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Familial risks of lung cancer are well-established, but whether lung cancer clusters with other discordant cancers is less certain, particularly beyond smoking-related sites, which may provide evidence on genetic contributions to lung cancer aetiology. We used a novel approach to search for familial associations in the Swedish Family-Cancer Database. This involved assessment of familial relative risk for cancer X in families with increasing numbers of lung cancer patients and, conversely, relative risks for lung cancer in families with increasing numbers of patients with cancers X. However, we lacked information on smoking. The total number of lung cancers in the database was 125 563. We applied stringent statistical criteria and found that seven discordant cancers were associated with lung cancer among family members, and six of these were known to be connected with smoking: oesophageal, upper aerodigestive tract, liver, cervical, kidney and urinary bladder cancers. A further novel finding was that cancer of unknown primary also associated with lung cancer. We also factored in histological evidence and found that anal and connective tissue cancers could be associated with lung cancer for reasons other than smoking. For endometrial and prostate cancers, suggestive negative associations with lung cancer were found. Although we lacked information on smoking it is prudent to conclude that practically all observed discordant associations of lung cancer were with cancers for which smoking is a risk factor.

Vaccination with dendritic cells (DCs), the most potent professional antigen-presenting cells in the body, is a promising approach in cancer immunotherapy. However, tumors induce immunosuppression in their microenvironment that suppresses and impairs the function of DCs. Therefore, human clinical trials with DC therapy have often been disappointing. To improve the therapeutic efficacy and to overcome the major obstacles of DC therapy, we generated a novel adenovirus, Ad3-hTERT-CMV-hCD40L, which is fully serotype 3 and expresses hCD40L for induction of antitumor immune response. The specific aim is to enhance DCs function. Data from a human cancer patient indicated that this capsid allows effective transduction of distant tumors through the intravenous route. Moreover, patient data suggested that virally produced hCD40L can activate DCs in situ. The virus was efficient in vitro and had potent antitumor activity in vivo. In a syngeneic model, tumors treated with Ad5/3-CMV-mCD40L virus plus DCs elicited greater antitumor effect as compared with either treatment alone. Moreover, virally coded CD40L induced activation of DCs, which in turn, lead to the induction of a Th1 immune response and increased tumor-specific T cells. In conclusion, Ad3-hTERT-CMV-hCD40L is promising for translation into human trials. In particular, this virus could enable successful dendritic cell therapy in cancer patients.

The purpose of this work was to carry out preclinical toxicity and bio-distribution studies required for regulatory approval of a clinical trial application for Phase I clinical studies of ONCOS-102 (Ad5/3-D24-GM-CSF) for therapy of advanced cancers (NCT01598129). The study design, route of administration and dosage differs from the clinical protocol and in more detail, investigate bio-distribution and toxicological profile of ONCOS-102 treatment in animal model. The study was carried out in 300 hamsters divided into nine test groups-three bio-distribution groups and six groups for analysis of toxicity. Hamsters received ONCOS-102 by intracardial, intraperitoneal or subcutaneous injections. Additionally, one group was administered twice a week with intraperitoneal injections of Cyclophosphamide. The control animals were administered with NaCl solution without ONCOS-102 in the same volume and the same way. No adverse effects of repeated administration of ONCOS-102 including body weight, food consumption, hematology and clinical chemistry parameters, histopathology and bio-accumulation were observed in the course of 6-month administration and following 3- month recovery period. All obtained findings indicate the treatment clinically safe.

In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer.

When I heard about the concept of immunotherapeutics, I immediately loved it. Everything I had learned about medicine, cancer biology, genetics and oncology indicated to me that this was a potent approach, and at the time, completely untapped. I figured that since we had been unable to cure most metastatic solid tumors, something completely different needed to be employed. Realistically, “magic bullets” are not easy to find and therefore something that can be combined with other therapies, for enhanced synergy without overlapping adverse events, would be appealing.

Cancer gene therapy consists of numerous approaches where the common denominator is utilization of vectors for achieving therapeutic effect. A particularly potent embodiment of the approach is virotherapy, in which the replication potential of an oncolytic virus is directed towards tumor cells to cause lysis, while normal cells are spared. Importantly, the therapeutic effect of the initial viral load is amplified through viral replication cycles and production of progeny virions. All cancer gene therapy approaches rely on a sufficient level of delivery of the anticancer agent into target cells. Thus,enhancement of delivery to target cells, and reduction of delivery to non-target cells, in an approach called transductional targeting, is attractive. Both genetic and non-genetic retargeting strategies have been utilized. However, in the context of oncolytic viruses, it is beneficial to have the specific modification included in progeny virions and hence genetic modification may be preferable. Serotype chimerism utilizes serotype specific differences in receptor usage, liver tropism and seroprevalence in order to gain enhanced infection of target tissue. This review will focus on serotype chimeric adenoviruses for cancer gene therapy applications.

Unfavorable ratios between the number and activation status of effector and suppressor immune cells infiltrating the tumor contribute to resistance of solid tumors to T-cell based therapies. Here, we studied the capacity of FDA and EMA approved recombinant cytokines to manipulate this balance in favor of efficient anti-tumor responses in B16.OVA melanoma bearing C57BL/6 mice. Intratumoral administration of IFN-α2, IFN-γ, TNF-α, and IL-2 significantly enhanced the anti-tumor effect of ovalbumin-specific CD8+ T-cell (OT-I) therapy, whereas GM-CSF increased tumor growth in association with an increase in immunosuppressive cell populations. None of the cytokines augmented tumor trafficking of OT-I cells significantly, but injections of IFN-α2, IFN-γ and IL-2 increased intratumoral cytokine secretion and recruitment of endogenous immune cells capable of stimulating T-cells, such as natural killer and maturated CD11c+ antigen-presenting cells. Moreover, IFN-α2 and IL-2 increased the levels of activated tumor-infiltrating CD8+ T-cells concomitant with reduction in the CD8+ T-cell expression of anergy markers CTLA-4 and PD-1. In conclusion, intratumoral administration of IFN-α2, IFN-γ and IL-2 can lead to immune sensitization of the established tumor, whereas GM-CSF may contribute to tumor-associated immunosuppression. The results described here provide rationale for including local administration of immunostimulatory cytokines into T-cell therapy regimens. One appealing embodiment of this would be vectored delivery which could be advantageous over direct injection of recombinant molecules with regard to efficacy, cost, persistence and convenience.

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.

Successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. Oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. However, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. In this article, we have explored if alternating the therapy virus could circumvent these problems. We demonstrate in two virus-resistant animal models a substantial delay in antiviral immune- and innate cellular response induction by alternating injections of two immunologically distinct oncolytic viruses, adenovirus, and vaccinia virus. Our results are in support of clinical development of heterologous adeno-/vaccinia virus therapy of cancer.

We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification.

Oncolytic adenoviruses (Onc.Ads) produce significant antitumor effects but as single agents they rarely eliminate tumors. Investigators have therefore incorporated sequences into these vectors that encode immunomodulatory molecules to enhance antitumor immunity. Successful implementation of this strategy requires multiple tumor immune inhibitory mechanisms to be overcome, and insertion of the corresponding multiple functional genes reduces the titer and replication of Onc.Ads, compromising their direct ant-tumor effects. By contrast, helper-dependent (HD) Ads are devoid of viral coding sequences, allowing inclusion of multiple transgenes. HDAds, however, lack replicative capacity. Since HDAds encode the adenoviral packaging signal, we hypothesized that the coadministration of Onc.Ad with HDAd would allow to be amplified and packaged during replication of Onc.Ad in transduced cancer cells. This combination could provide immunostimulation without losing oncolytic activity. We now show that coinfection of Onc.Ad with HDAd subsequently replicates HDAd vector DNA in trans in human cancer cell lines in vitro and in vivo, amplifying the transgenes the HDAd encode. This combinatorial treatment significantly suppresses the tumor growth compared to treatment with a single agent in an immunocompetent mouse model. Hence, combinatorial treatment of Onc.Ad with HDAd should overcome the inherent limitations of each agent and provide a highly immunogenic oncolytic therapy.

Following a century of preclinical and clinical work, oncolytic viruses are now proving themselves in randomized phase 3 trials. Interestingly, human data indicates that these agents have potent immunostimulatory activity, raising the possibility that the key consequence of oncolysis might be induction of antitumor immunity, especially in the context of viruses harboring immunostimulatory transgenes. While safety and efficacy of many types of oncolytic viruses, including adenovirus, herpes, reo, and vaccinia seem promising, few mechanisms of action studies have been performed with human substrates. Thus, the relative contribution of “pure” oncolysis, the immune response resulting from oncolysis, and the added benefit of adding a transgene remain poorly understood. Here, the available clinical data on oncolytic viruses is reviewed, with emphasis on immunological aspects.

Natural tropism to the liver is a major obstacle in systemic delivery of adenoviruses in cancer gene therapy. Adenovirus binding to soluble coagulation factors and to cellular heparan sulphate proteoglycans via the fiber shaft KKTK domain are suggested to cause liver tropism. Serotype 5 adenovirus constructs with mutated KKTK regions exhibit liver detargeting, but they also transduce tumors less efficiently, possibly due to altered fiber conformation. We constructed Ad5/3lucS*, a 5/3 chimeric adenovirus with a mutated KKTK region. The fiber knob swap was hypothesized to facilitate tumor transduction. This construct was studied with or without additional coagulation factor ablation. Ad5/3lucS* exhibited significantly reduced transduction of human hepatic cells in vitro and mouse livers in vivo. Combination of coagulation factor ablation by warfarinization to Ad5/3lucS* seemed to further enhance liver detargeting. Cancer cell transduction by Ad5/3lucS* was retained in vitro. In vivo, viral particle accumulation in M4A4-LM3 xenograft tumors was comparable to controls, but Ad5/3lucS* transgene expression was nearly abolished. Coagulation factor ablation did not affect tumor transduction. These studies set the stage for further investigations into the effects of the KKTK mutation and coagulation factor ablation in the context of 5/3 serotype chimerism. Of note, the putative disconnect between tumor transduction and transgene expression could prove useful in further understanding of adenovirus biology.

The down-regulation of miR-199 occurs in nearly all primary hepatocellular carcinomas (HCCs) and HCC cell lines in comparison with normal liver. We exploited this miR-199 differential expression to develop a conditionally replication-competent oncolytic adenovirus, Ad-199T, and achieve tumor-specific viral expression and replication. To this aim, we introduced four copies of miR-199 target sites within the 3' UTR of E1A gene, essential for viral replication. As consequence, E1A expression from Ad-199T virus was tightly regulated both at RNA and protein levels in HCC derived cell lines, and replication controlled by the level of miR-199 expression. Various approaches were used to asses in vivo properties of Ad-199T. Ad-199T replication was inhibited in normal, miR-199 positive, liver parenchyma, thus resulting in reduced hepatotoxicity. Conversely, the intrahepatic delivery of Ad-199T in newborn mice led to virus replication and fast removal of implanted HepG2 liver cancer cells. The ability of Ad-199T to control tumor growth was also shown in a subcutaneous xenograft model in nude mice and in HCCs arising in immune-competent mice. In summary, we developed a novel oncolytic adenovirus, Ad-199T, which could demonstrate a therapeutic potential against liver cancer without causing significant hepatotoxicity.

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

Oncolytic adenoviruses can be engineered for better tumor selectivity, gene delivery and be armed for imaging and concentrating radionuclides into tumors for synergistic oncolysis. We constructed Ad5/3-hTERT-hNIS where replication is controlled by hTERT-promoter. Ad5/3-hTERT-hNIS expresses hNIS for imaging of transgene expression and for treatment of infected tumors by radioiodine. Ad5/3-hTERT-hNIS efficiently killed prostate cancer cells and induced iodine uptake in vitro and in vivo after intratumoral virus administration. Survival of mice treated with intravenous Ad5/3-hTERT-hNIS significantly prolonged survival over mock or radioiodine only but the combination of virus with radioiodine was not more effective than virus alone. Temporal and spatial changes in hNIS-expression during therapy were detected with SPECT, demonstrating feasibility of evaluation of the combination therapy with hNIS-expressing adenoviruses and radioiodide.

In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherin(low)/cytoplasmic E-cadherin(high)/CD133(high), CD44(high), Tie2(low)) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.

Development of new cancer treatments focuses increasingly on the relation of cancer tissue with its microenvironment. A major obstacle for the development of new anti-cancer therapies has been the lack of relevant animal models that would reproduce all the events involved in disease progression from the early-stage primary tumor until the development of mature metastatic tissue. To this end, we have developed a readily imageable mouse model of colorectal cancer featuring highly reproducible formation of spontaneous liver metastases derived from intrasplenic primary tumors. We optimized several experimental variables, and found that the correct choice of cell line and the genetic background, as well as the age of the recipient mice, were critical for establishing a useful model system. Among a panel of colorectal cancer cell lines tested, the epithelial carcinoma HT29 line was found to be the most suitable in terms of producing homogeneous tumor growth and metastases. In our hands, SCID mice at the age of 125 days or older were the most suitable in supporting consistent HT29 tumor growth after splenic implantation followed by reproducible metastasis to the liver. A magnetic resonance imaging (MRI) protocol was optimized for use with this mouse model, and demonstrated to be a powerful method for analyzing the antitumor effects of an experimental therapy. Specifically, we used this system to with success to verify by MRI monitoring the efficacy of an intrasplenically administered oncolytic adenovirus therapy in reducing visceral tumor load and development of liver metastases. In summary, we have developed a highly optimized mouse model for liver metastasis of colorectal cancer, which allows detection of the tumor load at the whole body level and enables an accurate timing of therapeutic interventions to target different stages of cancer progression and metastatic development.

BackgroundCancer stem cells/initiating cells (CSC/CIC), are thought to exist as a small population in malignant tissues. They are resistant to conventional cancer treatments and possibly underlie post-treatment relapse. The CIC population can be targeted with capsid modified oncolytic adenoviruses.Methodology/principal findingsWe studied the mechanisms of innate immunity to oncolytic adenovirus Ad5/3-Delta24 in conventional treatment resistant non-CIC breast cancer cells, breast cancer CD44(+)/CD24(-/low) CIC population and normal breast tissue CD44(+)/CD24(-/low) stem cells. We compared virus recognition by pattern recognition receptors for adenovirus, Toll-like receptors (TLR) 2 and 9 and virus induced type I interferon (IFN) response regulation in these cell types. We show TLR mediated virus recognition in these non-immune cell types. Normal tissue stem cells have intact type I IFN signaling. Furthermore, TLR9 and TLR2 reside constantly in recognition sites, implying constant activation. In contrast, breast cancer CD44(+)/CD24(-/low) CIC have dysregulated innate immune responses featuring dysfunctional virus recognition caused by impaired trafficking of TLR9 and cofactor MyD88 and the absence of TLR2, having a deleterious impact on TLR pattern recognition receptor signaling. Furthermore, the CIC have increased inhibitory signaling via the suppressor of cytokine signaling/Tyro3/Axl/Mer receptor tyrosine kinase (SOCS/TAM) pathway. These defects in contribute to dysfunctional induction of type I IFN response in CIC and therefore permissivity to oncolytic adenovirus.Conclusions/significanceCICs may underlie the incurable nature of relapsed or metastatic cancers and are therefore an important target regarding diagnostic and prognostic aspects as well as treatment of the disease. This study addresses the mechanisms of innate infection immunity in stem cells deepening the understanding of stem cell biology and may benefit not only virotherapy but also immunotherapy in general.

Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline.

Clinical trials have confirmed the safety of selectively oncolytic adenoviruses for treatment of advanced cancers. However, increasingly effective viruses could result in more toxicity and therefore it would be useful if replication could be abrogated if necessary. We analyzed viruses containing the cyclooxygenase-2 (Cox-2) or vascular endothelial growth factor (VEGF) promoter for controlling replication. Anti-inflammatory agents can lower Cox-2 protein levels and therefore we hypothesized that also the promoter might be affected. As Cox-2 modulates expression of VEGF, also the VEGF promoter might be controllable. First, we evaluated the effect of anti-inflammatory agents on promoter activity or adenovirus infectivity in vitro. Further, we analyzed the oncolytic potency of the viruses in vitro and in vivo with and without the reagents. Moreover, the effect of on virus replication was analyzed. We found that RGD-4C or Ad5/3 modified fibers improved the oncolytic potency of the viruses in vitro and in vivo. We found that both promoters could be downregulated with dexamethasone, sodium salicylate, or salicylic acid. Oncolytic efficacy correlated with the promoter activity and in vitro virus production could be abrogated with the substances. In vivo, we saw good therapeutic efficacy of the viruses in a model of intravenous therapy of metastatic cervical cancer, but the inhibitory effect of dexamethasone was not strong enough to provide significant differences in a complex in vivo environment. Our results suggest that anti-inflammatory drugs may affect the replication of adenovirus, which might be relevant in case of replication associated side effects.