Immune Responses In Tumor-bearing Mice Research Articles

Listeria-vectored cervical cancer vaccine candidate strains reduce MDSCs via the JAK-STAT signaling pathway.

Immunosuppressive status is prevalent in cancer patients and increases the complexity of tumor immunotherapy. It has been found that Listeria-vectored tumor vaccines had the potential ability of two-side regulatory effect on the immune response during immunotherapy. The results show that the combined immunotherapy with the LM∆E6E7 and LI∆E6E7, the two cervical cancer vaccine candidate strains constructed by our lab, improves the antitumor immune response and inhibits the suppressive immune response in tumor-bearing mice in vivo, confirming the two-sided regulatory ability of the immune response caused by Listeria-vectored tumor vaccines. The immunotherapy reduces the expression level of myeloid-derived suppressor cells (MDSCs)-inducing factors and then inhibits the phosphorylation level of STAT3 protein, the regulatory factor of MDSCs differentiation, to reduce the MDSCs formation ability. Moreover, vaccines reduce the expression of functional molecules associated with MDSCs may by inhibiting the phosphorylation level of the JAK1-STAT1 and JAK2-STAT3 pathways in tumor tissues to attenuate the immunosuppressive function of MDSCs. Immunotherapy with Listeria-vectored cervical cancer vaccines significantly reduces the level and function of MDSCs in vivo, which is the key point to the destruction of immunosuppression. The study for the first to elucidate the mechanism of breaking the immunosuppression.

Maleimide as the PEG end-group promotes macrophage-targeted drug delivery of PEGylated nanoparticles in vivo by enhancing interaction with circulating erythrocytes

Radiotherapy (IR) is capable of enhancing antitumor immune responses. However, IR treatment also aggravates the infiltration of peripheral macrophages into the tumor, resulting in reversing the therapeutic effects of antitumor immunity. Thus, a strategy to effectively prevent tumor infiltration by macrophages may further improved the therapeutic efficacy of radiotherapy. Herein, we found that PEGylated solid lipid nanoparticles with maleimide as PEG end-group (SLN-PEG-Mal) show significantly enhanced adsorption onto RBCs through reacting with reactive sulfhydryl groups on RBCs’ surface both in vitro and in vivo, and caused significant changes in the surface properties and morphology of RBCs. These RBCs adsorbed by SLN-PEG-Mal were rapidly removed from circulation due to efficient engulfment by reticuloendothelial macrophages, supporting the usefulness of SLN-PEG-Mal for macrophage-targeted drug delivery. While lacking the use of radioisotope tracing (considered the gold standard for PK/BD studies), our data align with the expected pathway of host defense activation through surface-loaded RBCs. Importantly, injection of paclitaxel-loaded SLN-PEG-Mal effectively inhibited the tumor-infiltration by macrophages, and significantly improved the antitumor immune responses in tumor-bearing mice treated with low-dose irradiation. This study provides insights into the effects of maleimide as PEG end-group on enhancing the interaction between PEGylated nanoparticles and RBCs and offers an effective strategy to inhibit tumor infiltration by circulating macrophages.

Dressing Bacteria With a Hybrid Immunoactive Nanosurface to Elicit Dual Anticancer and Antiviral Immunity.

Approaches capable of simultaneously treating cancer and protecting susceptible patients from lethal infections such as coronavirus disease 2019, are highly desirable but prove to be difficult. Here, dressing bacteria with a hybrid immunoactive nanosurface is reported to elicit dual anticancer and antiviral immunity. A combination of a checkpoint blocking antibody and a virus-specific antigen is covalently conjugated to polydopamine nanoparticles, which can be anchored onto bacterial surface, by a one-step in situ polymerization of dopamine under a cell-friendly condition. By virtue of the ability to colonize and penetrate deep tumor tissue, dressed bacteria enable sustained release and expanded exposure of carried immunoactivators to stimulate immune cells. In addition to a carrier role, bacteria are able to further provoke innate immunity due to the native immunogenicity of the pathogen-associated molecular patterns. Immunization with dressed bacteria promotes the maturation, and activation of antigen-presenting cells, which induces robust humoral and cellular immune responses in tumor-bearing mice. As evidenced by efficient production of viral-antigen-specific immunoglobulin G antibody in serum and significantly suppressed tumor growth in different models, dressing bacteria with a hybrid immunoactive nanosurface paves an avenue to prepare next-generation therapeutics for synergistic treatment and prevention.

Effects of in-vitro modulation of TRPV1 activity on immune response of mice bearing metastatic breast carcinoma: Enhanced inflammatory response may hinder therapeutic potentials of TRPV1 agonists

AimsActivation of transient receptor potential vanilloid 1 (TRPV1) ion channels inhibits inflammation, enhance cytotoxic immune response, and may have therapeutic potential in treatment of cancer characterized by increased systemic inflammation. We here determined how activation of TRPV1 alters immune response of tumor-bearing mice. Main methodsThree different metastatic subset of 4 T1 breast carcinoma cells were used to induce tumors in Balb-c mice. Mix leukocyte cultures (MLCs) using spleens and draining lymph nodes were prepared and stimulated with various challenges. Effects TRPV1 agonists including capsaicin, antagonist (AMG9810) and Gambogic Amide (GA), a TrkA agonist that sensitizes TRPV1, on secreted levels of cytokines were determined. Key findingsMLCs of tumor-bearing mice secreted markedly higher levels of IL-6 and lower levels of IFN-γ compared to control mice. We observed differential effects of TRPV1 agonists in control and mice bearing different subset of metastatic cells. TRPV1 increased IFN–γ and IL-17 secretion in control mice while they markedly increased IL-6 secretion and suppressed IFN--γ secretion in tumor-bearing mice. Unexpectedly, AMG9810 acted as an inverse agonist and did not antagonize the effects of TRPV1 agonists. SignificanceOur results demonstrate constitutive activity of TRPV1 in immune cells, suggesting cross activation. To prevent excessive chronic activation of TRPV1 in immune cells in the presence of metastatic breast carcinoma, lower doses of TRPV1 agonist should be considered. Unexpected findings further document that a drug can have multiple intrinsic activities depending on surrounding factors can act on the same receptor as an agonist, antagonist or inverse agonist.

Abstract 1516: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors

Abstract Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 4-1BB, GITR, ICOS, or OX40 costimulatory domains in the chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10), chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1516.

A powerful ICOS agonist that enhances anti-tumor immune responses restored by immune checkpoint inhibitors

Abstract The Inducible T cell Costimulator (ICOS, CD278) is a receptor in the CD28 family of B7-binding proteins expressed mostly by activated T cells. Upon binding to its ligand ICOS-L expressed on APCs,T cells are further activated by ICOS, resulting in their expansion and production of effector cytokines. Clinical data in patients treated with anti-CTLA-4 or anti-PD-1 mAbs have shown that ICOShiT cells correlated with an increased treatment response in these patients. Here, we report the design of a high avidity pentavalent form of the human ICOS-L extracellular domain fused to a short α-helical peptide from the human cartilage oligomeric matrix protein (COMP). This ICOS agonist, termed ICOS-L.COMP, spontaneously assembles into stable pentamers, binds tightly to both human and murine ICOS (SPR; Kd<10 nM) and co-stimulates (with anti CD3 mAb) the proliferation and cytokine release of both murine and human CD4+ and CD8+ T cells when added as a soluble factor (unlike ICOS-L.Fc). Soluble ICOS-L.COMP also phosphorylates Akt in activated T cells and enhances anti-tumor immune responses in tumor-bearing mice [murine MC38 or CT26 tumors] treated with either anti-murine PD-1 or anti-murine CTLA-4 mAb. Impressively, most mice treated with ICOS-L.COMP combined with an immune checkpoint inhibitor were either cured or displayed a stable tumor burden in contrast to checkpoint monotherapies (delayed tumor progression) or when ICOS-L.COMP was given as a monotherapy (comparable to no treatment). Importantly, ICOS-L.COMP lacks a Fc domain thus eliminating Fc-associated off-target adverse effects. In summary, our results suggest that ICOS-L.COMP represents a new and powerful biologic to be used in combination with existing immune checkpoint inhibitors.

Abstract 3582: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors

Abstract Adoptive transfer of tumor-reactive T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 41BB, GITR, ICOS, or OX40 costimulatory domains in our chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10 and IL-5) as determined by ELISA and LegendPlex analysis, chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines, but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Amorette E. Barber, Geoffrey Parriott, Shane Crean, Hailey Kintz. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3582.

Anti-tumor Activity of Agaricus bisporus Extracts and its Relation with IL-2 in Tumor-Bearing Mice

Mushroom is widely used as a traditional medicinal fungus, and it has been known to exhibit immune-stimulatory and anti-cancer activity. However, the exact immune response in tumor-bearing mice after exposure to mushroom extract was unclear. The aim of this study was to investigate whether or not crude preparations extracted from Agaricus bisporus can promote type 1 immune response in tumor-bearing mice. Watery and ethanolic extracts of mushroom were intraperitoneally administered into tumor-bearing mice for 28 alternative days, tumor volume was measured twice weekly, also IL-2 level was determined in the sera of mice one day before the beginning of treatment and after 2 and 4 weeks of treatment. The results showed that both extracts significantly inhibit the growth of tumor up to 72.2% at the end of experiment, while only the ethanolic extract significantly increased the level of IL-2 production from 11.2 and 16.9 pg/ml up to 26.4 and 42.5 pg/ml after 2 and 4 weeks of treatment respectively. To confirm these results , further studies are required to investigate the ability of these extracts to promote the production of IL-12 & IFN-γ which are the major cytokines involved in immune response type 1 activation.

Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting TERT.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ Tcells within the tumor and decreased the frequency of regulatory Tcells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

Abstract 4706: Spherical nucleic acids targeting toll-like receptor 9 enhance antitumor activity in combination with anti-PD-1 antibody in mouse tumor models

Abstract Of the several immunotherapy approaches being developed, checkpoint inhibitors (CPI) represent a breakthrough for the treatment of cancer. A T cell-inflamed tumor microenvironment (TME) is required for antitumor efficacy of CPI. However, the evidence suggests that 70% of tumors do not possess T cell-inflamed TME limiting the potential of CPI treatments to a smaller population of cancer patients. Immunotherapies that enable CPI effectiveness in patients with non-T cell-inflamed phenotype are being studied. Oligonucleotide-based TLR9 agonists induce potent CD8+ T cell and plasmacytoid dendritic cell activation and limit immune suppressor cell phenotypes in TME. Therefore, the combination of TLR9 agonists with CPI would be a rational approach for achieving higher rates of success in cancer treatment. However, TLR9 agonists were unsuccessful in previous cancer clinical trials. Spherical Nucleic Acids (SNAs) are a novel class of agents with oligonucleotides densely packed and radially oriented around spherical liposomal nanoparticles. SNAs overcome limitations of therapeutic oligonucleotides through increased uptake and nuclease stability. We have evaluated a TLR9 targeted SNA for anticancer activity as a monotherapy and in combination with an anti-PD-1 (a-PD-1) antibody in both a-PD-1 sensitive and insensitive mouse tumor models. Mice bearing a-PD-1 sensitive MC38 tumors were treated with intratumoral dosing of TLR9 agonist SNA, intraperitoneal (IP) dosing of a-PD-1 or the combination of both agents when average tumor volume was 100 mm3, with twice weekly dosing for a total of 5 doses. SNA alone showed dose-dependent tumor growth inhibition (TGI) of up to 87% and increased survival of mice. The combination of SNA and a-PD-1 greatly enhanced TGI (92% vs SNA, 74% or a-PD-1, 77%) and mice survival compared with either monotherapy. Moreover, once weekly dosing of SNA showed similar TGI compared with twice weekly dosing. In a-PD-1 insensitive EMT-6 model, mice were treated with intravenous dosing of TLR9 agonist SNA, IP dosing of a-PD-1 or the combination of both agents starting on day 3 following subcutaneous tumor cell inoculation. SNA alone showed potent dose-dependent TGI of up to 80% and increased mice survival. The combination of SNA and a-PD-1 showed a synergistic TGI (86% vs SNA, 64% or a-PD-1, no effect) and increased mice survival. Further, no tumor growth was observed in surviving mice after subsequent challenge with EMT-6 cells, suggesting development of tumor-specific memory responses in SNA treated animals. These data demonstrate that the TLR9 agonist SNA studied herein induces innate and adaptive immune responses in tumor-bearing mice and shows synergistic TGI with a-PD-1 in both a-PD-1 sensitive and insensitive tumor models. A TLR9 agonist SNA, AST-008, is currently in clinical development. Citation Format: SubbaRao Nallagatla, Bart R. Anderson, Sagar Anantatmula, Ekambar R. Kandimalla. Spherical nucleic acids targeting toll-like receptor 9 enhance antitumor activity in combination with anti-PD-1 antibody in mouse tumor models [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 4706. doi:10.1158/1538-7445.AM2017-4706

Ascophyllan functions as an adjuvant to promote anti-cancer effect by dendritic cell activation.

Our previous study demonstrated that ascophyllan, a sulfated polysaccharide purified from brown alga, has immune-activating effects. In this study, we evaluated ascophyllan as an adjuvant for its therapeutic and preventive effect on tumor in a mouse melanoma model. Ascophyllan induced migration of DCs to spleen and tumor-draining lymph node (drLN) in a mouse B16 melanoma model. Moreover, ascophyllan induced activation of dendritic cells (DCs), and promoted IFN-γ- and TNF-α-producing Th1 immune responses in tumor-bearing mice. In addition, treatment with a combination of ascophyllan and ovalbumin (OVA) in the tumor-bearing mice promoted proliferation of OVA-specific CD4 and CD8 T cells and migration of those cells into the tumor, consequently inhibiting the tumor growth. Immunization with the combination of ascophyllan and OVA caused enhanced OVA-specific antibody production and memory T cell responses compared to OVA immunization alone, and almost completely prevented B16-OVA tumor growth upon subsequent tumor challenge. Finally, the combination of ascophyllan and OVA prevented B16-OVA tumor invasion and metastasis into the liver. Thus, these results demonstrate that ascophyllan can function as an adjuvant to induce DC activation, antigen specific CTL activation, Th1 immune response and antibody production, and hence may be useful as a therapeutic and preventive tumor vaccine.

Immune response modulation by Galectin-1 in a transgenic model of neuroblastoma

ABSTRACTGalectin-1 (Gal-1) has been described to promote tumor growth by inducing angiogenesis and to contribute to tumor immune escape by promoting apoptosis of activated T cells. We had previously identified upregulation of Gal-1 in preclinical models of aggressive neuroblastoma (NB), a solid tumor of childhood. However, the clinical and biological relevance of Gal-1 in this tumor entity is unclear. Here, the effect of Gal-1 on the immune system and tumorigenesis was assessed using modulation of Gal-1 expression in immune effector cells and in a transgenic NB model, designated TH-MYCN. The fraction of CD4+ T cells was decreased in tumor-bearing TH-MYCN mice compared to tumor-free littermates, while both CD4+ T cells as well as CD8+ T cells were less activated, compatible with a reduced immune response in tumor-bearing mice. Tumor incidence was not significantly altered by decreasing Gal-1/LGALS1 gene dosage in TH-MYCN mice, but TH-MYCN/Gal-1−/− double transgenic mice displayed impaired tumor angiogenesis, splenomegaly, and impaired T cell tumor-infiltration with no differences in T cell activation and apoptosis rate. Additionally, a lower migratory capacity of Gal-1 deficient CD4+ T cells toward tumor cells was observed in vitro. Transplantation of TH-MYCN-derived tumor cells into syngeneic mice resulted in significantly reduced tumor growth and elevated immune cell infiltration when Gal-1 was downregulated by shRNA. We therefore conclude that T cell-derived Gal-1 mediates T cell tumor-infiltration, whereas NB-derived Gal-1 promotes tumor growth. This opposing effect of Gal-1 in NB should be considered in therapeutic targeting strategies, as currently being developed for other tumor entities.

Development of inCVAX, In situ Cancer Vaccine, and Its Immune Response in Mice with Hepatocellular Cancer.

Manipulation of immune system toward the rejection of established cancers has become the standard of care in some patients. Here we propose the development of an in situ autologous cancer vaccine, inCVAX, for the treatment of hepatocellular cancer (HCC). inCVAX is based on the induction of local immunogenic cancer cell death combined with local dendritic cell stimulation by intratumoral injection of the immune-activator N-dihydro-galacto-chitosan (GC). In a first set of experiments, cellular and molecular studies were performed to investigate the effect of inCVAX on immune activation in a murine model of HCC that we previously developed. Once large tumors were formed in mice, the tumor is surgically exposed and a laser fiber was inserted into the center of an individual tumor mass. Using a 10 mm diffuser tip, laser irradiation of 1.5 W was applied to heat the tumor at different durations (6-10 min) to assess tolerability of photothermal application at different temperatures. The laser application was followed by immediate injection of GC, and each mouse received one laser treatment and one GC injection. ELISA was used to assess the level of cytokines; immunohistochemical staining was conducted to analyze the effect of inCVAX on immune cell tumor-filtration and expression of tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). Results indicate that survival correlated to thermal exposure. At lower temperatures the photothermal effect was sufficient to induce tumor necrosis, but without obvious complication to the mice, although at these temperatures the treatment didn’t alter the level of TSAs and TAAs, so further optimization is suggested. Nevertheless, in response to the inCVAX treatment, cytotoxic cytokine IFN-γ was significantly increased, but suppressive cytokine TGF-β was dramatically reduced. Furthermore, inCVAX prompted tumor infiltration of CD3+, CD4+, and CD8+ T cells; but modulated macrophage subsets differently. In conclusion, while the protocol needs further optimization, it would appear that inCVAX for the treatment of HCC activates an immune response in tumor-bearing mice, which in turn may have potential for the treatment of HCC.

Anti-tumor and immunomodulatory activity of iron hepta-tungsten phosphate oxygen clusters complex

Polyoxometalates (POMs) have attracted a considerable attention due to their unique structural characteristics, physicochemical properties and biological activities. In this study, iron hepta-tungsten phosphate oxygen clusters complex Na12H[Fe(HPW7O28)2]·44H2O (IHTPO) was synthesized and evaluated for in vitro cytotoxic activities on human hepatoma HepG2, leukemia K562, lung carcinoma A549, and large cell lung cancer NCI-H460 cells, therapeutic efficacies on mice transplantable tumor, and immunomodulatory potentials on the immune response in tumor-bearing mice. IHTPO exhibited lower in vitro cytotoxic activities against four human tumor cell lines, with the IC50 values being higher than 62.5μM (ca. 300μg/ml). IHTPO, however, significantly inhibited the growth of S180 sarcoma transplanted in mice. It was further showed that IHTPO could not only significantly promote splenocytes proliferation, NK cell and CTL activity from splenocytes, but remarkably enhance serum antigen-specific IgG, IgG2a and IgG2b antibody levels in S180-bearing mice. IHTPO also significantly promoted Th1 cytokines IFN-γ and IL-2 production, and up-regulated the mRNA expression levels of IFN-γ, IL-2 and Th1 transcription factors T-bet and STAT-4 in splenocytes from the S180-bearing mice. These results suggested that IHTPO significantly inhibited the growth of mice transplantable tumor, and that its in vivo antitumor activity might be achieved by improving Th1 protective cell-mediated immunity. IHTPO could act as antitumor agent with immunomodulatory activity.

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice.

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasmagondii (T.gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T.gondii which are able to mature DCs and consequently instruct immune responses in tumor-bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion-exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL-12p70 and IL-10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor-bearing mice. We found that ammonium sulfate fraction one (A1) matured-DCs produced higher IL-12 level and IL-12/IL-10 ratio; therefore, this fraction was selected for further fractionation by anion-exchange HPLC. The results showed that anion-exchange HPLC fraction 14 (C14) matured-DCs secrete higher levels of IL-12p70 and IL-12p70/IL-10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS-PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T.gondii are able to mature DCs more efficient.

Osthole promotes anti-tumor immune responses in tumor-bearing mice with hepatocellular carcinoma

Osthole, a natural coumarin derivative, has been shown to have anti-tumor and anti-inflammatory activity. However, the effect of osthole on anti-tumor immune responses in tumor-bearing mice has not yet been reported. In the present study, osthole treatment did not affect the weight and the coefficient of thymus and spleen in tumor-bearing mice with hepatocellular carcinoma (HCC). However, osthole administration significantly elevated the proportion and number of the splenic CD8+ T cells, the proportion of CD4+ T and CD8+ T cells in tumor tissues, and the levels of IL-2 and TNF-α in the serum of HCC tumor-bearing mice. Our results suggested that osthole could promote the activation of the tumor-infiltrating CD4+ T and CD8+ T cells, and elevate the proportion of CD4+ and CD8+ effector T cells. Osthole treatment also significantly decreased the proportion of CD4+CD25+Foxp3+ regulatory T cells in the spleen. Taken together, osthole could enhance the T cell mediated anti-tumor immune responses in the tumor-bearing mice with HCC.

Activation of antitumor immune responses by Ganoderma formosanum polysaccharides in tumor-bearing mice.

Fungi of the genus Ganoderma are basidiomycetes that have been used as traditional medicine in Asia and have been shown to exhibit various pharmacological activities. We recently found that PS-F2, a polysaccharide fraction purified from the submerged culture broth of Ganoderma formosanum, stimulates the maturation of dendritic cells and primes a T helper 1 (Th1)-polarized adaptive immune response in vivo. In this study, we investigated whether the immune adjuvant function of PS-F2 can stimulate antitumor immune responses in tumor-bearing mice. Continuous intraperitoneal or oral administration of PS-F2 effectively suppressed the growth of colon 26 (C26) adenocarcinoma, B16 melanoma, and sarcoma 180 (S180) tumor cells in mice without adverse effects on the animals' health. PS-F2 did not cause direct cytotoxicity on tumor cells, and it lost the antitumor effect in mice with severe combined immunodeficiency (SCID). CD4(+) T cells, CD8(+) T cells, and serum from PS-F2-treated tumor-bearing mice all exhibited antitumor activities when adoptively transferred to naïve animals, indicating that PS-F2 treatment stimulates tumor-specific cellular and humoral immune responses. These data demonstrate that continuous administration of G. formosanum polysaccharide PS-F2 can activate host immune responses against ongoing tumor growth, suggesting that PS-F2 can potentially be developed into a preventive/therapeutic agent for cancer immunotherapy.

ER stress response regulates the fate of myeloid-derived suppressor cells through TRAIL receptors mediated apoptosis (TUM4P.907)

Abstract We studied the fate of myeloid-derived suppressor cells (MDSC) in cancer. Unexpectedly, MDSC had lower viability and a shorter half-life than their control counterparts neutrophils and monocytes. This effect was due to increased apoptosis mediated by the changes in TRAIL receptors (TRAIL-R) expression. Targeting TRAIL-R did not affect myeloid cells from naïve mice, but dramatically reduced the presence of MDSC and improved the immune responses in tumor-bearing mice. Pro-inflammatory cytokines did not affect TRAIL-R expression. However, induction of ER stress recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. ER stress response was detected in MDSC isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes. Block of ER stress abrogated changes in TRAIL-R. Thus, MDSC pathophysiology is linked to ER stress, which shortens their lifespan in the periphery and promotes their expansion in bone marrow. In conclusion, TRAIL-R can be considered for selective targeting of MDSC.

ER stress regulates myeloid-derived suppressor cell fate through TRAIL-R-mediated apoptosis.

Myeloid-derived suppressor cells (MDSCs) dampen the immune response thorough inhibition of T cell activation and proliferation and often are expanded in pathological conditions. Here, we studied the fate of MDSCs in cancer. Unexpectedly, MDSCs had lower viability and a shorter half-life in tumor-bearing mice compared with neutrophils and monocytes. The reduction of MDSC viability was due to increased apoptosis, which was mediated by increased expression of TNF-related apoptosis-induced ligand receptors (TRAIL-Rs) in these cells. Targeting TRAIL-Rs in naive mice did not affect myeloid cell populations, but it dramatically reduced the presence of MDSCs and improved immune responses in tumor-bearing mice. Treatment of myeloid cells with proinflammatory cytokines did not affect TRAIL-R expression; however, induction of ER stress in myeloid cells recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. The ER stress response was detected in MDSCs isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes, and blockade of ER stress abrogated tumor-associated changes in TRAIL-Rs. Together, these data indicate that MDSC pathophysiology is linked to ER stress, which shortens the lifespan of these cells in the periphery and promotes expansion in BM. Furthermore, TRAIL-Rs can be considered as potential targets for selectively inhibiting MDSCs.

Gemcitabine chemotherapy induces phenotypic alterations of tumor cells that facilitate antitumor T cell responses in a mouse model of oral cancer

Gemcitabine (GEM) is a pyrimidine nucleoside analogue that is a new chemotherapeutic agent used for treating various cancers. Because accumulating evidence indicates that GEM may activate host immune responses, its potential as an immune modulator in cancer chemotherapy has generated considerable interest. In the present study, we investigated the antitumor effects of GEM using a mouse oral cancer model using immunological analyses. We examined apoptotic cell death of tumor cells with GEM treatment both in vitro and in vivo. We also investigated whether in vivo administration of GEM affected the distributions of immune cells, tumor-cell surface expression levels of immune accessory molecules and T cell immune responses in tumor-bearing mice. GEM induced significant oral cancer-cell apoptosis in vitro, and in vivo GEM administration markedly attenuated established mouse tumor growth. In vivo GEM administration decreased the numbers of both myeloid-derived suppressor cells (MDSCs) and B cells in tumor-bearing mice and enhanced dendritic cell maturation. Moreover, GEM treatment upregulated tumor-cell surface expressions of several immune accessory molecules and adhesion molecules, including CD80, CD86, CD40, ICAM-1, VCAM-1, and P-selectin. Remarkably, these tumor cells augmented tumor specific T-cell responses. These results suggest that GEM can induce host antitumor immune responses, which would facilitate antitumor effects in the treatment of oral cancer.