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1.  Mathematical modeling of tumor therapy with oncolytic viruses: Regimes with complete tumor elimination within the framework of deterministic models 
Biology Direct  2006;1:6.
Background
Oncolytic viruses that specifically target tumor cells are promising anti-cancer therapeutic agents. The interaction between an oncolytic virus and tumor cells is amenable to mathematical modeling using adaptations of techniques employed previously for modeling other types of virus-cell interaction.
Results
A complete parametric analysis of dynamic regimes of a conceptual model of anti-tumor virus therapy is presented. The role and limitations of mass-action kinetics are discussed. A functional response, which is a function of the ratio of uninfected to infected tumor cells, is proposed to describe the spread of the virus infection in the tumor. One of the main mathematical features of ratio-dependent models is that the origin is a complicated equilibrium point whose characteristics determine the main properties of the model. It is shown that, in a certain area of parameter values, the trajectories of the model form a family of homoclinics to the origin (so-called elliptic sector). Biologically, this means that both infected and uninfected tumor cells can be eliminated with time, and complete recovery is possible as a result of the virus therapy within the framework of deterministic models.
Conclusion
Our model, in contrast to the previously published models of oncolytic virus-tumor interaction, exhibits all possible outcomes of oncolytic virus infection, i.e., no effect on the tumor, stabilization or reduction of the tumor load, and complete elimination of the tumor. The parameter values that result in tumor elimination, which is, obviously, the desired outcome, are compatible with some of the available experimental data.
Reviewers
This article was reviewed by Mikhail Blagosklonny, David Krakauer, Erik Van Nimwegen, and Ned Wingreen.
doi:10.1186/1745-6150-1-6
PMCID: PMC1403749  PMID: 16542009
2.  Targeting of Interferon-Beta to Produce a Specific, Multi-Mechanistic Oncolytic Vaccinia Virus 
PLoS Medicine  2007;4(12):e353.
Background
Oncolytic viruses hold much promise for clinical treatment of many cancers, but a lack of systemic delivery and insufficient tumor cell killing have limited their usefulness. We have previously demonstrated that vaccinia virus strains are capable of systemic delivery to tumors in mouse models, but infection of normal tissues remains an issue. We hypothesized that interferon-beta (IFN-β) expression from an oncolytic vaccinia strain incapable of responding to this cytokine would have dual benefits as a cancer therapeutic: increased anticancer effects and enhanced virus inactivation in normal tissues. We report the construction and preclinical testing of this virus.
Methods and Findings
In vitro screening of viral strains by cytotoxicity and replication assay was coupled to cellular characterization by phospho-flow cytometry in order to select a novel oncolytic vaccinia virus. This virus was then examined in vivo in mouse models by non-invasive imaging techniques. A vaccinia B18R deletion mutant was selected as the backbone for IFN-β expression, because the B18R gene product neutralizes secreted type-I IFNs. The oncolytic B18R deletion mutant demonstrated IFN-dependent cancer selectivity and efficacy in vitro, and tumor targeting and efficacy in mouse models in vivo. Both tumor cells and tumor-associated vascular endothelial cells were targeted. Complete tumor responses in preclinical models were accompanied by immune-mediated protection against tumor rechallenge. Cancer selectivity was also demonstrated in primary human tumor explant tissues and adjacent normal tissues. The IFN-β gene was then cloned into the thymidine kinase (TK) region of this virus to create JX-795 (TK−/B18R−/IFN-β+). JX-795 had superior tumor selectivity and systemic intravenous efficacy when compared with the TK−/B18R− control or wild-type vaccinia in preclinical models.
Conclusions
By combining IFN-dependent cancer selectivity with IFN-β expression to optimize both anticancer effects and normal tissue antiviral effects, we were able to achieve, to our knowledge for the first time, tumor-specific replication, IFN-β gene expression, and efficacy following systemic delivery in preclinical models.
Stephen Thorne and colleagues describe, in a mouse model, an oncolytic vaccinia virus with interferon-dependent cancer selectivity that allows tumor-specific replication; it also expresses the IFN-β gene and hence has efficacy against tumors.
Editors' Summary
Background.
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. Cancers can develop anywhere in the body and, as they develop, their cells acquire other genetic changes that enable them to move and start new tumors (metastases) elsewhere. Chemotherapy drugs kill rapidly dividing cancer cells but, because some normal cells are also sensitive to these drugs, it is hard to destroy the cancer without causing serious side effects. Consequently, researchers are trying to develop “targeted” therapies that attack the changes in cancer cells that allow them to divide uncontrollably but leave normal cells unscathed. One promising class of targeted therapies is oncolytic viruses. These viruses make numerous copies of themselves inside cancer cells (but not inside normal cells). Eventually the cancer cell bursts open (lyses), releases more of the therapeutic agent, and dies.
Why Was This Study Done?
Existing oncolytic viruses have two major disadvantages: they have to be injected directly into tumors, and therefore they can't destroy distant metastases; and they don't kill cancer cells particularly efficiently. In this study, the researchers have tried to adapt vaccinia virus (a virus that infects humans and which has recently been shown to kill tumor cells when injected into the bloodstream) in two ways: to both infect cancer cells selectively and then to kill them effectively.
They hypothesized that putting a gene that causes expression of a protein called interferon-beta (IFN-β) in a particular virus strain that is itself incapable of responding to IFN-β might achieve these aims. Human cells infected with viruses usually release IFNs, which induce an antiviral state in nearby cells. But vaccinia virus makes anti-IFN proteins that prevent IFN release. If the viral genes that encode these proteins are removed from the virus, the virus cannot spread through normal cells. However, many cancer cells have defective IFN signaling pathways so the virus can spread through them. IFN-β expression by the virus, however, should improve its innate anticancer effects because IFN-β stops cancer cells dividing, induces an antitumor immune response, and stops tumors developing good blood supplies.
What Did the Researchers Do and Find?
The researchers selected a vaccinia virus strain called WR-delB18R in which the B18R gene, which encodes an anti-IFN protein, had been removed from the virus. (WR is a wild-type virus.) In laboratory experiments, IFN treatment blocked the spread of WR-delB18R in normal human cells but not in human tumor cells. After being injected into the veins of tumor-bearing mice, WR-delB18R was rapidly cleared from normal tissues but persisted in the tumors. A single injection of WR-delB18R directly into the tumor killed most of the tumor cells. A similar dose injected into a vein was less effective but nevertheless increased the survival time of some of the mice by directly killing the tumor cells, by targeting the blood supply of the tumors, and by inducing antitumor immunity. Finally, when the researchers inserted the IFN-β gene into this WR-delB18R, the new virus—JX-795—was much better at killing tumors after intravenous injection than either WR or WR-delB18R.
What Do These Findings Mean?
These findings indicate that the vaccinia virus can be adapted so that it replicates only in tumor cells and kills these cells effectively after intravenous injection. In particular, they show that the strategy adopted by the researchers both optimizes the anticancer effects of the virus and minimizes viral replication in normal tissues. JX-795 is a promising oncolytic virus, therefore, particularly since vaccinia virus has been safely used for many years to vaccinate people against smallpox. Nevertheless, it will be some years before JX-795 can be used clinically. Vaccinia virus constructs like this need to be tested extensively in the laboratory and in animals before any attempt is made to test them in people and, even if they passes all these preclinical tests with flying colors, only clinical trials will reveal whether they can treat human cancer. Several related strains of vaccinia virus are currently undergoing clinical testing.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/:10.1371/journal.pmed.0040353.
The US National Cancer Institute provides information on all aspects of cancer (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer (in several languages)
The UK charity Cancerbackup also provides information on all aspects of cancer
Wikipedia has a page on oncolytic viruses (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
A short interview about oncolytic viruses with researcher Dr. John Bell is available on the Insidermedicine Web site
The Oncolytic virus Web page provides lists of oncolytic viruses classified by type
doi:10.1371/journal.pmed.0040353
PMCID: PMC2222946  PMID: 18162040
3.  Towards Predictive Computational Models of Oncolytic Virus Therapy: Basis for Experimental Validation and Model Selection 
PLoS ONE  2009;4(1):e4271.
Oncolytic viruses are viruses that specifically infect cancer cells and kill them, while leaving healthy cells largely intact. Their ability to spread through the tumor makes them an attractive therapy approach. While promising results have been observed in clinical trials, solid success remains elusive since we lack understanding of the basic principles that govern the dynamical interactions between the virus and the cancer. In this respect, computational models can help experimental research at optimizing treatment regimes. Although preliminary mathematical work has been performed, this suffers from the fact that individual models are largely arbitrary and based on biologically uncertain assumptions. Here, we present a general framework to study the dynamics of oncolytic viruses that is independent of uncertain and arbitrary mathematical formulations. We find two categories of dynamics, depending on the assumptions about spatial constraints that govern that spread of the virus from cell to cell. If infected cells are mixed among uninfected cells, there exists a viral replication rate threshold beyond which tumor control is the only outcome. On the other hand, if infected cells are clustered together (e.g. in a solid tumor), then we observe more complicated dynamics in which the outcome of therapy might go either way, depending on the initial number of cells and viruses. We fit our models to previously published experimental data and discuss aspects of model validation, selection, and experimental design. This framework can be used as a basis for model selection and validation in the context of future, more detailed experimental studies. It can further serve as the basis for future, more complex models that take into account other clinically relevant factors such as immune responses.
doi:10.1371/journal.pone.0004271
PMCID: PMC2629569  PMID: 19180240
4.  ODE models for oncolytic virus dynamics 
Journal of theoretical biology  2010;263(4):530-543.
Replicating oncolytic viruses are able to infect and lyse cancer cells and spread through the tumor, while leaving normal cells largely unharmed. This makes them potentially useful in cancer therapy, and a variety of viruses have shown promising results in clinical trials. Nevertheless, consistent success remains elusive and the correlates of success have been the subject of investigation, both from an experimental and a mathematical point of view. Mathematical modeling of oncolytic virus therapy is often limited by the fact that the predicted dynamics depend strongly on particular mathematical terms in the model, the nature of which remain uncertain. We aim to address this issue in the context of ODE modeling, by formulating a general computational framework that is independent of particular mathematical expressions. By analyzing this framework, we find some new insights into the conditions for successful virus therapy. We find that depending on our assumptions about the virus spread, there can be two distinct types of dynamics. In models of the first type (the “fast spread” models), we predict that the viruses can eliminate the tumor if the viral replication rate is sufficiently high. The second type of models is characterized by a suboptimal spread (the “slow spread” models). For such models, the simulated treatment may fail, even for very high viral replication rates. Our methodology can be used to study the dynamics of many biological systems, and thus has implications beyond the study of virus therapy of cancers.
doi:10.1016/j.jtbi.2010.01.009
PMCID: PMC2839021  PMID: 20085772
5.  A Dynamical Systems Model for Combinatorial Cancer Therapy Enhances Oncolytic Adenovirus Efficacy by MEK-Inhibition 
PLoS Computational Biology  2011;7(2):e1001085.
Oncolytic adenoviruses, such as ONYX-015, have been tested in clinical trials for currently untreatable tumors, but have yet to demonstrate adequate therapeutic efficacy. The extent to which viruses infect targeted cells determines the efficacy of this approach but many tumors down-regulate the Coxsackievirus and Adenovirus Receptor (CAR), rendering them less susceptible to infection. Disrupting MAPK pathway signaling by pharmacological inhibition of MEK up-regulates CAR expression, offering possible enhanced adenovirus infection. MEK inhibition, however, interferes with adenovirus replication due to resulting G1-phase cell cycle arrest. Therefore, enhanced efficacy will depend on treatment protocols that productively balance these competing effects. Predictive understanding of how to attain and enhance therapeutic efficacy of combinatorial treatment is difficult since the effects of MEK inhibitors, in conjunction with adenovirus/cell interactions, are complex nonlinear dynamic processes. We investigated combinatorial treatment strategies using a mathematical model that predicts the impact of MEK inhibition on tumor cell proliferation, ONYX-015 infection, and oncolysis. Specifically, we fit a nonlinear differential equation system to dedicated experimental data and analyzed the resulting simulations for favorable treatment strategies. Simulations predicted enhanced combinatorial therapy when both treatments were applied simultaneously; we successfully validated these predictions in an ensuing explicit test study. Further analysis revealed that a CAR-independent mechanism may be responsible for amplified virus production and cell death. We conclude that integrated computational and experimental analysis of combinatorial therapy provides a useful means to identify treatment/infection protocols that yield clinically significant oncolysis. Enhanced oncolytic therapy has the potential to dramatically improve non-surgical cancer treatment, especially in locally advanced or metastatic cases where treatment options remain limited.
Author Summary
Novel cancer treatment strategies are urgently needed since currently available non-surgical methods for most solid malignancies have limited impact on survival rates. We used conditionally replicating adenoviruses as cancer-fighting agents since they are designed to target and lyse cells with specific aberrations, leaving healthy cells undamaged. Highly malignant cells, however, down-regulate the adenovirus receptor, impairing infection and subsequent cell death. We demonstrated that disruption of the MEK pathway (which is frequently activated in cancer) up-regulated this receptor, resulting in enhanced adenovirus entry. Although receptor expression was restored, disruption of signaling interfered with adenovirus replication due to cell cycle arrest, presenting an opposing trade-off. We developed a dynamical systems model to characterize the response of cancer cells to oncolytic adenovirus infection and drug treatment, providing a means to enhance therapeutic efficacy of combination treatment strategies. Our simulations predicted improved therapeutic efficacy when drug treatment and infection occurred simultaneously. We successfully validated predictions and found that a CAR-independent mechanism may be responsible for regulating adenovirus production and cell death. This work demonstrates the utility of modeling for accurate prediction and optimization of combinatorial treatment strategies, serving as a paradigm for improved design of anti-cancer combination therapies.
doi:10.1371/journal.pcbi.1001085
PMCID: PMC3040662  PMID: 21379332
6.  Complex Spatial Dynamics of Oncolytic Viruses In Vitro: Mathematical and Experimental Approaches 
PLoS Computational Biology  2012;8(6):e1002547.
Oncolytic viruses replicate selectively in tumor cells and can serve as targeted treatment agents. While promising results have been observed in clinical trials, consistent success of therapy remains elusive. The dynamics of virus spread through tumor cell populations has been studied both experimentally and computationally. However, a basic understanding of the principles underlying virus spread in spatially structured target cell populations has yet to be obtained. This paper studies such dynamics, using a newly constructed recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial cells, allowing us to track cell numbers and spatial patterns over time. The cells are arranged in a two-dimensional setting and allow virus spread to occur only to target cells within the local neighborhood. Despite the simplicity of the setup, complex dynamics are observed. Experiments gave rise to three spatial patterns that we call “hollow ring structure”, “filled ring structure”, and “disperse pattern”. An agent-based, stochastic computational model is used to simulate and interpret the experiments. The model can reproduce the experimentally observed patterns, and identifies key parameters that determine which pattern of virus growth arises. The model is further used to study the long-term outcome of the dynamics for the different growth patterns, and to investigate conditions under which the virus population eliminates the target cells. We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run. The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success. Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local neighborhoods in the agent-based model can predict the outcome of the spatial virus-cell dynamics, which has important practical implications. This analysis provides a first step towards understanding spatial oncolytic virus dynamics, upon which more detailed investigations and further complexity can be built.
Author Summary
Traditional chemotherapy of cancers is characterized by strong side effects, while showing a low success rate in the long term control of tumors. Besides small molecule inhibitors, which have shown great promise, oncolytic viruses present an emerging specific treatment approach. They are engineered viruses that spread from tumor cell to tumor cell, killing them in the process. Non-tumor cells are generally not infected. While clinical trials have given rise to promising results, reliable success remains elusive. Besides experiments, computational approaches provide a valuable tool to better understand the dynamics of virus spread through a growing population or tumor cells. Combining in vitro experimental approaches with computational models, we study the principles of virus spread through a spatially structured population of cells, which is of fundamental importance to understanding virus treatment of solid tumors. We describe different growth patterns that can occur, interpret them, and explore how they relate to the ability of the virus to induce tumor regression. We further define how these spatial dynamics relate to settings where cells and viruses mix more readily, such as in many cell culture experiments that are used to evaluate candidate viruses.
doi:10.1371/journal.pcbi.1002547
PMCID: PMC3375216  PMID: 22719239
7.  Intra-tumor Genetic Heterogeneity and Mortality in Head and Neck Cancer: Analysis of Data from The Cancer Genome Atlas 
PLoS Medicine  2015;12(2):e1001786.
Background
Although the involvement of intra-tumor genetic heterogeneity in tumor progression, treatment resistance, and metastasis is established, genetic heterogeneity is seldom examined in clinical trials or practice. Many studies of heterogeneity have had prespecified markers for tumor subpopulations, limiting their generalizability, or have involved massive efforts such as separate analysis of hundreds of individual cells, limiting their clinical use. We recently developed a general measure of intra-tumor genetic heterogeneity based on whole-exome sequencing (WES) of bulk tumor DNA, called mutant-allele tumor heterogeneity (MATH). Here, we examine data collected as part of a large, multi-institutional study to validate this measure and determine whether intra-tumor heterogeneity is itself related to mortality.
Methods and Findings
Clinical and WES data were obtained from The Cancer Genome Atlas in October 2013 for 305 patients with head and neck squamous cell carcinoma (HNSCC), from 14 institutions. Initial pathologic diagnoses were between 1992 and 2011 (median, 2008). Median time to death for 131 deceased patients was 14 mo; median follow-up of living patients was 22 mo. Tumor MATH values were calculated from WES results. Despite the multiple head and neck tumor subsites and the variety of treatments, we found in this retrospective analysis a substantial relation of high MATH values to decreased overall survival (Cox proportional hazards analysis: hazard ratio for high/low heterogeneity, 2.2; 95% CI 1.4 to 3.3). This relation of intra-tumor heterogeneity to survival was not due to intra-tumor heterogeneity’s associations with other clinical or molecular characteristics, including age, human papillomavirus status, tumor grade and TP53 mutation, and N classification. MATH improved prognostication over that provided by traditional clinical and molecular characteristics, maintained a significant relation to survival in multivariate analyses, and distinguished outcomes among patients having oral-cavity or laryngeal cancers even when standard disease staging was taken into account. Prospective studies, however, will be required before MATH can be used prognostically in clinical trials or practice. Such studies will need to examine homogeneously treated HNSCC at specific head and neck subsites, and determine the influence of cancer therapy on MATH values. Analysis of MATH and outcome in human-papillomavirus-positive oropharyngeal squamous cell carcinoma is particularly needed.
Conclusions
To our knowledge this study is the first to combine data from hundreds of patients, treated at multiple institutions, to document a relation between intra-tumor heterogeneity and overall survival in any type of cancer. We suggest applying the simply calculated MATH metric of heterogeneity to prospective studies of HNSCC and other tumor types.
In this study, Rocco and colleagues examine data collected as part of a large, multi-institutional study, to validate a measure of tumor heterogeneity called MATH and determine whether intra-tumor heterogeneity is itself related to mortality.
Editors’ Summary
Background
Normally, the cells in human tissues and organs only reproduce (a process called cell division) when new cells are needed for growth or to repair damaged tissues. But sometimes a cell somewhere in the body acquires a genetic change (mutation) that disrupts the control of cell division and allows the cell to grow continuously. As the mutated cell grows and divides, it accumulates additional mutations that allow it to grow even faster and eventually from a lump, or tumor (cancer). Other mutations subsequently allow the tumor to spread around the body (metastasize) and destroy healthy tissues. Tumors can arise anywhere in the body—there are more than 200 different types of cancer—and about one in three people will develop some form of cancer during their lifetime. Many cancers can now be successfully treated, however, and people often survive for years after a diagnosis of cancer before, eventually, dying from another disease.
Why Was This Study Done?
The gradual acquisition of mutations by tumor cells leads to the formation of subpopulations of cells, each carrying a different set of mutations. This “intra-tumor heterogeneity” can produce tumor subclones that grow particularly quickly, that metastasize aggressively, or that are resistant to cancer treatments. Consequently, researchers have hypothesized that high intra-tumor heterogeneity leads to worse clinical outcomes and have suggested that a simple measure of this heterogeneity would be a useful addition to the cancer staging system currently used by clinicians for predicting the likely outcome (prognosis) of patients with cancer. Here, the researchers investigate whether a measure of intra-tumor heterogeneity called “mutant-allele tumor heterogeneity” (MATH) is related to mortality (death) among patients with head and neck squamous cell carcinoma (HNSCC)—cancers that begin in the cells that line the moist surfaces inside the head and neck, such as cancers of the mouth and the larynx (voice box). MATH is based on whole-exome sequencing (WES) of tumor and matched normal DNA. WES uses powerful DNA-sequencing systems to determine the variations of all the coding regions (exons) of the known genes in the human genome (genetic blueprint).
What Did the Researchers Do and Find?
The researchers obtained clinical and WES data for 305 patients who were treated in 14 institutions, primarily in the US, after diagnosis of HNSCC from The Cancer Genome Atlas, a catalog established by the US National Institutes of Health to map the key genomic changes in major types and subtypes of cancer. They calculated tumor MATH values for the patients from their WES results and retrospectively analyzed whether there was an association between the MATH values and patient survival. Despite the patients having tumors at various subsites and being given different treatments, every 10% increase in MATH value corresponded to an 8.8% increased risk (hazard) of death. Using a previously defined MATH-value cutoff to distinguish high- from low-heterogeneity tumors, compared to patients with low-heterogeneity tumors, patients with high-heterogeneity tumors were more than twice as likely to die (a hazard ratio of 2.2). Other statistical analyses indicated that MATH provided improved prognostic information compared to that provided by established clinical and molecular characteristics and human papillomavirus (HPV) status (HPV-positive HNSCC at some subsites has a better prognosis than HPV-negative HNSCC). In particular, MATH provided prognostic information beyond that provided by standard disease staging among patients with mouth or laryngeal cancers.
What Do These Findings Mean?
By using data from more than 300 patients treated at multiple institutions, these findings validate the use of MATH as a measure of intra-tumor heterogeneity in HNSCC. Moreover, they provide one of the first large-scale demonstrations that intra-tumor heterogeneity is clinically important in the prognosis of any type of cancer. Before the MATH metric can be used in clinical trials or in clinical practice as a prognostic tool, its ability to predict outcomes needs to be tested in prospective studies that examine the relation between MATH and the outcomes of patients with identically treated HNSCC at specific head and neck subsites, that evaluate the use of MATH for prognostication in other tumor types, and that determine the influence of cancer treatments on MATH values. Nevertheless, these findings suggest that MATH should be considered as a biomarker for survival in HNSCC and other tumor types, and raise the possibility that clinicians could use MATH values to decide on the best treatment for individual patients and to choose patients for inclusion in clinical trials.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001786.
The US National Cancer Institute (NCI) provides information about cancer and how it develops and about head and neck cancer (in English and Spanish)
Cancer Research UK, a not-for-profit organization, provides general information about cancer and how it develops, and detailed information about head and neck cancer; the Merseyside Regional Head and Neck Cancer Centre provides patient stories about HNSCC
Wikipedia provides information about tumor heterogeneity, and about whole-exome sequencing (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information about The Cancer Genome Atlas is available
A PLOS Blog entry by Jessica Wapner explains more about MATH
doi:10.1371/journal.pmed.1001786
PMCID: PMC4323109  PMID: 25668320
8.  Dual E1A Oncolytic Adenovirus: Targeting Tumor Heterogeneity With Two Independent Cancer-specific Promoter Elements, DF3/MUC1 and hTERT 
Cancer gene therapy  2010;18(3):153-166.
The therapeutic utility of oncolytic adenoviruses controlled by a single, tumor-specific regulatory element may be limited by the intra- and inter-tumoral heterogeneity that characterizes many cancers. To address this issue, we constructed an oncolytic adenovirus that uses two distinct tumor-specific promoters (DF3/Muc1 and hTERT) to drive separate E1A expression cassettes, in combination with deletion of the viral E1B region, which confers additional tumor selectivity and increased oncolytic activity. The resultant virus, Adeno-DF3-E1A/hTERT-E1A, induced higher levels of E1A oncoprotein, enhanced oncolysis, and an earlier and higher apoptotic index in infected tumor cells than following infection with Adeno-hTERT-E1A, which harbors a single hTERT promoter-driven E1A cassette. In isolated U251 human gliosarcoma cell holoclones (putative cancer stem cells), where DF3/Muc1 expression is substantially enriched and hTERT expression is decreased compared to the parental U251 cell population, E1A production and oncolysis were strongly decreased following infection with Adeno-hTERT-E1A but not Adeno-DF3-E1A/hTERT-E1A. The strong oncolytic activity of Adeno-DF3-E1A/hTERT-E1A translated into superior anti-tumor activity over Adeno-hTERT-E1A in vivo in a U251 solid tumor xenograft model, where hTERT levels were >90% suppressed and the DF3/Muc1 to hTERT expression ratio was substantially increased compared to cultured U251 cells. The enhanced anti-tumor activity of the dual-targeted Adeno-DF3-E1A/hTERT-E1A was achieved despite premature viral host cell death and decreased production of functional viral progeny, which limited tumor cell spread of the viral infection. These findings highlight the therapeutic benefit of targeting oncolytic viruses to heterogeneous tumor cell populations.
doi:10.1038/cgt.2010.52
PMCID: PMC3010505  PMID: 20865021
Cancer gene therapy; replication-conditional; oncolytic adenovirus; tumor heterogeneity; holoclone; cytochrome P450 prodrug activation
9.  Choindroitinase ABC I-Mediated Enhancement of Oncolytic Virus Spread and Anti Tumor Efficacy: A Mathematical Model 
PLoS ONE  2014;9(7):e102499.
Oncolytic viruses are genetically engineered viruses that are designed to kill cancer cells while doing minimal damage to normal healthy tissue. After being injected into a tumor, they infect cancer cells, multiply inside them, and when a cancer cell is killed they move on to spread and infect other cancer cells. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove a major glioma ECM component, chondroitin sulfate glycosoamino glycans from proteoglycans without any deleterious effects in vivo. It has been shown that Chase-ABC treatment is able to promote the spread of the viruses, increasing the efficacy of the viral treatment. In this paper we develop a mathematical model to investigate the effect of the Chase-ABC on the treatment of glioma by oncolytic viruses (OV). We show that the model's predictions agree with experimental results for a spherical glioma. We then use the model to test various treatment options in the heterogeneous microenvironment of the brain. The model predicts that separate injections of OV, one into the center of the tumor and another outside the tumor will result in better outcome than if the total injection is outside the tumor. In particular, the injection of the ECM-degrading enzyme (Chase-ABC) on the periphery of the main tumor core need to be administered in an optimal strategy in order to infect and eradicate the infiltrating glioma cells outside the tumor core in addition to proliferative cells in the bulk of tumor core. The model also predicts that the size of tumor satellites and distance between the primary tumor and multifocal/satellite lesions may be an important factor for the efficacy of the viral therapy with Chase treatment.
doi:10.1371/journal.pone.0102499
PMCID: PMC4105445  PMID: 25047810
10.  The Utility of a Tissue Slice Model System to Determine Breast Cancer Infectivity by Oncolytic Adenoviruses 
The Journal of surgical research  2010;163(2):270-275.
Background
Due to advances in viral design, oncolytic adenoviruses have emerged as a promising approach for treatment of breast cancer. Tumor tissue slices offer a stringent model system for preclinical evaluation of adenovirus therapies, since the slices retain a morphology and phenotype that more closely resembles the in vivo setting than cell line cultures, and it has been shown to have utility in the evaluation of viral infectivity and replication. In this study, we evaluated the efficacy of viral infection and replication using a tropism-modified oncolytic adenovirus.
Methods
Breast tumor tissue slices were infected with a tropism-modified oncolytic adenovirus, and a wild-type adenovirus for comparison. Efficiency of infection was evaluated using fluorescent microscopy, as the viruses used have been modified to express red fluorescent protein. Replication of the viruses was evaluated with quantitative real-time PCR to assay viral E4 genome copy number, a surrogate indicator for the number of virions. The breast tumor tissue slices were evaluated for the expression of CD46 expression by immunohistochemistry.
Results
Infection and replication of our tropism modified oncolytic virus has been observed in breast cancer tissue slice model system and is comparative to wild-type virus. A qualitative increase in the number of cells showing RFP expression was observed correlating with increasing multiplicity of infection. Higher relative infectivity of the virus was observed in tumor tissue compared with normal breast tissue. Replication of the virus was demonstrated through increases in E4 copy number at 48 and 72 hours after infection in human breast tumor slices.
Conclusions
We have shown that a tropism modified oncolytic oncolytic adenovirus can infect and replicate in breast cancer tissue slices, which may be an important preclinical indicator for its therapeutic utility.
doi:10.1016/j.jss.2010.03.072
PMCID: PMC3015137  PMID: 20691986
adenovirus; breast cancer; CD46; CAR; oncolytic; tissue slice model
11.  Ex Vivo Infection of Live Tissue with Oncolytic Viruses 
Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.
doi:10.3791/2854
PMCID: PMC3197059  PMID: 21730946
12.  Switching a Replication-Defective Adenoviral Vector into a Replication-Competent, Oncolytic Adenovirus 
Journal of Virology  2014;88(1):345-353.
The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be “switched” into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad into fully oncolytic RC Ad for tumor therapy and is potentially applicable to a variety of gene therapy approaches.
doi:10.1128/JVI.02668-13
PMCID: PMC3911754  PMID: 24155386
13.  Oncolytic Virotherapy for Ovarian Carcinomatosis Using a Replication-Selective Vaccinia Virus Armed with a Yeast Cytosine Deaminase Gene* 
Cancer gene therapy  2007;15(2):115-125.
In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine (5-FC) conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy (GDEPT) is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian cancer.
doi:10.1038/sj.cgt.7701110
PMCID: PMC2975702  PMID: 18084242
poxvirus; ovarian cancer; peritoneal carcinomatosis; suicide gene; cytosine deaminase
14.  Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody 
PLoS ONE  2012;7(10):e47472.
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.
doi:10.1371/journal.pone.0047472
PMCID: PMC3473019  PMID: 23091626
15.  Immune Suppression during Oncolytic Virotherapy for High-Grade Glioma; Yes or No? 
Journal of Cancer  2015;6(3):203-217.
Oncolytic viruses have been seriously considered for glioma therapy over the last 20 years. The oncolytic activity of several oncolytic strains has been demonstrated against human glioma cell lines and in in vivo xenotransplant models. So far, four of these stains have additionally completed the first phase I/II trials in relapsed glioma patients. Though safety and feasibility have been demonstrated, therapeutic efficacy in these initial trials, when described, was only minor. The role of the immune system in oncolytic virotherapy for glioma remained much less studied until recent years. When investigated, the immune system, adept at controlling viral infections, is often hypothesized to be a strong hurdle to successful oncolytic virotherapy. Several preclinical studies have therefore aimed to improve oncolytic virotherapy efficacy by combining it with immune suppression or evasion strategies. More recently however, a new paradigm has developed in the oncolytic virotherapy field stating that oncolytic virus-mediated tumor cell death can be accompanied by elicitation of potent activation of innate and adaptive anti-tumor immunity that greatly improves the efficacy of certain oncolytic strains. Therefore, it seems the three-way interaction between oncolytic virus, tumor and immune system is critical to the outcome of antitumor therapy. In this review we discuss the studies which have investigated how the immune system and oncolytic viruses interact in models of glioma. The novel insights generated here hold important implications for future research and should be incorporated into the design of novel clinical trials.
doi:10.7150/jca.10640
PMCID: PMC4317755  PMID: 25663937
glioblastoma; oncolytic virotherapy; antitumor immunity
16.  Mathematical Modeling of Herpes Simplex Virus Distribution in Solid Tumors: Implications for Cancer Gene Therapy 
Purpose
Although oncolytic viral vectors show promise for the treatment of various cancers, ineffective initial distribution and propagation throughout the tumor mass often limit the therapeutic response. A mathematical model is developed to describe the spread of herpes simplex virus from the initial injection site.
Experimental Design
The tumor is modeled as a sphere of radius R. The model incorporates reversible binding, interstitial diffusion, viral degradation, and internalization and physiologic parameters. Three species are considered as follows: free interstitial virus, virus bound to cell surfaces, and internalized virus.
Results
This analysis reveals that both rapid binding and internalization as well as hindered diffusion contain the virus to the initial injection volume, with negligible spread to the surrounding tissue. Unfortunately, increasing the dose to saturate receptors and promote diffusion throughout the tumor is not a viable option: the concentration necessary would likely compromise safety. However, targeted modifications to the virus that decrease the binding affinity have the potential to increase the number of infected cells by 1.5-fold or more. An increase in the effective diffusion coefficient can result in similar gains.
Conclusions
This analysis suggests criteria by which the potential response of a tumor to oncolytic herpes simplex virus therapy can be assessed. Furthermore, it reveals the potential of modifications to the vector delivery method, physicochemical properties of the virus, and tumor extracellular matrix composition to enhance efficacy.
doi:10.1158/1078-0432.CCR-08-2082
PMCID: PMC2872130  PMID: 19318482
17.  Directed Evolution Generates a Novel Oncolytic Virus for the Treatment of Colon Cancer 
PLoS ONE  2008;3(6):e2409.
Background
Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term “Directed Evolution” for creating highly potent oncolytic viruses.
Methodology/Principal Findings
Taking the “Directed Evolution” approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2–3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1's efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent.
Conclusions/Significance
Using the “Directed Evolution” methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies.
doi:10.1371/journal.pone.0002409
PMCID: PMC2423470  PMID: 18560559
18.  Explicit targeting of transformed cells by VSV in ovarian epithelial tumor-bearing Wv mouse models 
Gynecologic oncology  2009;116(2):269.
Objective
Current treatment options for epithelial ovarian cancer are limited and therapeutic development for recurrent and drug-resistant ovarian cancer is an urgent agenda. We investigated the potential use of genetically engineered Vesicular Stomatitis Virus (VSV) to treat ovarian cancer patients who fail to respond to available therapies. Specifically, we examined the toxicity to hosts and specificity of targeting ovarian tumors using a Wv ovarian tumor model.
Methods
We first tested recombinant VSV for oncolytic activity in a panel of human ovarian epithelial cancer, immortalized, and primary ovarian surface epithelial cells in culture. Then, we test VSV oncolytic therapy using the immune competent Wv mice that develop tubular adenomas, benign tumor lesions derived from ovarian surface epithelial cells.
Results
The expression of GFP encoded by the recombinant VSV genome was detected in about 5% of primary ovarian surface epithelial cells (3 lines) up to 30 days without significantly altering the growth pattern of the cells, suggesting the lack of toxicity to the normal ovarian surface epithelial cells. However, VSV-GFP was detected in the majority (around 90%) of cells that are either “immortalized” by SV40 antigen expression or cancer lines. Some variation in killing time courses was observed, but all the transformed cell lines were killed within 3 days.
We found that regardless of the inoculation route (intra bursal, IP, or IV), VSV specifically infected and replicated in the in situ ovarian tumors in the Wv mice without significant activity in any other organs and tissues, and showed no detectable toxicity. The epithelial tumor lesions were greatly reduced in VSV-targeted ovarian tumors in the Wv mice.
Conclusions
VSV oncolytic activity depends on a cell autonomous property distinguishing primary and transformed cells. The efficient oncolytic activity of VSV for the “immortalized” non-tumorigenic ovarian surface epithelial cells suggests that the selective specificity extends from pre-neoplastic to overt cancer cells. The results demonstrated the explicit targeting of ovarian epithelial tumors by VSV in immune com petent, ovarian tumor-bearing mouse models, and further support the utility of VSV as an effective and safe anti-cancer agent.
doi:10.1016/j.ygyno.2009.10.086
PMCID: PMC2813895  PMID: 19932656
19.  Oncolytic Myxoma Virus: The path to clinic 
Vaccine  2013;31(39):4252-4258.
Many common neoplasms are still noncurative with current standards of cancer therapy. More therapeutic modalities need to be developed to significantly prolong the lives of patients and eventually cure a wider spectrum of cancers. Oncolytic virotherapy is one of the promising new additions to clinical cancer therapeutics. Successful oncolytic virotherapy in the clinic will be those strategies that best combine tumor cell oncolysis with enhanced immune responses against tumor antigens. The current candidate oncolytic viruses all share the common property that they are relatively nonpathogenic to humans, yet they have the ability to replicate selectively in human cancer cells and induce cancer regression by direct oncolysis and/or induction of improved anti-tumor immune responses. Many candidate oncolytic viruses are in various stages of clinical and preclinical development. One such preclinical candidate is myxoma virus (MYXV), a member of the Poxviridae family that, in its natural setting, exhibits a very restricted host range and is only pathogenic to European rabbits. Despite its narrow host range in nature, MYXV has been shown to productively infect various classes of human cancer cells. Several preclinical in vivo modeling studies have demonstrated that MYXV is an attractive and safe candidate oncolytic virus, and hence, MYXV is currently being developed as a potential therapeutic for several cancers, such as pancreatic cancer, glioblastoma, ovarian cancer, melanoma, and hematologic malignancies. This review highlights the preclinical cancer models that have shown the most promise for translation of MYXV into human clinical trials.
doi:10.1016/j.vaccine.2013.05.056
PMCID: PMC3755036  PMID: 23726825
Myxoma Virus; Oncolytic Virotherapy; Hematological Malignancies; Pancreatic Cancer
20.  Armed and targeted measles virus for chemovirotherapy of pancreatic cancer 
Cancer gene therapy  2011;18(8):598-608.
No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high- and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.
doi:10.1038/cgt.2011.30
PMCID: PMC3914720  PMID: 21701532
oncolysis; measles virus; prodrug-converting enzyme; single-chain antibody; pancreatic cancer
21.  Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by 19F-Magnetic Resonance Imaging (MRI) 
PLoS ONE  2013;8(2):e56317.
Background
Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate 19F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.
Methodology/Principal Findings
The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by 1H/19F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the 19F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the 19F signal hot spots and CD68+-macrophages. Thereby, the CD68+-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the 19F signal correlated with the extent of viral spreading within tumors.
Conclusions/Significance
These results suggest 19F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, 19F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.
doi:10.1371/journal.pone.0056317
PMCID: PMC3575337  PMID: 23441176
22.  Cancer Screening: A Mathematical Model Relating Secreted Blood Biomarker Levels to Tumor Sizes  
PLoS Medicine  2008;5(8):e170.
Background
Increasing efforts and financial resources are being invested in early cancer detection research. Blood assays detecting tumor biomarkers promise noninvasive and financially reasonable screening for early cancer with high potential of positive impact on patients' survival and quality of life. For novel tumor biomarkers, the actual tumor detection limits are usually unknown and there have been no studies exploring the tumor burden detection limits of blood tumor biomarkers using mathematical models. Therefore, the purpose of this study was to develop a mathematical model relating blood biomarker levels to tumor burden.
Methods and Findings
Using a linear one-compartment model, the steady state between tumor biomarker secretion into and removal out of the intravascular space was calculated. Two conditions were assumed: (1) the compartment (plasma) is well-mixed and kinetically homogenous; (2) the tumor biomarker consists of a protein that is secreted by tumor cells into the extracellular fluid compartment, and a certain percentage of the secreted protein enters the intravascular space at a continuous rate. The model was applied to two pathophysiologic conditions: tumor biomarker is secreted (1) exclusively by the tumor cells or (2) by both tumor cells and healthy normal cells. To test the model, a sensitivity analysis was performed assuming variable conditions of the model parameters. The model parameters were primed on the basis of literature data for two established and well-studied tumor biomarkers (CA125 and prostate-specific antigen [PSA]). Assuming biomarker secretion by tumor cells only and 10% of the secreted tumor biomarker reaching the plasma, the calculated minimally detectable tumor sizes ranged between 0.11 mm3 and 3,610.14 mm3 for CA125 and between 0.21 mm3 and 131.51 mm3 for PSA. When biomarker secretion by healthy cells and tumor cells was assumed, the calculated tumor sizes leading to positive test results ranged between 116.7 mm3 and 1.52 × 106 mm3 for CA125 and between 27 mm3 and 3.45 × 105 mm3 for PSA. One of the limitations of the study is the absence of quantitative data available in the literature on the secreted tumor biomarker amount per cancer cell in intact whole body animal tumor models or in cancer patients. Additionally, the fraction of secreted tumor biomarkers actually reaching the plasma is unknown. Therefore, we used data from published cell culture experiments to estimate tumor cell biomarker secretion rates and assumed a wide range of secretion rates to account for their potential changes due to field effects of the tumor environment.
Conclusions
This study introduced a linear one-compartment mathematical model that allows estimation of minimal detectable tumor sizes based on blood tumor biomarker assays. Assuming physiological data on CA125 and PSA from the literature, the model predicted detection limits of tumors that were in qualitative agreement with the actual clinical performance of both biomarkers. The model may be helpful in future estimation of minimal detectable tumor sizes for novel proteomic biomarker assays if sufficient physiologic data for the biomarker are available. The model may address the potential and limitations of tumor biomarkers, help prioritize biomarkers, and guide investments into early cancer detection research efforts.
Sanjiv Gambhir and colleagues describe a linear one-compartment mathematical model that allows estimation of minimal detectable tumor sizes based on blood tumor biomarker assays.
Editors' Summary
Background.
Cancers—disorganized masses of cells that can occur in any tissue—develop when cells acquire genetic changes that allow them to grow uncontrollably and to spread around the body (metastasize). If a cancer (tumor) is detected when it is small, surgery can often provide a cure. Unfortunately, many cancers (particularly those deep inside the body) are not detected until they are large enough to cause pain or other symptoms by pressing against surrounding tissue. By this time, it may be impossible to remove the original tumor surgically and there may be metastases scattered around the body. In such cases, radiotherapy and chemotherapy can sometimes help, but the outlook for patients whose cancers are detected late is often poor. Consequently, researchers are trying to develop early detection tests for different types of cancer. Many tumors release specific proteins—“cancer biomarkers”—into the blood and the hope is that it might be possible to find sets of blood biomarkers that detect cancers when they are still small and thus save many lives.
Why Was This Study Done?
For most biomarkers, it is not known how the amount of protein detected in the blood relates to tumor size or how sensitive the assays for biomarkers must be to improve patient survival. In this study, the researchers develop a “linear one-compartment” mathematical model to predict how large tumors need to be before blood biomarkers can be used to detect them and test this model using published data on two established cancer biomarkers—CA125 and prostate-specific antigen (PSA). CA125 is used to monitor the progress of patients with ovarian cancer after treatment; ovarian cancer is rarely diagnosed in its early stages and only one-fourth of women with advanced disease survive for 5 y after diagnosis. PSA is used to screen for prostate cancer and has increased the detection of this cancer in its early stages when it is curable.
What Did the Researchers Do and Find?
To develop a model that relates secreted blood biomarker levels to tumor sizes, the researchers assumed that biomarkers mix evenly throughout the patient's blood, that cancer cells secrete biomarkers into the fluid that surrounds them, that 0.1%–20% of these secreted proteins enter the blood at a continuous rate, and that biomarkers are continuously removed from the blood. The researchers then used their model to calculate the smallest tumor sizes that might be detectable with these biomarkers by feeding in existing data on CA125 and on PSA, including assay detection limits and the biomarker secretion rates of cancer cells growing in dishes. When only tumor cells secreted the biomarker and 10% of the secreted biomarker reach the blood, the model predicted that ovarian tumors between 0.11 mm3 (smaller than a grain of salt) and nearly 4,000 mm3 (about the size of a cherry) would be detectable by measuring CA125 blood levels (the range was determined by varying the amount of biomarker secreted by the tumor cells and the assay sensitivity); for prostate cancer, the detectable tumor sizes ranged from similar lower size to about 130 mm3 (pea-sized). However, healthy cells often also secrete small quantities of cancer biomarkers. With this condition incorporated into the model, the estimated detectable tumor sizes (or total tumor burden including metastases) ranged between grape-sized and melon-sized for ovarian cancers and between pea-sized to about grapefruit-sized for prostate cancers.
What Do These Findings Mean?
The accuracy of the calculated tumor sizes provided by the researchers' mathematical model is limited by the lack of data on how tumors behave in the human body and by the many assumptions incorporated into the model. Nevertheless, the model predicts detection limits for ovarian and prostate cancer that broadly mirror the clinical performance of both biomarkers. Somewhat worryingly, the model also indicates that a tumor may have to be very large for blood biomarkers to reveal its presence, a result that could limit the clinical usefulness of biomarkers, especially if they are secreted not only by tumor cells but also by healthy cells. Given this finding, as more information about how biomarkers behave in the human body becomes available, this model (and more complex versions of it) should help researchers decide which biomarkers are likely to improve early cancer detection and patient outcomes.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050170.
The US National Cancer Institute provides a brief description of what cancer is and how it develops and a fact sheet on tumor markers; it also provides information on all aspects of ovarian and prostate cancer for patients and professionals, including information on screening and testing (in English and Spanish)
The UK charity Cancerbackup also provides general information about cancer and more specific information about ovarian and prostate cancer, including the use of CA125 and PSA for screening and follow-up
The American Society of Clinical Oncology offers a wide range of information on various cancer types, including online published articles on the current status of cancer diagnosis and management from the educational book developed by the annual meeting faculty and presenters. Registration is mandatory, but information is free
doi:10.1371/journal.pmed.0050170
PMCID: PMC2517618  PMID: 18715113
23.  Permissiveness of Human Cancer Cells to Oncolytic Bovine Herpesvirus 1 Is Mediated in Part by KRAS Activity 
Journal of Virology  2014;88(12):6885-6895.
ABSTRACT
Oncolytic viruses (OVs) are attractive avenues of cancer therapy due to the absence of toxic side effects often seen with current treatment modalities. Bovine herpesvirus 1 (BHV-1) is a species-specific virus that does not induce cytotoxicity in normal primary human cells but can infect and kill various human immortalized and transformed cell lines. To gain a better understanding of the oncolytic breadth of BHV-1, the NCI panel of established human tumor cell lines was screened for sensitivity to the virus. Overall, 72% of the panel is permissive to BHV-1 infection, with corresponding decreases in cellular viability. This sensitivity is in comparison to a sensitivity of only 32% for a herpes simplex virus 1 (HSV-1)-based oncolytic vector. Strikingly, while 35% of the panel supports minimal or no BHV-1 replication, significant decreases in cellular viability still occur. These data suggest that BHV-1 is an OV with tropism for multiple tumor types and is able to induce cytotoxicity independent of significant virus replication. In contrast to other species-specific OVs, cellular sensitivity to BHV-1 does not correlate with type I interferon (IFN) signaling; however, mutations in KRAS were found to correlate with high levels of virus replication. The knockdown or overexpression of KRAS in human tumor cell lines yields modest changes in viral titers; however, overexpression of KRAS in normal primary cells elicits permissivity to BHV-1 infection. Together, these data suggest that BHV-1 is a broad-spectrum OV with a distinct mechanism of tumor targeting.
IMPORTANCE Cancer remains a significant health issue, and novel treatments are required, particularly for tumors that are refractory to conventional therapies. Oncolytic viruses are a novel platform given their ability to specifically target tumor cells while leaving healthy cells intact. For this strategy to be successful, a fundamental understanding of virus-host interactions is required. We previously identified bovine herpesvirus 1 as a novel oncolytic virus with many unique and clinically relevant features. Here, we show that BHV-1 can target a wide range of human cancer types, most potently lung cancer. In addition, we show that enhanced KRAS activity, a hallmark of many cancers, is one of the factors that increases BHV-1 oncolytic capacity. These findings hold potential for future treatments, particularly in the context of lung cancer, where KRAS mutations are a negative predictor of treatment efficacy.
doi:10.1128/JVI.00849-14
PMCID: PMC4054371  PMID: 24696490
24.  Oncolytic Measles Virus Encoding Thyroidal Sodium Iodide Symporter for Squamous Cell Cancer of the Head and Neck Radiovirotherapy 
Human Gene Therapy  2011;23(3):295-301.
Abstract
Oncolytic measles virus (MV) encoding the human thyroidal sodium iodide symporter (MV-NIS) has proved to be safe after intraperitoneal or intravenous administration in patients with ovarian cancer or multiple myeloma, respectively, but it has not yet been administered through intratumoral injection in humans. Squamous cell carcinoma (SCC) of the head and neck (SCCHN) usually is locally invasive and spreads to the cervical lymph nodes, which are suitable for the intratumoral administration of oncolytic viruses. To test whether oncolytic MV is an effective treatment for SCCHN, we used oncolytic MV-NIS to infect SCCHN in vitro and in vivo. The data show that SCCHN cells were infected and killed by MV-NIS in vitro. Permissiveness of the tumor cells to MV infection was not affected by irradiation after viral addition. Monitored noninvasively through radioiodine-based single-photon emission computed tomography/computed tomography, intratumorally virus-delivered NIS has concentrated the radioiodine in the MV-NIS–treated tumors in the FaDu mouse xenograft model of human SCCHN, and the antitumor effect could be boosted significantly (p<0.05) either with concomitant cyclophosphamide therapy or with appropriately timed administration of radioiodine 131I. MV-NIS could be a promising new anticancer agent that may substantially enhance the outcomes of standard therapy after intratumoral administration in patients with locally advanced SCCHN.
Li and colleagues investigate the use of oncolytic measles virus encoding human thyroidal sodium iodide symporter (MV-NIS) to treat squamous cell carcinoma of the head and neck (SCCHN) in vitro and in vivo. MV-NIS-treated tumors are able to concentrate administered radioiodine in a mouse xenograft model of human SCCHN, and the antitumor effect is significantly boosted by cyclophosphamide therapy.
doi:10.1089/hum.2011.128
PMCID: PMC3300082  PMID: 22235810
25.  Modeling Evolutionary Dynamics of Epigenetic Mutations in Hierarchically Organized Tumors 
PLoS Computational Biology  2011;7(5):e1001132.
The cancer stem cell (CSC) concept is a highly debated topic in cancer research. While experimental evidence in favor of the cancer stem cell theory is apparently abundant, the results are often criticized as being difficult to interpret. An important reason for this is that most experimental data that support this model rely on transplantation studies. In this study we use a novel cellular Potts model to elucidate the dynamics of established malignancies that are driven by a small subset of CSCs. Our results demonstrate that epigenetic mutations that occur during mitosis display highly altered dynamics in CSC-driven malignancies compared to a classical, non-hierarchical model of growth. In particular, the heterogeneity observed in CSC-driven tumors is considerably higher. We speculate that this feature could be used in combination with epigenetic (methylation) sequencing studies of human malignancies to prove or refute the CSC hypothesis in established tumors without the need for transplantation. Moreover our tumor growth simulations indicate that CSC-driven tumors display evolutionary features that can be considered beneficial during tumor progression. Besides an increased heterogeneity they also exhibit properties that allow the escape of clones from local fitness peaks. This leads to more aggressive phenotypes in the long run and makes the neoplasm more adaptable to stringent selective forces such as cancer treatment. Indeed when therapy is applied the clone landscape of the regrown tumor is more aggressive with respect to the primary tumor, whereas the classical model demonstrated similar patterns before and after therapy. Understanding these often counter-intuitive fundamental properties of (non-)hierarchically organized malignancies is a crucial step in validating the CSC concept as well as providing insight into the therapeutical consequences of this model.
Author Summary
Cancer is in essence a genetic disease that leads to uncontrolled cell proliferation, invasion and metastasis. The cancer stem cell (CSC) hypothesis states that tumors are not just a mass of uniform malignant cells but they are hierarchically organized, like normal tissues. At the top of such a hierarchy are cancer stem cells that fuel tumor growth in the long run, whereas the majority of other cells are able to divide only a few times. The experiments that support the CSC hypothesis are often criticized as being difficult to interpret. A novel approach to test the CSC paradigm is to integrate mathematical modeling with DNA variation data that carry the phylogenetic history of cells. We have developed a model that simulates the occurrence of such changes under both the CSC hypothesis and the classical, purely stochastic scenario. We found that although a CSC-driven tumor has a smaller number of tumorigenic cells, it triggers more malignant properties such as invasive growth, heterogeneity and evolutionary escape from peaks in the fitness landscape. These properties, that are unique to the CSC model, are enhanced even further when a treatment is applied to the tumor.
doi:10.1371/journal.pcbi.1001132
PMCID: PMC3088646  PMID: 21573198

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