each group to 0.9 Km and 2.2 Km, respectively, in mice at 28 months of age. All mice developed tumors. Tumor volume decreased in B6 mice from 4 months to 12 months to 450 mm and did not change with increasing age. Tumor volume in CB6 F1 mice remained constant at about 350 mm at all ages. In conclusion, CB6 F1 mice are more inclined to be physically active at a young age but less physically active at an old age compared to B6 mice, suggesting that running at a young age is not a predictor of physical aptitude for aging. Aging was not associated with increased tumor volume suggesting that non-genetic factors may be involved in the increased cancer incidence seen with increasing age. The aptitude for running may be associated with decreased tumor growth in young mice but not old mice. These preliminary data provide insight into the biological relevance of physiological assessment procedures in mice to help determine valid areas of focus in clinical trials of aging intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-107. doi:1538-7445.AM2012-LB-107

Abstract 395: Host ephrinB2 regulates T-cells in tumor microenvironments

Abstract Background: The tumor microenvironment consists of immune, stromal, and vascular cells. Proteins known to play roles in vascular development during embryogenesis also have been implicated in the control of leukocyte migration. The vascular patterning ligand ephrinB2, for example, influences T-cell migration in vitro. We therefore hypothesized that host ephrinB2 signaling regulates tumor immune microenvironments. Methods: EphrinB2+/lacZ mice were implanted with B16 melanoma, KR158 glioma, LLC lung, and EL4 T-lymphoma tumor cells. Macroscopic tumors were harvested and ephrinB2 expression assayed using x-gal staining. Mx1-cre/efnb2fl/fl mice were generated which allow for the post-natal deletion of ephrinB2 after pIpC treatment. Mx1-cre/efnb2+/+ mice served as controls. 1 million B16 or KR158 tumor cells were implanted in hindlimbs and tumor volume measured (V= 0.52*L*W2) at least twice weekly. Tumor tissues were stained with anti-CD3, F4/80, anti-CD34, and anti-NG2 antibodies. Experiments were repeated following T-cell depletion with i.p. injections of anti-CD4 and anti-CD8 antibodies 3x week. Finally, C57BL6 mice were transplanted with Mx1-cre/efnb2fl/fl or Mx1-cre/efnb2+/+ bone marrow and ephrinB2 was deleted after engraftment but prior to tumor growth experiments. Results: EphrinB2 was expressed in vascular structures all tumors. In addition we observed upregulation of ephrinB2 in microvascular structures in adjacent muscle during tumor invasion. Inducible deletion of host ephrinB2 significantly reduced B16 tumor growth (control tumor volume 2846 mm3 versus deleted tumor 869 mm3, p&amp;lt;0.01) and KR158 tumor growth (2028 mm3 versus 1403 mm3). EphrinB2 deletion did not affect CD34+ microvessel density, NG2+ pericyte or F4/80 macrophage infiltration. Rather, tumors grown in ephrinB2 deficient microenvironments demonstrated a significant increase in infiltrating CD3+ T-cells. In vivo depletion of T-cells abrogated the effect of ephrinB2 deletion on tumor growth delay demonstrating a role for immune cells in impairing tumor growth. EphrinB2 deletion in transplanted hematopoietic cells did not lead to tumor growth delay demonstrating a requirement for intact ephrinB2 signaling in vascular cells for T-cell regulation. Conclusion: EphrinB2 is upregulated in tumor vasculature and at sites of tumor invasion. Post-natal ephrinB2 deletion did not affect tumor angiogenesis but led to tumor growth delay via a T-cell mediated mechanism. These findings demonstrate a novel role for ephrinB2 specifically and vascular cells generally in regulating immune microenvironments. Therefore this study identifies a novel therapeutic strategy to increase the accumulation of T-cells in tumor tissues with relevance to combination immunotherapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 395. doi:1538-7445.AM2012-395

Abstract A68: An IL-12/Shh-C domain fusion protein based IL-12 autocrine loop for sustained natural killer cell activation

Abstract Background: It has been known that resting natural killer (NK) cells do not possess high levels of cytotoxicity or inflammatory cytokines that are needed for efficient tumor cell killings. Ex vivo activated NK cells, when transferred back in vivo, need a large dose of IL-2 or IL-12 to maintain such an activated status, which is a main concern for systemic toxicity. The aim of the present study is to establish an NK cell self-activation system by employing the autoprocessing domain of mouse sonic hedgehog gene to form an IL-12/Shh-C fusion gene. This fusion protein can go through autocatalytic processing to form cholesterol-modified IL-12 molecules, which would in turn bind back to the NK cells in which it is produced in an autocrine fashion. Methods: EGFP and IL-12 sequences were fused in frame with mouse sonic hedgehog C-terminal domain (Shh-C) to form eGFP/shh-C and IL-12/shh-C fusions. IL-12 without shh-C tail was used as control. Pseudo-lentiviral particles were produced and used to transduce purified murine NK cells. Subsequently, cell surface markers, cell proliferation rates, and IFN-Y secretion of NK cells transduced with different viral particles were analyzed with flow cytometry, alamar blue cell proliferation assay, and cytokine ELISA respectively. For in vivo studies, C57BL/6 mice received transduced NK cells intravenously without IL-2 support 3 days after receiving tail vein injection of B16 melanoma tumor cells and lung tissues were analyzed to determine the infiltration of modified NK cells into the tumor microenvironment. Results: The transduction rates of the three (eGFP/shh-C, IL-12, IL-12/shh-C) lentivirus-transduced NKcell populations 5 days after infection are ranging from 20% to 32%, and were maintained within this range at least for up to 10 days after transduction. CD25 (IL-2 receptor) was found to be substantially higher in eGFP/shh-C infected (2.59-fold), IL-12 infected (2.67-fold), and IL-12/shh-C (2.04-fold) infected NK cells respectively, compared to untreated NK cells. EGFP/shh-C infected, IL-12 infected, and IL-12/shh-C infected NK cells were also observed to have significantly increased Mac-1 expressions (25%, 27%, and 18% respectively). Perforin production in IL-12/shh-C infected NK cells was doubled compared to that in untreated NK cells. In the presence of 60 lU/mL and 6000 lU/mL IL-2, IL-12/shh-C infected NK cells have 63.9% (p&amp;lt;0.05) and 216.7% increase of proliferation rate respectively when compared to eGFP/shh-C infected NK cells, suggesting that the fusion protein decreased the requirement of NK cells for IL-2. The amounts of IFN-Y in the supernatant of IL-12/shh-C virus transduced NK cells 5 days and 7 days after transduction were 40% (p=0.008) and 48% (p=0.00045) higher respectively compared to the untreated group. Results from the cytotoxicity assay indicated that lentivirus transduction did not alter NK cell-associated cytotoxicity. Immunofluorescent staining in the lung tissue slides from B16-bearing mice suggested that donor NK cells successfully infiltrated into tumor nodules in lung tissues. Conclusion: The IL-12/Shh-C domain fusion protein could function, once autoprocessed to form cholesterol anchored IL-12, to maintain the activated status of NK cells in vitro, as evidenced by the greatly reduced amount of IL-2 required as well as the greatly increased secretion of IFN-Y by NK cells in vitro. In addition, IL-12/shh-C virus transduced NK cells successfully infiltrated into B16 tumor nodules in lung tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A68.

Boronated unnatural cyclic amino acids as potential delivery agents for neutron capture therapy

Boron delivery characteristics of cis and trans isomers of a boronated unnatural amino acid, 1-amino-3-boronocyclopentanecarboxylic acid (ABCPC) were tested in the B16 mouse model for human melanoma. Both ABCPC isomers delivered comparable boron to B16 melanoma tumor cells as L-p-boronophenylalanine (BPA). Secondary ion mass spectrometry (SIMS) analysis revealed the presence of boron throughout the tumor from these compounds, and a near homogeneous distribution between the nucleus and cytoplasm of B16 cells grown in vitro. These encouraging observations support further studies of these new boron carriers in BNCT.

Immunotherapy of Epstein-Barr Virus Associated Malignancies Using Mycobacterial HSP70 and LMP2A356–364 Epitope Fusion Protein

Epstein-Barr virus infection is strongly associated with a number of malignancies. The EBV latent membrane protein 2A has been implicated as one of the most attractive candidates for immunotherapy of related malignancies. In previous studies, the T cell epitopes of LMP2A have been identified systematically. However, the epitope-based vaccine generally meets inefficient immunogenicity when used in vivo directly, which could be overcome by combination with appropriate adjuvants. Heat shock protein is a natural chaperon, which is able to activate the classical major histocompatibility complex class I antigen-processing pathway (cross-presentation). In this study, a minigene encoding LMP2A(356-364) (FLYALALLL) was genetically fused to the carboxy-terminal of mycobacterial heat shock protein 70. The epitope fusion protein was expressed and purified, and the cross-presentation of LMP2A(356-364) by monocyte-derived dendritic cells pulsed with the epitope fusion protein was evaluated. Results showed that the epitope fusion protein-pulsed mDCs were much more efficient than the single peptide-pulsed mDCs on CTL activation. Immunization of HLA-A2.1 transgenic mice with MtHsp70-LMP2A(356-364) generated peptide specific CTL more effectively than a single peptide plus incomplete Freund's adjuvant (IFA). Growth of LMP2A expressing B16 melanoma tumor cells was suppressed in the vaccinated groups. Our results suggested that MtHsp70-LMP2A(356-364) fusion protein was more effective than the CD8(+) T cell epitope alone on anti-tumor immunity. As a result, the MtHsp70-LMP2A(356-364) fusion protein is considered to be a promising candidate vaccine for EBV related malignancies.

Both CD4+ and CD8+ T cell epitopes fused to heat shock cognate protein 70 (hsc70) can function to eradicate tumors

Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP-associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant-free manner and is mediated by CD8(+) T cells, indicating that HSP can act as a natural adjuvant and cross-prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8(+) T cell epitope fused to the carboxyl-terminus of hsc70 and demonstrated efficient generation of antigen-specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70-CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4(+) T cell epitope to the amino terminus of the hsc70-CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)-derived 8 mer peptide, OVA(257-264), and 16mer peptide, OVA(265-280), were used as CD8(+) and CD4(+) T cell epitopes, respectively. Vaccination with hsc70-OVA(257-264) generated peptide specific CTL more effectively than a peptide plus incomplete Freund's adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA(265-280) to the amino-terminus of hsc70-OVA(257-264) (OVA(265-280)-hsc70-OVA(257-264)) enhanced the generation of the OVA(257-264)-specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70-OVA(257-264). Our results suggest that fusion of both CD4(+) and CD8(+) T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8(+) T cell epitope alone.

Covalent coupling of a novel TLR7 agonist to antigen elicits an effective immune response in a murine melanoma tumor model

3019 Background: Toll-like receptor (TLR) signaling triggers innate immune stimulation capable of generating an adaptive immune response under appropriate conditions. TLR agonists have shown efficacy in preclinical and clinical cancer therapies. However, one limitation to this approach has been the use of TLR agonists delivered at distances away from the target (ie-subcutaneous injections for general immune stimulation) or the inability to effectively couple antigen with adjuvant. Methods: We have synthesized a novel TLR7 agonist capable of covalent coupling to primary amines under physiologic conditions. In vitro activity of adjuvant alone or antigen-adjuvant complexes were tested for innate immune activation by utilizing bone marrow-derived murine or peripheral blood mononuclear cell-derived dendritic cells (DC) with respect to DC maturation and cytokine secretion (IL12, IL6, TGFbeta and IFNgamma). Immunocompetent syngeneic C57/b6 mice were prophylactically vaccinated with intradermal with either antigen, antigen mixed with adjuvant or antigen- adjuvant coupled complexes and challenged with B16 melanoma tumor cells expressing the cOVA transgene. Results: The effective concentration (EC50) for each compound generally followed a bell shaped distribution with higher doses being inhibitory. Maximal stimulation occurred between 10 and 1000nM. Coupled adjuvant molecules retained activity with generally lower EC50 values. Coupling 1V199 to chicken ovalbumin nearly doubled median survival from 22 to 35 days following subcutaneous tumor challenge compared with chicken ovalbumin alone. Conclusions: Covalent linkage of a TLR7 agonist to antigen stimulates DC cytokine production and protects mice from tumor challenge. Use of a soluble TLR7 agonist which retains its immune stimulating properties following coupling to antigen under physiologic conditions may be useful in the development of an in situ vaccine in solid tumor therapy. No significant financial relationships to disclose.

Tumor Cell Loaded Type-1 Polarized Dendritic Cells Induce Th1-Mediated Tumor Immunity

Dendritic cells are professional antigen-presenting cells capable of inducing and regulating innate and antigen-specific immune responses. Therapeutic cancer vaccines using ex vivo engineered or in vivo targeted dendritic cells are being evaluated in clinical trials. T-helper type-1 (Th1)-skewed immune responses are characterized by the preferential induction of antigen-specific IFN-gamma-secreting CD4+ T cells and correlate with effector mechanisms important for tumor and viral immunity. Methods to "polarize" human monocyte-derived dendritic cells for the preferential induction of Th1-skewed immune responses have been developed, and polarized dendritic cells (DC1s) are being evaluated in preclinical and clinical studies. Here, we show that stimulation of bone marrow-derived murine dendritic cell populations with poly(I:C) and CpGs results in phenotypic maturation of dendritic cells and synergistic induction of durable, high-level IL-12p70 secretion characteristic of human type-1 polarized dendritic cells. Functionally, these dendritic cells induce antigen-specific Th1-type CD4+ T-cell activation in vitro and in vivo. Dendritic cell maturation and polarization are not inhibited by the presence of live B16 melanoma tumor cells, and tumor-loaded DC1s induce delayed-type hypersensitivity responses in vivo. DC1s loaded with B16 melanoma cells and injected into tumor-bearing mice induce Th1-skewed tumor-specific CD4+ T cells and a significant reduction in tumor growth. Tumor infiltrates in DC1-immunized animals are characterized by the presence of CD4+ T cells and activated macrophages. These results show a murine model of DC1 function and suggest an important role for CD4+ T cells and macrophages in DC1-induced antitumor immune responses. They have implications for the future development of DC1-based immunotherapies and strategies for clinical immune monitoring of their effectiveness.