Interleukin-37 promotes colitis-associated carcinogenesis via SIGIRR-mediated cytotoxic T cells dysfunction.

Interleukin-37b (hereafter called IL-37) was identified as fundamental inhibitor of natural and acquired immunity. The molecular mechanism and function of IL-37 in colorectal cancer (CRC) has been elusive. Here, we found that IL-37 transgenic (IL-37tg) mice were highly susceptible to colitis-associated colorectal cancer (CAC) and suffered from dramatically increased tumor burdens in colon. Nevertheless, IL-37 is dispensable for intestinal mutagenesis, and CRC cell proliferation, apoptosis, and migration. Notably, IL-37 dampened protective cytotoxic T cell-mediated immunity in CAC and B16-OVA models. CD8+ T cell dysfunction is defined by reduced retention and activation as well as failure to proliferate and produce cytotoxic cytokines in IL-37tg mice, enabling tumor evasion of immune surveillance. The dysfunction led by IL-37 antagonizes IL-18-induced proliferation and effector function of CD8+ T cells, which was dependent on SIGIRR (single immunoglobulin interleukin-1 receptor-related protein). Finally, we observed that IL-37 levels were significantly increased in CRC patients, and positively correlated with serum CRC biomarker CEA levels, but negatively correlated with the CD8+ T cell infiltration in CRC patients. Our findings highlight the role of IL-37 in harnessing antitumor immunity by inactivation of cytotoxic T cells and establish a new defined inhibitory factor IL-37/SIGIRR in cancer-immunity cycle as therapeutic targets in CRC.

Treatment of dextran sodium sulfate-induced experimental colitis by adoptive transfer of peritoneal cells.

The adoptive transfer of the natural regulatory B cells and macrophages should be a useful treatment for inflammation and autoimmune disease. However, it is usually difficult to isolate these cells from the tissues and expand them. Here, we investigated the feasibility of adoptively transferring peritoneal cells (PCs) as a treatment for DSS-induced colitis. We found that peritoneal cavity can provide an easily accessible site for harvesting enough number of PCs, namely, two-dose PCs for the treatment from a mouse in one operation. Adoptive therapy of these cells from healthy mice or those with disease is effectively in reducing the disease activity score. The natural B cells and macrophages of the infused PCs can selectively migrate to lesion sites and regulate the expression of Stat3, NF-κB, Smad3 and Smad7. Additionally, PCs exert dual activity of IL-10 and TGF-ß secreted spontaneously by both peritoneal B cells and macrophages, which in turn enhance the induction of regulatory B cells and Macrophages in microenvironment of inflammation. Moreover, PCs can re-establish immunological tolerance in the OVA-immunized mice. Thus, our findings provide a new strategy for colitis therapy and could be of importance in additional exploration of other inflammation and autoimmune diseases therapy.

Down-regulation of MFG-E8 by RNA interference combined with doxorubicin triggers melanoma destruction.

The pathogenic mechanism of malignant melanoma involves the dynamic interplay of transformed cell and normal host cell, but cancer treatments always target each partition separately. In the tumor microenvironment, milk fat globule epidermal growth factor-8 (MFG-E8) is a secreted glycoprotein highly expressed in the vertical growth phase of melanoma, leading to tumor progression through coordinated αvß3 and αvß5 integrin signaling in tumor cells and host cells. Doxorubicin (Dox) is one of the most widely used antitumor drugs against a lot of solid tumors, including melanoma. In this work, Dox was used to combine with down-regulation of MFG-E8 by RNA interference (RNAi) in order to determine the synergistic effect of the antitumor activity in vivo. And the possible mechanisms were investigated. Results showed that combination group (MFG-E8 RNAi plus Dox) could inhibit the growth of melanoma more effectively than monotherapy or control groups. We found that the combination treatment induced more tumor cell apoptosis and inhibited more neovascularization than other groups. Moreover, this combination treatment attenuated CD4(+) CD25(+) Foxp3(+) Treg cells in tumor-infiltrating lymphocytes compared with other groups. Our findings suggested that MFG-E8 down-regulation enhanced the antitumor function of chemotherapy through coordinated cell apoptosis and immune-mediated mechanisms, which might be a feasible way for cancer therapy.

Accumulation of FLT3(+) CD11c (+) dendritic cells in psoriatic lesions and the anti-psoriatic effect of a selective FLT3 inhibitor.

Psoriasis is a common chronic T-cell-mediated autoimmune skin disease, and traditional immunotherapies for psoriasis have focused on the direct inhibition of T cells, which often causes toxicity and lacks long-term effectiveness. Safe and effective therapeutic strategies are strongly needed for psoriasis. In this study, we show for the first time a significant accumulation of FLT3(+) CD11c(+) dendritic cells (DCs) in human psoriatic lesions and in the skin of experimental preclinical K14-VEGF transgenic homozygous mice, our animal model, although not an exact match for human psoriasis, displays many characteristics of inflammatory skin inflammation. SKLB4771, a potent and selective FLT3 inhibitor that we designed and synthesised, was used to treat cutaneous inflammation and psoriasis-like symptoms of disease in mice and almost completely cured the psoriasis-like disease without obvious toxicity. Mechanistic studies indicated that SKLB4771 treatment significantly decreased the number and activation of pDCs and mDCs in vitro and in vivo, and subsequent T-cell cascade reactions mediated by Th1/Th17 pathways. These findings show that targeted inhibition of FLT3, and hence direct interference with DCs, may be a novel therapeutic approach for the treatment of psoriasis.

Dimethyl Sulfoxide Suppresses Mouse 4T1 Breast Cancer Growth by Modulating Tumor-Associated Macrophage Differentiation.

PURPOSE: The universal organic solvent dimethyl sulfoxide (DMSO) can be used as a differentiation inducer of many cancer cells and has been widely used as a solvent in laboratories. However, its effects on breast cancer cells are not well understood. The aim of this study is to investigate the effect and associated mechanisms of DMSO on mouse breast cancer. METHODS: We applied DMSO to observe the effect on tumors in a mouse breast cancer model. Tumor-associated macrophages (TAMs) were tested by flow cytometry. Ex vivo tumor microenvironment was imitated by 4T1 cultured cell conditioned medium. Enzyme-linked immunosorbent assays were performed to detect interleukin (IL)-10 and IL-12 expression in medium. To investigate the cytotoxicity of DMSO on TAMs, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed. RESULTS: We found that DMSO produced tumor retardation when injected into mouse peritoneal cavities in a certain concentration range (0.5-1.0 mg/g). Furthermore, as detected by flow cytometry, TAM subtypes were found to be transformed. We further imitated a tumor microenvironment in vitro by using 4T1 cultured cell conditioned medium. Similarly, by using low concentration DMSO (1.0%-2.0% v/v), TAMs were induced to polarize to the classically activated macrophage (M1-type) and inhibited from polarizing into the alternatively activated macrophage (M2-type) in the conditioned medium. IL-10 expression in tumors was reduced, while IL-12 was increased compared with the control. Furthermore, we reported that 2.0% (v/v) DMSO could lead to cytotoxicity in peritoneal macrophages after 48 hours in MTT assays. CONCLUSION: Our findings suggest that DMSO could exert antitumor effects in 4T1 cancer-bearing mice by reversing TAM orientation and polarization from M2- to M1-type TAMs. These data may provide novel insight into studying breast cancer immunotherapy.

IL-7 inhibits tumor growth by promoting T cell-mediated antitumor immunity in Meth A model.

Immune suppression is well documented during tumor progression, which includes loss of effect of T cells and expansion of T regulatory (Treg) cells. IL-7 plays a key role in the proliferation, survival and homeostasis of T cells and displays a potent antitumor activity in vivo. In the present study, we investigated the antitumor effect of IL-7 in Meth A model. IL-7 inhibited tumor growth and prolonged the survival of tumor-bearing mice with corresponding increases in the frequency of CD4 and CD8 T cells, Th1 (CD4(+)IFN-γ(+)), Tc1 (CD8(+)IFN-γ(+)) and T cells cytolytic activity against Meth A cells. Neutralization of CD4 or CD8 T cells reversed the antitumor benefit of IL-7. Furthermore, IL-7 decreased regulatory T Foxp3 as well as cells suppressive activity with a reciprocal increase in SMAD7. In addition, we observed an increase of the serum concentrations of IL-6 and IFN-γ, and a significant decrease of TGF-ß and IL-10 after IL-7 treatment. Taken together, these results indicate that IL-7 augments T cell-mediated antitumor immunity and improves the effect of antitumor in Meth A model.

Synergic antitumor effect of SKLB1002 and local hyperthermia in 4T1 and CT26.

A de novo VEGFR2-inhibited compound SKLB1002 which is independently developed in our laboratory has been described for antiangiogenesis and displays a potent antitumor activity in vivo and in vitro. In the present investigation, we aim to prove that combination therapy of SKLB1002 with hyperthermia plays a synergy as an antitumor agent in solid tumor. In this study, we analyzed their synergetic inhibitory action on human umbilical vein endothelial cells (HUVEC), murine mammary cancer 4T1, murine colon carcinoma CT26 in vitro. Multiply-table tournament was performed to detect cell proliferation in vitro. 4T1 implantation and CT26 implantation in BALB/c mice were used to examine the activity of combination therapy of SKLB1002 with hyperthermia in vivo. Vascular density was determined by CD31 immunohistochemistry. TUNEL was used to measure apoptosis in tumor tissue. Metastasis assay was investigated via measurement of pulmonary metastasis nodules under the microscope. Potential toxicity of combination therapy was observed by histologic analysis of main organs stained with H&E. In vitro, the combination therapy significantly inhibited cell proliferation of HUVEC, 4T1 and CT26. In vivo, 4T1 and CT26 model experiments showed that combination therapy remarkably inhibited tumor growth and prolonged life span. When compared with controls, combination therapy reached 61 % inhibition index of tumor growth against CT26 and 51 % against 4T1. Moreover, it reduced angiogenesis and increased tumor apoptosis and necrosis. It was further found that combination therapy could efficiently prevent tumor from metastasizing to lung. Importantly, it had no toxicity to main organs including heart, liver, spleen, lung and kidney. Combination treatment has been proved to be a novel and strong strategy in clinical antitumor therapy. Our findings suggest that the combination therapy of SKLB1002 with hyperthermia has a synergistic antiangiogenesis, anticancer and promotion of apoptosis efficacy compared with controls. These findings could pave a new way in clinical tumor therapy.

Inhibition of tumor growth and alteration of associated macrophage cell type by an HO-1 inhibitor in breast carcinoma-bearing mice.

Heme oxygenase-1 [HO-1, also called heat shot protein 32 (HSP32)] can specifically metabolize heme to carbon monoxide, biliverdin, and ferrous iron and plays an important role in the processes of anti-inflammation, tissue protection, and antioxidative stress reaction. It has been reported that HO-1 can promote tumorigenesis and metastasis of many tumors. However, the detailed mechanisms of how HO-1 affects tumor progress are not clear. Here, we used ZnPPIX (a specific inhibitor of HO-1) to evaluate its potential effects on mouse breast cancer and tumor-associated macrophages (TAMs). We found out that mouse 4T1 breast cancer growth can be effectively suppressed through inhibition of HO-1 in vitro and in vivo. Moreover, in the 4T1 mouse model, when HO-1 was suppressed in TAMs, alternatively activated macrophages (M2 type) switched to classically activated macrophages (M1 type). In conclusion, 4T1 breast cancer growth was modulated by HO-1 expression. Furthermore, inhibition of HO-1 may induce tumor-associated immune response by activating TAMs' alternative proliferation. These data suggest that HO-1 may be an important target of breast cancer treatment.

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts.

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p<0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.

Active immunotherapy for mouse breast cancer with irradiated whole-cell vaccine expressing VEGFR2.

As tumor-associated antigens are not well characterized for the majority of human tumors, polyvalent vaccines prepared with whole-tumor antigens are an attractive approach for tumor vaccination. Vascular endothelial growth factor receptor-2 (VEGFR2), as a model antigen with which to explore the feasibility of immunotherapy, has shown great promise as a tumor vaccine. However, the efficacy of immunotherapy is often not ideal when used alone. In this study, we explored the therapeutic efficacy of an irradiated AdVEGFR2-infected cell vaccine-based immunotherapy in the weakly immunogenic and highly metastatic 4T1 murine mammary cancer model. An adenovirus encoding the VEGFR2 gene (AdVEGFR2) was constructed. Lethally irradiated, virus-infected 4T1 cells were used as vaccines. Vaccination with lethally irradiated AdVEGFR2-infected 4T1 cells inhibited subsequent tumor growth and pulmonary metastasis compared with challenge inoculations. Angiogenesis was inhibited, and the number of CD8+ T lymphocytes was increased within the tumors. Antitumor activity was also caused by the adoptive transfer of isolated spleen lymphocytes. In vitro, the expression of HMGB1 and HSP70 in the AdVEGFR2­infected 4T1 cells was increased, and was involved in the activation of tumor antigen-specific T-cell immunity. Our results indicate that the immunotherapy based on irradiated AdVEGFR2-infected whole-cancer cell vaccines may be a potentially effective strategy for 4T1 cancer treatment.

Orlistat, a novel potent antitumor agent for ovarian cancer: proteomic analysis of ovarian cancer cells treated with Orlistat.

Orlistat is an orally administered anti-obesity drug that has shown significant antitumor activity in a variety of tumor cells. To identify the proteins involved in its antitumor activity, we employed a proteomic approach to reveal protein expression changes in the human ovarian cancer cell line SKOV3, following Orlistat treatment. Protein expression profiles were analyzed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. More than 110 differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue staining. Furthermore, 71 proteins differentially expressed proteins were positively identified via mass spectrometry (MS)/MS analysis. In particular, PKM1/2, a key enzyme involved in tumorigenesis, was found to be significantly downregulated in SKOV3 cells following treatment with Orlistat. Moreover, PKM1/2 was proved to be downregulated in SKOV3 cells by western blot analysis after treatment with Orlistat. Taken together, using proteomic tools, we identified several differentially expressed proteins that underwent Orlistat-induced apoptosis, particularly PKM2. These changes confirmed our hypothesis that Orlistat is a potential inhibitor of ovarian cancer and can be used as a novel adjuvant antitumor agent.