Search

Conditionally replicating adenoviruses (CRAd) have been under extensive investigations as anticancer agents. Previously, we found that ZD55, an adenovirus serotype 5-based CRAd, infected and killed the leukemia cells expressing coxsackie adenovirus receptor (CAR). However, majority of leukemic cells lack CAR expression on their cell surface, resulting in resistance to CRAd infection. In this study, we showed that SG235, a novel fiber chimeric CRAd that has Ad35 tropism, permitted CAR-independent cell entry, and this in turn produced selective cytopathic effects in a variety of human leukemic cells in vitro and in vivo. Moreover, SG235 expressing exogenous tumor necrosis factor-related apoptosis-inducing ligand (SG235-TRAIL) effectively induced apoptosis of leukemic cells via the activation of extrinsic and intrinsic apoptotic pathway and elicited a superior antileukemia activity compared with SG235. In addition, normal hematopoietic progenitors were resistant to the inhibitory activity of SG235 and SG235-TRAIL. Our data suggest that these novel oncolytic agents may serve as useful tools for the treatment of leukemia. [Mol Cancer Ther 2009;8(5):1387–97]

Supplementary Fig. S1 from Effective gene-viral therapy of leukemia by a new fiber chimeric oncolytic adenovirus expressing TRAIL: in vitro and in vivo evaluation

Recently, mesenchymal stem cell (MSC) therapy has been suggested as an effective alternative approach for the treatment of hepatic diseases. MSCs have potential therapeutic value, because they have high self-renewal ability, are capable of multipotent differentiation, and have low immunogenicity. Furthermore, MSCs have the potential to differentiate into hepatocytes, and the therapeutic value exists in their immune-modulatory properties and secretion of trophic factors, such as growth factors and cytokines. Moreover, MSCs can suppress inflammatory responses, reduce hepatocyte apoptosis, increase hepatocyte regeneration, regress liver fibrosis, and enhance liver functionality.

Both ZeBrafish (

Background: Malignant pleural/peritoneal mesothelioma (MPM) is a rare, aggressive cancer with poor prognosis and high mortality of 65%-70% for pleural and 30% for peritoneal MPM. Patients who fail the standard therapy often survive less than 1 year, so it is urgent to develop new effective therapies for MPM patients. Chimeric antigen receptor (CAR)-T cells have been applied in MPM, but the efficacy was still limited due to immunosuppressive tumor microenvironment (TME). To overcome these obstacles, we developed armored CAR-T cells with nanobody targeting mesothelin (MSLN) and IFN-γ-activated secretion of PD-1 nanobody in a non-viral transposon system, named as BZDS1901. Preclinical studies have demonstrated cytotoxicity of the BZDS1901 in NCI-H226 lung/mesothelioma xenograft mouse model. To verify the safety and efficacy of BZDS1901, we conducted a single-arm, open label, dose-escalating clinical trials (NCT04503980, 05089266, 03615313) in solid tumors. Methods: Eligible patients were those who failed prior standard therapies with MSLN expression (≥50%) and PD-L1 positive in tumor specimen and voluntarily signed the informed consent. After apheresis and lymphodepletion with cyclophosphamide and fludarabine. BZDS1901 was administered intravenously in dose cohorts (1 × 106-2 × 107/kg) and the second infusion was given if no disease progression. After infusion, safety was evaluated during 28 days by the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0, efficacy was assessed by RECIST 1.1 or mRECIST with CT scan. Blood CAR copies were measured by qPCR, PD-1 nanobodies and cytokines by Meso Scale Discovery method, and T cell subtypes by flow cytometry. Patients’ progression-free survival (PFS) and overall survival (OS) were measured from the day of infusion to progression or death. Results: From July 20, 2020 to December 31, 2022, 11 MPM patients were enrolled and completed the assessment, while most patients received two infusions. BZDS1901 was safe, demonstrated by 54% grade 3 and 15% grade adverse events (AEs) that were hematological side effects due to lymphodepletion and reversible with supportive care. No on-target, off-tumor toxicity and dose-limiting toxicity were observed. All patients showed expansion of CAR-T cells and increased PD-1 nanobodies in circulation. CAR-T Cmax (cp/μg) copies number was 20062, and continually detectable in blood over 4 months. PD-1 Cmax (pg/ml) was 82841, and continually detectable in blood for up to 9 months. IFN- γ and IL-6 also increased at day 4 or Day 7. All patients obtained objective tumor response, one with complete response, six with partial response, and four with stable disease. The total objective response rate was 63.64%. All enrolled patients are still alive, and mPFS and mOS are not reached. The longest PFS was up to 26 months. Median follow-up was four months. Conclusions: PD-1 nanobody secreted and MSLN targeting CAR-T cells have demonstrated promising efficacy on MPM patients. Besides CAR-T direct tumor killing activity, secreted PD-1 nanobodies may provide additional clinical benefit by invigorating CAR-T from PD-L1 inhibition, activating TILs and relieving local immunosuppression. Citation Format: Zhuqing Liu, Yong Xia, Linlin Li, Yan Sun, Zhicai Lin, Lijie Rong, Zhongzheng Zhu, Zongchang Song, Hui Xue, Jianchun Duan, Shujing Shen, Jing Wang, Linjie Lv, Yaping Yang, Xue Tan, Liping Han, Wei Zhao, Jie Wang, Wenfeng Xu, Weimin Zhu, Zhong Li, Xingya Li, Jinxing Lou, Qing Xu, Qijun Qian. Non-viral mesothelin-targeted CAR-T cells armored with IFNg-induced secretion of PD-1 nanobody in treatment of malignant mesothelioma in phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT134.

Immunotherapies, such as monoclonal antibody therapy and checkpoint inhibitor therapy, have shown inspiring clinical effects for the treatment of cancer. Chimeric antigen receptor T (CAR-T) cells therapy was an efficacious therapeutic approach treating hematological malignancies and encouraging results have been achieved. Three kinds of CAR-T cell therapies, Kymriah (tisagenlecleucel), Yescarta (axicabtagene ciloleucel), were approved for clinical application in 2017 and Tecartus (brexucabtagene autoleucel) was approved in 2020. Despite some progress have been made in treating multiple hematologic tumors, threats still remain for the application of CAR-T cell therapy considering its toxicities and gaps in knowledge. To further comprehend present research status and trends, the review concentrates on CAR-T technologies, applications, adverse effects and safety measures about CAR-T cell therapy in hematological neoplasms. We believe that CAR-T cell therapy will exhibit superior safety and efficacy in the future and have potential to be a mainstream therapeutic choice for the elimination of hematologic tumor.

Objective The evaluation of lymphocyte subsets is widely regarded as an important factor for monitoring tumor progression and response to therapy. This study was designed to establish a comprehensive and detailed assessment of peripheral lymphocyte subsets with a multi-parametric flow cytometry assay for response prediction and prognosis evaluation of cancer patients. Methods Peripheral blood samples collected from 40 cancer patients and 23 age- and sex-matched healthy volunteers were tested for 29 lymphocyte subsets by flow cytometry. The univariate analysis was applied to establish the reference interval of healthy samples, and the ratio and proportion of 29 lymphocyte subsets between patient samples and healthy controls were compared to evaluate their clinical significance by Mann-Whitney U-test model. Results The reference ranges of 29 lymphocyte subsets were established with a normal distribution and no significant differences were observed between genders. Compared with healthy control group, lower proportion and ratio of specific parameters, such as Naive Th cells (p

“On-target off-tumor” toxicity is a major challenge to the use of chimeric antigen receptor (CAR)-engineered T cells in the treatment of solid malignancies, because of the expression of target antigens in normal tissues. Mesothelin overexpression is associated with poor prognosis of multiple solid tumors, and would therefore appear to be a suitable antigen target. To understand the risk of toxicity to different organs on anti-mesothelin CAR T cell therapy, single-cell RNA sequencing (scRNA-seq) datasets derived from major human physiological systems were analyzed in this study, including the respiratory, cardiovascular, digestive, and urinary systems. According to scRNA-seq datasets, the organs were stratified into high or low risk based on the level of mesothelin expression. We report that the proportion of mesothelin-positive cells was 7.71%, 2.40% and 2.20% of myocardial cells, pulmonary cells and stomach cells, respectively, indicating that these organs could be at high risk of “on-target off-tumor” toxicity on anti-mesothelin CAR T cell therapy. By contrast, esophagus, ileum, liver, kidney and bladder exhibited low mesothelin expression (

Mesothelin (MSLN) is an attractive antigen for chimeric antigen receptor (CAR) T therapy and the epitope selection within MSLN is essential. In this study, we constructed two types of CARs targeting either region I of MSLN (meso1 CAR, also known as a membrane-distal region) or region III of MSLN (meso3 CAR, also known as a membrane-proximal region) using a modified piggyBac transposon system. We reported that, compared with meso1 CAR T cells, meso3 CAR T cells express higher levels of CD107α upon activation and produce increased levels of interleukin-2, TNF-α, and IFN-γ against multiple MSLN-expressing cancer cells in vitro. In a real-time cell analyzer system and a three-dimensional spheroid cancer cell model, we also demonstrated that meso3 CAR T cells display an enhanced killing effect compared with that of meso1 CAR T cells. More importantly, in a gastric cancer NSG mice model, meso3 CAR T cells mediated stronger antitumor responses than meso1 CAR T cells did. We further identified that meso3 CAR T cells can effectively inhibit the growth of large ovarian tumors in vivo. Collectively, our study provides evidences that meso3 CAR T-cell therapy performs as a better immunotherapy than meso1 CAR T-cell therapy in treating MSLN-positive solid tumors.

Epithelial ovarian cancer (EOC) is the leading cause of death among gynecological malignancies in China. In particular, advanced/refractory ovarian cancer lacks effective targeted therapies due to the immunosuppressive and proangiogenic tumor microenvironment. Mesothelin (MSLN) has been found to be highly expressive in most EOC. Targeting MSLN by antibodies or chimeric antigen receptor-modified T (CAR-T) cells and immune checkpoint blockades as well as apatinib, an anti-angiogenic drug, have been used in patients with refractory ovarian cancer. Apatinib was reported to promote the infiltration of CD8+T cells in lung cancer. However, the combination therapy of CAR-T secreting anti-PD-1 antibody with apatinib in EOC has not been reported.Case presentationHere we report a case of refractory EOC in a patient who had relapsed after multiline chemotherapy. The patient received autologous T cells that contained sequences encoding single-chain variable fragments specific for MSLN and full-length antibody for PD-1 (αPD-1). The modified T cells were called αPD-1-mesoCAR-T cells. After infusion, the copy number and PD-1 antibody secretion of the CAR-T cells were increased in the blood. By application of multimodality tumor tracking, MRI of the liver showed shrinkage of metastatic nodules from average diameter of 71.3–39.1 mm at month 2. The patient achieved partial response and survived more than 17 months. IL-6 levels in the patient fluctuated from the baseline to 2–4-folds after treatment, but side effects were mild with only grade 1 hypertension and fatigue.ConclusionαPD-1-mesoCAR-T cell therapy combined with apatinib demonstrates a potential therapeutic effect on advanced refractory ovarian cancer.Trial registration numberNCT03615313.

Background Although chimeric antigen receptor (CAR)-T cell therapy has been remarkably successful for haematological malignancies, its efficacy against solid tumors is limited. The combination of CAR-T cell therapy with immune checkpoint inhibitors (CPIs), such as PD-1, PD-L1, and CTLA-4 antibodies, is a promising strategy for enhancing the antitumor efficacy of CAR-T cells. However, because most patients acquire resistance to CPIs, investigating other strategies is necessary to further improve the antitumor efficacy of CAR-T cell therapy for solid tumors. Recently, CD40 agonist antibodies showed potential antitumor efficacy by activating the CD40 pathway. Results Based on the piggyBac transposon system, rather than the widely used viral vectors, we constructed a meso3-CD40 CAR-T targeting region III of mesothelin (MSLN) that possessed the ability to secrete anti-CD40 antibodies. Compared with meso3 CAR-T cells, which did not secrete the anti-CD40 antibody, meso3-CD40 CAR-T cells secreted more cytokines and had a relatively higher proportion of central memory T (TCM) cells after stimulation by the target antigen. In addition, compared with meso3 CAR-T cells, meso3-CD40 CAR-T cells had a more powerful cytotoxic effect on target cells at a relatively low effector-to-target ratio. More importantly, we demonstrated that the antitumor activity of meso3-CD40 CAR-T cells was enhanced in a human ovarian cancer xenograft model in vivo. Conclusions In conclusion, these results highlight anti-CD40-secreting CAR-T cells generated by nonviral vectors as a potential clinical strategy for improving the efficacy of CAR-T cell therapies.

T cells modified to co-express cytokine or other factors with chimeric antigen receptor (CAR) can induce substantial and persistent increases in antitumor capacity in vivo. However, the uncontrolled expression of cytokines or factors can lead to the overactivation of immune cells, causing severe adverse events such as cytokine release syndrome (CRS) and neurotoxicity by CAR T cells with excessive growth potential. Conventional promoters are unregulated, and their expression is unlimited in T cells. In this study, by connecting the cytomegalovirus (CMV) enhancer, core interferon gamma (IFN-γ) promoter, and a T-lymphotropic virus long terminal repeat sequence (TLTR), we constructed and screened the chimeric promoter CIFT, which was highly expressed in some cell lines secreting IFN-γ and silenced in others. We placed this promoter upstream of the anti-programmed cell death protein 1 (anti-PD-1) antibody gene, and this construct was co-transfected with the CAR construct into T cells. In vitro or in vivo, CAR T cells showed increased secretion of anti-PD-1 antibody under control of the chimeric promoter CIFT. pS-CIFT-αPD-1/CAR T also had similar or lower PD-1 expression, higher levels of T cell activation, more release of IFN-γ, and better antitumor activity specifically against mesothelin-positive and PD-1 ligand 1 (PD-L1)-positive cell lines. The chimeric promoter may be a promising strategy to manipulate the content of immune checkpoint inhibitors or other proteins in future therapeutic approaches for cancer treatment., Graphical Abstract, Chimeric antigen receptor (CAR) T cell excessive growth always leads to severe adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity. The source of this issue is considered to be the unbridled expression of cytokines or immune checkpoint inhibitors, which is caused by ineligible chimeric promoters. In this study, Qian and colleagues developed a chimeric promoter that is able to regulate anti-PD-1 antibody expression in CAR T cells.

As non-viral transgenic vectors, the piggyBac transposon system represents an attractive tool for gene delivery to achieve a long-term gene expression in immunotherapy applications due to its large cargo capacity, its lack of a trace of transposon and of genotoxic potential, and its highly engineered structure. However, further improvements in transpose activity are required for industrialization and clinical applications. Herein, we established a one-plasmid effective screening system and a two-step high-throughput screening process in yeast to isolate hyperactive mutants for mammalian cell applications. By applying this screening system, 15 hyperactive piggyBac transposases that exhibited higher transpose activity compared with optimized hyPBase in yeast and four mutants that showed higher transpose activity in mammalian cells were selected among 3000 hyPBase mutants. The most hyperactive transposase, bz-hyPBase, with four mutation sites showed an ability to yield high-efficiency editing in Chinese hamster ovarian carcinoma (CHO) cells and T cells, indicating that they could be expanded for gene therapy approaches. Finally, we tested the potential of this screening system in other versions of piggyBac transposase.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The success of peptide-based dendritic cell (DC) cancer vaccines mainly depends on the utilized peptides and selection of an appropriate adjuvant. Herein, we aimed to evoke a broad immune response against multiple epitopes concurrently in the presence of immunoadjuvant. Three synthetic HLA-A∗0201-restricted peptides were separately linked with HMGB1-derived peptide (SAFFLFCSE, denoted as HB100-108) as immunoadjuvant via double arginine (RR) linker and loaded onto human monocyte-derived DCs. Peptide uptake was detected by immunofluorescence microscopy and flow cytometry. The maturation and activation status of pulsed DCs were monitored by detection of the expression of specific markers and released cytokines. The ability of peptide-pulsed DCs to activate allogeneic T cells has been assessed by a degranulation assay and detection of secreted cytokines. The lytic activity of effector T cells against cancer cells in vitro was analyzed by a lactate dehydrogenase (LDH) assay. Results revealed that DCs efficiently take up peptides+HB100-108 and expressed higher levels of surface markers (HLA-ABC, HLA-DR, CD80, CD86, CD83, CD40, and CCR7) and proinflammatory cytokines (IL-6, IFN-γ, TNF-α, and IL-12) than control DCs, free peptide-pulsed DCs, and free HB100-108-pulsed DC groups. Moreover, peptides+HB100-108/pulsed DCs were capable of activating allogeneic T cells and enhance their lytic activity against a pancreatic cancer cell line (PANC-1) in vitro. These findings suggest that antigenic peptides covalently linked with HB100-108/pulsed DCs could be a promising strategy to improve the current DC-based cancer vaccines.

Cancer vaccines have been exclusively studied all through the past decades, and have made exceptional achievements in cancer treatment. Few cancer vaccines have been approved by the US Food and Drug Administration (FDA), for instance, Provenge, which was approved for the treatment of prostate carcinoma in 2012. Moreover, more recently, T-VEC got approval for the treatment of melanoma. While, the overall therapeutic effects of cancer vaccines have been taken into consideration as below expectations, low antigenicity of targeting antigen and tumor heterogeneity are the two key limiting barriers encountered by the cancer vaccines. Nonetheless, recent developments in cancer immune-therapies together with associated technologies, for instance the unparalleled achievements bagged by immune checkpoint inhibitor based therapies and neo-antigen identification tools, envisage potential improvements in cancer vaccines in respect to the treatments of malignancies. This review brings forth measures for the purpose of refining therapeutic cancer vaccines by learning lessons from the success of PD-1 inhibitor based immune-therapies.

Purpose The aim of this study was to evaluate the clinical response of immunotherapy with dendritic cell-cytotoxic T lymphocytes (DC-CTLs) in patients with hepatocellular carcinoma (HCC). Method Sixty-eight patients with a confirmed diagnosis of HCC and who received follow-up until December 2015 were enrolled. We measured immune phenotypes of DCs and activated T cells using flow cytometry and clinical indexes using an electrochemiluminescence method. Results DCs exhibited up-regulation of the maturation markers CD83, CD80, CD11c, and CD86 on day8. Levels of IFN-γ and TNF-α were higher in the DCs pulsed with tumor-associated antigens (TAAs) than in DCs with a non-proliferative recombinant adenovirus. The percentage of regulatory T cells (Tregs) decreased in patients after DC-CTLs therapy. In addition, serum levels of AFP, AFP-L3, ALT, and CA19-9 were significantly reduced in these patients. Quality of life was improved, especially on physical functioning scales. Median overall survival (OS) and progression-free survival (PFS) were 8.2 months and 4.3 months, respectively, for the control group and 12.8 months and 9 months, respectively, for the DC-CTL group. Patients treated with DC-CTLs therapy showed a statistically significant PFS and OS curve (OS: p=0.016; PFS: p

Background: Infiltration of immune cells and immune microenvironment determine the proliferative activity of the tumor and metastasis. The aim of this study was to analyze the influence of activation or suppression of the immune response mediators on the prognosis of biliary tract cancer (BTC). Methods: We searched Pubmed, Web of Science, Embase and The Cochrane Library for relevant literatures until June 2016. The quality of studies was assessed by QUADAS-2 and NOS tools. Forest and funnel plots and all statistical analyses were generated by using Review Manager 5.3. The bias of included studies was estimated by Egger's test using Meta R package. Results: A total of 2339 patients from 12 studies were finally enrolled in this meta-analysis. Patients with high expression of immune active factors, intraepithelial tumor-infiltrating CD4+ , CD8+, and Foxp3+ T lymphocytes, MHC I, NKG2D, showed a better overall survival (OS) than those with low expression (HR=0.52, 95% CI=0.41-0.67, P

Objective The evaluation of lymphocyte subsets is widely regarded as an important factor for monitoring tumor progression and response to therapy. This study was designed to establish a comprehensive and detailed assessment of peripheral lymphocyte subsets with a multi-parametric flow cytometry assay for response prediction and prognosis evaluation of cancer patients. Methods Peripheral blood samples collected from 40 cancer patients and 23 age- and sex-matched healthy volunteers were tested for 29 lymphocyte subsets by flow cytometry. The univariate analysis was applied to establish the reference interval of healthy samples, and the ratio and proportion of 29 lymphocyte subsets between patient samples and healthy controls were compared to evaluate their clinical significance by Mann–Whitney U-test model. Results The reference ranges of 29 lymphocyte subsets were established with a normal distribution and no significant differences were observed between genders. Compared with healthy control group, lower proportion and ratio of specific parameters, such as Naïve Th cells (p

Immunotherapy has become the fourth cancer therapy after surgery, chemotherapy, and radiotherapy. In particular, immune checkpoint inhibitors are proved to be unprecedentedly in increasing the overall survival rates of patients with refractory cancers, such as advanced melanoma, non-small cell lung cancer, and renal cell carcinoma. However, inhibitor therapies are only effective in a small proportion of patients with problems, such as side effects and high costs. Therefore, doctors urgently need reliable predictive biomarkers for checkpoint inhibitor therapies to choose the optimal therapies. Here, we review the biomarkers that can serve as potential predictors of the outcomes of immune checkpoint inhibitor treatment, including tumor-specific profiles and tumor microenvironment evaluation and other factors.

Effective control of non-small-cell lung cancer (NSCLC) remains clinically challenging, especially during advanced stages of the disease. This study developed an adoptive T-cell treatment through expression of a chimeric antigen receptor (CAR) to target human epidermal growth factor receptor (EGFR) in NSCLC. We optimized the non-viral piggyBac transposon system to engineer human T cells for the expression of EGFR-CAR, consisting of EGFR scFv, transmembrane domain, and intracellular 4-1BB-CD3ζ signaling domains. The modified CAR T cells exhibited expansion capability and anticancer efficacy in a time- and antigen-dependent manner in vitro as well as regression of EGFR-positive human lung cancer xenografts in vivo. EGFR-CAR T therapy is a promising strategy to improve the efficacy and potency of the adoptive immunotherapy in NSCLC. Moreover, EGFR-CAR T therapy could become a clinical application for NSCLC patients in the future.

Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis. Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis. In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors. A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.

Hepatocellular carcinoma (HCC) is a major primary liver malignancy in adults. Despite the progress made, the outcome of the treatment to this disease is less than satisfactory as the post therapy tumor recurrence is almost inevitable. Accumulating pieces of evidence have suggested that the recurrence is due to the existence of a subpopulation of the HCC cells that possess the properties of stem cells and are resistant to radiation and chemotherapy. It is therefore important to understand the characteristics of this subpopulation of HCC cells, and which requires the establishment of an in vitro system to study these stem-like cancer cells. However, despite extensive efforts, the progress in establishing such an in vitro system has been slow largely due to the lack of definitive biomarkers in the isolation and expansion of these cells. In order to successfully maintain and expand HCC CSCs, we first optimized the culture system. We establish a novel medium system that allows the culture and enrichment of these hepatic stem-like cancer cells from both hepatoma cells and human primary HCC cells. These cells exhibited typical stem cell properties, such as enhanced stem cell markers, gain of EMT properties and drug resistance, and more importantly, stronger tumor-initiating capabilities. The medium may help to establish an in vitro model for hepatic cancer stem cell (HCSC) studies, which may contribute to the development of novel cell therapies and new drugs for the treatment of HCC.

AGO2 (Argonaute 2, EIF2C2) is the only member in AGO family with catalytic activity and of extreme importance during small RNAs guided gene silencing processes. The structural investigations have provided insights into details and functional mechanisms of the four major domains within AGO2. As a multifunction player, AGO2 has been revealed involved in tumorgenesis through miRNAs-dependent or independent ways. And nowadays, AGO2 has also been more importantly found ectopically over-expressed in carcinomas and closely associated with aspects of cancers in means of interacting with well-known tumor factors. Here, we provide a review on structural insights, functional mechanisms, novel roles and relationship with carcinomas of AGO2.

Cirrhosis is the long‑term outcome of chronic hepatic injury and no effective therapy is currently available for this disease. Mesenchymal stromal cells (MSCs) are multipotent cells that are easily acquired and amplified, and may be potential candidates for cell therapy against cirrhosis. This study aimed to determine the therapeutic effects of human umbilical cord‑derived MSCs (hUCMSCs) for the treatment of liver cirrhosis and identify an effective method for engrafting MSCs. The model of liver cirrhosis was established by induction of diethylnitrosamine (DEN) in rats. The isolated hUCMSCs were identified by morphology, flow cytometry and multilineage differentiation; they were injected into the vein of DEN‑induced rats at varied cell doses and infusion times. Biochemical analyses of the serum and histopathological analysis of the liver tissues were performed to evaluate the therapeutic effects of hUCMSCs in all treatment groups. The results indicated that isolated hUCMSCs were capable of self‑replication and differentiated into multiple lineages, including osteoblast‑, adipocyte‑ and hepatocyte‑like cells. Compared with the control group, administration of hUCMSCs at different cell doses and infusion times relieved DEN‑induced cirrhosis to varying degrees. The therapeutic effects of hUCMSCs on liver cirrhosis gradually improved with increased cell dose and infusion times. The improvement of cirrhosis was due to the capacity of hUCMSCs to breakdown collagen fibers in the liver. It was demonstrated that infusion of hUCMSCs effectively relieved liver cirrhosis by facilitating the breakdown of collagen fibers in a dose‑dependent manner and multiple infusions caused a relatively greater improvement in cirrhosis compared with a single infusion of hUCMSCs.

// Yin Tong 1,* , Liangshun You 1,* , Hui Liu 1 , Lu Li 1 , Haitao Meng 1 , Qijun Qian 2 , Wenbin Qian 1* 1. Institute of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University, P.R. China 2. Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, P.R. China * Contributed equally to this work Correspondence: Wenbin Qian, email: // Keywords : leukemia; oncolytic adenovirus; autophagy; Beclin-1; autophagic cell death; UVRAG Received : May 15, 2013, Accepted : May 31, 2013, Published : June 3, 2013 Abstract An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis.

Purpose Cellular immunotherapy has appeared to be a promising modality for the treatment of malignant tumor. The objective of this study was to evaluate the efficacy of cellular immunotherapy combined with minimally invasive therapy. Methods We searched PubMed, Web of Science and The Cochrane Library through March 2016 for relevant studies. Short-term efficacy (the disease control rate, the control rate of quality life and the AFP descent rate) and long-term efficacy (overall survival (OS) and progression-free survival (PFS) rate) were compared as the major outcome measures. The meta-analysis was performed using Review Manager 5.3. Results A total of 1174 references in 3 databases were found of which 19 individual studies with 1774 HCC patients enrolled in this meta-analysis. Meta-analysis results showed that cellular immunotherapy combined with minimally-invasive treatment significantly improved the measures of short-term response (the disease control rate (OR = 5.91, P = 0.007), the control rate of quality lift (OR = 3.38, P = 0.003) and the AFP descent rate (OR = 4.48, P = 0.02)). Also higher 6-month PFS (OR = 2.78, P = 0.05), ≥12-month PFS (OR = 3.56, P

Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.

Aberrant JAK/STAT3 pathway has been reported to be related to hepatocellular carcinoma (HCC) in many cell lines. In this study, a double-regulated oncolytic adenovirus vector that can replicate and induce a cytopathic effect in alpha-fetoprotein (AFP)-positive HCC cell lines with p53 dysfunction was successfully constructed. Two therapeutic genes, suppressor of cytokine signaling 3 (SOCS3) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were chosen and incorporated into this vector system, respectively. The combined treatment of AFP-D55-SOCS3 and AFP-D55-TRAIL (2:3 ratio) exhibited potent antitumor activity in AFP-positive HCC cell lines compared with any other treatment both in vitro and in vivo. Specific replication and low progeny yield in AFP-positive HCC cell lines rendered these double-regulated oncolytic adenoviruses remarkably safe. Our data demonstrated that restoration of SOCS3, which inhibits the JAK/STAT3 pathway, by AFP-D55-SOCS3 not only could antagonize HCC therapeutic resistance to TRAIL and adenoviruses, but could also induce cell cycle arrest in HCC cell lines. SOCS3 could down-regulate Cyclin D1 and anti-apoptotic proteins such as XIAP, Survivin, Bcl-xL, and Mcl-1, which are responsible for the synergistic inhibitory effects of AFP-D55-SOCS3 and AFP-D55-TRAIL. Dual gene and double-regulated oncolytic adenoviruses may provide safety and excellent antitumor effects for liver cancer, which is the advantage of a cancer-targeting gene virotherapy strategy.

Using conditionally replicating adenoviral vectors (CRAds) is a promising strategy in the treatment of solid tumors. The prospective of this study was to design a novel CRAd for the treatment of gastrointestinal cancer and show its efficacy in vitro, as well as in vivo. To determine if aberrant wnt signaling in tumor cells can be used to selectively drive viral replication, we analyzed six colorectal and hepatocellular cell lines, as well as 13 colorectal tumors and 17 gastric tumors, for β-catenin mutation status or aberrant wnt signaling, both of which were found frequently. Based on these findings, a novel CRAd (Ad5F11.wnt-E1A-hIL24) containing an E1A expression cassette driven by an artificial wnt promoter and delivering an apoptosis-inducing gene, interleukin-24 (IL24), was engineered. To enhance infection efficiency, the virus was pseudotyped by replacing adenovirus serotype 5 (Ad5) with Ad11 fiber. Ad5F11.wnt-E1A-hIL24 virus exhibited high selectivity toward cells with aberrant wnt signaling both in vitro and in mouse xenograft tumors. Transduction efficiency was significantly improved compared with that of nonpseudotyped control viruses. The proliferation of tumor cell lines, as well as tumor growth, in mouse xenografts could be profoundly inhibited by viral infection with Ad5F11.wnt-E1A-hIL24. The therapeutic effect was associated with increased apoptosis through caspase-3 activation. In addition, Ad5F11b vector exhibited a more favorable biodistribution, blood clearance, and transgene expression compared with conventional Ad5 vector after systemic or intratumoral injection in human gastrointestinal cancer xenografts. We think that our approach is a promising strategy in the treatment of gastrointestinal cancer, warranting further clinical investigation.

Conditionally replicative adenoviruses (CRAds) are widely used for cancer biotherapy and show a significant growth-suppressing effect on many types of cancer. However, it was reported that breast cancer was highly resistant to the infection of traditionally used adenovirus of serotype 5 (Ad5)-based CRAds. Although partial substitution of the fiber protein of replication-deficient Ad5 with that of adenovirus of serotype 35 (Ad35) facilitated infection of breast cancer cells by adenoviral vectors, it is still unknown whether this modification can improve CRAds in their tumor-eliminating capacity. We generated a 5/35 fiber-modified CRAd with a p53 cDNA construct and investigated whether this alteration in fiber region can make CRAds suppress the growth of breast cancer more effectively. Our data reinforced the proposal that 5/35-modified fiber conferred higher adenovirus infectivity for breast cancer cells than natural Ad5 fiber. Interestingly, 5/35 fiber-modified CRAd replicated more efficiently in breast cancer cells than Ad5-based CRAd. We also found 5/35 fiber-modified CRAd mediated higher expression of p53 in breast cancer cells. In vitro, 5/35 fiber-modified CRAd eliminated breast cancer cells more efficiently. Growth of xenograft tumors in nude mice was also significantly retarded by 5/35 fiber-modified CRAd. The 5/35 fiber-modified CRAd suppressed the growth of breast cancer cells more effectively than Ad5-based CRAd, both in vitro and in vivo. Thus CRAd with 5/35 hybrid fiber may be a promising vector for breast cancer treatment.

Conditionally replicating adenoviruses (CRAd) have been under extensive investigations as anticancer agents. Previously, we found that ZD55, an adenovirus serotype 5-based CRAd, infected and killed the leukemia cells expressing coxsackie adenovirus receptor (CAR). However, majority of leukemic cells lack CAR expression on their cell surface, resulting in resistance to CRAd infection. In this study, we showed that SG235, a novel fiber chimeric CRAd that has Ad35 tropism, permitted CAR-independent cell entry, and this in turn produced selective cytopathic effects in a variety of human leukemic cells in vitro and in vivo. Moreover, SG235 expressing exogenous tumor necrosis factor-related apoptosis-inducing ligand (SG235-TRAIL) effectively induced apoptosis of leukemic cells via the activation of extrinsic and intrinsic apoptotic pathway and elicited a superior antileukemia activity compared with SG235. In addition, normal hematopoietic progenitors were resistant to the inhibitory activity of SG235 and SG235-TRAIL. Our data suggest that these novel oncolytic agents may serve as useful tools for the treatment of leukemia. [Mol Cancer Ther 2009;8(5):1387–97]

Conditionally replicative adenovirus (CRAD) represents a promising approach for cancer therapy. Several CRADs controlled by the human telomerase reverse transcriptase promoter have been developed. However, because of their replicative capacity, the importance of cancer specificity for CRADs needs to be further emphasized. In this study, we have developed a novel dual-regulated CRAD, CNHK500-mE, which has its E1a and E1b gene controlled by the human telomerase reverse transcriptase promoter and the hypoxia response element, respectively. It also carries a mouse endostatin expression cassette controlled by the cytomegalovirus promoter. These properties allow for increased cancer cell targeting specificity and decreased adverse side effects. We showed that CNHK500-mE preferentially replicated in cancer cells. Compared with a replication-defective vector carrying the same endostatin expression cassette, CNHK500-mE–mediated transgene expression level was markedly increased via viral replication within cancer cells. In the nasopharyngeal tumor xenograft model, CNHK500-mE injection resulted in antitumor efficacy at day 7 after therapy. Three weeks later, it led to significant inhibition of xenograft tumor growth due to the combined effects of viral oncolytic therapy and antiangiogenesis gene therapy. Pathologic examination showed that most cancer cells were positive for adenoviral capsid protein and for apoptotic terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling in the CNHK500-mE–treated tumor tissues, and the microvessels in these tumor tissues were diminished in quantity and abnormal in morphology. These results suggest that, as a potential cancer therapeutic agent, the CNHK500-mE is endowed with higher specificity to cancer cells and low cytotoxicity to normal cells. (Mol Cancer Res 2008;6(4):568–75)