Tumor ablation plus co-administration of CpG and saponin adjuvants affects IL-1 production and multifunctional T cell numbers in tumor draining lymph nodes.

BACKGROUND: Tumor ablation techniques, like cryoablation, are successfully used in the clinic to treat tumors. The tumor debris remaining in situ after ablation is a major antigen depot, including neoantigens, which are presented by dendritic cells (DCs) in the draining lymph nodes to induce tumor-specific CD8+ T cells. We have previously shown that co-administration of adjuvants is essential to evoke strong in vivo antitumor immunity and the induction of long-term memory. However, which adjuvants most effectively combine with in situ tumor ablation remains unclear. METHODS AND RESULTS: Here, we show that simultaneous administration of cytidyl guanosyl (CpG) with saponin-based adjuvants following cryoablation affects multifunctional T-cell numbers and interleukin (IL)-1 induced polymorphonuclear neutrophil recruitment in the tumor draining lymph nodes, relative to either adjuvant alone. The combination of CpG and saponin-based adjuvants induces potent DC maturation (mainly CpG-mediated), antigen cross-presentation (mainly saponin-based adjuvant mediated), while excretion of IL-1ß by DCs in vitro depends on the presence of both adjuvants. Most strikingly, CpG/saponin-based adjuvant exposed DCs potentiate antigen-specific T-cell proliferation resulting in multipotent T cells with increased capacity to produce interferon (IFN)γ, IL-2 and tumor necrosis factor-α in vitro. Also in vivo the CpG/saponin-based adjuvant combination plus cryoablation increased the numbers of tumor-specific CD8+ T cells showing enhanced IFNγ production as compared with single adjuvant treatments. CONCLUSIONS: Collectively, these data indicate that co-injection of CpG with saponin-based adjuvants after cryoablation induces an increased amount of tumor-specific multifunctional T cells. The combination of saponin-based adjuvants with toll-like receptor 9 adjuvant CpG in a cryoablative setting therefore represents a promising in situ vaccination strategy.

Combined sialic acid and histone deacetylase (HDAC) inhibitor treatment up-regulates the neuroblastoma antigen GD2.

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue. GD2 is a primary target for the development of immunotherapy for neuroblastoma. Immunotherapy with monoclonal anti-GD2 antibodies has proven safety and efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. Strategies to modulate GD2 expression in neuroblastoma could further improve anti-GD2-targeted immunotherapy. Here, we report that the cellular sialylation pathway, as well as epigenetic reprogramming, strongly modulates GD2 expression in human and mouse neuroblastoma cell lines. Recognition of GD2 by the 14G2a antibody is sialic acid-dependent and was blocked with the fluorinated sialic acid mimetic Ac53FaxNeu5Ac. Interestingly, sialic acid supplementation using a cell-permeable sialic acid analogue (Ac5Neu5Ac) boosted GD2 expression without or with minor alterations in overall cell surface sialylation. Furthermore, sialic acid supplementation with Ac5Neu5Ac combined with various histone deacetylase (HDAC) inhibitors, including vorinostat, enhanced GD2 expression in neuroblastoma cells beyond their individual effects. Mechanistic studies revealed that Ac5Neu5Ac supplementation increased intracellular CMP-Neu5Ac concentrations, thereby providing higher substrate levels for sialyltransferases. Furthermore, HDAC inhibitor treatment increased mRNA expression of the sialyltransferases GM3 synthase (ST3GAL5) and GD3 synthase (ST8SIA1), both of which are involved in GD2 biosynthesis. Our findings reveal that sialic acid analogues and HDAC inhibitors enhance GD2 expression and could potentially be employed to boost anti-GD2 targeted immunotherapy in neuroblastoma patients.

Sialic Acid Blockade Suppresses Tumor Growth by Enhancing T-cell-Mediated Tumor Immunity.

Sialic acid sugars on the surface of cancer cells have emerged as potent immune modulators that contribute to the immunosuppressive microenvironment and tumor immune evasion. However, the mechanisms by which these sugars modulate antitumor immunity as well as therapeutic strategies directed against them are limited. Here we report that intratumoral injections with a sialic acid mimetic Ac53FaxNeu5Ac block tumor sialic acid expression in vivo and suppress tumor growth in multiple tumor models. Sialic acid blockade had a major impact on the immune cell composition of the tumor, enhancing tumor-infiltrating natural killer cell and CD8+ T-cell numbers while reducing regulatory T-cell and myeloid regulatory cell numbers. Sialic acid blockade enhanced cytotoxic CD8+ T-cell-mediated killing of tumor cells in part by facilitating antigen-specific T-cell-tumor cell clustering. Sialic acid blockade also synergized with adoptive transfer of tumor-specific CD8+ T cells in vivo and enhanced CpG immune adjuvant therapy by increasing dendritic cell activation and subsequent CD8+ T-cell responses. Collectively, these data emphasize the crucial role of sialic acids in tumor immune evasion and provide proof of concept that sialic acid blockade creates an immune-permissive tumor microenvironment for CD8+ T-cell-mediated tumor immunity, either as single treatment or in combination with other immune-based intervention strategies.Significance: Sialic acid sugars function as important modulators of the immunosuppressive tumor microenvironment that limit potent antitumor immunity.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3574/F1.large.jpg Cancer Res; 78(13); 3574-88. ©2018 AACR.

Lipid Droplets as Immune Modulators in Myeloid Cells.

Lipid droplets (LDs) were initially described as fat storage organelles in adipocytes, but are increasingly recognized as dynamic players in lipid metabolism, with important roles not only in diseases such as diabetes and cancer, but also in immune regulation. Alterations in immune cell function, such as myeloid cell activation, are connected to profound changes in LD numbers and LD protein composition. Thus, these organelles appear to be essential to metabolically support immune responses, and have a vital role in antigen crosspresentation, interferon (IFN) responses, production of inflammatory mediators, and pathogen clearance. Here, we review recent studies that report on the role of LDs in the modulation of immune cell function, primarily focusing on myeloid cells, such as macrophages and dendritic cells (DCs).

Impact of MR-guided boiling histotripsy in distinct murine tumor models.

Interest in mechanical high intensity focused ultrasound (HIFU) ablation is rapidly growing. Boiling histotripsy (BH) is applied for mechanical fragmentation of soft tissue into submicron fragments with limited temperature increase using the shock wave and cavitation effects of HIFU. Research on BH has been largely limited to ex vivo experiments. As a consequence, the in vivo pathology after BH treatment and the relation to preexistent tissue characteristics are not well understood. This study reports on in vivo MR guided BH treatment, either with 100 or 200 pulses per focal spot, in three different subcutaneous mouse tumor models: a soft-tissue melanoma (B16OVA), a compact growing thymoma (EL4), and a highly vascularized neuroblastoma (9464D). Extensive treatment evaluation was performed using MR imaging followed by histopathology 2h after treatment. T2 weighted MRI allowed direct in vivo visualization of the BH lesions in all tumor models. The 100-pulse treated area in the B16OVA tumors was larger than the predicted treatment volume (500±10%). For the more compact growing EL4 and 9464D tumors this was 95±13% and 55±33%, respectively. Histopathology after the 100-pulse treatment revealed completely disintegrated lesions in the treated area with sharp borders in the compact EL4 and 9464D tumors, while for B16OVA tumors the lesion contained a mixture of discohesive (partly viable) clusters of cells, micro-vessel remainings, and tumor cell debris. The treatment of B16OVA with 200 pulses increased the fragmentation of tumor tissue. In all tumor types only micro-hemorrhages were detected after ablation (slightly higher after 200-pulse treatment for the highly vascularized 9464D tumors). Collagen staining revealed that the collagen fibers were to a greater or lesser extent still intact and partly clotted together near the lesion border in all tumor models. In conclusion, this study reveals effective mechanical fragmentation of different tumor types using BH without major hemorrhages. However, treatment settings may need to be adjusted to the tissue characteristics for optimal tissue fragmentation.

Steering Siglec-Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry.

Sialic acid sugars that terminate cell-surface glycans form the ligands for the sialic acid binding immunoglobulin-like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross-reactivity and led to the discovery of three selective Siglec-5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec-3 dampened the activation of Siglec-3+ monocytic cells through the NF-κB and IRF pathways.

Thermal and mechanical high-intensity focused ultrasound: perspectives on tumor ablation, immune effects and combination strategies.

Tumor ablation technologies, such as radiofrequency-, cryo- or high-intensity focused ultrasound (HIFU) ablation will destroy tumor tissue in a minimally invasive manner. Ablation generates large volumes of tumor debris in situ, releasing multiple bio-molecules like tumor antigens and damage-associated molecular patterns. To initiate an adaptive antitumor immune response, antigen-presenting cells need to take up tumor antigens and, following activation, present them to immune effector cells. The impact of the type of tumor ablation on the precise nature, availability and suitability of the tumor debris for immune response induction, however, is poorly understood. In this review, we focus on immune effects after HIFU-mediated ablation and compare these to findings using other ablation technologies. HIFU can be used both for thermal and mechanical destruction of tissue, inducing coagulative necrosis or subcellular fragmentation, respectively. Preclinical and clinical results of HIFU tumor ablation show increased infiltration and activation of CD4+ and CD8+ T cells. As previously observed for other types of tumor ablation technologies, however, this ablation-induced enhanced infiltration alone appears insufficient to generate consistent protective antitumor immunity. Therapies combining ablation with immune stimulation are therefore expected to be key to boost HIFU-induced immune effects and to achieve systemic, long-lasting, antitumor immunity.

Metabolic sialic acid blockade lowers the activation threshold of moDCs for TLR stimulation.

Sialic acid sugars cover the surface of dendritic cells (DCs) and have been suggested to impact several aspects of DC biology. Research into the role of sialic acids in DCs, however, is complicated by the limited number of tools available to modulate sialic acid expression. Here we report on a synthetic, fluorinated sialic acid mimetic, Ac53FaxNeu5Ac, which potently blocks sialic acid expression in human monocyte-derived DCs (moDCs). Sialic acid blockade enhanced the responsiveness of moDCs to Toll-like receptor (TLR) stimulation as measured by increased maturation marker expression and cytokine production. Consequently, the T-cell activation capacity of Ac53FaxNeu5Ac-treated moDCs was strongly increased. In addition to sialic acids, moDCs also expressed the sialic acid-binding immunoglobulin-like lectins (Siglecs) -3, -5, -7, -9 and -10, immune inhibitory receptors recognizing these sialic acids. Treatment with Ac53FaxNeu5Ac abrogated putative cis and trans interactions between sialic acids and Siglec-7/-9. Together, these data indicate that sialic acids limit the activation of moDCs via the TLR pathway, potentially by interacting with Siglec-7 or Siglec-9. Metabolic sialic acid blockade with Ac53FaxNeu5Ac could therefore potentially be used to generate more potent DC-based vaccines for induction of robust anti-viral or anti-tumor immune responses.

Saponin-based adjuvants induce cross-presentation in dendritic cells by intracellular lipid body formation.

Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is induced is not clear. Here we show that SBAs uniquely induce intracellular lipid bodies (LBs) in the CD11b+ DC subset in vitro and in vivo. Using genetic and pharmacological interference in models for vaccination and in situ tumour ablation, we demonstrate that LB induction is causally related to the saponin-dependent increase in cross-presentation and T-cell activation. These findings link adjuvant activity to LB formation, aid the application of SBAs as a cancer vaccine component, and will stimulate development of new adjuvants enhancing T-cell-mediated immunity.

Anti-GD2 mAb and Vorinostat synergize in the treatment of neuroblastoma.

Neuroblastoma (NBL) is a childhood malignancy of the sympathetic nervous system. For high-risk NBL patients, the mortality rate is still over 50%, despite intensive multimodal treatment. Anti-GD2 monoclonal antibody (mAB) in combination with systemic cytokine immunotherapy has shown clinical efficacy in high-risk NBL patients. Targeted therapy using histone deacetylase inhibitors (HDACi) is currently being explored in cancer treatment and already shows promising results. Using our recently developed transplantable TH-MYCN NBL model, we here report that the HDAC inhibitor Vorinostat synergizes with anti-GD2 mAb therapy in reducing NBL tumor growth. Further mechanistic studies uncovered multiple mechanisms for the observed synergy, including Vorinostat-induced specific NBL cell death and upregulation of the tumor antigen GD2 on the cell surface of surviving NBL cells. Moreover, Vorinostat created a permissive tumor microenvironment (TME) for tumor-directed mAb therapy by increasing macrophage effector cells expressing high levels of Fc-receptors (FcR) and decreasing the number and function of myeloid-derived suppressor cells (MDSC). Collectively, these data imply further testing of other epigenetic modulators with immunotherapy and provide a strong basis for clinical testing of anti-GD2 plus Vorinostat combination therapy in NBL patients.

In vivo MR guided boiling histotripsy in a mouse tumor model evaluated by MRI and histopathology.

Boiling histotripsy (BH) is a new high intensity focused ultrasound (HIFU) ablation technique to mechanically fragmentize soft tissue into submicrometer fragments. So far, ultrasound has been used for BH treatment guidance and evaluation. The in vivo histopathological effects of this treatment are largely unknown. Here, we report on an MR guided BH method to treat subcutaneous tumors in a mouse model. The treatment effects of BH were evaluated one hour and four days later with MRI and histopathology, and compared with the effects of thermal HIFU (T-HIFU). The lesions caused by BH were easily detected with T2 w imaging as a hyper-intense signal area with a hypo-intense rim. Histopathological evaluation showed that the targeted tissue was completely disintegrated and that a narrow transition zone (<200 µm) containing many apoptotic cells was present between disintegrated and vital tumor tissue. A high level of agreement was found between T2 w imaging and H&E stained sections, making T2 w imaging a suitable method for treatment evaluation during or directly after BH. After T-HIFU, contrast enhanced imaging was required for adequate detection of the ablation zone. On histopathology, an ablation zone with concentric layers was seen after T-HIFU. In line with histopathology, contrast enhanced MRI revealed that after BH or T-HIFU perfusion within the lesion was absent, while after BH in the transition zone some micro-hemorrhaging appeared. Four days after BH, the transition zone with apoptotic cells was histologically no longer detectable, corresponding to the absence of a hypo-intense rim around the lesion in T2 w images. This study demonstrates the first results of in vivo BH on mouse tumor using MRI for treatment guidance and evaluation and opens the way for more detailed investigation of the in vivo effects of BH. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a high-field MR-guided HIFU setup for thermal and mechanical ablation methods in small animals.

BACKGROUND: Thermal and mechanical high intensity focused ultrasound (HIFU) ablation techniques are in development for non-invasive treatment of cancer. However, knowledge of in vivo histopathologic and immunologic reactions after HIFU ablation is still limited. This study aims to create a setup for evaluation of different HIFU ablation methods in mouse tumors using high-field magnetic resonance (MR) guidance. An optimized MR-guided-HIFU setup could be used to increase knowledge of the different pathologic and immunologic reactions to different HIFU ablation methods. METHODS: Three different HIFU treatment strategies were applied in mouse melanomas (B16): a thermal (continuous wave), a mechanical (5 ms pulsed wave), and an intermediate setting (20 ms pulsed wave) for HIFU ablation, all under MR guidance using a 7 tesla animal MR system. Histopathologic evaluation was performed 3 days after treatment. RESULTS: The focus of the ultrasound transducer could accurately be positioned within the tumor under MR image guidance, without substantial damage to the surrounding tissue and skin. All mice retained complete use of the treated leg after treatment. Temperatures of >60, <50, and <44 °C were reached during thermal, intermediate, and mechanical HIFU ablation, respectively. Thermal-treated tumors showed large regions of coagulative necrosis. Tumors of both the mechanical and intermediate groups showed fractionated tissue with islands of necrosis and some pseudocysts with hemorrhage. CONCLUSION: A stable small animal MR-guided HIFU setup was designed and evaluated for follow-up MR imaging and histopathologic responses of the treated tumors. This will facilitate further studies with a larger number of mice for detailed evaluation of the pathologic and immunologic response to different HIFU strategies.

Mechanical high-intensity focused ultrasound destruction of soft tissue: working mechanisms and physiologic effects.

The best known method of high-intensity focused ultrasound is thermal ablation, but interest in non-thermal, mechanical destruction is increasing. The advantages of mechanical ablation are that thermal protein denaturation remains limited and less damage is created to the surrounding tissue by thermal diffusion. The two main techniques for mechanical fragmentation of tissue are histotripsy and boiling histotripsy. These techniques can be used for complete liquefaction of tumor tissue into submicron fragments, after which the fragmented tissue can be easily removed by natural (immunologic) responses. Interestingly it seems that there is a correlation between the degree of destruction and tissue specific characteristics based on the treatment settings used. In this review article, the technical aspects of these two techniques are described, and an overview of the in vivo pathologic and immunologic responses is provided.

Intra-adrenal murine TH-MYCN neuroblastoma tumors grow more aggressive and exhibit a distinct tumor microenvironment relative to their subcutaneous equivalents.

In around half of the patients with neuroblastoma (NBL), the primary tumor is located in one of the adrenal glands. We have previously reported on a transplantable TH-MYCN model of subcutaneous (SC) growing NBL in C57Bl/6 mice for immunological studies. In this report, we describe an orthotopic TH-MYCN transplantable model where the tumor cells were injected intra-adrenally (IA) by microsurgery. Strikingly, 9464D cells grew out much faster in IA tumors compared to the subcutis. Tumors were infiltrated by equal numbers of lymphocytes and myeloid cells. Within the myeloid cell population, however, tumor-infiltrating macrophages were more abundant in IA tumors compared to SC tumors and expressed lower levels of MHC class II, indicative of a more immunosuppressive phenotype. Using 9464D cells stably expressing firefly luciferase, enhanced IA tumor growth could be confirmed using bioluminescence. Collectively, these data show that the orthotopic IA localization of TH-MYCN cells impacts the NBL tumor microenvironment, resulting in a more stringent NBL model to study novel immunotherapeutic approaches for NBL.

Targeted delivery of a sialic acid-blocking glycomimetic to cancer cells inhibits metastatic spread.

Sialic acid sugars are overexpressed by cancer cells and contribute to the metastatic cascade at multiple levels. Therapeutic interference of sialic acids, however, has been difficult to pursue because of the absence of dedicated tools. Here we show that a rationally designed sialic acid-blocking glycomimetic (P-3F(ax)-Neu5Ac) successfully prevents cancer metastasis. Formulation of P-3F(ax)--Neu5Ac into poly(lactic-co-glycolic acid nanoparticles coated with antityrosinase-related protein-1 antibodies allowed targeted delivery of P-3F(ax)--Neu5Ac into melanoma cells, slow release, and long-term sialic acid blockade. Most importantly, intravenous injections of melanoma-targeting P-3F(ax)--Neu5Ac nanoparticles prevented metastasis formation in a murine lung metastasis model. These findings stress the importance of sialoglycans in cancer metastasis and advocate that sialic acid blockade using rationally designed glycomimetics targeted to cancer cells can effectively prevent cancer metastases. This targeting strategy to interfere with sialic acid-dependent processes is broadly applicable not only for different types of cancer but also in infection and inflammation.

Sweet escape: sialic acids in tumor immune evasion.

Sialic acids represent a family of sugar molecules derived from neuraminic acid that frequently terminate glycan chains and contribute to many biological processes. Already five decades ago, aberrantly high expression of sialic acids has been proposed to protect cancer cells from recognition and eradication by the immune system. Today, increased understanding at the molecular level demonstrates the broad immunomodulatory capacity of tumor-derived sialic acids that is, at least in part, mediated through interactions with immunoinhibitory Siglec receptors. Here we will review current studies from a sialic acid sugar perspective showing that tumor-derived sialic acids disable major killing mechanisms of effector immune cells, trigger production of immune suppressive cytokines and dampen activation of antigen-presenting cells and subsequent induction of anti-tumor immune responses. Furthermore, strategies to modulate sialic acid expression in cancer cells to improve cancer immunotherapy will be discussed.

Sialic acids sweeten a tumor's life.

Over four decades ago, specific tumor characteristics were ascribed to the increased expression of sialic acid sugars on the surface of cancer cells, and this led to the definition of sialic acids as potential therapeutic targets. Recent advances in glycobiology and cancer research have defined the key processes underlying aberrant expression of sialic acids in cancer, and its consequences, more precisely. These consequences include effects on tumor growth, escape from apoptosis, metastasis formation, and resistance to therapy. Collectively, these novel insights provide further rationale for the design and development of therapeutic approaches that interfere with excessively high expression of sialic acids in cancer cells. Strategies to target aberrant sialylation in cancer, however, have evolved comparatively slowly. Here, we review recent findings that emphasize the detrimental effects of hypersialylation on multiple aspects of tumor growth and behavior. We also discuss novel therapeutic strategies.

A transplantable TH-MYCN transgenic tumor model in C57Bl/6 mice for preclinical immunological studies in neuroblastoma.

Current multimodal treatments for patients with neuroblastoma (NBL), including anti-disialoganglioside (GD2) monoclonal antibody (mAb) based immunotherapy, result in a favorable outcome in around only half of the patients with advanced disease. To improve this, novel immunocombinational strategies need to be developed and tested in autologous preclinical NBL models. A genetically well-explored autologous mouse model for NBL is the TH-MYCN model. However, the immunobiology of the TH-MYCN model remains largely unexplored. We developed a mouse model using a transplantable TH-MYCN cell line in syngeneic C57Bl/6 mice and characterized the immunobiology of this model. In this report, we show the relevance and opportunities of this model to study immunotherapy for human NBL. Similar to human NBL cells, syngeneic TH-MYCN-derived 9464D cells endogenously express the tumor antigen GD2 and low levels of MHC Class I. The presence of the adaptive immune system had little or no influence on tumor growth, showing the low immunogenicity of the NBL cells. In contrast, depletion of NK1.1+ cells resulted in enhanced tumor outgrowth in both wild-type and Rag1(-/-) mice, showing an important role for NK cells in the natural anti-NBL immune response. Analysis of the tumor infiltrating leukocytes ex vivo revealed the presence of both tumor associated myeloid cells and T regulatory cells, thus mimicking human NBL tumors. Finally, anti-GD2 mAb mediated NBL therapy resulted in ADCC in vitro and delayed tumor outgrowth in vivo. We conclude that the transplantable TH-MYCN model represents a relevant model for the development of novel immunocombinatorial approaches for NBL patients.

Targeting aberrant sialylation in cancer cells using a fluorinated sialic acid analog impairs adhesion, migration, and in vivo tumor growth.

Cancer cells decorate their surface with a dense layer of sialylated glycans by upregulating the expression of sialyltransferases and other glycogenes. Although sialic acids play a vital role in many biologic processes, hypersialylation in particular has been shown to contribute to cancer cell progression and metastasis. Accordingly, selective strategies to interfere with sialic acid synthesis might offer a powerful approach in cancer therapy. In the present study, we assessed the potential of a recently developed fluorinated sialic acid analogue (P-3F(ax)-Neu5Ac) to block the synthesis of sialoglycans in murine melanoma cells and the consequences on cell adhesion, migration, and in vivo growth. The results showed that P-3F(ax)-Neu5Ac readily caused depletion of α2,3-/α2,6-linked sialic acids in B16F10 cells for several days. Long-term inhibition of sialylation for 28 days was feasible without affecting cell viability or proliferation. Moreover, P-3F(ax)-Neu5Ac proved to be a highly potent inhibitor of sialylation even at high concentrations of competing sialyltransferase substrates. P-3F(ax)-Neu5Ac-treated cancer cells exhibited impaired binding to poly-l-lysine, type I collagen, and fibronectin and diminished migratory capacity. Finally, blocking sialylation of B16F10 tumor cells with this novel sialic acid analogue reduced their growth in vivo. These results indicate that P-3F(ax)-Neu5Ac is a powerful glycomimetic capable of inhibiting aberrant sialylation that can potentially be used for anticancer therapy.

Saponin-based adjuvants create a highly effective anti-tumor vaccine when combined with in situ tumor destruction.

Today's most commonly used microbial vaccines are essentially composed of antigenic elements and a non-microbial adjuvant, and induce solid amounts of antibodies. Cancer vaccines mostly aim to induce anti-tumor CTL-responses, which require cross-presentation of tumor-derived antigens by dendritic cells (DCs). Adjuvants that improve DC function and antigen cross-presentation are therefore advantageous for inducing anti-tumor immunity. Previously, we have reported that in situ tumor destruction of established murine tumors by ablation efficiently delivers antigens to DC for the in vivo induction of anti-tumor immunity. Yet, tumor ablation alone resulted in only partial protection against a subsequent tumor-challenge. In this article, the ability of various non-microbial vaccine adjuvants to modulate the immune response following cryo-ablation was tested. The data show that tumor ablation with co-injection of saponin-based adjuvants, but not oil-in-water, water-in-oil or alum-based adjuvants, creates a highly effective in situ vaccine. Draining lymph node CD11c+ DCs acquire antigens more efficiently and become increasingly activated following ablation with saponin adjuvants relative to ablation alone. Moreover, our data reveal that the saponin-based adjuvants facilitate an in this model unprecedented level of antigen cross-presentation, induction of tumor-specific CTL and long-lasting tumor protection. Collectively, combining saponin-based adjuvants with in situ tumor destruction leads to an extremely potent systemic anti-tumor response. This combination approach forms a powerful in situ DC vaccine for which no prior knowledge of tumor antigens is required. As saponin-based adjuvants are currently clinically available, they represent attractive tools for various human and veterinary settings where in situ tumor destruction is applied.