Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a “theragnostic” agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world.
Citation: Browne AW, Leddon JL, Currier MA, Williams JP, Frischer JS, Collins MH, et al. (2011) Cancer Screening by Systemic Administration of a Gene Delivery Vector Encoding Tumor-Selective Secretable Biomarker Expression. PLoS ONE 6(5): e19530. https://doi.org/10.1371/journal.pone.0019530
Editor: Syed A. Aziz, Health Canada, Canada
Received: March 1, 2011; Accepted: March 31, 2011; Published: May 11, 2011
Copyright: © 2011 Browne et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the CancerFree Kids Pediatric Cancer Research Alliance (AWB) and NIH award 1R01-CA114001-01A2 (TPC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Early cancer detection is vital to improve cure rates because cancer stage predicts prognosis. Cancer-associated blood biomarkers have been identified in a few cancers such as prostate specific antigen (PSA) in prostate cancer and alpha fetoprotein in some liver and germline cancers. Biomarkers have not been identified for most pediatric cancers and many adult cancers. New innovations in systemic gene transfer raise the prospect of selectively delivering and activating genes encoding easily detectable biomarkers into tumor cells that do not produce known serum biomarkers. We sought to develop a prototypical cancer screening strategy whereby genetic information encoding a universal serum biomarker for cancer would be injected into a patient systemically, delivered to and expressed within tumor cells in a tumor-selective manner. Tumors would effectively be forced to secrete a serum biomarker, which could then be measured in the blood or urine as a screening test while tumor-free patients would show no or only low levels of biomarker following systemic administration of the gene delivery vector (Fig. 1a).
(a) Steps for cancer screening strategy are as follows: 1) Cancer-targeting Herpes Simplex Virus (HSV) is injected systemically. 2) Engineered HSV selectively replicates in tumors while being cleared from healthy non-cancerous tissues. 3) Biomarker is selectively produced in tumors. 4) Blood samples are collected and analyzed for biomarker levels. 5) Serum levels of exogenously delivered biomarker are higher in tumor bearing mice than healthy tumor-free mice. (b) Gene maps for wild-type HSV and novel recombinant rQ-M38G.
Exogenously administered gene-encoded biomarkers require tumor-targeted gene delivery and/or expression. Targeting small molecules and particles to tumors can be achieved by passive Enhanced Permeability and Retention (EPR) , , , ligand-guided active targeting , , , , tumor microenvironment-dependent targeting , , , or a combination of each . Viruses are naturally occurring nanoparticles optimized to deliver genetic information into target cells. The major advantage of viruses over non-viral vectors for DNA delivery is their inherent predilection for replication in tumors and consequent amplification of signal.
The Herpes Simplex Virus (HSV) type 1 is a model gene delivery vector because it can infect a wide range of human cell types, transduce both dividing and quiescent cells efficiently, be engineered to express transgene products, accept transgenes driven by heterologous or homologous promoters, is epigenomic, has scalable production, and can safely be controlled with antiviral drugs . Selective mutations in HSV genes confer cancer-selective viral replication. Mutation in the HSV genes encoding the ribonucleotide reductase large subunit (ICP6/UL39) and the late viral protein ICP34.5 (γ134.5) limits robust viral replication to tumors , , ,  and have been shown to be safe in clinical trials. Activation of the strict late viral UL38 gene is dependent upon preceding sequential activation of immediate early and early viral genes and cellular transcription factors  and the UL38 promoter (UL38p) has been demonstrated to be selectively activated in cancer cells in the context of replication competent γ134.5−/− HSV mutants . The dependence of late gene expression upon activation by early genes makes UL38p a strong candidate for delivery of transgenes to cancer cells with selective expression in the context of a double mutant HSV lacking ICP6 and γ134.5.
We developed an HSV as an exemplar gene delivery vector for inducing biomarker secretion selectively from tumors. Others have incorporated genes encoding secretable biomarkers to oncolytic viruses as reporters for virus activity , , ,  and gene cassettes for non-invasive monitoring viral delivered genes . Sodium-iodide symporter genes encoded in oncolytic viruses also facilitate nuclear medicine imaging and treatment in infected tumors , . Recently, Gaussia luciferase (GLuc) was identified and developed as a powerful new reporter molecule  that is readily secreted from cells making it useful for both in vitro and in vivo applications where expression kinetics are of interest. We employed GLuc as a sample biomarker for this proof of principle because GLuc is 1000 times brighter than other luciferases, is more sensitive than secretable alkaline phosphatase, and is detectable in blood and urine in vivo , . Using a directed recombination approach , we engineered an HSV mutant, rQ-M38G, with GLuc under the control of the late viral promoter UL38 (Fig. 1b).
We sought to evaluate our engineered viral gene delivery vector in animal models for several different tumor types formed different locations: intraperitoneal, subcutaneous, intramuscular and orthotopic intrarenal tumors. Following systemic injection of our gene delivery vector (rQ-M38G) into the blood of tumor-bearing mice, we observed rQ-M38G producing cancer cell-dependent cytotoxicity and biomarker production. We also noted several instances where rQ-M38G was able to force microscopic tumor burdens to produce detectable blood biomarker. These experimental trials demonstrated the principle of detecting tumors by forcing them to express a secretable biomarker.
In vitro characterization of mutant HSV rQ-M38G
rQ-M38G-mediated GLuc transduction, replication and cytotoxicity were tested by infecting a range of cell types with various virus concentrations and assessing GLuc levels in culture media (Fig. 2a, Fig. S1), virus genome copy number (Fig. 2b, Fig. S2) and cytotoxicity (Fig. 2c, Fig. S3) on days 2, 4, and 6 after infection. Cell replication-dependent cytotoxicity was observed in replicating human foreskin keratinocytes (HFK-r) while cytotoxicity was attenuated or absent in differentiated/quiescent human foreskin keratinocytes (HFK-q) (Fig. S3). Vero, an African green monkey kidney cell line that is known for permissive HSV replication, showed high GLuc expression and rQ-M38G replication following low dose rQ-M38G infection (MOI = 0.001, 1 virus per 1000 cells).
(a) Gaussia luciferase (GLuc) transgene expression, (b) virus replication, and (c) cytotoxicity following infection of Vero cells and a panel of human tumor cell lines with rQ-M38G (MOI = 0.001).
We assessed transgene expression, virus replication and cytotoxicity following rQ-M38G infection of 5 human tumor cell lines. SK-NEP_Luc (Ewing sarcoma) demonstrated elevated GLuc expression, virus replication and cytotoxic susceptibility similar to Vero cells. Osteomet (Osteosarcoma), STS26T_dsRed (MPNST), and S462.TY (MPNST) each demonstrated lower sensitivity in all three assays. Finally, we assessed cytotoxicity in 3 well established mouse tumor models derived from a C57/Bl6 background (Fig. S3) and several spontaneous murine thyroid or small cell lung tumor lines generated by a collaborator (data not shown). HGF116 (rhabdomyosarcoma) was the only cell line showing any measurable cytotoxicity, but only at a high virus dose (MOI = 1, 1 infectious virus per cell). These data identified SK-NEP_Luc as a prime target for in vivo screening using rQ-M38G while other human tumor cell lines were predicted to be less amenable to screening with rQ-M38G.
Systemic administration of rQ-M38G to identify tumor presence
We tested the suitability of rQ-M38G as a gene delivery vector to force tumor-specific secretion of a biomarker following systemic administration of rQ-M38G in mice with and without tumors. Eleven mice were injected orthotopically with 106 SK-NEP_Luc cells into their renal subcapsule. Tumor-implanted mice and control tumor-free mice were subsequently injected intravenously (i.v) with 1.2×107 pfu of rQ-M38G 5 weeks after tumor implantation. On days 1, 4 and 7 following virus injection mice were imaged to identify firefly luciferase-positive tumors and blood samples were collected by retro-orbital eye bleed and assayed for serum GLuc (Fig. 3a). In vivo imaging identified 10 out of 11 mice which received tumor cell injections had formed tumors (Fig. 3b). One mouse (#9) never formed a tumor and one mouse (#11) had a tumor that was scarcely detectable. In all mice where tumor burden was apparent (9 out of 11), serum GLuc levels were 15- to 440-fold higher than tumor-free mice, whereas serum GLuc from the other two mice remained below control mouse serum GLuc levels (Fig. 3). Therefore, rQ-M38G administered systemically to induce biomarker production resulted in a detection sensitivity of 90% for SK-NEP_Luc-bearing mice. Similar results were for tumors in different anatomical sites (Fig. S4).
(a) Time course of GLuc expression in renal subcapsule (r.s.c) SK-NEP_Luc-bearing and tumor-free mice injected systemically with 1.2×107 pfu or rQ-M38G. Numbers in legend represent individual mice. (b) GLuc expression and in vivo imaging of SK-NEP_Luc-bearing mice four days following systemic injection of 1.2×107 pfu of rQ-M38G. Serum GLuc levels for mice injected with tumor and control that did not receive tumor injections (bar graph), and in vivo luciferase imaging of mice that received tumor cell injections.
To determine the location of rQ-M38G in tissues following i.v. injection and confirm that the serum GLuc signal was being expressed from tumors and not normal organs, organs were harvested from mice 7 days after virus infection and subjected to immunofluorescence for GFP expression and quantitative PCR for virus genomes. Only tumors demonstrated GFP expression in both control (data not shown) and experimental mice (Fig. 4a) injected systemically with rQ-M38G. Quantitative PCR for virus genome copies reflected the same distribution of virus in tumor-bearing mice as was seen with GFP expression where virus copies were at least 2100–fold higher in the tumors than healthy tissues (Fig. 4b). Immunofluorescence and qPCR indicate that virus infection predominated in the tumor while being minimal in healthy tissues. Therefore, systemically administered rQ-M38G successfully identified the presence of SK-NEP_Luc tumors by forcing tumors to produce a secretable biomarker. We demonstrated similar findings in tumor models that are less susceptible to virus infection including models of malignant peripheral nerve sheath tumors (in both subcutaneous and intraperitoneal locations) and osteosarcoma (Figures S5, S6, S7).
(a) GFP immunofluoresence in SK-NEP_Luc-bearing mice with and without systemic virus administration. Mice receiving virus (#1 and #2) showed selective GFP expression in tumor, while mice receiving no virus (No Virus Control) showed global GFP-negativity. Scale bar = 15 microns. (b) Biodistribution of virus in mice with SK-NEP_Luc tumors following systemic administration as determined by qPCR for viral genomes.
Induction of GLuc expression in tumors by intratumoral virus injection
A clear difference was observed between SK-NEP_Luc and the three other tumor models tested which each showed lower in vitro cytotoxicity and GLuc expression coupled with lower biomarker expression in vivo. To identify whether delivering larger doses of virus to tumors could increase in vivo biomarker expression, we administered rQ-M38G directly into tumors by intratumoral injection in models of S462.TY and Osteomet.
Mice bearing subcutaneous flank Osteomet tumors greater than 200 mm3 were injected with 1.9×105 pfu of virus. Blood samples were collected and analyzed for GLuc at each time point (Fig. S7a) while two mice were sacrificed at each time point and analyzed for viral genomic copies by qPCR (Fig. S7b). Serum GLuc levels in blood from mice bearing tumors injected with rQ-M38G was higher than GLuc levels in control uninfected mice, and increasing GLuc over time traced a profile similar to viral copy number in tumors. Despite a 4000-fold genomic amplification in tumors, serum GLuc levels rose only 10-fold above uninfected controls. Intratumoral injection with 8×103 pfu or 8×107 pfu of virus was repeated in S462.TY tumors when tumors were larger than 500 mm3. Four days after i.t. injection serum GLuc levels were assayed as plotted in Fig. S8. All mice receiving intratumoral injection of virus demonstrated higher GLuc levels than tumor-free controls without a significant difference in GLuc concentration between the low dose and 10,000-fold high virus dose, suggesting saturation at the low dose (at least via the intratumoral route).
Small animal models used to detect minimal tumor burdens present theoretical scalability challenges when translating findings to human-scale practicality. To address these challenges we sought to evaluate theoretical detection limits for our biomarker based on an in vitro assay and detect small tumor burdens in vivo by 3 methods: IVIS luminescence imaging, virus-mediated GLuc expression in serum and post-mortem histological analysis. The blood volume in a mouse is approximately 2 mL (8% of the 25 g total body weight ). Spherical SKNEP-Luc tumor comprised of 106 cells was determined to be 1.83 mm in diameter by measuring volume occupied in a centrifuged sample of an equal number of cells, assuming small tumors are comprised predominantly of tumor cells. Ten to 1 million cells were plated in 2 mL of cell culture media and co-infected with rQ-M38G at an MOI of 3 to ensure every cell would be infected. Two days after infection cell culture media was collected and analyzed for GLuc concentration. Virus-mediated GLuc expression could be reliably resolved by simultaneously transducing 1,000 cells, which approximates a spherical tumor of diameter 183 microns (Fig. S9).
Ewings sarcoma tumors (SKNEP-Luc) of different sizes were established in 11 mice by injecting 106 cells into the left kidney subcapsularly in 3 groups of mice on 3 consecutive weeks. Mice with tumors that had developed over 2, 3 and 4 weeks and 4 tumor-free control mice were infected systemically with 1×107 pfu of rQ-M38G. Four days following infection mice were imaged via IVIS, blood samples collected for GLuc quantitation, and mice were sacrificed for histological tumor sizing. In vivo imaging for luciferase expression revealed mice baring tumors drastically different in size at the time of infection and sacrifice (extreme examples shown in Fig. 5a). Serum GLuc concentration in tumor bearing mice 4 days after infection revealed GLuc levels greater than similarly infected tumor-free control mice (Fig. 5b). Histological evaluation of tumors in each mouse revealed a macroscopic tumor and microscopic tumor foci in mouse i and ii respectively (Fig. 5c).
a) in vivo imaging of two mice (i and ii) with vastly different SKNEP-Luc tumor burdens. b) Serum GLuc concentration in mouse i, ii and four tumor-free control mice 4 days following systemic infection with 1×107 pfu of rQ-M38G. c) Hematoxylin and eosin macroscopic images of tumor bearing kidneys (T = tumor, scale bars = 1 mm) and insets of microscopic tumor foci in the renal parenchyma of mouse ii (scale bar = 200 µm).
We developed a novel cancer screening strategy whereby tumors are forced to secrete a biomarker. We developed a conditionally replicative HSV, rQ-M38G, to selectively force tumor cells to secrete Gaussia luciferase as a demonstration of this screening strategy. Tumor-bearing animals given intravenous rQ-M38G expressed higher levels of biomarker compared with tumor-free animals, which showed only low, background expression. The utility of rQM-38G for screening appeared to be a function of the capability of a given tumor to support HSV replication. Regardless of tumor type or location, tumor screening by rQ-M38G was highly sensitive to tumor presence.
A critical feature for cancer screening is the ability to detect small tumors, ultimately in people. One a priori concern was that small tumors may be associated with less vascular surface area available for HSV entry. This issue did not appear to be a limitation in mice, because in some of the models we were able to detect tumors as small as 4–5 mm in diameter (50 mm3) and in another model we detected microscopic tumor burden. Scalability to humans (with larger blood volumes) is difficult to predict, but some estimates are possible. In vitro evaluation of increasing numbers of SKNEP-Luc tumor cells indicates that infecting as few as 1,000 cells in a volume of cell culture media equal to a mouse blood volume yields detectable GLuc secretion. Therefore, simultaneous expression of GLuc from 1,000 cells at any time in a mouse is theoretically detectable. Under these conditions infecting 10% of the cells in a tumor of diameter 400 µm (104 cells) would be detectable in a mouse. When scaled from a mouse to a human the theoretical limit of detection is increased by a ratio of 1∶2600 (25 mg mouse∶65 kg human) and diluting the biomarker in a larger average human blood volume of 4.7 L. Using these numbers, the theoretical limit of detection when infecting 10% of the cells in a tumor is changed from a 394 um diameter mass in a mouse to a 1–4 mm tumor diameter in a human. If only 1% of tumor cells are transduced by virus, tumors as small as 8.5 mm in diameter might still be detectable using these calculations. This sensitivity suggests strong potential for identifying minimal tumor burdens even when scaled to human proportions.
The ability of this screening strategy to reveal tumors is dependent upon viral factors, tumor microenvironment, and biomarker detection limitations each of which can be enhanced for real world practicality. Here we employed a doubly attenuated HSV to effect a tumor specific transduction and gene expression. Such vectors have already been documented to be safe for human use . Alternative viruses with single or less-attenuating mutations demonstrate greater oncolytic capacity are also already in clinical trials (e.g., HSV1716, see www.clinicaltrials.gov, NCT00931931). Coupling this screening strategy to less attenuated viruses will likely diversify its utility among the various solid malignancies as well as enhance the therapeutic component. Agents that are simultaneously both diagnostic and therapeutic have been dubbed “theragnostic.” It is possible our strategy could be further refined by the use of a more robust cancer-dependent promoter to drive biomarker expression. Improved vector delivery to the tumor may also be achieved using tumor targeting small molecule and nanoparticles uptake-enhancement strategies as recently described with the use of internalizing RGD peptides . Eventual commercialization of our proposed screening strategy would benefit from using biomarkers that are already in widespread use such as βHCG (pregnancy test), PSA (prostate cancer) and αFP (liver and germ cell malignancies). Biomarker assay sensitivity in the context of this screening strategy can still improve exponentially as is being realized with microfluidic technologies , , , .
A significant concern for the prototypical cancer screening method developed in this work is the development of an immune response to the gene delivery agent or the transduced biomarker that would preclude repeated use of the screening tool. Previous work in the field has shown that pre-immunized animals still experience therapeutic benefit from HSV with sustained efficacy . Because 80% of the general population has been exposed to HSV, implementing this detection strategy with HSV will depend upon the efficacy of engineered HSVs administered to immunocompetent subjects who have previously been exposed to wild-type HSV strains. In vitro screening for mouse tumor cell lines revealed that all mouse lines we tested exhibited comparably low susceptibility to infection by our attenuated mutant virus, on par with normal quiescent human cells. In vivo models of HGF116 demonstrated no virus sensitivity following i.t. or i.v. injection. Thus, we are unable to assess our cancer screening strategy in an immunocompetent model until the identification of mouse models that are more susceptible to human HSV. Implementation of this strategy may evolve with non-viral vectors  or viral vectors masked in liposomes .
The challenge of population-wide cancer screening is the development of clinical assays that are fiscally practical, universal across a spectrum of cancer types, and easy to implement. Physical examination, while affordable, often fails to identify malignancies that are deep or asymptomatic. Imaging benefits from high sensitivity, but may not always differentiate nonspecific or benign masses from malignant disease (eg, lung nodules may be granuloma or cancer), is financially challenging and difficult to broadly implement. With the identification of appropriate biomarkers, cancer screening could potentially be economically viable with automated point of care testing (POCT) technologies (www.i-stat.com, www.biosite.com, www.siloambio.com ).
This work demonstrates the principle of inducing expression of a secretable transgene in cancer using a systemically administered gene delivery agent as a biomarker to screen for tumor presence. The novel screening principle proposed and demonstrated in this work could hold immediate implications for patients with known cancer risks such as genetic predispositions (BRCA-1, Nf1 mutations) or patients with a history of cancer who are at high risk for recurrence. Ultimately, exogenous administration of a cancer targeting gene delivery agent (infectious or not) could force any malignancy to secrete a biomarker and could be used as a universal first step for population-wide cancer screening. The impact of this approach would revolutionize the technology for cancer detection in industrialized nations and the developing world where imaging and biopsy-based diagnostics may not be as readily available.
Materials and Methods
Cells and Viruses
Human tumor cell lines have been described previously ,  or were purchased from American Type Culture Collection (ATCC, Manassas, VA) except SK-NEP_Luc, which was a kind gift from Jason Frischer (CCHMC, Cincinnati, OH). Cells were grown in DMEM or McCoys 5A medium (ATCC, SK-OV-3, SK-NEP_Luc) with 10% fetal bovine serum (Hyclone, Logan, UT) and penicillin/streptomycin (Life Technologies, Carlsbad, CA). Mouse C57/Bl6 cell lines LLCGFP  (Lewis lung carcinoma) and B16-BL6 (melanoma) were kind gifts from Joe Palumbo (CCHMC, Cincinnati, OH) and HGF116 (rhabdomyosarcoma) was derived from a genetically engineered mouse. Primary human foreskin keratinocytes (HFK) were a kind gift from Susa Wells (CCHMC, Cincinnati, OH), grown in EpiLife Media (Cascade Biologics, Portland, OR) and differentiated into quiescent keratinocytes with 10% FBS and 1 mmol/L CaCl2 .
rQ-M38G was constructed as follows: The UL38 promotor was isolated from HSV rRp450 by PCR using primers designed previously  that were modified to include a 5′ BglII and 3′ HindIII sites (F1, R1 in Table S1) and cloned into the BglII and HindIII sites of pCMV-GLuc (Invitrogen), replacing the CMV promoter. The UL38p-GLuc cassette was amplified by PCR from pUL38p-GLuc with inclusion of a 5′SpeI and 3′ EcoRI and cloned into the SpeI-EcoRI sites of the “HSV-Quick” shuttle plasmid, pT-OriSIE4/5  (a kind gift from Yoshinaga Saeki, The Ohio State University, Columbus, Ohio), in which the oriS(E4/5 Kpn I fragment had been removed. The resulting plasmid, pT-UL38p-GLuc, was used in the HSVQuick BAC system to generate rQ-M38G . Primers for PCR analysis and sequencing are shown in Table S2. Viruses were propagated and titered  by plaque assay on Vero cells. Cell cytotoxicity was determined in 96-well tissue culture plates using a modified MTS/PMS assay (Promega, Madison, WI).
Gaussia luciferase assay
GLuc activity was assessed on 10 µL samples of tissue culture media or serum by chemiluminescent assay  on an autoinjecting luminometer by injecting 50 µL of 50 µM coelenterazine (Prolume, Pinetop, AZ) to each sample in triplicate and integrating the signal for 2.5 seconds .
Animal studies were approved by the Cincinnati Children's Hospital Institutional Animal Care and Use Committee (Animal protocol #9D12095). 5–6-week old female Balb/c athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN) were injected with 1–5 million cells. Cells were prepared in 33% Matrigel (Becton Dickinson, Bedford, MA) and 66% phosphate buffered saline (PBS, pH 7.4) for subcutaneous (s.q.) injection and PBS only for renal subcapsule (r.s.c) or intraperitoneal (i.p.) injection. Virus doses are described in the text. Virus was suspended in 100–150 µL of PBS for tail vein injections and 50–100 µL of PBS for intratumoral injections, which were distributed into 5 fractions throughout the tumor.
Tissue preparation and immunofluorescence
Tissues were fixed, embedded, and cut into 12-micron sections using standard procedures. Sections were permeablized with 0.2% Triton-X in PBS for 10 minutes, blocked in 10% normal goat serum for 60–90 minutes, and incubated with Chicken anti-GFP (#16901, Millipore/Chemicon, Billerica, MA) for 60 minutes, rinsed three times with PBS, and incubated with secondary antibody (Goat anti-Chicken FITC, Jackson ImmunoResearch, West Grove, PA), rinsed with PBS, and mounted with Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA). All slides were then imaged with OpenLab Imaging Software (Improvision, Waltham, MA) on an inverted fluorescent Zeiss microscope.
HSV genome quantitation
DNA was isolated from mouse tissues using the Gentra Puregene DNA isolation kit (Qiagen, Valencia, CA) and quantified using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). Real-time PCR was performed using the ABI 7500 system (Applied Biosystem, Foster City, CA) . Standards were made from purified HSV1716 viral DNA.
rQ-M38G gene expression and replication studies in cell lines
Cells were infected in 12-well dishes for 1 hour and harvested at times indicated. 100 uL of the cell suspension was used for measuring Gaussia luciferase, while the remaining cell suspension was subjected to three cycles of freeze–thaw and centrifuged at 20,000× g. Pellets were resuspended in 200 uL PBS. DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA). Fast real-time PCR was performed using the 7900HT Fast Real Time PCR System (Applied Biosystem, Foster City, CA). Standard DNA or DNA extracted from infected cells (40 ng) was added to 10 µL of SYBR Premix Ex Taq II Kit (TaKaRa, Otsu, Shiga, Japan), 1.6 µL of a thymidine kinase primer mixture (TK 290-F: 5′ TCG CGA ACA TCT ACA CCA CAC AAC; TK 400-R: 5′ CGG CAT AAG GCA TGC CCA TTG TTA; each at 400 nM), 0.4 µL Rox (TaKaRa) and PCR-grade water to a final volume of 20 µl per reaction. PCR was 1 cycle of 95°C for 30 seconds, 40 cycles of 95°C for 3 seconds, 60°C for 15 seconds, and 72°C for 25 seconds.
GLuc excretion following infection of Vero cells and 4 human tumor cell lines (100,000 cells/well) with rQ-M38G across a range of MOIs.
Virus replication as measured by qPCR following infection of Vero cells and 4 human tumor cell lines (100,000 cells/well) with rQ-M38G across a range of MOIs.
In vitro cytotoxicity of rQ-M38G in a well characterized virus permissive cell line (Vero), replicating and non-replicating human keratinocytes (HFK-r and HFK-q respectively), 5 human tumor cell lines (S462.TY, Osteomet, SK-OV-3, STS26T_dsRed, and SK-NEP_Luc) and 3 mouse tumor cell lines (LLCGFP, B16-Bl6, HGF116).
Serum GLuc levels following i.v. infection with 1×107 pfu of rQ-M38G in mice bearing intraperitoneal, intramuscular and subcutaneous tumors.
GLuc expression in two MPNST tumor models (S462.TY and STS26T_dsRed) with low in vitro virus sensitivity and GFP expression in S462.TY-bearing mice. (a) Serum GLuc following systemic injection of 5×107 pfu of rQ-M38G by tail vein into mice bearing S462.TY subcutaneous tumors. Shaded gray represent the range of GLuc levels for tumor-free mice which received the same dose of virus; (b) Serum GLuc levels following systemic administration of 1×107 pfu of rQ-M38G in mice bearing subcutaneous STS26T_dsRed tumors; and (c) GFP expression in 2 S462.TY-bearing mice (#iii and #vii) demonstrating few punctate GFP positive cells in tumors and no GFP positive cells in healthy tissues. Tumor volume (mm3) at time of virus injection is noted for each mouse in the plot legends.
Serum GLuc levels following systemic administration of 1×107 pfu of rQ-M38G in mice which were injected with 2×106 STS26T_dsRed cells intraperitoneally.
Serum GLuc levels (a), and number of virus copies in tumors as determined by qPCR (b) following direct intratumoral injection of rQ-M38G into subcutaneous Osteomet tumors larger than 200 mm3. c) Serum GLuc levels in a subcutaneous in vivo model for Osteomet following systemic injection of 1.9×107 pfu or 1.9×105 pfu of rQ-M38G. Gray shaded area represents serum GLuc levels in tumor free mice also receiving 1.9×107 pfu of rQ-M38G. Key identifies mouse number, virus dose, and tumor size at time of injection respectively.
Serum GLuc levels from S462.TY bearing mice four days following i.t. injection of 8×103 pfu or 8×107 pfu of rQ-M38G.
In vitro modeling of increasing numbers of cells (SKNEP-Luc), their theoretical tumor volume and GLuc production from infection of every cell.
Primers used in the production of HSV-UL38p-GLuc (rQ-M38G).
Special thanks to Kelly Crawford for assistance with SK-NEP_Luc cells, Joe Palumbo for providing B16-BL6 and LLCGFP, Katherine Wikenheiser-Brokamp for providing a mouse thyroid cancer line, Yoni Mahller for academic discussion and support, and Chuck Klanke for help with HSV quick production.
Conceived and designed the experiments: AWB CHA TPC. Performed the experiments: AWB MAC JLL JPW JSF MHC. Analyzed the data: AWB TPC. Contributed reagents/materials/analysis tools: TPC JSF MHC. Wrote the paper: AWB TPC.
- 1. Maeda H (2001) The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Advan Enzyme Regul 41: 189–207.H. Maeda2001The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting.Advan Enzyme Regul41189207
- 2. Maeda H, Fanga J, Inutsuka T, Kitamotoc Y (2003) Vascular permeability enhancement in solid tumor: various factors, mechanisms involved and its implications. International Immunopharmacology 3: 319–328.H. MaedaJ. FangaT. InutsukaY. Kitamotoc2003Vascular permeability enhancement in solid tumor: various factors, mechanisms involved and its implications.International Immunopharmacology3319328
- 3. Heneweer C, Holland J, Divilov V, Carlin S, Lewis J (2011) Magnitude of Enhanced Permeability and Retention Effect in Tumors with Different Phenotypes: 89Zr-Albumin as a Model System. J Nucl Med. C. HeneweerJ. HollandV. DivilovS. CarlinJ. Lewis2011Magnitude of Enhanced Permeability and Retention Effect in Tumors with Different Phenotypes: 89Zr-Albumin as a Model System.J Nucl Med[Epub ahead of print]. [Epub ahead of print].
- 4. Kirpotin DB, Drummond DarylC, Shao Yi, Shalaby M Refaat, Hong Keelung, Nielsen UlrikB, Marks JamesD, Benz ChristopherC, Park JohnW (2006) Antibody Targeting of Long-Circulating Lipidic Nanoparticles Does Not Increase Tumor Localization but Does Increase Internalization in Animal Models. Cancer Res 66: 6732–6740.DB KirpotinDarylC Drummond Yi ShaoM Refaat ShalabyKeelung Hong UlrikB Nielsen JamesD Marks ChristopherC Benz JohnW Park 2006Antibody Targeting of Long-Circulating Lipidic Nanoparticles Does Not Increase Tumor Localization but Does Increase Internalization in Animal Models.Cancer Res6667326740
- 5. Kocbek P, Obermajer N, Cegnar M, Kos J, Kristl J (2007) Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody. Journal of Controlled Release 120: 18–26.P. KocbekN. ObermajerM. CegnarJ. KosJ. Kristl2007Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody.Journal of Controlled Release1201826
- 6. Brannon-Peppas L, Blanchette JO (2004) Nanoparticle and targeted systems for cancer therapy. Advanced Drug Delivery Reviews 56: 1649–1659.L. Brannon-PeppasJO Blanchette2004Nanoparticle and targeted systems for cancer therapy.Advanced Drug Delivery Reviews5616491659
- 7. Hatakeyama H, Akita H, Ito E, Hayashi Y, Oishi M, et al. (2011) Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid. Biomaterials. H. HatakeyamaH. AkitaE. ItoY. HayashiM. Oishi2011Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid.Biomaterials[Epub ahead of print]. [Epub ahead of print].
- 8. Sugahara KN, Teesalu T, Karmali PP, Kotamraju VR, Agemy L, et al. (2010) Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science 328: 1031–1035.KN SugaharaT. TeesaluPP KarmaliVR KotamrajuL. Agemy2010Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.Science32810311035
- 9. Ko J, Park K, Kim Y-S, Kim MS, Han JK, et al. (2007) Tumoral acidic extracellular pH targeting of pH-responsive MPEG-poly([beta]-amino ester) block copolymer micelles for cancer therapy. Journal of Controlled Release 123: 109–115.J. KoK. ParkY-S KimMS KimJK Han2007Tumoral acidic extracellular pH targeting of pH-responsive MPEG-poly([beta]-amino ester) block copolymer micelles for cancer therapy.Journal of Controlled Release123109115
- 10. Qi X, Chu Z, Mahller YY, Stringer KF, Witte DP, et al. (2009) X. QiZ. ChuYY MahllerKF StringerDP Witte2009Cancer-Selective Targeting and Cytotoxicity by Liposomal-Coupled Lysosomal Saposin C Protein Clinical Cancer Research: in press. Cancer-Selective Targeting and Cytotoxicity by Liposomal-Coupled Lysosomal Saposin C Protein Clinical Cancer Research: in press.
- 11. Gao X, Cui Y, Levenson RM, Chung LWK, Nie S (2004) In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnology 22: 969–976.X. GaoY. CuiRM LevensonLWK ChungS. Nie2004In vivo cancer targeting and imaging with semiconductor quantum dots.Nature Biotechnology22969976
- 12. Shen Y, Nemunaitis J (2006) Herpes simplex virus 1 (HSV-1) for cancer treatment. Cancer Gene Ther 7: 7.Y. ShenJ. Nemunaitis2006Herpes simplex virus 1 (HSV-1) for cancer treatment.Cancer Gene Ther77
- 13. Martuza R (2000) Conditionally replicating herpes vectors for cancer therapy. J Clin Invest 105: 841–846.R. Martuza2000Conditionally replicating herpes vectors for cancer therapy.J Clin Invest105841846
- 14. Aghi M, Visted T, Depinho RA, Chiocca EA (2008) Oncolytic herpes virus with defective ICP6 specifically replicates in quiescent cells with homozygous genetic mutations in p16. Oncogene 27: 4249–4254.M. AghiT. VistedRA DepinhoEA Chiocca2008Oncolytic herpes virus with defective ICP6 specifically replicates in quiescent cells with homozygous genetic mutations in p16.Oncogene2742494254
- 15. Harland J, Dunn P, Cameron E, Conner J, Brown SM (2003) The herpes simplex virus (HSV) protein ICP34.5 is a virion component that forms a DNA-binding complex with proliferating cell nuclear antigen and HSV replication proteins. J Neurovirol 9: 477–488.J. HarlandP. DunnE. CameronJ. ConnerSM Brown2003The herpes simplex virus (HSV) protein ICP34.5 is a virion component that forms a DNA-binding complex with proliferating cell nuclear antigen and HSV replication proteins.J Neurovirol9477488
- 16. He B, Gross M, Roizman B (1997) The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A 94: 843–848.B. HeM. GrossB. Roizman1997The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase.Proc Natl Acad Sci U S A94843848
- 17. Guzowski JF, Singh J, Wagner EK (1994) Transcriptional activation of the herpes simplex virus type 1 UL38 promoter conferred by the cis-acting downstream activation sequence is mediated by a cellular transcription factor. J Virol 68: 7774–7789.JF GuzowskiJ. SinghEK Wagner1994Transcriptional activation of the herpes simplex virus type 1 UL38 promoter conferred by the cis-acting downstream activation sequence is mediated by a cellular transcription factor.J Virol6877747789
- 18. Fu X, Meng F, Tao L, Jin A, Zhang X (2003) A strict-late viral promoter is a strong tumor-specific promoter in the context of an oncolytic herpes simplex virus. Gene Therapy 10: 1458–1464.X. FuF. MengL. TaoA. JinX. Zhang2003A strict-late viral promoter is a strong tumor-specific promoter in the context of an oncolytic herpes simplex virus.Gene Therapy1014581464
- 19. Iankov ID, Hillestad ML, Dietz AB, Russell SJ, Galanis E (2009) Converting Tumor-specific Markers Into Reporters of Oncolytic Virus Infection. Mol Ther 17: 1395–1403.ID IankovML HillestadAB DietzSJ RussellE. Galanis2009Converting Tumor-specific Markers Into Reporters of Oncolytic Virus Infection.Mol Ther1713951403
- 20. Kanerva A, Zinn K, Peng K, Ranki T, Kangasniemi L, et al. (2005) Noninvasive dual modality in vivo monitoring of the persistence and potency of a tumor targeted conditionally replicating adenovirus. Gene Ther 12: 87–94.A. KanervaK. ZinnK. PengT. RankiL. Kangasniemi2005Noninvasive dual modality in vivo monitoring of the persistence and potency of a tumor targeted conditionally replicating adenovirus.Gene Ther128794
- 21. Rajecki M, Kanerva A, Stenman U-H, Tenhunen M, Kangasniemi L, et al. (2007) Treatment of prostate cancer with Ad5/3d24hCG allows non-invasive detection of the magnitude and persistence of virus replication in vivo. Molecular Cancer Therapy 6: 742–751.M. RajeckiA. KanervaU-H StenmanM. TenhunenL. Kangasniemi2007Treatment of prostate cancer with Ad5/3d24hCG allows non-invasive detection of the magnitude and persistence of virus replication in vivo.Molecular Cancer Therapy6742751
- 22. Peng K, Facteau S, Wegman T, O'Kane D, Russell S (2002) Non-invasive in vivo monitoring of trackable viruses expressing soluble marker peptides. Nature Medicine 8: 527–531.K. PengS. FacteauT. WegmanD. O'KaneS. Russell2002Non-invasive in vivo monitoring of trackable viruses expressing soluble marker peptides.Nature Medicine8527531
- 23. Barton ES, Youree BE, Ebert DH, Forrest JC, Connolly JL, et al. (2003) Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease. J Clin Invest 111: 1823–1833.ES BartonBE YoureeDH EbertJC ForrestJL Connolly2003Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease.J Clin Invest11118231833
- 24. Penheiter A, Wegman T, Classic K, Dingli D, Bender C, et al. (2010) Sodium iodide symporter (NIS)-mediated radiovirotherapy for pancreatic cancer. AJR Am J Roentgenol 195: 341–349.A. PenheiterT. WegmanK. ClassicD. DingliC. Bender2010Sodium iodide symporter (NIS)-mediated radiovirotherapy for pancreatic cancer.AJR Am J Roentgenol195341349
- 25. Li H, Peng K, Dingli D, Kratzke R, Russell S (2010) Oncolytic measles viruses encoding interferon beta and the thyroidal sodium iodide symporter gene for mesothelioma virotherapy. Cancer Gene Ther 17: 550–558.H. LiK. PengD. DingliR. KratzkeS. Russell2010Oncolytic measles viruses encoding interferon beta and the thyroidal sodium iodide symporter gene for mesothelioma virotherapy.Cancer Gene Ther17550558
- 26. Tannous BA, Kim D-E, Fernandez JL, Weissleder R, Breakefield XO (2005) Codon-Optimized Gaussia Luciferase cDNA for Mammalian Gene Expression in Culture and in Vivo. Mol Ther 11: 435–443.BA TannousD-E KimJL FernandezR. WeisslederXO Breakefield2005Codon-Optimized Gaussia Luciferase cDNA for Mammalian Gene Expression in Culture and in Vivo.Mol Ther11435443
- 27. Wurdinger T, Badr C, Pike L, de Kleine R, Weissleder R, et al. (2008) A secreted luciferase for ex vivo monitoring of in vivo processes. Nat Meth 5: 171–173.T. WurdingerC. BadrL. PikeR. de KleineR. Weissleder2008A secreted luciferase for ex vivo monitoring of in vivo processes.Nat Meth5171173
- 28. Marquardt A, Halle S, Seckert C, Lemmermann N, Veres T, et al. (2011) Single cell detection of latent cytomegalovirus reactivation in host tissue. J Gen Virol. A. MarquardtS. HalleC. SeckertN. LemmermannT. Veres2011Single cell detection of latent cytomegalovirus reactivation in host tissue.J Gen Virol[Epub ahead of print]. [Epub ahead of print].
- 29. Terada K, Wakimoto H, Tyminski E, Chiocca EA, Saeki Y (2006) Development of a rapid method to generate multiple oncolytic HSV vectors and their in vivo evaluation using syngeneic mouse tumor models. Gene Ther 19: 19.K. TeradaH. WakimotoE. TyminskiEA ChioccaY. Saeki2006Development of a rapid method to generate multiple oncolytic HSV vectors and their in vivo evaluation using syngeneic mouse tumor models.Gene Ther1919
- 30. Hwang KJ, Luk KF, Beaumier PL (1980) Hepatic uptake and degradation of unilamellar sphingomyelin/cholesterol liposomes: a kinetic study. Proc Natl Acad Sci U S A 77: 4030–4034.KJ HwangKF LukPL Beaumier1980Hepatic uptake and degradation of unilamellar sphingomyelin/cholesterol liposomes: a kinetic study.Proc Natl Acad Sci U S A7740304034
- 31. Markert JM, Liechty PG, Wang W, Gaston S, Braz E, et al. (2009) Phase Ib trial of mutant herpes simplex virus G207 inoculated pre-and post-tumor resection for recurrent GBM. Mol Ther 17: 199–207.JM MarkertPG LiechtyW. WangS. GastonE. Braz2009Phase Ib trial of mutant herpes simplex virus G207 inoculated pre-and post-tumor resection for recurrent GBM.Mol Ther17199207
- 32. Stoeva SI, Lee J-S, Smith JE, Rosen ST, Mirkin CA (2006) Multiplexed Detection of Protein Cancer Markers with Biobarcoded Nanoparticle Probes. Journal of the American Chemical Society 128: 8378–8379.SI StoevaJ-S LeeJE SmithST RosenCA Mirkin2006Multiplexed Detection of Protein Cancer Markers with Biobarcoded Nanoparticle Probes.Journal of the American Chemical Society12883788379
- 33. Henares TG, Mizutani F, Hisamoto H (2008) Current development in microfluidic immunosensing chip. Analytica Chimica Acta 611: 17–30.TG HenaresF. MizutaniH. Hisamoto2008Current development in microfluidic immunosensing chip.Analytica Chimica Acta6111730
- 34. Fan R, Vermesh O, Srivastava A, Yen BKH, Quin L, et al. (2008) Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood. Nature Biotechnology 26: 1373–1378.R. FanO. VermeshA. SrivastavaBKH YenL. Quin2008Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood.Nature Biotechnology2613731378
- 35. Roda A, Cevenini L, Michelini E, Branchin iB (2011) A portable bioluminescence engineered cell-based biosensor for on-site applications. Biosens Bioelectron. A. RodaL. CeveniniE. MicheliniiB Branchin2011A portable bioluminescence engineered cell-based biosensor for on-site applications.Biosens Bioelectron[Epub ahead of print]. [Epub ahead of print].
- 36. Lambright ES, Kang EH, Force S, Lanuti M, Caparrelli D, et al. (2000) Effect of preexisting anti-herpes immunity on the efficacy of herpes simplex viral therapy in a murine intraperitoneal tumor model. Mol Ther 2: 387–393.ES LambrightEH KangS. ForceM. LanutiD. Caparrelli2000Effect of preexisting anti-herpes immunity on the efficacy of herpes simplex viral therapy in a murine intraperitoneal tumor model.Mol Ther2387393
- 37. Yu W, Pirollo KF, Yu B, Rait A, Xiang L, et al. (2004) Enhanced transfection eficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide. Nucleic Acids Research 32: W. YuKF PirolloB. YuA. RaitL. Xiang2004Enhanced transfection eficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide.Nucleic Acids Research32
- 38. Shikano T, Kasuya H, Sahin T, Nomura N, Kanzaki A, et al. (2010) High Therapeutic Potential for Systemic Delivery of a Liposome-conjugated Herpes Simplex Virus. Curr Cancer Drug Targets 11: 111–122.T. ShikanoH. KasuyaT. SahinN. NomuraA. Kanzaki2010High Therapeutic Potential for Systemic Delivery of a Liposome-conjugated Herpes Simplex Virus.Curr Cancer Drug Targets11111122
- 39. Do J, Ahn CH (2008) A polymer lab-on-a-chip for magnetic immunoassay with on-chip sampling and detection capabilities. Lab Chip 8: 542–549.J. DoCH Ahn2008A polymer lab-on-a-chip for magnetic immunoassay with on-chip sampling and detection capabilities.Lab Chip8542549
- 40. Mahller YY, Vaikunth SS, Ripberger MC, Baird WH, Saeki Y, et al. (2008) Tissue inhibitor of metalloproteinase-3 via oncolytic herpesvirus inhibits tumor growth and vascular progenitors. Cancer Res 68: 1170–1179.YY MahllerSS VaikunthMC RipbergerWH BairdY. Saeki2008Tissue inhibitor of metalloproteinase-3 via oncolytic herpesvirus inhibits tumor growth and vascular progenitors.Cancer Res6811701179
- 41. Bharatan N, Currier M, Cripe T (2001) Differential susceptibility of pediatric sarcoma cells to oncolysis by conditionally replication-competent herpes simplex viruses. J Ped Hem Onc 24: 447–453.N. BharatanM. CurrierT. Cripe2001Differential susceptibility of pediatric sarcoma cells to oncolysis by conditionally replication-competent herpes simplex viruses.J Ped Hem Onc24447453
- 42. Palumbo JS, Talmage KE, Liu H, La Jeunesse CM, Witte DP, et al. (2003) Plasminogen supports tumor growth through a fibrinogen-dependent mechanism linked to vascular patency. Blood 102: 2819–2827.JS PalumboKE TalmageH. LiuCM La JeunesseDP Witte2003Plasminogen supports tumor growth through a fibrinogen-dependent mechanism linked to vascular patency.Blood10228192827
- 43. Boyce ST, Ham RG (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81: 33s–40s.ST BoyceRG Ham1983Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.J Invest Dermatol8133s40s
- 44. Mahller YY, Rangwala F, Ratner N, Cripe TP (2006) Malignant peripheral nerve sheath tumors with high and low Ras-GTP are permissive for oncolytic herpes simplex virus mutants. Pediatr Blood Cancer 46: 745–754.YY MahllerF. RangwalaN. RatnerTP Cripe2006Malignant peripheral nerve sheath tumors with high and low Ras-GTP are permissive for oncolytic herpes simplex virus mutants.Pediatr Blood Cancer46745754
- 45. Tannous BA (2009) Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nature Protocols 582–591.BA Tannous2009Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.Nature Protocols582591
- 46. Currier MA, Gillespie RA, Sawtell NM, Mahller YY, Stroup G, et al. (2008) Efficacy and safety of the oncolytic herpes simplex virus rRp450 alone and combined with cyclophosphamide. Mol Ther 16: 879–885.MA CurrierRA GillespieNM SawtellYY MahllerG. Stroup2008Efficacy and safety of the oncolytic herpes simplex virus rRp450 alone and combined with cyclophosphamide.Mol Ther16879885