Clinical Cancer Research Grants Advances in Breast Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, T.-C.
Right arrow Articles by Rabkin, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, T.-C.
Right arrow Articles by Rabkin, S. D.
Clinical Cancer Research Vol. 12, 6791-6799, November 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Dominant-Negative Fibroblast Growth Factor Receptor Expression Enhances Antitumoral Potency of Oncolytic Herpes Simplex Virus in Neural Tumors

Ta-Chiang Liu1, Tingguo Zhang1, Hiroshi Fukuhara1, Toshihiko Kuroda1, Tomoki Todo1, Xavier Canron2, Andreas Bikfalvi2, Robert L. Martuza1, Andreas Kurtz1 and Samuel D. Rabkin1

Authors' Affiliations: 1 Molecular Neurosurgery Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts and 2 Institut National de la Santé et de la Recherche Médicale E0113, Molecular Mechanisms of Angiogenesis, University Bordeaux I, Talence, France

Requests for reprints: Samuel D. Rabkin, Molecular Neurosurgery Laboratory, Massachusetts General Hospital, CPZN-3800 Simches Research Building, 185 Cambridge Street, Boston, MA 02114. Phone: 617-726-6817; Fax: 617-643-3422; E-mail: rabkin{at}helix.mgh.harvard.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Oncolytic herpes simplex viruses (HSV) appear to be a promising platform for cancer therapy. However, efficacy as single agents has thus far been unsatisfactory. Fibroblast growth factor (FGF) signaling is important for the growth and migration of endothelial and tumor cells. Here, we examine the strategy of arming oncolytic HSV with a dominant-negative FGF receptor (dnFGFR) that targets the FGF signaling pathway.

Experimental Design: A mouse Nf1:p53 malignant peripheral nerve sheath tumor (MPNST) cell line expressing dnFGFR was generated by transfection. The effects of dnFGFR expression on cell growth and migration in vitro and tumor formation in vivo were determined. The dnFGFR transgene was then inserted into oncolytic HSV G47{Delta} using a bacterial artificial chromosome construction system. Antitumoral and antiangiogenic activities of bG47{Delta}-dnFGFR were examined.

Results: MPNST 61E4 cells expressing dnFGFR grew less well than parental control cells. bG47{Delta}-dnFGFR showed enhanced killing of both tumor (human U87 glioma and F5 malignant meningioma cells and murine MPNST 61E4 and 37-3-18-4 cells) and proliferating endothelial cells (human umbilical vascular endothelial cell and Py-4-1) in vitro compared with the control vector bG47{Delta}-empty without inhibiting viral replication. In vivo, bG47{Delta}-dnFGFR was more efficacious than its nonexpressing parent bG47{Delta}-empty at inhibiting tumor growth and angiogenesis in both human U87 glioma and mouse 37-3-18-4 MPNST tumors in nude mice.

Conclusions: By using multiple therapeutic mechanisms, including destruction of both tumor cells and tumor endothelial cells, an oncolytic HSV encoding dnFGFR enhances antitumor efficacy. This strategy can be applied to other oncolytic viruses and for clinical translation.

Replication-selective oncolytic herpes simplex virus (HSV) vectors have emerged as a new platform for cancer therapy (1). Most of the engineered HSV mutants currently being used as oncolytic agents have deletions in the neurovirulence gene {gamma}34.5 and/or ICP6, the large subunit of HSV ribonucleotide reductase (RR). Consequently, these HSV mutants replicate selectively in and kill dividing tumor cells, possibly due to the fact that dividing cells express mammalian RR and growth arrest DNA damage 34 (GADD34). Although not fully characterized, these gene products regulate the cell cycle and complement the mutations of these viruses (2). In particular, mammalian RR generates deoxyribonucleotides in place of the HSV RR, and the COOH terminus of GADD34 substitutes for the homologous region of {gamma}34.5.

Several oncolytic HSV mutants (e.g., 1716, G207, NV1020, and OncoVexGM-CSF) have entered phase I to II clinical trials with various solid tumors (3–7). However, despite the significant efficacy in preclinical models and safety in humans, therapeutic benefits have been limited (4, 8). It is therefore prudent to incorporate mechanisms in addition to direct oncolysis for tumor cell destruction. To this end, we have previously shown that HSV mutant G47{Delta} (9), in addition to enhanced viral replication, also possesses an immunoregulatory function in which MHC class I presentation was increased compared with its parent, G207, while maintaining the safety profile of G207. This provides for the possibility of an enhanced cytotoxic lymphocyte response toward tumor cells and increased efficacy of the virus.

To improve efficacy, oncolytic viruses have been armed with therapeutic transgenes. Armed oncolytic HSV expressing cytokines (e.g., granulocyte macrophage colony-stimulating factor, interleukin-12, and interleukin-18), prodrug-activating enzymes (e.g., thymidine kinase and cytosine deaminase), and other therapeutic transgene products have been shown to enhance the antitumoral effect (10–14). However, we propose that the targets of armed oncolytic viruses should be expanded to include tumor stromal cells and vasculature to maximize the therapeutic effect, using fibroblast growth factor (FGF) as an example.

The FGF family contains 22 members, which interact with four receptors (FGFR1-FGFR4; ref. 15). Prototypical FGF1 (acidic FGF) and FGF2 (basic FGF) both interact with FGFR1. FGFs play critical roles in tumorigenesis (15–17). The binding of FGF to FGFR activates a variety of signal transduction cascades through its intrinsic tyrosine kinase, including Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and phospholipase C-{gamma}, which in turn regulate cell proliferation, survival, differentiation, and migration (17, 18). FGF therefore acts as a mitogenic factor, angiogenic factor, and antiapoptotic factor. Overexpression of FGF, by either tumor cells or surrounding stromal cells, can result in autocrine and/or paracrine effects and is found to occur in tumors, such as gliomas (19). On the other hand, overexpression or activating mutations of FGFR have been shown in brain, breast, prostate, thyroid, and melanoma tumors (15, 20). Thus, dysregulated FGF signaling is present in most human cancers. Of note, two nervous system tumors [i.e., gliomas and malignant peripheral nerve sheath tumors (MPNST)] are well known to be vascular and aggressive, characteristics that have been attributed to angiogenesis and dysregulated FGF signaling pathways (21, 22). In particular, FGFR1 is generally elevated in gliomas and correlates with the degree of malignancy (19, 23).

Blocking FGF signaling for cancer treatment therefore seems appealing due to its ability to target not only tumor cells but also surrounding stromal cells and tumor vasculature. Indeed, inhibition of FGF/FGFR signaling with dominant-negative FGFR (dnFGFR), antisense, soluble FGFR, etc., as well as downstream pathways can have substantial antitumoral effects via multiple mechanisms (16, 24–27). We therefore hypothesized that combining oncolytic virotherapy with FGF signaling blockade would enhance antitumor efficacy. In particular, both tumor vascular endothelial cells as well as tumor cells would be targeted by this approach. The concept was tested by engineering an oncolytic HSV with a dnFGFR1, a truncated receptor that can dimerize, forming heterodimers with endogenous receptors, but not signal (28). This vector (bG47{Delta}-dnFGFR) shows enhanced antitumoral effects both in vitro and in vivo via direct antitumoral and antiangiogenic mechanisms.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells. Human glioblastoma cell lines U87 and T98 and African monkey kidney cell line Vero were obtained from the American Type Culture Collection (Manassas, VA) and grown in DMEM plus 10% calf serum. Human umbilical vascular endothelial cells (HUVEC) and its culture medium EGM-2 were obtained from Cambrex (Walkersville, MD) and maintained as described by the vendor. Human malignant meningioma cell line F5 (29) was provided by Dr. Anil Menon (University of Cincinnati, Cincinnati, OH). Murine MPNST cell lines 61E4 and 37-3-18-4, isolated from spontaneously arising tumors in Nf1+/–:p53+/– mice (30), were provided by Dr. Luis Parada (University of Texas, Southwestern Medical Center, Dallas, TX) and cultured in DMEM plus 10% calf serum. Murine endothelial cell line Py-4-1, isolated from hemangiomas of polyomavirus transgenic mice (31), was provided by Dr. Victoria Bautch (University of North Carolina, Chapel Hill, NC) and maintained in DMEM plus 10% calf serum.

Transfection of cells. For stable constitutive expression of dnFGFR, 61E4 cells were transfected with mouse dnFGFR1 (tyrosine kinase domain deleted, two immunoglobulin-like loop form, IIIc splice variant) cDNA (provided by Dr. Lewis Williams, University of California at San Francisco, San Francisco, CA; ref. 28) subcloned into pRC/CMV (Invitrogen, Carlsbad, CA) using LipofectAMINE (Life Technologies, Carlsbad, CA). Control cells (61E4-Zeo) were transfected with pRC/CMV alone. Clones were selected with geneticin (Life Technologies).

In vitro wound-healing assay. 61E4-Zeo and 61E4-dnFGFR cells were seeded into six-well plates, and an artificial wound was created with a scalpel when the cells were confluent. Images were captured before and 12 hours after scratching.

Chicken chorioallantoic membrane assay. On embryonic day 10, a plastic ring was placed on the chicken chorioallantoic membrane as described before (32), and 5 million 61E4-Zeo or 61E4-dnFGFR tumor cells in 20 µL of medium were deposited after gentle laceration of surface. Digital photos were taken under a stereomicroscope (Nikon SMZ800, Tokyo, Japan). One week after deposition, tumor volume was determined by the formula V = 4/3r3, with r = 1/2 (d1 x d2), and compared as described before (33).

Virus construction. The backbone for the constructed viruses is G47{Delta} (9), which was cloned into a bacterial artificial chromosome (BAC) vector for propagation in Escherichia coli. The details of G467{Delta}-BAC plasmid and virus constructions have been described (14). For the generation of the vectors described here, the Hind III blunt-ended/BamHI dnFGFR cDNA fragment from pSW2 was ligated into StuI/BamHI-digested pVec91 (containing LacZ, loxP, and FRT sites, a cytomegalovirus promoter, and multiple cloning site) to generate pVec91-dnFGFR. pVec91-dnFGFR or pVec91 was inserted into the ICP6 region of pG47{Delta}-BAC using Cre recombinase to generate pG47{Delta}-BAC-dnFGFR and pG47{Delta}-BAC-empty, respectively. Cotransfection of pG47{Delta}-BAC-dnFGFR or pG47{Delta}-BAC-empty and a FLPe recombinase expression plasmid (pCAGGSFlpeIRES; provided by Dr. Pedro Lowenstein, Cedars-Sinai Medical Center, University of California at Los Angeles, Los Angeles, CA; ref. 34) into Vero cells results in the removal of FRT-flanked BAC sequences and generation of bG47{Delta}-dnFGFR or bG47{Delta}-empty viruses.

Western blotting. Vero cells were mock infected or infected with viruses at 1 plaque-forming unit (pfu)/cell for 20 hours and harvested. Proteins (30 µg) were subjected to SDS-PAGE, transferred to polyvinylidene difluoride Plus membrane (MSI Micron Separations, Westborough, MA), and blotted overnight with FGFR primary antibody McAb6 (diluted 1:250; provided by Dr. Pamela Maher, The Scripps Research Institute, La Jolla, CA; ref. 35), anti-ICP4 (diluted 1:6,000; U.S. Biological, Swampscott, MA), or anti-actin (diluted 1:500; Sigma, St. Louis, MO). The membrane was then washed, blotted with either anti-mouse secondary antibody (horseradish peroxidase conjugated; diluted 1:10,000; Amersham, Piscataway, NJ) or anti-rabbit secondary antibody (horseradish peroxidase conjugated; diluted 1:10,000; Amersham), washed, exposed to ECL Plus, and developed.

For FGF signaling, U87, 61E4, and 37-3-18-4 cells were seeded into six-well plates (3 x 105 per well). The cells were cultured in serum-free DMEM (U87) or DMEM plus 0.3% heat-inactivated FCS (61E4 and 37-3-18-4) for 48 hours before treatment. Cells were then mock infected or infected with bG47{Delta}-empty or bG47{Delta}-dnFGFR (5 pfu/cell). Sixteen hours after infection, cells were treated with (61E4 and 37-3-18-4) or without (U87) acidic FGF (1 ng/mL; PeproTech, Rocky Hill, NJ) for 5 minutes. Acidic FGF was not added to U87 cells because these cells endogenously secrete FGF. Cells were then harvested, subjected to SDS-PAGE, transferred to polyvinylidene difluoride Plus membrane, and blotted overnight with antibody to total extracellular signal-regulated kinase (ERK) or phosphorylated ERK (diluted 1:1,000; Cell Signaling, Danvers, MA). The membrane was then washed, blotted with anti-rabbit secondary antibody (diluted 1:10,000; horseradish peroxidase conjugated), washed, exposed to ECL Plus, and developed. The same membrane blotted for phosphorylated ERK was stripped and blotted for total ERK.

Cell survival assay. Cells were seeded into 96-well plates at 5,000 to 7,500 per well. After 24 hours, cells were infected with the indicated viruses at various concentrations (3-fold dilution from 30 to 0.001 pfu/cell) and incubation was continued for a further 72 hours when a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Sigma) was done and dose-response curves were obtained. Experiments were repeated at least four times with each condition in quadruplicate.

Virus replication assays. Cells were seeded into 12-well plates (1 x 105 per well) and infected at 70% to 80% confluency with different viruses, and the inoculum was removed after 2 hours and replaced with medium (DMEM/1% inactivated FCS). Cells and medium were harvested at the indicated times after infection, processed with three freeze/thaw cycles, sonicated, and titered on Vero cells. Experiments were repeated at least thrice with each condition in duplicate.

Migration assay. HUVECs (1 x 106/mL) were seeded into 0.45-µm pore Transwell inserts (Becton Dickinson, Franklin Lakes, NJ) in 45 µL. Conditioned medium from PBS-infected, bG47{Delta}-empty-infected, or bG47{Delta}-dnFGFR-infected U87 cells was collected and placed in culture wells. HUVECs migrated through the insert membrane were fixed, stained with Giemsa, and counted. Baseline numbers (i.e., in the absence of conditioned medium) were obtained and subtracted.

In vivo efficacy studies. 37-3-18-4 or U87 cells were implanted into the flanks of 6- to 8-week-old nu/nu mice (1 x 106 per implantation). Once tumor size reached 50 to 100 mm3, animals were randomized into three groups (n = 10 per group): PBS, bG47{Delta}-empty, or bG47{Delta}-dnFGFR. For 37-3-18-4 tumors, four intratumoral injections (three directions per injection) of virus (1 x 107 pfu/injection) or PBS were given every 3 days; for U87 tumors, two intratumoral injections of virus (1 x 106 pfu/injection) or PBS were given. Tumor volumes were monitored two to three times weekly.

Intratumoral biological end point studies. Mice with tumors (50-100 mm3) implanted as above were randomized into three groups (n = 7 per group): PBS, bG47{Delta}-empty, or bG47{Delta}-dnFGFR. Two intratumoral injections with 1 x 107 pfu/injection (37-3-18-4 tumors) or 5 x 105 pfu/injection (U87 tumors) were given on days 1 and 4. Animals were sacrificed at indicated time points, and tumors were harvested, snap frozen, and processed for immunohistochemistry.

Immunohistochemistry. Cryosections (5-7 µm) were obtained from snap-frozen tissues, fixed in cold methanol for 10 minutes, blocked with serum-free blocking agent (DAKO, Carpinteria, CA) for 10 minutes, quenched for endogenous hydroxide (DAKO) for 5 minutes, and incubated with primary antibody [rabbit anti-mouse von Willebrand factor (VWF); 1:100; DAKO] overnight. Sections were then washed, incubated with secondary antibody (donkey anti-rabbit; 1:500; Amersham), developed with 3,3'-diaminobenzidine substrate-chromagen system (DAKO), and counterstained with hematoxylin. VWF-positive structures were analyzed under light microscopy.

Microvessel counting. Areas of vascularization after VWF staining were randomly chosen for microvessel counting at a low optical power (40x). Microvessel counting was done on 200x fields (area of a 200x field, 0.724 mm2). The final microvessel density was the mean value of five individual, nonoverlapping fields in the area of vascularization.

Statistical analysis. Student's t test or one-way ANOVA was used for statistical analysis. Kaplan-Meier curves for survival were compared by log-rank tests.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
dnFGFR expression reduced tumor cell proliferation and migration. To investigate the effect of blocking FGF/FGFR signaling in tumor cells, murine MPNST 61E4 cells were stably transfected with dnFGFR. dnFGFR-expressing cells (61E4-dnFGFR) showed slower proliferation in vitro compared with the parental line transfected with a control plasmid Zeo (61E4-Zeo; P = 0.035; Fig. 1A ). In an in vitro wound-healing assay, dnFGFR expression decreased migration of cells into a scratch in a cell monolayer (Fig. 1B). The growth of tumor cells 61E4-Zeo and 61E4-dnFGFR was compared in vivo with two different models. We first tested tumor growth in a novel model on chicken chorioallantoic membrane that we recently described, which assays tumor growth and invasion in addition to angiogenesis (33). Control cells formed a homogeneous well-organized tumor mass with marked angiogenesis, whereas dnFGFR-expressing cells formed small tumors with reduced angiogenesis (Fig. 1C). One week after implanting the tumor cells onto chorioallantoic membrane, 61E4-dnFGFR tumors had significantly smaller volumes than the 61E4-Zeo tumors (P = 0.042; Fig. 1C and D). Furthermore, angiogenesis is reduced in 61E4-dnFGFR tumors by ~30% [vessel density, 100% (61E4-Zeo) versus 73% (61E4-dnFGFR)]. Finally, 61E4-dnFGFR s.c. tumors in nude mice grew more slowly than 61E4-Zeo tumors (P = 0.019 at day 16 and P = 0.021 at day 20; Fig. 1E). These inhibitory effects of dnFGFR in the transduced MPNST cells provided the basis for constructing an oncolytic HSV vector expressing this transgene.


Figure 1
View larger version (61K):
[in this window]
[in a new window]
  		Fig. 1. dnFGFR-transfected cell lines exhibited reduced proliferation in vitro and in vivo. Murine MPNST cell line 61E4 was transfected with dnFGFR cDNA, and the transfected cell line (61E4-dnFGFR) was compared with control cell line 61E4-Zeo. A, equal numbers of 61E4-dnFGFR and 61E4-Zeo cells were plated in dishes and counted after 48 hours at 37°C. Columns, mean (n = 3); bars, SD. *, P < 0.05. B, 61E4-dnFGFR (right) showed reduced migration compared with 61E4-Zeo (left) in a wound-healing assay. C, tumor growth of 61E4-dnFGFR (bottom) and 61E4-Zeo (top) 1 week after cell deposition on the chick chorioallantoic membrane when tumors were fixed. D, tumor volumes of 61E4-dnFGFR were significantly smaller than 61E4-Zeo (n = 6 per group) after 1 week of growth in the chick chorioallantoic membrane. *, P < 0.05. E, tumor growth in vivo in athymic mice after s.c. implantation of 104 cells (n = 4 per group). {circ}, 61E4-dnFGFR; bullet, 61E4-Zeo. *, P < 0.05.

 
Construction and characterization of bG47{Delta}-dnFGFR. We previously showed that G47{Delta}, a triple-mutated oncolytic HSV mutant, has enhanced antitumoral efficacy in neural tumors compared with its parental oncolytic virus (9). We hypothesized that arming G47{Delta} with the dnFGFR transgene would enhance efficacy. Using the Flip-Flop HSV-BAC system (14), we inserted the dnFGFR transgene and LacZ into the ICP6 region to generate bG47{Delta}-dnFGFR or LacZ alone to generate bG47{Delta}-empty (Fig. 2A ). Expression of dnFGFR in bG47{Delta}-dnFGFR-infected Vero cells lacking FGFR was confirmed by Western blot analysis (Fig. 2B).


Figure 2
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 2. bG47{Delta}-dnFGFR construction. A, schema of bG47{Delta}-dnFGFR and bG47{Delta}-empty structure. LacZ is driven by the HSV ICP6 promoter, whereas dnFGFR is driven by the CMVIE promoter. bG47{Delta}-empty is identical to bG47{Delta}-dnFGFR, except that the transgene is missing. Note that there are no BAC sequences present in the viruses. B, expression of dnFGFR was confirmed by Western blotting. Vero cells were mock infected (lane 1) or infected with bG47{Delta}-empty (lane 2) or bG47{Delta}-dnFGFR (lane 3). dnFGFR runs as three bands of approximately 80 to 100 kDa, whereas native FGFR1 present in uninfected 37-3-18-4 cells (lane 4) is ~130 kDa (27). The expression of HSV immediate-early ICP4 in lanes 2 and 3 shows that similar numbers of infected cells were loaded on the gel, and the actin levels show that similar amounts of protein were loaded in all the lanes.

 
bG47{Delta}-dnFGFR showed enhanced potency on tumor and vascular endothelial cells. Expression of dnFGFR enhanced the potency of bG47{Delta} in a variety of neural tumor and endothelial cells in vitro. In each of the human and murine cells tested, bG47{Delta}-dnFGFR had a lower ED50 value than bG47{Delta}-empty (i.e., more potent; P = 0.033 to P < 0.001; Fig. 3A ). The inhibitory effect of dnFGFR expression was larger in the endothelial cells. Therefore, bG47{Delta}-dnFGFR showed enhanced potency on both tumor and vascular endothelial cells in vitro.


Figure 3
View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. bG47{Delta}-dnFGFR showed enhanced cell killing, blocked FGF signaling, and enhanced endothelial cell migration inhibition. A, the ED50 values for bG47{Delta}-dnFGFR and bG47{Delta}-empty were determined in the cell lines indicated and the ratio was plotted. bG47{Delta}-dnFGFR showed significantly enhanced potency over bG47{Delta}-empty in all the tumor and endothelial cell lines (P < 0.05; n = 4). B, U87, 61E4, and 37-3-18-4 cells were treated with PBS, bG47{Delta}-empty, or bG47{Delta}-dnFGFR, and cell lysates were electrophoresed on SDS-PAGE, Western blotted, and probed with anti-phosphorylated ERK (Phospho-ERK) or total ERK. Lanes 1, 4, and 7, PBS treated; lanes 2, 5, and 8, bG47{Delta}-empty treated; lanes 3, 6, and 9, bG47{Delta}-dnFGFR treated. bG47{Delta}-dnFGFR infection blocked ERK phosphorylation, whereas bG47{Delta}-empty did not. C, bG47{Delta}-dnFGFR inhibits endothelial cell migration to a greater extent than bG47{Delta}-empty. Numbers of migrated HUVEC in the presence of virus or mock infected U87 conditioned medium in a Transwell assay were counted and compared (n = 3). *, P < 0.05; **, P < 0.001.

 
Expression of dnFGFR blocks FGFR signaling. Molecular pathways by which FGF signaling promotes angiogenesis have been studied (15, 35). Among those downstream targets, ERK in the mitogen-activated protein kinase pathway has been shown to play a critical role in migration and proliferation of endothelial cells (36). We therefore tested whether expression of dnFGFR by oncolytic HSV could block activation of FGFR signaling using phosphorylation of ERK as a readout. Indeed, bG47{Delta}-dnFGFR infection strongly inhibited ERK phosphorylation in all three cell lines tested (U87, 61E4, and 37-3-18-4), whereas bG47{Delta}-empty infection did not alter ERK phosphorylation compared with PBS treatment (Fig. 3B). Therefore, the expression of dnFGFR after bG47{Delta}-dnFGFR infection inhibits FGFR signaling.

Expression of dnFGFR inhibits migration of endothelial cells. The FGF/FGFR signaling pathway has been shown to play a critical role in angiogenesis, particularly for migration of vascular endothelial cells. We therefore tested if blocking FGF signaling would affect endothelial cell migration using a Transwell coculture system with conditioned medium from infected U87 cells. Conditioned medium from bG47{Delta}-empty-infected U87 cells significantly reduced the number of migrated HUVEC compared with mock-infected medium (P = 0.042; Fig. 3C); however, the number was further reduced with conditioned medium from bG47{Delta}-dnFGFR-infected U87 cells (P < 0.001; Fig. 3C). Therefore, dnFGFR expression in tumor cells significantly inhibits HUVEC migration.

dnFGFR expression did not inhibit viral replication. To investigate whether the expression of dnFGFR inhibits viral replication, cells were infected with 1 pfu/cell, and viral yield was determined at 24 and 48 hours after infection. As seen in Fig. 4A , bG47{Delta}-dnFGFR showed a comparable viral burst with bG47{Delta}-empty at both time points in all tumor and endothelial cell lines tested (P > 0.05). Of note, replication of both bG47{Delta}-empty and bG47{Delta}-dnFGFR was significantly lower in quiescent HUVEC compared with proliferating HUVEC (P = 0.005-0.017; Fig. 4A), suggesting that these viruses would not cause significant damage to normal vasculature as opposed to tumor vasculature. Virus replication and spread was also determined at a low multiplicity of infection (0.01 pfu/cell), which allows for multiple replication cycles. bG47{Delta}-dnFGFR showed comparable viral growth kinetics with bG47{Delta}-empty in both human U87 glioma and HUVEC (P > 0.05; Fig. 4B). Therefore, dnFGFR expression did not inhibit bG47{Delta} viral replication.


Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4. Expression of dnFGFR did not impede virus replication. A, after infection of cells (U87, F5, 37-3-18-4, and HUVEC) with 1 pfu/cell, single-step growth curves showed that the virus yield of bG47{Delta}-dnFGFR was similar to bG47{Delta}-empty. HUVECs were made quiescent by fluid shear stress. White columns, 24 hours after infection; black columns, 48 hours after infection (n = 3). B, when U87 (left) and HUVEC (right) were infected at low multiplicity of infection (0.01 pfu/cell), bG47{Delta}-dnFGFR had similar growth kinetics to bG47{Delta}-empty. bullet, bG47{Delta}-empty; {circ}, bG47{Delta}-dnFGFR.

 
Antitumoral efficacy of bG47{Delta}-dnFGFR. To examine the effects of dnFGFR expression on neural tumor growth in vivo, human U87 or murine 37-3-18-4 tumor cells were implanted into the flanks of nude mice, and intratumoral treatments were started when tumors grew to 50 to 100 mm3. In both tumor models, the bG47{Delta}-empty treatment group showed a significant delay in tumor growth compared with that of the PBS treatment group (P < 0.001; Fig. 5 ), as expected. However, tumor growth in the bG47{Delta}-dnFGFR treatment group was further inhibited (P < 0.001, compared with bG47{Delta}-empty; Fig. 5). Therefore, dnFGFR expression significantly enhanced the efficacy of G47{Delta} in both a human glioma and murine MPNST tumor model.


Figure 5
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 5. bG47{Delta}-dnFGFR has enhanced antitumoral efficacy in athymic mice. A, U87 s.c. tumors were inoculated with PBS or virus (1 x 106 pfu) on days 1 and 4. B, 37-3-18-4 s.c. tumors were inoculated with PBS or virus (1 x 107 pfu) on days 1, 4, 7, and 10. More virus was inoculated into the 37-3-18-4 tumors because these cells are less permissive to G47{Delta} replication than U87 (see Fig. 2B). bullet, PBS treatment groups; {circ}, bG47{Delta}-empty treatment groups; {blacktriangleup}, bG47{Delta}-dnFGFR treatment groups. *, P < 0.01 between PBS and bG47{Delta}-empty treatment groups and P < 0.05 between bG47{Delta}-empty and bG47{Delta}-dnFGFR treatment groups (n = 10).

 
In vivo biological end points. To explore the antiangiogenic effects of bG47{Delta}-empty and bG47{Delta}-dnFGFR vectors in vivo, tumor tissues were harvested 8 days after treatment and tumor vasculature was assessed by VWF staining (Fig. 6A ). Similar to the in vitro results, bG47{Delta}-empty treatment significantly reduced the number of VWF-positive structures compared with PBS treatment (P = 0.013; Fig. 6B). Importantly, dnFGFR expression further reduced the number (P < 0.001) so that there were >4-fold fewer vascular structures than in the mock-treated tumors (Fig. 6B). This large decrease in tumor vasculature likely plays an important role in the inhibition of tumor growth.


Figure 6
View larger version (60K):
[in this window]
[in a new window]
  		Fig. 6. bG47{Delta}-dnFGFR reduces tumor vasculature. A, representative fields of stained tumors from each treatment group. Arrows, VWF-positive structures are stained brown (10 x 20). B, microvessel density of U87 tumors harvested at day 8 after treatment. *, P < 0.05; **, P < 0.001.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Herein, we describe the effect of incorporating a therapeutic transgene, dnFGFR, which inhibits the FGF signaling pathway, into an oncolytic HSV vector. We first tansfected dnFGFR into mouse MPNST cell line 61E4 and showed that dnFGFR-transfected tumor cells had decreased proliferation and invasion in vitro and reduced growth in vivo in two tumor models. An oncolytic HSV vector expressing dnFGFR was then constructed. dnFGFR expression from G47{Delta} greatly enhanced potency in both neural tumor and proliferating endothelial cells in vitro. In addition to direct oncolysis, the expression of dnFGFR blocks the FGF autocrine/paracrine loop, thereby enhancing the antitumoral effect. The autocrine effects were apparent in human glioma and malignant meningioma cells, murine MPNST cells isolated from spontaneously arising tumors in Nf1+/–:p53+/– mice, and human and murine proliferating endothelial cells. Paracrine effects were shown by the ability of conditioned medium from bG47{Delta}-dnFGFR-infected U87 cells to inhibit the migration of HUVECs. In vivo, bG47{Delta}-dnFGFR treatment significantly inhibited the growth and angiogenesis of human glioma (U87) and murine MPNST (37-3-18-4) tumors in nude mice compared with bG47{Delta}-empty.

The selectivity of the currently tested oncolytic HSV mutants is based in part on defective IFN signaling and mutant complementation with cellular GADD34 and/or RR. Although this provides for sufficient selectivity and safety, the genetic manipulations also reduce the potency of the virus. To counteract the reduced growth properties of {gamma}34.5 mutants, a suppressor mutation can be introduced that places the late Us11 gene under the control of the immediate-early {alpha}47 promoter, as in G47{Delta} (9), or {gamma}34.5 can be expressed under the control of a tumor-specific promoter, as in Myb34.5 (37), or tumor cells can be treated to induce GADD34 or RR expression, as occurs after temezolomide treatment (38). Another approach is to insert therapeutic transgene(s) that will enhance antitumoral potency independent of the viral mutants without losing replication efficiency. This study used a combination of both approaches by engineering dnFGFR into G47{Delta} and showing that this indeed enhanced the efficacy.

Although replication-deficient adenovirus vectors have been used to deliver dnFGFR to tumor cells (27, 39), this is the first report of an oncolytic virus (adenovirus, HSV, or otherwise) targeting FGF signaling. One potential limitation of the current approach would be the reduction/limitation in viral replication due to decreased cell proliferation. However, the expression of dnFGFR does not affect viral yield. That G47{Delta} replicates equally well with dnFGFR suggests that FGF signaling pathways are not critical to providing tumor cell permissivity/selectivity for oncolytic HSV. It has been reported that activation of the Ras pathway, one of the downstream targets of FGFR, is important in enabling oncolytic HSV vectors with {gamma}34.5 mutations to replicate (40). It is therefore critical to determine if strategies targeting multiple levels of FGF signaling pathway will enhance therapeutic effect. Indeed, the MPNST 37-3-18-4 cells have constitutive Ras activation due to the loss of Nf1 and G47{Delta} efficacy was further enhanced by blocking FGFR signaling.

Our data indicated that inhibition of FGF activity by dnFGFR can disrupt both angiogenesis-dependent and angiogenesis-independent pathways required for tumor growth and invasion, consistent with previous studies (24). Furthermore, as the FGF signaling pathway is critical to tumor growth and angiogenesis, the enhanced potency should be seen in both FGF-secreting and FGF-nonsecreting tumors, thus broadening the application of the vector. Of note, our data support previous publications that oncolytic HSV mutants with deletions in {gamma}34.5 and/or ICP6 have antiangiogenic activities by themselves, inhibiting capillary tube formation in vitro and in vivo and replicating in tumor endothelial cells but not in normal vasculature in vivo (41, 42). We showed that G47{Delta} is a potent antiangiogenic virus; it significantly reduced endothelial cell proliferation by direct cell killing as well as HUVEC migration in a Transwell coculture system, presumably due to a reduction in angiogenic factors secreted by infected tumor cells. Expression of dnFGFR further enhanced this effect, showing that different mechanisms work cooperatively in inhibiting angiogenesis.

The FGF signaling pathway is complex and involves multiple molecules and interactions with other signaling pathways (18). The approach taken in this study is to block FGF signaling, whether because of mutant FGFR or elevated FGF, at the receptor level. It will be interesting to see whether blocking the pathway at other checkpoints will result in a similar effect. In addition, apart from the FGF/FGFR pathway, other signaling pathways are implicated in tumorigenesis and/or angiogenesis (vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor signaling pathways, etc.). The approach taken in this study can also be applied to these pathways. Of note, it has been shown that tumors are able to use different angiogenic pathways and may switch, for example, from the vascular endothelial growth factor pathway to FGF (43). The switch from vascular endothelial growth factor to FGF after vascular endothelial growth factor inhibition has been experimentally shown but never the opposite. Although tumors often use multiple redundant angiogenic pathways and treatment might select for FGF-independent tumor cells, the endothelial cells are less likely to loose responsiveness to FGF. Because oncolytic HSV vectors can accommodate large and/or multiple transgenes, construction of vectors that express transgenes targeting multiple pathways is possible. As tumors evolve, they secrete a variety of angiogenic factors (44, 45); targeting these pathways should enhance the therapeutic efficacy of oncolytic HSV and also benefit late-stage cancer patients (46).

These studies indicate that dnFGFR is an excellent therapeutic transgene for oncolytic vectors, affecting not only infected tumor and endothelial cells but also the surrounding environment. Therefore, even tumors that do not support robust oncolytic virus replication, such as the mouse MPNST cells, can be effectively treated. This strategy should be applicable to other oncolytic viruses and considered for clinical translation.


   Acknowledgments
 
We thank Drs. Luis Parada, Anil Menon, and Victoria Bautch for providing cells; Dr. Lewis Williams for providing cDNA; Dr. Pedro Lowenstein for providing plasmid; and Dr. Pamela Maher for providing the antibody.


   Footnotes
 
Grant support: U.S. Department of Defense grants DAMD17-02-1-0648 (A. Kurtz and S.D. Rabkin) and W81XWH-04-1-0237 (R.L. Martuza) and NIH grant RO1 NS032677 (R.L. Martuza).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A. Kurtz and S.D. Rabkin contributed equally to this work. Current address for T. Zhang: Department of Pathology, Shandong University Hospital, Jinan, China; H. Fukuhara: Department of Urology, University of Tokyo, Tokyo, Japan; T. Todo: Department of Neurosurgery, University of Tokyo, Tokyo, Japan; A. Kurtz: Robert Koch-Institute, Berlin, Germany.

Received 2/ 3/06; revised 7/28/06; accepted 8/15/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kirn D, Martuza RL, Zwiebel J. Replication-selective virotherapy for cancer: biological principles, risk management, and future directions. Nat Med 2001;7:781–7.[CrossRef][Medline]
  2. Mineta T, Rabkin SD, Yazaki T, Hunter WD, Martuza RL. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat Med 1995;1:938–43.[CrossRef][Medline]
  3. Markert JM, Medlock MD, Rabkin SD, et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther 2000;7:867–74.[CrossRef][Medline]
  4. Varghese S, Rabkin SD. Oncolytic herpes simplex virus vectors for cancer virotherapy. Cancer Gene Ther 2002;9:967–78.[CrossRef][Medline]
  5. Rampling R, Cruickshank G, Papanastassiou V, et al. Toxicity evaluation of replication-competent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma. Gene Ther 2000;7:859–66.[CrossRef][Medline]
  6. Harrow S, Papanastassiou V, Harland J, et al. HSV1716 injection into the brain adjacent to tumour following surgical resection of high-grade glioma: safety data and long-term survival. Gene Ther 2004;11:1648–58.[CrossRef][Medline]
  7. MacKie RM, Stewart B, Brown SM. Intralesional injection of herpes simplex virus 1716 in metastatic melanoma. Lancet 2001;357:525–6.[CrossRef][Medline]
  8. Aghi M, Martuza RL. Oncolytic viral therapies—the clinical experience. Oncogene 2005;24:7802–16.[CrossRef][Medline]
  9. Todo T, Martuza RL, Rabkin SD, Johnson PA. Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing. Proc Natl Acad Sci U S A 2001;98:6396–401.[Abstract/Free Full Text]
  10. Aghi M, Chou TC, Suling K, Breakefield XO, Chiocca EA. Multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine/rat cytochrome P450 2B1 and ganciclovir/herpes simplex virus thymidine kinase gene therapies. Cancer Res 1999;59:3861–5.[Abstract/Free Full Text]
  11. Nakamura H, Mullen JT, Chandrasekhar S, Pawlik TM, Yoon SS, Tanabe KK. Multimodality therapy with a replication-conditional herpes simplex virus 1 mutant that expresses yeast cytosine deaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil. Cancer Res 2001;61:5447–52.[Abstract/Free Full Text]
  12. Liu BL, Robinson M, Han ZQ, et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther 2003;10:292–303.[CrossRef][Medline]
  13. Wong RJ, Patel SG, Kim S, et al. Cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma. Hum Gene Ther 2001;12:253–65.[CrossRef][Medline]
  14. Fukuhara H, Ino Y, Kuroda T, Martuza RL, Todo T. Triple gene-deleted oncolytic herpes simplex virus vector double-armed with interleukin 18 and soluble b7-1 constructed by bacterial artificial chromosome-mediated system. Cancer Res 2005;65:10663–8.[Abstract/Free Full Text]
  15. Grose R, Dickson C. Fibroblast growth factor signaling in tumorigenesis. Cytokine Growth Factor Rev 2005;16:179–86.[CrossRef][Medline]
  16. Compagni A, Wilgenbus P, Impagnatiello MA, Cotten M, Christofori G. Fibroblast growth factors are required for efficient tumor angiogenesis. Cancer Res 2000;60:7163–9.[Abstract/Free Full Text]
  17. Auguste P, Javerzat S, Bikfalvi A. Regulation of vascular development by fibroblast growth factors. Cell Tissue Res 2003;314:157–66.[CrossRef][Medline]
  18. Wiedlocha A, Sorensen V. Signaling, internalization, and intracellular activity of fibroblast growth factor. Curr Top Microbiol Immunol 2004;286:45–79.[Medline]
  19. Morrison RS, Yamaguchi F, Saya H, et al. Basic fibroblast growth factor and fibroblast growth factor receptor I are implicated in the growth of human astrocytomas. J Neurooncol 1994;18:207–16.[CrossRef][Medline]
  20. Zumkeller W, Schofield PN. Growth factors, cytokines, and soluble forms of receptor molecules in cancer patients. Anticancer Res 1995;15:343–8.[Medline]
  21. Bello L, Giussani C, Carrabba G, Pluderi M, Costa F, Bikfalvi A. Angiogenesis and invasion in gliomas. Cancer Treat Res 2004;117:263–84.[Medline]
  22. Mashour GA, Ratner N, Khan GA, Wang HL, Martuza RL, Kurtz A. The angiogenic factor midkine is aberrantly expressed in NF1-deficient Schwann cells and is a mitogen for neurofibroma-derived cells. Oncogene 2001;20:97–105.[CrossRef][Medline]
  23. Wang W, Zhu NL, Chua J, et al. Retargeting of adenoviral vector using basic fibroblast growth factor ligand for malignant glioma gene therapy. J Neurosurg 2005;103:1058–66.[Medline]
  24. Auguste P, Gursel DB, Lemiere S, et al. Inhibition of fibroblast growth factor/fibroblast growth factor receptor activity in glioma cells impedes tumor growth by both angiogenesis-dependent and -independent mechanisms. Cancer Res 2001;61:1717–26.[Abstract/Free Full Text]
  25. Aoki T, Kato S, Fox JC, et al. Inhibition of autocrine fibroblast growth factor signaling by the adenovirus-mediated expression of an antisense transgene or a dominant negative receptor in human glioma cells in vitro. Int J Oncol 2002;21:629–36.[Medline]
  26. Lappi DA. Tumor targeting through fibroblast growth factor receptors. Semin Cancer Biol 1995;6:279–88.[CrossRef][Medline]
  27. Saiki M, Mima T, Takahashi JC, et al. Adenovirus-mediated gene transfer of a truncated form of fibroblast growth factor receptor inhibits growth of glioma cells both in vitro and in vivo. J Neurooncol 1999;44:195–203.[CrossRef][Medline]
  28. Werner S, Weinberg W, Liao X, et al. Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation. EMBO J 1993;12:2635–43.[Medline]
  29. Yazaki T, Manz HJ, Rabkin SD, Martuza RL. Treatment of human malignant meningiomas by G207, a replication-competent multimutated herpes simplex virus 1. Cancer Res 1995;55:4752–6.[Abstract/Free Full Text]
  30. Vogel KS, Klesse LJ, Velasco-Miguel S, Meyers K, Rushing EJ, Parada LF. Mouse tumor model for neurofibromatosis type 1. Science 1999;286:2176–9.[Abstract/Free Full Text]
  31. Dubois NA, Kolpack LC, Wang R, Azizkhan RG, Bautch VL. Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas. Exp Cell Res 1991;196:302–13.[CrossRef][Medline]
  32. Hagedorn M, Zilberberg L, Wilting J, et al. Domain swapping in a COOH-terminal fragment of platelet factor 4 generates potent angiogenesis inhibitors. Cancer Res 2002;62:6884–90.[Abstract/Free Full Text]
  33. Hagedorn M, Javerzat S, Gilges D, et al. Accessing key steps of human tumor progression in vivo by using an avian embryo model. Proc Natl Acad Sci U S A 2005;102:1643–8.[Abstract/Free Full Text]
  34. Umana P, Gerdes CA, Stone D, et al. Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination. Nat Biotechnol 2001;19:582–5.[CrossRef][Medline]
  35. Hanneken A, Maher PA, Baird A. High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells—implications for the modulation of FGF-2. J Cell Biol 1995;128:1221–8.[Abstract/Free Full Text]
  36. Javerzat S, Auguste P, Bikfalvi A. The role of fibroblast growth factors in vascular development. Trends Mol Med 2002;8:483–9.[CrossRef][Medline]
  37. Chung RY, Saeki Y, Chiocca EA. B-myb promoter retargeting of herpes simplex virus {gamma}34.5 gene-mediated virulence toward tumor and cycling cells. J Virol 1999;73:7556–64.[Abstract/Free Full Text]
  38. Aghi M, Rabkin S, Martuza RL. Effect of chemotherapy-induced DNA repair on oncolytic herpes simplex viral replication. J Natl Cancer Inst 2006;98:38–50.[Abstract/Free Full Text]
  39. Ozen M, Giri D, Ropiquet F, Mansukhani A, Ittmann M. Role of fibroblast growth factor receptor signaling in prostate cancer cell survival. J Natl Cancer Inst 2001;93:1783–90.[Abstract/Free Full Text]
  40. Farassati F, Yang AD, Lee PW. Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1. Nat Cell Biol 2001;3:745–50.[CrossRef][Medline]
  41. Cinatl JJ, Michaelis M, Driever P, et al. Multimutated herpes simplex virus g207 is a potent inhibitor of angiogenesis. Neoplasia 2004;6:725–35.[CrossRef][Medline]
  42. Benencia F, Courreges MC, Conejo-Garcia JR, et al. Oncolytic HSV exerts direct antiangiogenic activity in ovarian carcinoma. Hum Gene Ther 2005;16:765–78.[CrossRef][Medline]
  43. Casanovas O, Hicklin DJ, Bergers G, Hanahan D. Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 2005;8:299–309.[CrossRef][Medline]
  44. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249–57.[CrossRef][Medline]
  45. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 2005;23:1011–27.[Abstract/Free Full Text]
  46. Kong HL, Crystal RG. Gene therapy strategies for tumor antiangiogenesis. J Natl Cancer Inst 1998;90:273–86.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
H. Wakimoto, S. Kesari, C. J. Farrell, W. T. Curry Jr., C. Zaupa, M. Aghi, T. Kuroda, A. Stemmer-Rachamimov, K. Shah, T.-C. Liu, et al.
Human Glioblastoma-Derived Cancer Stem Cells: Establishment of Invasive Glioma Models and Treatment with Oncolytic Herpes Simplex Virus Vectors
Cancer Res., April 15, 2009; 69(8): 3472 - 3481.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, T.-C.
Right arrow Articles by Rabkin, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, T.-C.
Right arrow Articles by Rabkin, S. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online