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The Gilt-bronze seated Buddha at Sinamsa temple in Dangin has an uneven in the shape the gilt, poor gloss and cracks were damaged. Such damage to the gold-gilding layer may degrade the corrosion resistance of the Buddha statue, thereby promoting corrosion of the metal compound. The Buddha statue was a restoration in the 1980s, and it was highly likely that modern materials such as cashew were used as adhesive materials for gold-gilding. In the study, a conservation management plan was proposed by comprehensively examining the material characteristics of the gold-gilding layer and adhesives. As the composition analysis, all gold-gilding layers show high Au content, so it seems that pure gold was used, and copper and tin were detected as the main alloying materials for the base metal, corresponding to a bronze. Various analyses of the gilt adhesive material, it was found that gilt was recovered using cashew. The cashew has a weaker adhesion than the lacquer, so the blistering of gold powder and gilt proceeds faster, and can cause various damages such as metal corrosion and whitening. In order to stably preserve the Gilt-bronze seated Buddha at Sinamsa temple and restore the authenticity, it is necessary to remove the cashew covering layer and perform lacquer gold-gilding.

Supplementary Figure S5 PDF file - 27K, Patient year of birth, disease type, stage and sites of metastasis where applicable, for samples which were evaluated in Figure 6 for KL-1 mediated NK cell expansion are shown

upplementary Data Legend PDF file - 94K, Supplementary data legend

The tomb complex of the royal family from the period of the Ungjin Baekje Kingdom (475 to 538 AD) in Gongju, Korea, contains the tomb of King Muryeong and other royal tombs. After the excavation of the tomb of King Muryeong in 1971, these tombs were opened up to the public, without the establishment of systems for their safety, conservation and management. The tombs have consequently experienced rapid environmental changes and suffered various damages. In this study, specific vulnerable parts inside the tombs were selected for deviation analysis using 3D scanning, and 3D image models were constructed on this basis. Progressive displacement was identified in tomb No. 5, and basic data for future investigations was acquired from tomb No. 6 and the tomb of King Muryeong. In the deviation analysis for the southern plastered wall of tomb No. 5, the damage was not found to exceed the ranges of ±18 mm and ±2 mm. However, the lintel stone was found to be sagging by 0.32 mm on average, and the distance between the walls to have increased by 0.36 mm on average. Direct water seepage occurring in tomb No. 5 is considered to be increasing the damage within the tomb, such as the dropping and sagging of the lintel. The 3D image models constructed in this study will play an important role as baseline data for future research, and can be used to discuss a secure conservation scheme for the tombs through cross-validation with precise measurement monitoring.

Childhood cancer is on the rise in high-income countries. Epidemiological studies suggest that reduced exposure to common infections in early life is to blame. However, no specific infection responsible for protection against cancer has been identified, and the underlying mechanisms remain a matter of speculation. Recent findings that Epstein-Barr virus (EBV) can induce antitumor immunity lead us to hypothesize that the delay in EBV infection in such countries might contribute to the increase in childhood cancers.

Tumour-associated antigens (TAAs) comprise a large set of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as targets for immunotherapy has been explored for more than two decades1, yet the origins of TAA-specific T cells remain unclear. While tumour cells may be an important source of TAAs for T cell priming2, several recent studies suggest that infection with some viruses, including Epstein–Barr virus and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs3,4. However, the cellular and molecular basis of such responses remains undefined. Here we show that expression of the Epstein–Barr virus signalling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signalling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on major histocompatibility complex classes I (MHC-I) and II (MHC-II) (mainly through the endogenous pathway) and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4+ and CD8+ T cell responses. These findings delineate a mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in tumour B cells from patients with cancer and thereby enabling them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4+ T cells against a wide range of endogenous tumour antigens, such as TAAs and neoantigens, for treating B cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches to treatment of cancers. Expression of the Epstein–Barr virus protein LMP1 in B cells increases expression of—and promotes T cell responses to—tumour-associated antigens, delineating a mechanism of infection-induced anti-tumour immunity, which could inform immune-based approaches to cancer treatment.

Epstein–Barr virus (EBV), the first identified human tumor virus, infects and takes up residency in almost every human. However, EBV genome–positive tumors arise in only a tiny minority of infected people, presumably when the virus-carrying tumor cells are able to evade immune surveillance. Traditional views regard viral antigens as the principal targets of host immune surveillance against virus-infected cells. However, recent findings indicate that EBV-infected/-transformed B cells elicit both cytotoxic CD8+ and CD4+ T-cell responses against a wide range of overexpressed cellular antigens known to function as tumor-associated antigens (TAA), in addition to various EBV-encoded antigens. This not only broadens the ways by which the immune system controls EBV infection and prevents it from causing cancers, but also potentially extends immune protection toward EBV-unrelated cancers by targeting shared TAAs. The goal of this review is to incorporate these new findings with literature data and discuss future directions for improved understanding of EBV-induced antitumor immunity, as well as the hopes for rational immune strategies for cancer prevention and therapy.

Multiple immunomodulatory pathways shape the development of anti-tumor immune responses to lymphoid malignancies. We previously defined the recurrent genetic alterations in diffuse large B-cell lymphoma (DLBCL) and identified associated substructure and additional potential genetic bases for immune escape. CD70 was the most commonly perturbed immune response pathway component in our cohort of primary DLBCLs; alterations included inactivating mutations and copy loss. CD70 co-stimulation of CD27+ T cells induces antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Given the frequent co-association of CD70 alterations and BCL6 translocations in our DLBCL patient series, we assessed the consequences of Cd70 deficiency on Bcl6-driven lymphomagenesis in a murine model. We crossed previously generated Cd70 −/- and Bcl6 tg/+ mice to obtain Cd70 −/−; Bcl6 tg/+ animals. In our aging cohorts, Cd70− / −; Bcl6tg/+ mice developed significantly increased numbers of histopathologically confirmed DLBCLs at earlier timepoints, compared to Bcl6 tg/+ animals. Both the Cd70 −/−; Bcl6 tg/+ and Bcl6 tg/+ mice that were euthanized for symptoms exhibited massive splenomegaly and lymphomatous splenic infiltration. None of the wild-type (WT) and Cd70 −/- animals developed lymphoma. To characterize potential differences in anti-tumor responses in Cd70 −/−; Bcl6 tg/+ versus Bcl6 tg/+ mice, we harvested spleens from asymptomatic animals in each cohort at 6, 14 and 18 months (mo). Cd70 −/−; Bcl6 tg/+ mice exhibited significantly earlier onset splenomegaly than Bcl6 tg/+ animals (both in comparison with WT mice). We performed single cell RNA sequencing of splenic cell suspensions from each murine cohort at the above-mentioned predetermined timepoints (6, 14 and 18 mo) and describe genotype-related changes in splenic CD8+ T-cell infiltration in this abstract. Our study revealed an age-related decline in the percentages of naive CD8+ T cells in all genotypes, with more striking and earlier changes in Cd70 −/−; Bcl6 tg/+ animals. Cd70 −/−; Bcl6 tg/+ and Bcl6 tg/+ mice exhibited a selective and significant expansion of CD8+ cytotoxic T cells (CTLs), which expressed Gzmb and Prf1 and the exhaustion markers, Pdcd1, Lag3, Tigit, Tox and Tim3, and exhibited clonal expansion. At 6 mo, prior to splenic enlargement and the development of symptoms, CD8+ CTLs in Cd70 −/−; Bcl6 tg/+ animals expressed significantly higher levels of exhaustion markers than those in Bcl6 tg/+ mice. Consistent with this finding, there was a more limited expansion and a subsequent contraction of these splenic CD8+ CTLs in Cd70 −/−; Bcl6 tg/+ mice, in comparison to Bcl6 tg/+ animals. Taken together, these findings suggest that initial anti-tumor immune responses are less effective in Cd70 −/−; Bcl6 tg/+ mice than in Bcl6 tg/+ animals and highlight the likely importance of CD70/CD27 co-stimulation in CD8+ T-cell response to Bcl6-driven DLBCL. Citation Format: Elisa Mandato, Eleonora Calabretta, Gali Bai, Li Song, Yanbo Sun, Vignesh Shanmugam, Julia Paczkowska, Il-Kyu Choi, Robert Redd, Ming Tang, Lee N Lawton, Donna Neuberg, Scott Rodig, Franziska Michor, Baochun Zhang, Margaret A Shipp. Cd70 genetic perturbation limits the development of an effective CD8+ T-cell immune response to Bcl6-driven diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A38.

Multiple co-stimulatory and co-inhibitory pathways modulate T-cell dependent anti-tumor immune responses in lymphoid malignancies. We recently defined the recurrent genetic alterations and associated substructure of diffuse large B-cell lymphoma (DLBCL), including five distinct clusters (1-5), and identified potential genetic bases for immune evasion [Nature Medicine 2018; 24:679-690]. In our series, 26% of tumors had inactivating somatic mutations or copy loss of CD70 and likely disruption of CD70/CD27 co-stimulation. CD70 and CD27 are homotrimer type II and homodimer type I transmembrane proteins and members of the TNF and TNF receptor superfamilies, respectively. CD70 is transiently expressed on certain normal B-cell and dendritic cell populations upon activation and constitutively expressed on multiple B-cell tumors. CD70 activation of CD27 + T cells promotes antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Patients with germline deficiencies of either CD70 or CD27 have an increased incidence of EBV-associated lymphoid malignancies, underscoring the importance of this co-stimulatory pathway. In our series, CD70 alterations were most common in Cluster 1 DLBCLs, which also exhibited recurrent BCL6 chromosomal translocations. The co-occurrence of CD70 and BCL6 genetic alterations was noteworthy because of the established role of CD8 + T-cell dependent immune surveillance in murine models of Bcl6-driven DLBCL [Nature Medicine 2014; 20:283-290]. To assess the consequences of Cd70 deficiency and perturbed CD70/CD27 co-stimulation on Bcl6-driven lymphomagenesis, we crossed the previously described Bcl6tg/+ and Cd70-/- mice to generate Cd70-/-; Bcl6tg/+ animals. In the aging cohorts, Cd70-/-; Bcl6tg/+ mice were more likely than Bcl6tg/+ animals (or the Cd70-/- or wild-type [WT] groups) to be euthanized for symptoms before the study endpoint (18 months [mo]) (5 of 18 Cd70-/-; Bcl6tg/+ euthanized for symptoms prior to the first of 9 Bcl6tg/+). Additionally, significantly greater total numbers and percentages of Cd70-/-; Bcl6tg/+ mice, in comparison to Bcl6tg/+ or Cd70-/- animals, were euthanized for symptoms (64% Cd70-/-; Bcl6tg/+ vs. 29% Bcl6tg/+, p=0.005 and 64% Cd70-/-; Bcl6tg/+ vs. 11% Cd70-/-, p=0.002). Almost all euthanized Cd70-/; Bcl6tg/+ and Bcl6tg/+ micehad massively enlarged spleens infiltrated with histopathologically confirmed DLBCLs characterized by clonal Ig gene rearrangements. Our findings indicate that genetic perturbation of Cd70 accelerates the onset and significantly increases the incidence of Bcl6-driven DLBCL. To characterize potential differences in the anti-tumor immune responses in Cd70-/-; Bcl6tg/+ and Bcl6tg/+ mice (and Cd70-/- and WT controls), we also harvested spleens from 6 animals in each of the aging cohorts at predetermined intervals (2, 6, 14 and 18 mo). We analyzed the composition of splenic-cell suspensions by flow cytometry and evaluated the intact splenic architecture and morphology with expert hematopathologists (VS, KW and SR). At 14 and 18 mo, spleens from WT and Cd70-/- animals were largely normal in appearance and size. In contrast, spleens from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ mice exhibited disordered architecture and abnormal pre-malignant lymphoid proliferation with expanded white pulp including morphologically and immunophenotypically aberrant B cells of small to large size and increased infiltrating T-cells. Additionally, significantly higher fractions of splenic CD8 + T cells from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ animals expressed the CD69 activation marker and exhibited terminal differentiation, consistent with an ongoing anti-tumor immune response. Interestingly, Bcl6tg/+ animals had significantly higher percentages of splenic TCRβ + T cells at the earlier time point (14 mo) with delayed-onset splenomegaly (at 18 mo), which is in marked contrast to the Cd70-/-; Bcl6tg/+ mice that had significantly lower percentages of splenic TCRβ + T cells at the earlier time point (14 mo) and early-onset splenomegaly (14 mo). These findings suggest that the initial T-cell mediated immune response was more effective in Bcl6tg/+ than Cd70-/-; Bcl6tg/+ animals. Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective anti-tumor immune response in Bcl6tg/+-driven DLBCL. Disclosures Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Rodig: Affimed: Research Funding; Bristol-Myers-Squibb: Research Funding; Merck: Research Funding; Immunitas: Membership on an entity's Board of Directors or advisory committees; KITE/Gilead: Research Funding. Shipp: Bristol Myers Squibb: Research Funding; Immunitas Therapeutics: Consultancy; AstraZeneca: Consultancy, Research Funding; Merck: Research Funding; Abbvie: Other: Institution: Research Grant/Funding; Bayer: Other: Institution: Research Grant/Funding.

// Eonju Oh 1 , Il-Kyu Choi 2 , JinWoo Hong 1 , Chae-Ok Yun 1 1 Department of Bioengineering, College of Engineering, Hanyang University, Seongdong-gu, Seoul 133-791, Korea 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA Correspondence to: Chae-Ok Yun, email: chaeok@hanyang.ac.kr Keywords: oncolytic adenovirus, IL-12, decorin, TGF-β, Treg Received: July 25, 2016 Accepted: December 01, 2016 Published: December 16, 2016 ABSTRACT Interleukin (IL)-12 is a potent antitumor cytokine. However, immunosuppressive tumor microenvironments containing transforming growth factor-β (TGF-β) attenuate cytokine-mediated antitumor immune responses. To enhance the efficacy of IL-12-mediated cancer immunotherapy, decorin (DCN) was explored as an adjuvant for overcoming TGF-β-mediated immunosuppression. We designed and generated a novel oncolytic adenovirus (Ad) coexpressing IL-12 and DCN (RdB/IL12/DCN). RdB/IL12/DCN-treated tumors showed significantly greater levels of interferon (IFN)-γ, tumor necrosis factor-α, monocyte chemoattractant protein-1, and IFN-γ-secreting immune cells than tumors treated with cognate control oncolytic Ad expressing a single therapeutic gene (RdB/DCN or RdB/IL12). Moreover, RdB/IL12/DCN attenuated intratumoral TGF-β expression, which positively correlated with reduction of Treg cells in draining lymph nodes and tumor tissues. Furthermore, tumor tissue treated with RdB/IL12/DCN showed increases infiltration of CD8 + T cells and proficient viral spreading within tumor tissues. These results demonstrated that an oncolytic Ad co-expressing IL-12 and DCN induces a potent antitumor immune response via restoration of antitumor immune function in a weakly immunogenic murine 4T1 orthotopic breast cancer model. These findings provide new insights into the therapeutic mechanisms of IL-12 plus DCN, making it a promising cancer immunotherapeutic agent for overcoming tumor-induced immunosuppression.

The B-lymphotropic Epstein–Barr virus (EBV), pandemic in humans, is rapidly controlled on initial infection by T cell surveillance; thereafter, the virus establishes a lifelong latent infection in the host. If surveillance fails, fatal lymphoproliferation and lymphomagenesis ensue. The initial T cell response consists of predominantly CD8+ cytotoxic T cells and a smaller expansion of CD4+ cells. A major approach to treating EBV-associated lymphomas is adoptive transfer of autologous or allogeneic T cells that are stimulated/expanded on EBV-transformed B cells. Strikingly, the clinical response correlates with the frequency of CD4 cells in the infused T cells. Although in vitro studies suggested that EBV-specific CD4 cells develop cytotoxicity, they have not been comprehensively characterized and the molecular mechanism underlying their formation remains unknown. Our recent work, using a transgenic approach in mice, has revealed a central role for the EBV signaling molecule LMP1 in immune surveillance and transformation of EBV-infected B cells. The mouse model offers a unique tool for uncovering basic features of EBV immunity. Here, we show that LMP1 expression in B cells induces potent cytotoxic CD4 and CD8 T cell responses, by enhancing antigen presentation and costimulation by CD70, OX40 ligand, and 4-1BB ligand. Our data further suggest that cytotoxic CD4 cells hold superior therapeutic value for LMP1 (EBV)-driven lymphomas. These findings provide insights into EBV immunity, demonstrating that LMP1 signaling alone is sufficient to induce a prominent cytotoxic CD4 response, and suggest strategies for immunotherapy in EBV-related and other cancers.

Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.

Decorin is a natural transforming growth factor-β1 (TGF-β1) antagonist. Reduced decorin synthesis is associated with dermal scarring, and increased decorin expression appears to reduce scar tissue formation. To investigate the therapeutic potential of decorin for keloids, human dermal fibroblasts (HDFs) and keloid-derived fibroblasts (KFs) were transduced with decorin-expressing adenovirus (dE1-RGD/GFP/DCN), and we examined the therapeutic potential of decorin-expressing Ad for treating pathologic skin fibrosis. Decorin expression was examined by immunofluorescence assay on keloid tissues. HDFs and KFs were transduced with dE1-RGD/GFP/DCN or control virus, and protein levels of decorin, epidermal growth factor receptor (EGFR) and secreted TGF-β1 were assessed by Western blotting and ELISA. And type I and III collagen, and matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) mRNA levels were measured by real-time RT-PCR. Additionally, we immunohistochemically investigated the expression levels of the major extracellular matrix (ECM) proteins in keloid spheroids transduced with dE1-RGD/GFP/DCN. Lower decorin expression was observed in the keloid region compared to adjacent normal tissues. After treatment with dE1-RGD/GFP/DCN, secreted TGF-β1 and EGFR protein expressions were decreased in TGF-β1-treated HDFs and KFs. Also, type I and III collagen mRNA levels were decreased, and the expression of MMP-1 and MMP-3 mRNA was strongly upregulated. In addition, the expression of type I and III collagen, fibronectin and elastin was significantly reduced in dE1-RGD/GFP/DCN-transduced keloid spheroids. These results support the utility of decorin-expressing adenovirus to reduce collagen synthesis in KFs and keloid spheroid, which may be highly beneficial in treating keloids.

The past century's description of oncolytic virotherapy as a cancer treatment involving specially-engineered viruses that exploit immune deficiencies to selectively lyse cancer cells is no longer adequate. Some of the most promising therapeutic candidates are now being engineered to produce immunostimulatory factors, such as cytokines and co-stimulatory molecules, which, in addition to viral oncolysis, initiate a cytotoxic immune attack against the tumor. This study addresses the combined effects of viral oncolysis and T-cell-mediated oncolysis. We employ a mathematical model of virotherapy that induces release of cytokine IL-12 and co-stimulatory molecule 4-1BB ligand. We found that the model closely matches previously published data, and while viral oncolysis is fundamental in reducing tumor burden, increased stimulation of cytotoxic T cells leads to a short-term reduction in tumor size, but a faster relapse. In addition, we found that combinations of specialist viruses that express either IL-12 or 4-1BBL might initially act more potently against tumors than a generalist virus that simultaneously expresses both, but the advantage is likely not large enough to replace treatment using the generalist virus. Finally, according to our model and its current assumptions, virotherapy appears to be optimizable through targeted design and treatment combinations to substantially improve therapeutic outcomes.

Pancreatic cancer is a highly lethal disease for which limited therapeutic options are available. Pancreatic cancer exhibits a pronounced collagen-rich stromal reaction, which induces chemoresistance by inhibiting drug diffusion into the tumor. Complementary treatment with oncolytic virus such as an oncolytic adenovirus expressing relaxin (YDC002) is an innovative treatment option for combating chemoresistant pancreatic cancer. Here, we examined the ability of combined treatment with gemcitabine and YDC002, which degrades extracellular matrix (ECM), to efficiently treat chemoresistant and desmoplastic pancreatic cancer. Gemcitabine alone exhibited similarly low cytotoxicity toward pancreatic cancer cells throughout the concentration range (1-50 μM) used, whereas the combination of YDC002 and a subtherapeutic dose of gemcitabine (0.01-0.05 μM) resulted in potent anticancer effects through effective induction of apoptosis. Importantly, YDC002 combined with gemcitabine significantly attenuated the expression of major ECM components including collagens, fibronectin, and elastin in tumor spheroids and xenograft tumors compared with gemcitabine alone, resulting in potent induction of apoptosis, gemcitabine-mediated cytotoxicity, and an oncolytic effect through degradation of tumor ECM. Our results demonstrate that YDC002 can selectively degrade aberrant ECM and attenuate the ECM-induced chemoresistance observed in desmoplastic pancreatic tumor, resulting in a potent antitumor effect through effective induction of apoptosis.

Dengue fever is a tropical endemic disease; however, because of climate change, it may become a problem in South Korea in the near future. Research on vaccines for dengue fever and outbreak preparedness are currently insufficient. In addition, because there are no appropriate animal models, controversial results from vaccine efficacy assessments and clinical trials have been reported. Therefore, to study the mechanism of dengue fever and test the immunogenicity of vaccines, an appropriate animal model is urgently needed. In addition to mouse models, more suitable models using animals that can be humanized will need to be constructed. In this report, we look at the current status of model animal construction and discuss which models require further development.

The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.

Recurrent or metastatic cancer in most cases remains an incurable disease, and thus alternative treatment strategies, such as oncolytic virotherapy, are of great interest for clinical application. Oncolytic adenoviruses (Ads) have many advantages as virotherapeutic agents and have been safely employed in the clinics. However, the efficacy of oncolytic Ads is insufficient to eradicate tumors and current clinical applications are restricted to local administration against primary tumors because of immunological obstacles and poor tumor-cell targeting. Thus, alternative viable approaches are needed to establish therapies based on oncolytic Ad that will eliminate both primary and metastatic cancers. To this end, rational design of oncolytic Ads that express immunostimulatory genes has been employed. Even when restricted to local viral delivery, these oncolytic Ad-based immunotherapeutics have been shown to exert systemic antitumor immunity and result in eradication of both primary and metastatic cancers. Moreover, oncolytic Ad-based immunotherapeutics in combination with either dendritic cell-based vaccine or radiotherapy further strengthen the systemic tumor-specific immunity, resulting in complete suppression of both local and distant tumor metastatic growth. This review will focus on the most recent updates in strategies to develop potent oncolytic Ad-based immunotherapeutics for use in cancer gene therapy.

Intact p53 function is essential for responsiveness to cancer therapy. However, p53 activity is attenuated by the proto-oncoprotein Mdm2, the adenovirus protein E1B 55kD, and the p53 C-terminal domain. To confer resistance to Mdm2, E1B 55kD, and C-terminal negative regulation, we generated a p53 variant (p53VPΔ30) by deleting the N-terminal and C-terminal regions of wild-type p53 and inserting the transcriptional activation domain of herpes simplex virus VP16 protein. The oncolytic adenovirus vector Ad-mΔ19 expressing p53VPΔ30 (Ad-mΔ19/p53VPΔ30) showed greater cytotoxicity than Ad-mΔ19 expressing wild-type p53 or other p53 variants in human cancer cell lines. We found that Ad-mΔ19/p53VPΔ30 induced apoptosis through accumulation of p53VPΔ30, regardless of endogenous p53 and Mdm2 status. Moreover, Ad-mΔ19/p53VPΔ30 showed a greater antitumor effect and increased survival rates of mice with U343 brain cancer xenografts that expressed wild-type p53 and high Mdm2 levels. To our knowledge, this is the first study reporting a p53 variant modified at the N terminus and C terminus that shows resistance to degradation by Mdm2 and E1B 55kD, as well as negative regulation by the p53 C terminus, without decreased trans-activation activity. Taken together, these results indicate that Ad-mΔ19/p53VPΔ30 shows potential for improving p53-mediated cancer gene therapy.

Oncolytic viruses (OVs) are used to treat cancer, as they selectively replicate inside of and lyse tumor cells. The efficacy of this process is limited and new OVs are being designed to mediate tumor cell release of cytokines and co-stimulatory molecules, which attract cytotoxic T cells to target tumor cells, thus increasing the tumor-killing effects of OVs. To further promote treatment efficacy, OVs can be combined with other treatments, such as was done by Huang et al., who showed that combining OV injections with dendritic cell (DC) injections was a more effective treatment than either treatment alone. To further investigate this combination, we built a mathematical model consisting of a system of ordinary differential equations and fit the model to the hierarchical data provided from Huang et al. We used the model to determine the effect of varying doses of OV and DC injections and to test alternative treatment strategies. We found that the DC dose given in Huang et al. was near a bifurcation point and that a slightly larger dose could cause complete eradication of the tumor. Further, the model results suggest that it is more effective to treat a tumor with immunostimulatory oncolytic viruses first and then follow-up with a sequence of DCs than to alternate OV and DC injections. This protocol, which was not considered in the experiments of Huang et al., allows the infection to initially thrive before the immune response is enhanced. Taken together, our work shows how the ordering, temporal spacing, and dosage of OV and DC can be chosen to maximize efficacy and to potentially eliminate tumors altogether.

We developed a three-dimensional organotypic multicellular spheroid scar model to mimic the microenvironment of human keloid tissues. Keloid tissues were cultured for 7 days. Changes in total cellularity and apoptotic index in the primary keloid spheroid cultures were evaluated histologically and with a TUNEL assay, respectively. The expression profiles of transforming growth factor-β (TGF-β), collagen I, collagen III, elastin, fibronectin, matrix metalloproteinase-2, and matrix metalloproteinase-9 were examined with immunohistochemistry. In addition, these expression profiles were investigated after treating primary keloid spheroids with triamcinolone acetonide. Cell viability and morphology of ex vivo cultured keloid spheroids were maintained, and the apoptotic index did not increase for up to 1 week in culture. Keloid spheroids cultivated ex vivo retained the major characteristics of keloids, such as high levels of collagen I and TGF-β expression for up to 7 days. The biological activity of keloids responding to TGF-β was also maintained during ex vivo culture. Moreover, ex vivo triamcinolone acetonide treatment of cultivated keloid spheroids significantly reduced collagen I, collagen III, elastin, and fibronectin expression levels, in accordance with clinical observations. The three-dimensional organotypic multicellular spheroid keloid culture will allow investigators to study keloid pathogenesis and test potential keloid therapeutic agents.

The p53 gene plays a determinant role in radiation-induced cell death and its protein product is negatively regulated by MDM2. We investigated whether adenovirus-mediated modified p53 gene transfer, which blocks p53-MDM2 binding, is effective for radiation-induced cell death in hepatocellular carcinoma (HCC) at different MDM2 cellular levels. Human hepatocellular carcinoma cell lines expressing MDM2 at low levels (Huh7) and high levels (SK-Hep1) were used. Ad-p53 and Ad-p53vp are replication-deficient adenoviral vectors containing human wild-type or modified p53, respectively. The anti-tumor effect was highest for Ad-p53 + radiotherapy (RT) in the low-level MDM2 cells, whereas this effect was highest for Ad-p53vp + RT in the MDM2-overexpressing cells. In Huh-7 cells, Ad-p53 + RT decreased cell viability (32%) in vitro and inhibited tumor growth (enhancement factor, 1.86) in vivo. Additionally, p21 expression and apoptosis were increased. In contrast, in SK-Hep1 cells, Ad-p53vp + RT showed decreased cell viability (51%) in vitro and inhibition of tumor growth (enhancement factor, 3.07) in vivo. Caspase-3 expression and apoptosis were also increased. Adenovirus-expressing modified p53, which blocks p53-MDM2 binding, was effective in killing tumor cells overexpressing MDM2. Furthermore, the combination strategy for disruption of the p53-MDM2 interaction with RT demonstrated enhanced anti-tumor effects both in vitro and in vivo.

Relaxin (RLX) has multiple vascular actions, including vasodilation and angiogenesis, which occur via induction of vascular endothelial growth factor (VEGF) expression. We generated a RLX-expressing (dE1-RGD/lacZ/RLX) adenovirus and investigated whether it enhances skin flap survival. Thirty Sprague-Dawley rats were divided into three groups: RLX-expressing adenovirus group, control virus group, and phosphate-buffered saline (PBS) group. Two days before surgery and immediately after flap elevation, the caudally based flap that was 3 × 9 cm in size was subdermally injected with the dE1-RGD/lacZ/RLX virus (10⁷ PFU), dE1-RGD/lacZ virus (10⁷ PFU), or PBS. The surviving area of the flap and the amount of blood flow were measured. On postoperative day 10, CD31-positive vessels and VEGF protein expression were examined. We observed a significant increase in the survival area of the flap in the RLX group. Doppler measurement also showed significantly increased blood flow immediately after the operation and on postoperative days 7 and 10. CD31-positive vessels and VEGF protein expression were significantly greater in the RLX group. Thus, administration of RLX-expressing adenovirus into elevated skin flaps increased VEGF expression, the number of capillaries, and blood flow to the flap, thereby improving skin flap survival.

Summary Background Keloids or hypertrophic scars are pathological proliferations of the dermal skin layer resulting from excessive collagen deposition. Because the hormone relaxin (RLX) inhibits collagen synthesis and expression in stimulated fibroblasts, an adenovirus expressing RLX (dE1-RGD/lacZ/RLX) was generated. Objectives To investigate the effect of RLX-expressing adenovirus on expression of various extracellular matrix (ECM) components in primary keloid spheroids. Methods The expression levels of type I and III collagen, fibronectin and elastin were investigated by immunohistochemistry in primary keloid spheroids transduced with the RLX-expressing adenovirus. Results Immunohistochemical analysis showed that expression of major ECM components (e.g. type I and III collagen, elastin and fibronectin) was markedly reduced in primary keloid spheroids transduced with dE1-RGD/lacZ/RLX. Conclusions These results suggest that the antifibrotic effect of RLX-expressing adenovirus may have therapeutic effects on keloids by reversing pathological fibrosis and preventing keloid recurrence after surgical excision.

Dendritic cell (DC)-based vaccination is a promising strategy for cancer immunotherapy. However, clinical trials have indicated that immunosuppressive microenvironments induced by tumors profoundly suppress antitumor immunity and inhibit vaccine efficacy, resulting in insufficient reduction of tumor burdens. To overcome these obstacles and enhance the efficiency of DC vaccination, we generated interleukin (IL)-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-coexpressing oncolytic adenovirus (Ad-ΔB7/IL12/GMCSF) as suitable therapeutic adjuvant to eliminate immune suppression and promote DC function. By treating tumors with Ad-ΔB7/IL12/GMCSF prior to DC vaccination, DCs elicited greater antitumor effects than in response to either treatment alone. DC migration to draining lymph nodes (DLNs) dramatically increased in mice treated with the combination therapy. This result was associated with upregulation of CC-chemokine ligand 21 (CCL21(+)) lymphatics in tumors treated with Ad-ΔB7/IL12/GMCSF. Moreover, the proportion of CD4(+)CD25(+) T-cells and vascular endothelial growth factor (VEGF) expression was decreased in mice treated with the combination therapy. Furthermore, combination therapy using immature DCs also showed effective antitumor effects when combined with Ad-ΔB7/IL12/GMCSF. The combination therapy had a remarkable therapeutic efficacy on large tumors. Taken together, oncolytic adenovirus coexpressing IL-12 and GM-CSF in combination with DC vaccination has synergistic antitumor effects and can act as a potent adjuvant for promoting and optimizing DC vaccination.

The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte–macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.

The pressing challenge for contemporary gene therapy is to deliver enough therapeutic genes to enough cancer cells in vivo. With the aim of improving viral distribution and tumor penetration, we explored the use of decorin to enhance viral spreading and tumor tissue penetration. We generated decorin-expressing replication-incompetent (dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK) and replication-competent (Ad-DeltaE1B-DCNG, Ad-DeltaE1B-DCNQ and Ad-DeltaE1B-DCNK) adenoviruses (Ads). Point mutants of decorin gene (DCNG), DCNK and DCNQ, have a negative and moderate binding affinity to type-I collagen fibril, respectively. In both tumor spheroids and established solid tumors in vivo, tissue penetration potency of dl-LacZ-DCNG was greatly enhanced than those of dl-LacZ, dl-LacZ-DCNQ and dl-LacZ-DCNK, and this enhanced tissue penetration effect derived from decorin-expressing Ad was dependent on the binding affinity of decorin to collagen fibril. Expression of DCNG enhanced viral spread of replicating Ad, leading to improved tumor reduction and survival benefit. Moreover, the tumoricidal effects of Ad-DeltaE1B-DCNQ and Ad-DeltaE1B-DCNK were lessened, as the binding affinity to collagen was decreased, showing that the increased cancer cell cytotoxicity was driven by the action of decorin on extracellular matrix (ECM). Furthermore, Ad-DeltaE1B-DCNG substantially decreased ECM components within the tumor tissue. Finally, intratumoral injection of Ad-DeltaE1B-DCNG in primary tumor site greatly reduced the formation of B16BL6 melanoma cell pulmonary metastases in mice. Taken together, these data show the utility of decorin as a dispersion agent and highlight its utility and potential in improving the efficacy of replicating Ad-mediated cancer gene therapy.

After myocardial infarction (MI), the heart undergoes a series of structural changes, termed post-infarct cardiac remodeling, at the organ, cellular, and molecular levels. During decades, diverse efforts in experimental and clinical trials have been made to investigate promising cardio-protective strategies. In our previous work, the aggressive rescue for the central fibrotic lesion in left ventricle (LV) after MI is emerging as one of key therapeutic targets to overcome limited functional and prognostic improvements.

Purpose: We investigated whether an armed viral platform, where lytic property of a viral infection is coupled to viral-mediated delivery of therapeutic genes, could increase the therapeutic potential of a viral-based therapy.Experimental Design: We generated interleukin (IL)-12-expressing oncolytic adenovirus (YKL-IL-12) and IL-12- and B7-1-expressing (YKL-IL12/B7) oncolytic adenovirus. Therapeutic efficacy of these newly engineered adenoviruses was then evaluated in vivo using an immunocompetent mouse bearing murine melanoma B16-F10 tumors. Overall survival was assessed with the Kaplan-Meier method. The induction of immune cell cytotoxicity was assessed by CTL assay, IFN-γ enzyme-linked immunospot assay, and immunohistochemical studies.Results: YKL-IL12/B7 oncolytic adenovirus, expressing both IL-12 and B7-1, showed a higher incidence of complete tumor regression compared with the analogous oncolytic adenovirus, YKL-1, or IL-12-expressing, YKL-IL12. Significant survival advantage was also seen in response to YKL-IL12/B7. Moreover, IL-12 and IFN-γ levels produced in tumors treated with YKL-IL12/B7 was significantly greater than those treated with YKL-IL12. The enhanced survival advantage was mediated by the induction of immune cell cytotoxicity. In agreement with these results, massive infiltration of CD4+ and CD8+ T cells into tissues surrounding the necrotic area of the tumor was observed following in situ delivery of YKL-IL12/B7.Conclusion: Combination of oncolysis and the enhancement of antitumor immune response by oncolytic adenovirus expressing both IL-12 and B7-1 elicits potent antitumor effect and survival advantage.

Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.

Background: Decorin is a natural transforming growth factor-b1 (TGF-b1) antagonist. Reduced decorin synthesis is associated with dermal scarring and increased decorin expression appears to reduce scar tissue formation. To investigate the therapeutic potential of decorin for keloids, human dermal fibroblasts (HDFs) and keloid-derived fibroblasts (KFs) were transduced with decorin-expressing adenovirus (dE1-RGD/GFP/DCN), and we examined the therapeutic potential of decorin-expressing Ad for treating pathologic skin fibrosisMethod: Decorin expression was examined by immunofluorescence assay on keloid tissues. HDFs and KFs were transduced with dE1-RGD/GFP/DCN or control virus, and protein levels of decorin, epidermal growth factor receptor (EGFR), and secreted TGF-β1 were assessed by western blotting and ELISA. And collagen type I, III, and MMP-1, 3 mRNA levels were measured by real-time RT-PCR. Additionally, we immunohistochemically investigated expression levels of the major extracellular matrix (ECM) proteins in keloid spheroids transduced with dE1-RGD/GFP/DCN.Results: Lower decorin expression was observed in the keloid region compared to adjacent normal tissues. After treatment with dE1-RGD/GFP/DCN, secreted TGF-βl and EGFR protein expression were decreased in TGF-b1-treated HDFs and KFs. Also, type I, III collagen mRNA levels were decreased and expression of MMP-1, 3 mRNA was strongly upregulated. In addition, expression of type I, III collagen, fibronectin, and elastin was significantly reduced in dE1-RGD/GFP/DCN-transduced keloid spheroids.Conclusion: These results support the utility of decorin-expressing adenovirus to reduce collagen synthesis in KFs and keloid spheroid, which may be highly beneficial in treating keloids.

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I–negative or –mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1–mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1–lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation. Cancer Res; 73(8); 2598–607. ©2012 AACR.

The local milieu of malignant tumor cells has key roles in cancer progression. A major component of the niche is the extracellular matrix (ECM), a complex interdigitating meshwork of macromolecules with multiple biophysical and biochemical characteristics. Although tightly controlled during normal tissue development and homeostasis, the ECM is mostly deregulated and becomes disorganized in cancer. Abnormal ECM has an impact on cancer progression by promoting tumor malignancy and metastatic dissemination. Importantly, the altered ECM in tumor is associated with deregulated ECM-regulating enzymes (matrix metalloproteinases, lysyl oxidase, urokinase plasminogen activator, and cysteine cathepsin). Excess expression of ECM-regulating enzymes alters behavior of cancer cells in the tumor niche, and its sustained upregulation results in the progressive breakdown of normal ECM which is replaced by tumor-derived ECM, thereby allowing tumor malignancy and cancer cell dissemination. Thus, ECM-regulating enzymes act as essential mediators of deregulating and disorganizing ECM. In this chapter, we will review and discuss how ECM-regulating enzymes generate disruption of ECM homeostasis and contribute to cancer progression, especially cancer metastasis.

The hormone relaxin has been shown to affect the extracellular matrix by inhibiting collagen synthesis and expression in fibroblasts stimulated with a profibrotic agent. It also increases matrix metalloproteinase (MMP) expression. To investigate its effect on expression of collagen and MMPs in keloid fibroblasts and human dermal fibroblasts, the authors introduced a relaxin-expressing adenovirus (dE1-RGD/lacZ/RLX) into a human dermal fibroblast cell line and keloid fibroblasts.Both fibroblasts were infected with dE1-RGD/lacZ/RLX or control virus, and protein levels of relaxin and secreted transforming growth factor (TGF)-β1 were assessed by enzyme-linked immunosorbent assay, and mRNA levels of collagen type I, collagen type III, MMP-1, and MMP-3 were assessed by real-time reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Expression of Smad3 and phosphorylated Smad3 was also examined, and relaxin's effect on Smad2/3 complex localization was evaluated.When human dermal fibroblasts and keloid fibroblasts were transduced with dE1-RGD/lacZ/RLX or dE1-RGD/lacZ (control), mRNA expression of type I and type III collagen was markedly decreased by relaxin regardless of TGF-β (10 ng/ml) treatment. Expression of Smad3 and phosphorylated Smad3 was reduced in keloid fibroblasts and decreased translocation of Smad 2/3 complex from cytosols to the nucleus of the human dermal fibroblasts with TGF-β after dE1-RGD/lacZ/RLX transduction, suggesting that relaxin reduces collagen synthesis by blocking TGF-β signaling. Analyses revealed that MMP-1 and MMP-3 expression were significantly increased in human dermal fibroblasts and keloid fibroblasts after dE1-RGD/lacZ/RLX transduction.These results suggest that the antifibrotic effect of relaxin-expressing adenovirus may have therapeutic effects on keloids.

We developed a three-dimensional organotypic multicellular spheroid scar model to mimic the microenvironment of human keloid tissues. Keloid tissues were cultured for 7 days. Changes in total cellularity and apoptotic index in the primary keloid spheroid cultures were evaluated histologically and with a TUNEL assay, respectively. The expression profiles of transforming growth factor-β (TGF-β), collagen I, collagen III, elastin, fibronectin, matrix metalloproteinase-2, and matrix metalloproteinase-9 were examined with immunohistochemistry. In addition, these expression profiles were investigated after treating primary keloid spheroids with triamcinolone acetonide. Cell viability and morphology of ex vivo cultured keloid spheroids were maintained, and the apoptotic index did not increase for up to 1 week in culture. Keloid spheroids cultivated ex vivo retained the major characteristics of keloids, such as high levels of collagen I and TGF-β expression for up to 7 days. The biological activity of keloids responding to TGF-β was also maintained during ex vivo culture. Moreover, ex vivo triamcinolone acetonide treatment of cultivated keloid spheroids significantly reduced collagen I, collagen III, elastin, and fibronectin expression levels, in accordance with clinical observations. The three-dimensional organotypic multicellular spheroid keloid culture will allow investigators to study keloid pathogenesis and test potential keloid therapeutic agents.

Interleukin (IL)-12 and granulocyte-monocyte colony-stimulating factor (GM-CSF) have recently been used as immunotherapeutic agents in cancer gene therapy. IL-12 and GM-CSF have differential roles in the antitumor immune response, as IL-12 targets T, NK and natural killer T (NKT) cells and GM-CSF principally targets antigen-presenting cells (APCs). To strengthen the therapeutic efficacy of these two cytokines, we generated an oncolytic adenovirus (Ad), Ad-ΔB7/IL12/GMCSF, coexpressing IL-12 and GM-CSF. Using a murine B16-F10 syngeneic tumor model, we show that Ad-ΔB7/IL12/GMCSF promoted antitumor responses and increased survival compared with an oncolytic Ad expressing IL-12 or GM-CSF alone (Ad-ΔB7/IL12 or Ad-ΔB7/GMCSF, respectively). By measuring cytotoxic T lymphocyte activity and interferon-γ production, we show that the enhanced therapeutic effect was mediated by the induction of immune cell cytotoxicity. In situ delivery of Ad-ΔB7/IL12/GMCSF resulted in massive infiltration of CD4(+) T cells, CD8(+) T cells, NK cells and CD86(+) APCs into the tissue surrounding the necrotic area of the tumor. Moreover, GM-CSF effectively promoted antitumor immune memory, which was significantly augmented by IL-12. Lastly, IL12-expressing oncolytic Ads prevented tumor-induced thymic atrophy and was associated with reduced apoptosis and increased proliferation in the thymus. Taken together, these data demonstrate that an oncolytic Ad coexpressing IL-12 and GM-CSF is a potential therapeutic tool for the treatment of cancer.

MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21(WAF1) (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3'UTR, and the Hu binding site of p21-3'UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21(WAF1) pathway.

Relaxin (RLX) has multiple vascular actions, including vasodilation and angiogenesis, which occur via induction of vascular endothelial growth factor (VEGF) expression. We generated a RLX-expressing (dE1-RGD/lacZ/RLX) adenovirus and investigated whether it enhances skin flap survival. Thirty Sprague-Dawley rats were divided into three groups: RLX-expressing adenovirus group, control virus group, and phosphate-buffered saline (PBS) group. Two days before surgery and immediately after flap elevation, the caudally based flap that was 3 × 9 cm in size was subdermally injected with the dE1-RGD/lacZ/RLX virus (10⁷ PFU), dE1-RGD/lacZ virus (10⁷ PFU), or PBS. The surviving area of the flap and the amount of blood flow were measured. On postoperative day 10, CD31-positive vessels and VEGF protein expression were examined. We observed a significant increase in the survival area of the flap in the RLX group. Doppler measurement also showed significantly increased blood flow immediately after the operation and on postoperative days 7 and 10. CD31-positive vessels and VEGF protein expression were significantly greater in the RLX group. Thus, administration of RLX-expressing adenovirus into elevated skin flaps increased VEGF expression, the number of capillaries, and blood flow to the flap, thereby improving skin flap survival.

Recently, gene-based cytokine treatment has been actively pursued as a new promising approach in treating cancer. In an effort to augment the efficiency of antitumor effect by cytokine-mediated immunotherapy, we selected both interleukin (IL)-12 and 4-1BB ligand (4-1BBL) as suitable cytokines to fully activate the type-1 immune response. Coexpression of IL-12 and 4-1BBL mediated by oncolytic adenovirus (Ad) greatly enhanced the antitumor effect. Further, synergistic enhancement in interferon (IFN)-gamma levels were seen in mice treated with oncolytic Ad expressing both IL-12 and 4-1BBL. Next, to improve the overall antitumor immune response, we coadministered IL-12- and 4-1BBL-coexpressing oncolytic Ad with dendritic cells (DCs). Combination treatment of IL-12- and 4-1BBL-coexpressing oncolytic Ad and DCs elicited greater antitumor and antimetastatic effects than either treatment alone. Moreover, enhanced type-1 antitumor immune response and higher migratory abilities of DCs in tumors were also observed in the combination arms. The nature of the enhanced antitumor immune response seems to be mediated through the enhanced cytolytic activity of cytotoxic T lymphocytes (CTLs) and IFN-gamma-releasing immune cells. Taken together, these data highlight the potential therapeutic benefit of combining IL-12- and 4-1BBL-coexpressing oncolytic Ad with DCs and warrants further evaluation in the clinic.

Aging is a complex process accompanied by a decreased capacity of cells to cope with random damages induced by reactive oxygen species, the natural by-products of energy metabolism, leading to protein aggregation in various components of the cell. Chaperones are important players in the aging process as they prevent protein misfolding and aggregation. Small chaperones, such as small heat shock proteins, are involved in the refolding and/or disposal of protein aggregates, a feature of many age-associated diseases. In Drosophila melanogaster, mitochondrial Hsp22 (DmHsp22), is localized in the mitochondrial matrix and is preferentially up-regulated during aging. Its overexpression results in an extension of life span (30%) (Morrow, G., Samson, M., Michaud, S., and Tanguay, R. M. (2004) FASEB J. 18, 598-599 and Morrow, G., Battistini, S., Zhang, P., and Tanguay, R. M. (2004) J. Biol. Chem. 279, 43382-43385). Long lived flies expressing Hsp22 also have an increased resistance to oxidative stress and maintain locomotor activity longer. In the present study, the cross-species effects of Hsp22 expression were tested. DmHsp22 was found to be functionally active in human cells. It extended the life span of normal fibroblasts, slowing the aging process as evidenced by a lower level of the senescence associated beta-galactosidase. DmHsp22 expression in human cancer cells increased their malignant properties including anchorage-independent growth, tumor formation in nude mice, and resistance to a variety of anticancer drugs. We report that the DmHsp22 interacts and inactivates wild type tumor suppressor protein p53, which may be one possible way of its functioning in human cells.

With the aim of improving viral distribution and tumor penetration, we have engineered decorin expressing replication-incompetent (dl-LacZ-DCNG) and -competent (Ad-[DELTA]E1B-DCNG) adenoviruses. In both tumor spheroids and established solid tumors in vivo, administration of dl-LacZ-DCNG resulted in greater transduction efficiency and viral spread throughout the tumor mass. Ad-[DELTA]E1B-DCNG also enhanced viral distribution and tumor spread, leading to an increased anti-tumor effect and survival advantage. Upon histological analysis, Ad-[DELTA]E1B-DCNG also elicited greater percentage of apoptotic cells and extensive necrosis compared to those from untreated or control virus-treated tumors. Furthermore, Ad-[DELTA]E1B-DCNG substantially decreased extracellular matrix components within the tumor tissue, while normal tissue adjacent to the tumor was not affected. Finally, intratumoral administration of Ad-[DELTA]E1B-DCNG did not enhance but inhibited the formation of pulmonary metastases of B16BL6 melanoma cells in mice. Taken together, these data demonstrate the utility of decorin as a dispersion agent and suggest its utility and potential in improving the efficacy of replicating adenovirus-mediated cancer gene therapy.

Interleukin (IL)-12 has been shown to be one of the most promising antitumor cytokines. The major antitumor activities of IL-12 rely on its ability to promote T helper type 1 (Th1) immune responses and CTL responses. However, immune suppression-dominated microenvironments in advanced tumors hinder the effect of IL-12 gene therapy for cancer. Most tumor cells produce large amounts of transforming growth factor-β (TGF-β), which plays crucial roles of tumor-induced immune suppression. The TGF-β inhibits proliferation of immune cells such as T, NK, and dendritic cells, resulting in suppression of antitumor immunity. Moreover, TGF-β is also known to regulate the maintenance and induction of regulatory T (Treg) cells. Thus, it has been hypothesized that inhibiting TGF-β might be an apt strategy for generating more effective cancer immunotherapy. Therefore, to enhance the efficiency of IL-12-mediated cancer immunotherapy, we generated oncolytic adenovirus co-expressing IL-12 and Decorin (hDCN) (RdB/IL12/hDCN) as suitable therapeutic adjuvant to overcome immune suppression by down-regulation of TGF-β through hDCN. Intratumoral administration of RdB/IL12/hDCN promoted enhanced antitumor effects compared with an oncolytic adenovirus expressing IL-12 or hDCN alone (RdB/IL12 or RdB/hDCN, respectively). We showed markedly elevated levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α and IFN-γ secreting cells in RdB/IL12/hDCN-treated tumors. Moreover, Treg in draining lymph nodes (DLNs) dramatically decreased in mice treated with RdB/IL12/hDCN, suggesting an association with the down-regulation of TGF-β in tumor treated with RdB/IL12/hDCN. Consistent with these data, tumor tissue injected with RdB/IL12/hDCN showed increased infiltration of CD4+ T and CD8+ T cells infiltration. Furthermore, hDCN enhanced viral distribution and tumor penetration, leading to improved virus-mediated cancer gene therapy by overcoming the extracellular matrix barrier within tumor masses. Together, these results provide new insight into the ability of hDCN to eliminate immune suppression and demonstrate that adenovirus co-expressing IL-12 and hDCN is a promising therapeutic tool for cancer treatment than adenovirus expressing IL-12 alone. Note: This abstract was not presented at the meeting. Citation Format: Eonju Oh, Il-Kyu Choi, June Kyu Hwang, Chae-Ok Yun. Overcoming TGF-β-related immunosuppression for cancer immunotherapy by oncolytic adenovirus co-expressing Interleukin-12 and decorin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 298. doi:10.1158/1538-7445.AM2015-298

We developed a three-dimensional organotypic multicellular spheroid scar model to mimic the microenvironment of human keloid tissues. Keloid tissues were cultured for 7 days. Changes in total cellularity and apoptotic index in the primary keloid spheroid cultures were evaluated histologically and with a TUNEL assay, respectively. The expression profiles of transforming growth factor-b (TGF-b), collagen I, collagen III, elastin, fibronectin, matrix metalloproteinase-2, and matrix metalloproteinase-9 were examined with immunohistochemistry. In addition, these expression profiles were investigated after treating primary keloid spheroids with triamcinolone acetonide. Cell viability and morphology of ex vivo cultured keloid spheroids were maintained, and the apoptotic index did not increase for up to 1 week in culture. Keloid spheroids cultivated ex vivo retained the major characteristics of keloids, such as high levels of collagen I and TGF-bexpression for up to 7 days. The biological activity of keloids responding to TGF-b was also maintained during ex vivo culture. Moreover, ex vivo triamcinolone acetonide treatment of cultivated keloidspheroidssignificantlyredu cedcollagenI, collagen III, elastin, andfibronectin expression levels, in accordance with clinical observations. The three-dimensional organotypic multicellular spheroid keloid culture will allow investigators to study keloid pathogenesis and test potential keloid therapeutic agents.