Abstract Liver hepatocellular carcinoma (LIHC) is one of the most common malignant tumors in the world with a poor prognosis. FoxM1, a known transcription factor, plays a principal role in the progression and development of multiple tumors. However, the relationships between FoxM1 expression, tumor immunity, and prognosis in LIHC remain unclear. Here we investigated the expression of FoxM1, its prognostic value and immune cell infiltration in LIHC via Oncomine, TIMER, GEPIA, HCCDB and Kaplan-Meier plotter databases. FoxM1 expression was significantly higher in LIHC and correlated with tumor stage, grade, and TP53 mutation status. The high expression of FoxM1 was related to the poor prognosis of LIHC patients, which was influenced by the abundance of immune infiltrates including B cells, CD4+T cells, CD8+T cells and Tregs. FoxM1 showed strong correlations with the infiltrating levels of different immune cell subtypes including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells. FoxM1 expression was also significantly positively correlated with the expression of immune checkpoint molecules such as PD-1, CTLA-4, LAG3 and HAVCR2. What’s more, we found that inhibition of FoxM1 enhanced T-cell-mediated cytotoxicity against liver cancer cells via modulating PD-L1 expression. In conclusion, our results demonstrated that FoxM1 could be a potential prognostic target involved in its immune regulatory function in LIHC.

The aim of this study The purpose of this study is to investigate whether the DC cells combined with CIK cells (DC/CIK) and DC activated cytotoxic T cells (DC-ACT) treatment can promote antitumor response and change the immune indicators by targeting the heterogeneous tumor cell populations at a system level.Methods In this study, 112 patients with cancer were assigned to the DC/CIK treatment and 116 patients received the DC-ACT therapy. We detected the lymphocyte subsets and other immune indicators pre- and post-treatment to evaluate the changes of patient's immunity and compare the differences in immune status between two adoptive cellular immunotherapies.Results DC/CIK therapy elevated the percentage of CD3+ HLA-DR+ T cells, NK cells and several serological cytokines such as IL-2, IL-6 after cell infusion (p < .05). DC-ACT therapy could increase the total CD3 + T cells, CD8 + T cells, CD3+ HLA-DR+ cells and IL-12 cytokines after cell infusion (p < .05). The levels of IL-4/IFN-γ, IL-4/IL-12 and IL-6/IL-12 were reduced significantly in the DC-ACT group compared with DC/CIK group. These observations suggested that DC-ACT therapy has more dominance to induce Th1 cytokine response instead of skewing toward the Th2 cytokine profile based on the immunomodulatory properties.Conclusions These results indicated that DC, CIK, and DC-ACT cells exert anti-tumor activity through the different pathways. Thus, this work may provide valuable insights into the clinical curative effect evaluation of immunocyte therapy and the design of combined immunotherapeutic strategies for malignant tumors.

The present study was performed to explore the prognostic significance of periostin expression in a cohort of patients with early-stage breast cancer treated with breast conserving surgery following radiotherapy. A tissue microarray of tumor samples from 259 patients with early-stage breast cancer was assayed for periostin, estrogen receptor (ER), progesterone receptor (PR), ErbB2 receptor tyrosine kinase 2 and Ki-67 expression by immunohistochemistry. The association of periostin with other clinicopathological parameters and clinical outcomes, including local recurrence free survival (RFS), distant metastasis free survival (DFS) and overall survival (OS), were assessed through log-rank tests and univariate and multivariate analysis. Periostin expression was identified in 91 of the 259 tissue samples (35%). The periostin status was significantly associated with histological grade (P=0.001), nodal status (P=0.023), molecular subtype (P<0.01), ER status (P<0.01), PR status (P<0.01) and Ki-67 expression (P=0.011). Furthermore, periostin expression was associated with an increased risk of five-year local recurrence (95.8% vs. 89.0%; P=0.017) and distant metastasis (92.3% vs. 79.1%; P=0.001) in patients with early stage breast cancer. Multivariate analysis using Cox's proportional hazards model demonstrated that periostin expression was an independent predictor of all clinical outcomes in breast cancer (RFS, P=0.018; DFS, P=0.025; OS, P=0.047). Therefore, it was concluded that periostin is associated with an increased risk of local relapse and distant metastasis in early-stage breast cancer treated with conserving surgery and radiotherapy. This association should be further investigated in larger cohorts to validate the clinical significance of periostin expression.

Croton, a large genus of Euphorbiaceae, is widely distributed in tropical regions of South-East Asia and China. Many researchers previously reported the pharmacological and physiological actions of Croton species on anti-inflammatory, anticonvulsant and wound healing properties. Mature Croton tiglium contains large amounts of natural medical components, in which croton alkaloid, flavonoids and diterpenes are anticancer agents. The study on application of Croton in lung cancer is lacking. In this study, we investigated the regulating effects of Croton tiglium extract on A549 cell proliferation and apoptosis. Preparation of Croton tiglium seed extract. A549 human lung cancer cell cultures Cell viability assay Flow cytometric analysis of apoptosis. The MTT assay showed that Croton tiglium extract could exert a significant inhibitory effect on the proliferation of A549 cells. (Figure 1, P<0.05). It is notable that the highest proportion of early apoptotic cells were 45.94% with 100 μg/ml Croton tiglium extract treatment. (Figure 2, P<0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our results demonstrate that the Croton tiglium extract could inhibit the proliferation of A549 cells by regulating apoptosis in vitro. It has potential to provide biologically active compounds for treating NSCLC and deserves additional evaluation criteria as a new plant-derived anticancer agent.

Recurrence of breast cancer after radiotherapy may be partly explained by the presence of radioresistant cells. Thus, it would be desirable to develop an effective therapy against radioresistant cells. In this study, we demonstrated the intense antitumor activity of cytokine-induced killer cells against MCF-7 and radioresistant MCF-7 cells, as revealed by cytokine-induced killer-mediated cytotoxicity, tumor cell proliferation, and tumor invasion. Radioresistant MCF-7 cells were more susceptible to cytokine-induced killer cell killing. The stronger cytotoxicity of cytokine-induced killer cells against radioresistant MCF-7 cells was dependent on the expression of major histocompatibility complex class I polypeptide-related sequence A/B on radioresistant MCF-7 cells after exposure of cytokine-induced killer cells to sensitized targets. In addition, we demonstrated that cytokine-induced killer cell treatment sensitized breast cancer cells to chemotherapy via the downregulation of TK1, TYMS, and MDR1. These results indicate that cytokine-induced killer cell treatment in combination with radiotherapy and/or chemotherapy may induce synergistic antitumor activities and represent a novel strategy for breast cancer.

Objective: To investigate the impact of a Croton tiglium extract on cellular proliferation and apoptosis in a nonsmallcell lung cancer cell line (A549) in vitro. Methods: A Croton tiglium seed methanol extract was prepare andassessed for effects on A549 cells regarding cellular proliferation, apoptotic rates, and expression of apoptosis relatedgenes and proteins using real-time PCR and immunofluorescence. Results: The tested Croton tiglium extract inhibitedA549 cell proliferation in a dose- and time-dependent manner, with significant elevation of apoptotic indexes atvarious concentrations after 24 h. In addition, rates in both early and late stages were higher in treated than untreatedgroups, the 100 μg/ml dose causing the highest levels of apoptosis. RT-PCR showed that A549 cells treated with 100μg/ml Croton tiglium extract for 24 h has markedly higher Bax mRNA expression levels and obviously lower Bcl-2expression levels than controls, equivalent results being observed for proteins by immunofluorescence. However, themRNA expression levels of Fas and caspase-8 were not significantly altered. Conclusion: A Croton tiglium extractcan inhibit proliferation of A549 cells and promote apoptosis though Bax/Bcl-2 pathways.

Cytokine induced killer (CIK) cells have a powerful tumor cells killing activity both in vitro and in vivo and transfusion of these cells have become an adjuvant treatment for tumors. CIK cells are induced and amplified from peripheral blood mononuclear cells (PBMCs) with multiple cytokines. As CD4+CD25bri regulatory T cells can be also induced by high dose of interleukin 2 (IL-2) which is used for CIK cells amplification in the CIK cell culture system, the anti-tumor activity of CIK cells was suppressed to some extent. In order to overcome this unwanted suppressive factor, we found that low dose of gemcitabine could reduce the proportion of CD4+CD25bri regulatory T cells in the CIK cell culture system and significantly enhance the anti-tumor activity of CIK cells in vitro. The levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were also reduced significantly following the depletion of CD4+CD25bri regulatory T cells in gemcitabine treated CIK cell culture system. In vivo experiment showed that low dose of gemcitabine treated CIK cells significantly suppressed tumor growth and prolonged their lifespan in tumor-bearing nude mice, with the proportion of CD4+CD25bri regulatory T cells reduced. Meanwhile, we detected lower levels of IL-10, TGF-β and a higher level of interferon-γ (IFN-γ) in tumor-bearing nude mice that received gemcitabine treated CIK cells transfusion than those in other groups. The possible mechanism involved in the enhanced anti-tumor activity in vivo was that gemcitabine treated CIK cells created a strengthened anti-tumor immune microenvironment with the changed levels of cytokines such as IL-10, TGF-β and IFN-γ. These results suggested a strategy to improve the adoptive immune therapy in recent use by removing the suppressive factors and a more effective tumor treatment combining chemotherapy and immunotherapy.

OBJECTIVE To investigate the mechanisms underlying the ability ofheparin-treated dendritic cells (DCs) to promote Th0 to Th1 differentiation in chronic hepatitis B (CHB). METHODS Peripheral blood mononuclear cells (PBMCs) were isolated from CHB patients and cultured in RPMI-1640 with recombinant GM-CSF and IL-4 with or without heparin to obtain DCs for study. The levels of Toll-like receptors (TLRs) on the DCs were measured using FACS and qPCR techniques.DC subsets with high expression of TLRs were selected for analysis of functional changes by treatment with the corresponding TLR-siRNA. The CD4+ T cell subpopulation was purified from peripheral blood by Dynal immunomagnetic beads, and then the production of IL-12 by DCs in the presence of poly(I:C) or R848 and ofIFN and IL-4 by Th cells co-cultured with DCs was evaluated by ELISA. The t-test was used for statistical analysis. RESULTS TLR3 expression, and not expression of TLR 7 or TLR8,was significantly increased in heparin-treated DCs as compared to levels detected in the DCs without heparin treatment (t =2.849,P less than 0.05;t =3.027,P less than 0.05). The level of IL-12 produced by heparin-treated DCs stimulated with poly(I:C) was obviously higher than that produced by DCs without heparin treatment and stimulated with poly(I: C) (t =8.68,P less than 0.01) or with R848 (t =19.01,P less than 0.01). However, the IL-12 production by TLR3-siRNA transfected-DCs was significantly reduced (t =31.49, P less than 0.01).When Th cells from allogenic patients with CHB were co-cultured with the TLR3-siRNA transfectedDCs, the frequency ofCD4+ IFN+ cells was significantly reduced (1.64+/-0.57% vs.6.31+/-0.88%,P less than 0.01),as was the capability of Thl to generate IFNg (t =20.83,Pless than 0.01). CONCLUSION Heparin may have up-regulated the TLR3 expression level of DCs, and sequentially promoted Th0 to Th1 differentiation.

Dendritic cell (DC) vaccination and cytokine-induced killer (CIK) cell therapy (DC/CIK) have shown limited success in the treatment of advanced non-small cell lung cancer (NSCLC). To investigate the reason for this limited success, the effects of DC/CIK cell therapy on the immune responses of tumor-bearing patients and patients with resected NSCLC were evaluated. In the total 50 patients studied, the serum concentrations of the Th2 cytokines (IL-4 and IL-10) in tumor-bearing patients were significantly higher than those with resected NSCLC before immunotherapy. The post-therapy Th1 cytokine (IFN-γ) level in patients with resected NSCLC significantly increased from the pre-therapy level. In contrast, significantly enhanced post-therapy Th2 cytokine (IL-4 and IL-10) levels were found in tumor-bearing patients. The intracellular staining assay revealed that DC/CIK cell therapy increased the IFN-γ-producing T lymphocyte (CD8+IFN-γ+) frequency in patients with resected NSCLC, but these lymphocytes were not found in tumor-bearing patients. Furthermore, overproduction of vascular endothelial growth factor (VEGF) in tumor-bearing patients showed a statistically positive correlation with IL-4, suggesting that VEGF might be responsible for the predominance of serum Th2 cytokines. In a word, tumor-bearing patients developed a Th2-dominant status that could not be reversed toward Th1 following immunotherapy. A combined regiment of DC vaccination and CIK cell therapy with other treatments to overcome systemic Th2-dominant immune response might improve the current clinical benefit.

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objective:The aim of the study was to evaluate the safety and therapeutic efficacy of autologous dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) for the treatment of lung cancer patients. Methods:In the present study, we recruited a group of 40 lung cancer patients after surgery registered in the Biotherapy Center during 2005-2011 and randomly divided patients into two groups: Twenty patients treated with DC-CIK therapy as the treatment group and the other twenty patients served as controls.Subjects were administrated with 1×106 units of IL-2 daily for 5 days before mononuclear cell isolation from peripheral blood. For the treatment group, after primary tumors were removed by surgery, 1-3 doses of DCs and CIKs were perfused via peripheral veins. The immunologic function,short-term therapeutic effects, 5-years overall survival rate (OS), and disease free survival rate (DFS) were compared between the two groups.In addition, the peripheral blood samples were extracted from all patients before and after DC-CIK therapy,analyzed for T cell subsets by flow cytometry,and the intracellular cytokines of T cells were detected simultaneously. Results:OS for 1, 3, and 5 years of DC-CIK therapy group were 100%, 88%, and 88% respectively which contrasted with 78%,66%, and 25% in the control group. DFS 1, 3, and 5 years of DC-CIK therapy groups were 100%, 85%,65% respectively compared to 73%,58%, 23% in the control groups. Although there were no changes in the subsets of T cells in peripheral blood before and after DC-CIK therapy, the intrace1lular cytokines IFN-γ,IL-2,IL-4, and IL-6 of the immune cells were noticeably improved in the treatment group. Conclusion:DC-CIK therapy is a potential effective therapeutic modality to improve OS and DFS in post-surgical lung cancer patients. Citation Format: Daiqing Gao, Changyou Li, Peng Zhao, Xiaofang Wei, Xihe Xie, Weihong Sun, Qingming Guo, Danni Zhu, Tianyi Gao, Aris T. Alexandrou, Jian Jian Li. The clinical effects of dendritic cell and cytokine-induced killer cell therapy for lung cancer after surgery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2812. doi:10.1158/1538-7445.AM2014-2812

Gastric and colorectal cancers (GC and CRC) have poor prognosis and are resistant to chemo- and/or radiotherapy. In the present study, the prophylactic effects of dendritic cell (DC) vaccination are evaluated on disease progression and clinical benefits in a group of 54 GC and CRC patients treated with DC immunotherapy combined with cytokine-induced killer (CIK) cells after surgery with or without chemo-radiotherapy. DCs were prepared from the mononuclear cells isolated from patients using IL-2/GM-CSF and loaded with tumor antigens; CIK cells were prepared by incubating peripheral blood lymphocytes with IL-2, IFN-γ, and CD3 antibodies. The DC/CIK therapy started 3 days after low-dose chemotherapy and was repeated 3–5 times in 2 weeks as one cycle with a total of 188.3±79.8×106 DCs and 58.8±22.3×108 CIK cells. Cytokine levels in patients' sera before and after treatments were measured and the follow-up was conducted for 98 months to determine disease-free survival (DFS) and overall survival (OS). The results demonstrate that all cytokines tested were elevated with significantly higher levels of IFN-γ and IL-12 in both GC and CRC cohorts of DC/CIK treated patients. By Cox regression analysis, DC/CIK therapy reduced the risk of post-operative disease progression (p<0.01) with an increased OS (<0.01). These results demonstrate that in addition to chemo- and/or radiotherapy, DC/CIK immunotherapy is a potential effective approach in the control of tumor growth for post-operative GC and CRC patients.