Attenuated Salmonella Carrying IL-21-siRNA-CCR2 Co-expression Plasmid Enhances Anti-tumor Immune Response in Lung Cancer-bearing Mice.

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.

Chemotherapy is the main strategy for the treatment of lung cancer. However, sensitivity to chemotherapy, one of the main factors affecting the survival rate of patients with lung cancer, is extremely poor. Forkhead box P3 (Foxp3) is the key regulatory gene for the development and function of CD4+CD25+ regulatory T cells (Tregs). Increased levels of Tregs and Foxp3 expression in the peripheral blood and tumour specimens of cancer patients are associated with tumour progression and poor prognosis. In addition, certain studies have suggested that Tregs may be resistant to conventional chemotherapy and thus, enhance tumour immune evasion. Previous studies have demonstrated that Foxp3 is also expressed within tumour cells and that it may mimic the function of Tregs. Currently, the correlation between the tumour cell expression of Foxp3 and sensitivity to chemotherapy is unclear. Therefore, it was hypothesised that Foxp3 causes resistance to chemotherapeutic agents in lung cancer cells and that it may consequently promote the progression of lung cancer. In the current study, the expression of Foxp3 in mouse Lewis lung cancer (LLC) cells was detected using RT-PCR and immunocytochemistry. The overexpression of Foxp3, which was accomplished by the transient transfection of recombinant pcDNA3.1-Foxp3 or empty plasmids into LLC cells, was confirmed by RT-PCR and western blot analysis. The inhibition of cell proliferation was measured using MTT assay. The expression of multidrug resistance protein1 (mdr1) mRNA and its protein product, P-glycoprotein (P-gp), were detected by RT-PCR and flow cytometry, respectively. The results revealed that Foxp3 was expressed by LLC cells. The inhibitory rate of cell proliferation in Foxp3-overexpressing LLC cells compared with those transfected with an empty plasmid was significantly decreased following adriamycin (ADM) and mitomycinC (MMC) treatment. The IC50 values of ADM and MMC in Foxp3-overexpressing LLC cells were increased. The expression levels of mdr1 mRNA and P-gp were significantly upregulated in Foxp3 overexpressing LLC cells. These results suggest that Foxp3 reduces the sensitivity of LLC cells to ADM and MMC, thus promoting tumour progression, by upregulating the expression of mdr1 mRNA and P-gp.

Identification of secreted proteins of lung cancer could provide new candidates of serum biomarkers for cancer diagnosis or targets for therapeutic intervention. In this study, we developed a novel strategy that combined functional monoclonal antibody library screening technique and mass spectrometry to identify functional secreted proteins. BALB/c mice were immunized with cancer cells isolated from fresh human lung cancer tissues. The monoclonal antibody library containing 1160 mAbs was established with the mouse spleen cells, whose serum had most anti-proliferative effect on lung cancer cells. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with secreted proteins in condition medium and lung cancer tissues with high inhibotion of cell proliferation were selected. Antigens that recognized by antibodies were obtained by immunoprecipitation and then identified by mass spectrometry. Mac-2-binding protein (Mac-2BP), the antigen of 13H3 antibody, was identified using this approach. Functional studies demonstrated that the 13H3 antibody suppressed lung cancer cell lines ANIP-973 and A549 proliferation in vitro and inhibit ANIP973 xenograft tumors growth in vivo by inducing cell-cycle arrest at G1 phase, with up-regulation of p27 and down-regulation of cyclin D1. Moreover, the serum level of Mac-2BP was significantly higher in lung cancer patients than healthy controls. At a cutoff value of 6 μg/ml, Mac-2BP might be a diagnostic biomarker of lung cancer, especially for SCLC. Mac-2BP concentrations of 6 μg/ml or higher was associated with poor overall survival in univariate analysis, and was an independent predictor in the multivariate COX analysis. Together, these results firstly demonstrated that Mac-2BP can be used as a therapeutic target and potential biomarker for lung cancer. Our strategy is feasible, which may facilitate the identification of novel secreted biomarkers of lung cancer.

Antitumor and immunomodulatory activity of polysaccharides from Sargassum fusiforme

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.

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.

This study describes a unique strategy designed to identify breast cancer antigens (TAA) that induce therapeutic immune responses in tumor-bearing mice. In a mouse model, the strategy led to the identification of growth factor receptor-bound protein 10 (Grb10) as a newly identified TAA. Grb10 is a signal transduction molecule associated with multiple transmembrane tyrosine kinase receptors. It was discovered by comparing microarrays of cellular breast cancer vaccines highly enriched for cells that induced breast cancer immunity with non-enriched vaccines. The vaccines were prepared by transferring a cDNA expression library derived from SB5b cells, a breast cancer cell line derived from a neoplasm that arose spontaneously in the mammary gland of a C3H/He mouse into LM cells, a mouse fibroblast cell line. As the transferred cDNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide, and is expressed, the vaccine could be prepared from a little as 10 micrograms of tumor tissue. Relatively few cells in the transfected cell population, however, incorporated cDNA fragments that included genes specifying TAA. (The vast majority specified normal cellular constituents.) A unique strategy was developed, therefore, to enrich the vaccine for immunotherapeutic cells. Comparative microarrays of enriched and non-enriched vaccines resulted in the identification of twenty overrepresented genes. One, the gene for Grb10, was approximately 100-fold overrepresented. To determine if Grb10 in the enriched vaccine was partly responsible for its therapeutic benefits, the gene was incorporated into an expression plasmid that was then transfected into the fibroblast cell line, which was then used as a vaccine. Mice with established breast cancer treated solely by immunization with the fibroblasts modified to express Grb10 developed robust immunity to the breast cancer cells, which, in some instances, was sufficient to result in tumor rejection. 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 1583. doi:1538-7445.AM2012-1583

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.

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

Internal radiotherapy holds a greater potential than external radiotherapy for precisely destroying tumors and minimizing side effects. 125I seeds are routinely used as radioactive sources in clinical brachytherapy for patients with various types of cancers. However, 125I seeds are losing ground to flashier cancer therapies, mainly due to their limited therapeutic efficacy, uneven dose distribution, and negligible antitumor immune response. Here, we present porphyrin-engineered 125I-nanoseeds as a prototype for immunogenic brachytherapy. 125I-nanoseeds were rationally designed as a core-shell structure, in which Au@Ag cores enhance the energy deposition of photons to produce more ·OH, while porphyrin shells transfer the energy of Auger electrons to generate 1O2. Benefiting from improving energy utilization efficiency, 125I-nanoseeds can efficiently produce ·OH and 1O2 in tumors, enhancing antitumor efficacy and inducing immunogenic cell death in both murine tumor models and human tumor tissues. When combined with checkpoint blockade immunotherapy, 125I-nanoseeds elicit a systemic immune response in tumor-bearing mice, inhibiting both distant and metastatic tumors. This work demonstrates that porphyrin-engineered 125I-nanoseeds can synergize brachytherapy and dynamic therapy, resulting in enhanced antitumor efficacy and antitumor immune response compared to those of clinical 125I seeds, which is expected to improve the applied prospect of clinical brachytherapy.

High-Intensity Focused Ultrasound Tumor Ablation Activates Autologous Tumor-Specific Cytotoxic T Lymphocytes

Tertiary Lymphoid Structures Target the Antitumor Immune Response to Lung Cancer

Con: The Case for Expanded Lung Cancer Research Support

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

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.