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Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery [W. A., E. C., M. W., D. T. C., J. G. N.], Departments of Pathology, Cell Biology and Surgery [G. P. S.], Department of Obstetrics and Gynecology [R. D. A.], and the Gene Therapy Center [W. A., M. W., D. T. C., J. G. N., G. P. S., R. D. A.], University of Alabama at Birmingham, Birmingham, AL 35294; and Department of Molecular Genetics and Biochemistry, University of Pittsburgh, PA 15261 [J. C. G.]
ABSTRACT
We and others have proposed mammalian cells as gene delivery vehicles with the potential for overcoming physiological barriers to viral vectors. To that end, we previously have shown the potential of CD34+ endothelial progenitors for systemic gene delivery in a primate angiogenesis model. Here we seek to explore the utility of CD34+ cells of human origin as vehicles for toxin genes and, in particular, to measure their capacity to effect a cytotoxic bystander effect in human endothelium and tumor cells. To this end, CD34+ cells were transduced with TOZ.1, a nonreplicative herpes simplex vector encoding thymidine kinase. To test the capacity of CD34+ cells to induce a cytotoxic bystander effect in target cells, we performed mixing experiments, whereby TOZ.1-transduced CD34+ cells were mixed with either human vascular endothelial cells or human ovarian tumor cells (SKOV3.ip1). Cell viability was measured by the MTS assay. Lastly, mixtures of TOZ.1-transduced CD34+ cells and SKOV3.ip1 tumor cells were injected s.c. to evaluate the bystander effect in vivo. After transduction of CD34+ cells with TOZ.1, treatment with ganciclovir induced the killing of 99% of cells. In cell-mixing experiments, a linear correlation was observed between the percentages of TOZ.1-transduced CD34+ cells and total cell killing. For example, when 50% of CD34+ transduced cells were mixed with nontransduced SKOV3.ip1, >70% of all cells died. Similarly, when the same percentage was mixed with human vascular endothelial cells, >80% of the total number of cells died. In vivo studies showed an abrogation of tumor formation when TOZ.1-transduced CD34+ cells and ganciclovir were administered. Our observations establish the feasibility of a method for cell-based toxin gene delivery into disseminated areas of tumor angiogenesis.
INTRODUCTION
A number of strategies have been developed to accomplish cancer gene therapy. One of these is molecular chemotherapy. Although similar in principle to conventional chemotherapeutic interventions for cancer, molecular chemotherapy is designed to achieve selective expression of a toxin gene in the tumor site by transferring the corresponding DNA. Typically, the approach involves both the transfer into the tumor cells of a gene encoding an enzyme and the administration to the patient of an inactive, innocuous prodrug. When the delivered enzyme is expressed, it transforms the prodrug into a cytotoxic metabolite. Purportedly this happens exclusively within the genetically modified tumor cell, thus establishing the basis for a favorable therapeutic index and for a definite advantage with respect to conventional chemotherapy (1) .
Fundamental to the clinical implementation of this strategy in patients with advanced cancer is the development of a vector system that allows direct, extensive, and efficient in vivo gene transfer into disseminated tumors. In this regard, both viral and nonviral vectors previously have been used clinically for the treatment of cancer, mostly in a loco-regional context. Among the vector systems that have been used successfully are viruses (retrovirus, adenovirus, and adeno-associated virus, poxvirus, herpesvirus), naked DNA, and lipids. In general, despite their utility in selected applications, these vectors have been limited in their ability to accomplish highly efficient gene delivery to target cells in clinically relevant models (1) . Furthermore, for disseminated cancer, vectors with consistent capacities for systemic gene delivery have not been available (2) . Thus, shortcomings in the present generation of vectors have limited the overall efficacy and implementation of gene-based molecular chemotherapy for advanced cancer.
As an alternative to these aforementioned vector approaches, cells have also been used as vectors. In this approach, cells are removed from the body, and therapeutic genes are transferred to the cells extracorporally, followed by their re-implantation back into the patient. In this manner, the genetically modified cell itself becomes the ultimate vector for gene delivery. For example, the pioneering clinical use of primary cells as so-called "cellular vehicles" is based on T lymphocytes (3, 4, 5, 6) . Conceivably, a variety of genes can be introduced in candidate cellular vehicles, and expression of these payloads can be obtained in an environment where these cells localize. A loco-regional effect subsequent to the expression of the therapeutic gene can thus be achieved in areas otherwise inaccessible to direct gene transfer. In this regard, our group has shown that mature human endothelial cells can be used in vivo in a murine model as cellular vehicles to deliver a toxin gene regionally and induce cytotoxicity in i.p. ovarian cancer cells (7) .
For the clinical application of cellular vehicles in the context of disseminated malignant disease, however, a cellular vector should have the attributes of systemic distribution, adequate tumor tropism, ready availability, and lack of immunogenicity. Interestingly, a novel role has been described recently for stem cells, known as EPCs,2 obtained from the bone marrow, peripheral circulation, or cord blood (8, 9, 10) . Phenotypically, these cells are characterized by the expression of the cellular surface markers CD34 and KDR/Flk-1 (8 , 9) , which is a receptor for vascular endothelial growth factor. Another possible marker described recently is AC133 (11) . A very intriguing aspect of the EPCs’ behavior, originally described in animal models of limb ischemia, is their capacity to localize into areas of angiogenesis after their systemic administration (8) . Thus, endothelial progenitors might be further developed as a novel cellular vector with unique features, based on their capacity for systemic circulation and natural tropism to areas of active angiogenesis. Previously, we have demonstrated the ability of genetically modified CD34+ cells to localize into areas of angiogenesis in a nonhuman primate skin model (12) . Furthermore, in rhesus animals infused with autologous CD34+ EPCs transduced with a TK-encoding herpes virus, skin autografts underwent necrosis or accelerated regression after administration of GCV (12) . Thus, EPCs not only localize into areas of angiogenesis but are also able to express a therapeutic gene and induce therein a meaningful biological effect.
In this report, we further explore the capacity of human CD34+ EPCs to serve as vehicles for toxin gene delivery into the tumor vasculature and their capacity to exert an effective cytotoxic bystander effect over human tumor and primary endothelial cells.
MATERIALS AND METHODS
Cells.
The human ovarian carcinoma cell line SKOV3.ip1 was a kind gift
from Janet Price (University of Texas M.D. Anderson Cancer Center,
Houston, TX). Cells were maintained in DMEM:F12 [50:50 (v/v)
mixture; Cellgro, Herndon, VA] supplemented with
L-glutamine (2 µM), penicillin (100 mg/ml),
streptomycin (25 mg/ml), and 10% heat-inactivated fetal bovine
serum (Hyclone Laboratories, Logan, UT) at 37°C in a
humidified, 5% CO2 atmosphere.
HUVECs were obtained from the laboratory of F. M. Booyse (University of Alabama at Birmingham, Birmingham, AL) from freshly obtained umbilical cords, as described previously (13) . Cells were grown on 1% gelatin-coated flasks with Medium 199 (Cellgro, Herndon, VA) supplemented with L-glutamine (200 mg/ml), penicillin (100 mg/ml), streptomycin (25 mg/ml), heparin sodium (10 units/ml; Elikins-Sinn, Incorporate, Cherry Hill, NJ), endothelial mitogen (0.1 mg/ml; Biomedical Technologies, Stoughton, MA), and 10% FBS (Hyclone Laboratories, Logan UT). The medium was changed every 3 days, and the cells were maintained at 37°C in a humidified, 5% CO2 atmosphere.
Human Normal CD34+ Cells.
Human peripheral blood mononuclear cells were obtained by leukapheresis
of a normal donor after approval by the Institutional Review Board at
University of Alabama at Birmingham. Mobilization was performed by
treatment with 15 µg/kg/day of granulocyte colony-stimulating
factor for 5 consecutive days. Human CD34+ cells were purified
to >95% purity by immunoadsorption (Systemix, Palo Alto, CA). Cells
were then frozen at -70°C until use. CD34+
cells were cultured in StemPro-34 SFM Complete Medium supplemented with
StemPro-34 Nutrient Supplement (Life Technologies, Inc., Gaithersburg,
MD), 2 mM L-glutamine, interleukin 3 (5 ng/ml),
interleukin 6 (10 ng/ml), and stem cell factor (25 ng/ml; all from R&D
Systems, Minneapolis, MN).
Recombinant Herpes Simplex Vectors and Cell Infection.
For genetic modification of CD34+ cells, we used
the nonreplicative herpes virus vector TOZ.1, which is deleted of the
genes ICP4, ICP22, ICP27, and
UL41, and encodes the reporter gene lacZ.
As a control, we used a similar vector lacking the lacZ
gene, T3. Both vectors encode the HSV-TK gene. The
construction and preparation of these vectors, derived from a KOS
strain mutant, has been described elsewhere (14)
. Viral
stocks were purified by centrifugation in OptiPrep (Life Technologies,
Inc.), and titered using 7B cells, which express both the
ICP4 and ICP27 genes.
Before infection, cells were washed twice with PBS and resuspended within microcentrifuge tubes with appropriate cell-specific media. The suspended cells were incubated with HSV vectors using MOI doses of 0.3, 3, and 10 plaque-forming units, with gentle rocking for 2 h at 37°C.
Detection of Transduction Efficiency by Flow Cytometry Analysis of
ß-Galactosidase Activity
Aliquots of transduced cells or peripheral blood mononuclear cells were
stained with antihuman CD34+-biotin antibody
(Cellpro, Bothell, WA) or an irrelevant antibody conjugated with
streptavidin-PerCP (Becton Dickinson, San Jose, CA). After 20 min of
incubation in the dark, the cells were washed with PBS and then stained
with FDG (Sigma, St. Louis, MO) by the FACS-gal technique of Fiering
et al. (15)
. Briefly, after incubation
in a 37°C water bath for 5 min, 50 µl of distilled water containing
2 mM FDG were mixed thoroughly with an equal
volume of cells suspended in staining medium [10
mM HEPES, and 4% FCS in 1x PBS (pH 7.3)]. The
cells were then returned to the water bath for exactly 1 min. FDG cell
loading was stopped by adding 500 µl of ice-cold staining medium. The
cells were maintained on ice until FACS analysis was performed within
the following 30 min. Cells not incubated with FDG served as the
negative control. Flow cytometry data acquisition was performed using a
FACS caliber flow cytometer (Becton Dickinson, Mountain View, CA), and
analysis was supported by Cell Quest software.
In Vitro Analysis of the Cytotoxic Bystander Effect.
Cell mixing experiments were performed to assess for a bystander
effect, as described previously (16)
. Briefly,
CD34+ cells were transduced with the recombinant
herpes virus TOZ.1 and then mixed at different ratios with
non-HSV-infected cells (SKOV3.ip1 and HUVECs). As a control,
TOZ.1-transduced SKOV3.ip1 cells were mixed with nontransduced
SKOV3.ip1. The ratios of TOZ.1-infected cells to uninfected cells were
as follows: 0:100, 10:90, 50:50, 90:10, and 100:0. Cell mixtures were
plated in triplicate in 96-well plates at a total density of 20,000
cells/well. Twenty-four h later, half of the samples were treated with
GCV (20 mM). Cell viability was determined 6 days later by
a colorimetric cell proliferation assay measuring the conversion of a
tetrazolium salt to formazan by viable cells, as described by the
manufacturer (MTS assay; Cell Titer 96 Aqueous Nonradioactive Cell
Proliferation Assay; Promega, Madison, WI). To this end, the
plates were incubated for 4 h at 37°C after the addition of 20
µl of assay solution. Subsequently, the absorbance was measured at
490 nm by an ELISA reader (Molecular Devices, Menlo Park, CA). To
quantify the cell numbers, a standard curve was created by plating
nontreated cells in triplicate wells at the following concentrations:
0; 2,000; 10,000; 20,000; and 50,000 cells/well. The total numbers of
cells in the experimental groups were calculated based on the standard
curve, using SOFT max computer software (Molecular Devices).
In Vivo Analysis of the Cytotoxic Bystander Effect.
To study the ability of the cytotoxic bystander effect to inhibit tumor
growth, female nude mice (ages 6–8 weeks; obtained from the National
Cancer Program, Frederic, MD) were treated as follows. Animals
(n = 24) received injections in each flank of a mixture
of tumor and CD34+ cells (four s.c.
injections/mouse), including (a)
CD34+/TK+
(CD34+ cells transduced with HSV-TK) mixed with
ovarian cancer SKOV3.ip1 cells; (b)
CD34+/TK- (nontransduced)
mixed with SKOV3.ip1 cells; or (c) SKOV3.ip1 alone. Each
animal received a total of 2.5 x 106 tumor
cells/nodule. The CD34+ cells had been infected
in vitro with the recombinant herpes virus TOZ.1 at a MOI of
10 plaque-forming units. Two h post infection, the cells were washed
twice and resuspended with PBS. The infected
CD34+ cells were then mixed with SKOV3.ip1 cells
at a 1:1 ratio (2.5 x 106
CD34+ and 2.5 x 106
SKOV3.ip1 cells), and injected s.c. into the
CD34+/TK+ and
CD34+/TK- groups.
Twenty-four h after injection of the cellular mixture, half of the animals were started on a treatment of daily i.p. injections of GCV (50 mg/kg) for 10 days. Tumor volumes were measured every 3 days during treatment for 1 month, using the formula: volume = (width x height x diameter)/2. Animal experiments were performed according to institutional guidelines and under the supervision of the Animal Resources Program at the University of Alabama at Birmingham.
RESULTS
Our molecular chemotherapy approach is predicated upon the capacity of the proposed cellular vehicles to induce cytotoxicity, mediated by the TK gene, within angiogenic vessels and in the immediate vicinity of surrounding tumor deposits. Testing this feature amounts to determining the potency of the toxin gene as delivered by the cellular vehicle on bystander human endothelial and tumor cells.
A Recombinant HSV Vector Efficiently Transduces Human CD34 Cells.
To determine the efficiency of transduction of human
CD34+ cells by a herpes virus vector,
CD34+ cells were incubated with various MOI doses
(0, 0.3, 3, or 10) of TOZ.1, a nonreplicative, recombinant
lacZ gene-encoding HSV. Twelve h later, FACS analysis was
carried out using FDG staining. More than 95% of cells were positive
in this analysis with a MOI of 3 (Fig. 1
A). Fewer than 1% of cells
were positive when infected with a control HSV vector. Additionally,
TOZ.1-infected cells developed an intense blue pigmentation after
staining with X-gal, confirming that CD34+ cells
can be efficiently transduced with a herpes virus vector (data not
shown).
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B). Of note, GCV had no demonstrable toxic
effect on uninfected CD34+ cells at the dose
tested (data not shown).
TOZ.1-transduced Human CD34+ Cells Can Elicit a
Bystander Effect on Human Cells.
Fundamental to the efficiency of the molecular chemotherapy strategy is
the bystander effect, whereby toxin gene-expressing cells exert a
noxious effect on surrounding untransduced cells. We, thus,
investigated whether the HSV-TK-expressing CD34+
cells could functionally accomplish a bystander effect with a series of
human ovarian carcinoma cell lines and with human endothelial cells
(HUVECs) in vitro. A mixing experiment was performed in
which TOZ.1-transduced CD34+ cells were mixed at
various ratios with nontransduced CD34+ cells,
human ovarian cancer cells (SKOV3.ip1), and HUVECs. GCV-mediated
killing was measured using a MTS assay. Studies were performed with
CD34+ cells to determine whether they could
induce bystander cytotoxicity into homologous, nonadherent cells. In
effect, as few as 10% of HSV-TK-transduced CD34+
cells achieved killing of 
60% of the total population of
CD34+ cells (Fig. 2
A). A similar effect was
observed when CD34+ cells were mixed with ovarian
cancer SKOV3.ip1 cells (Fig. 2
B). Lastly, when 50% of
HSV-TK-transduced CD34+ cells were mixed with
HUVECs, >80% of the total number of cells died (Fig. 2
C).
Of note, CD34+ cells remained in the dish mostly
as unattached, floating cells, which restricted the contact of
HSV-TK-transduced CD34+ cells over other target
cells. Interestingly, the highest levels of bystander effect were
observed when the cell mixtures were incubated with gentle rocking,
suggesting the importance of intercellular contact between the
suspended CD34+ cells and the other cell types.
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A). After 6 weeks of
observation, nodules were still visible in animals not receiving GCV
and in those treated with nonmodified CD34+ cells
(Fig. 3
B). However, no nodules were detected in the animals
that received HSV-TK-transduced CD34+ cells and
treatment with GCV (Fig. 3
B). Thus, genetically
modified human CD34+ cells can effectively induce
a toxic bystander effect in murine endothelium and human cancer cells
in vivo.
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Our ultimate goal is to design a viable vector system that allows systemic gene delivery into multiple areas of tumor angiogenesis. To this end, we report here on our further attempts to develop an autologous cellular vehicle based on human EPCs, which purportedly localize into areas of angiogenesis. A basic requirement for our proposed cellular system is the ability to determine an antitumoral effect by affecting cells in the vicinity via expression of an exogenous therapeutic gene, i.e., to induce a bystander effect.
As a first iteration, we explored the utility of human CD34+ cells genetically modified with the TK gene from HSV. The use of human CD34+ cells, which are typically hard to transduce at high levels with the vectors available at present, places a high level of stringency on this approach, which we wanted given our orientation toward therapeutic applicability. In this regard, we consider the use of autologous cells a potentially critical advantage of our strategy to assure safety and a long enough survival of the cell vehicles (17) . The other determinant of the efficacy of our proposed utilization of cellular vehicles for inducing tumor regression is the potency for exerting cytotoxicity in surrounding tumor cells, host cells, and the stroma, including the vasculature. Indeed, we have found that the genetically modified cells themselves are sensitive to the toxic effect of GCV and that they exert a bystander cytotoxic effect on both adjacent tumor cells and human endothelium. Using the strategy of cell-based induction of cytotoxicity, we have accomplished a killing effect on tumor cells comparable to that obtained by direct viral vector-mediated toxin gene delivery. In addition, we have observed a comparable toxin sensitivity in primary human endothelial cells, which suggests that the bystander effect similarly applies to both cell types, arguably the key targets for our cytotoxic intervention. Thus, we have advanced the evaluation of our system as regards to the capacities of the proposed cellular substrate, i.e., human CD34+ cells.
In our previous studies, we established the initial proof-of-concept with respect to the strategy of using circulating EPCs as cellular vehicles for systemic gene delivery (12) . The initial goal was to develop a vehicle able to systemically reach multiple disseminated areas of angiogenesis. To this end, we sought to exploit the natural tropism of cells recently described as EPCs and existent within the CD34+ population of mononuclear cells from mobilized peripheral blood (8 , 9) . In this regard, multiple studies in embryos and a variety of transplantation studies have supported the existence of EPCs, i.e., cells localized in the bone marrow that would have the capacity to migrate, proliferate, and differentiate into mature endothelial cells. Additional recent evidence suggests that these cells can be characterized by the expression of the immunophenotypic marker AC133 within the CD34+, FLK-1+ population (11) . Although we did not select any subpopulation of cells among those positive for CD34, the obtainment of very high levels of gene transfer argues for the genetic modification of a significant fraction from the general population. Our previous results in the rhesus model support this hypothesis. Eventually, the optimal cell subpopulation, endowed with a more specific angiogenic tropism behavior, will be selected by flow cytometry, genetically modified, and used for systemic gene delivery. Thus, we contend that ultimately, a defined subset of circulating human cells can be conveniently exploitable as "Trojan horses" targeted into areas of angiogenesis.
A requisite property of our proposed cellular vehicle in its current TK gene configuration, however, is to exert a potent enough cytotoxic bystander effect. Although our results in the rhesus model suggest that such an effect occurred, as did the dramatic toxic effect induced in skin grafts with only a limited number of modified EPCs detected in situ, we sought here to formally examine this hypothesis. We have here demonstrated that the transduced CD34+ cells express the TK gene with high efficiency and, thus, become sensitive to the effect of the prodrug GCV, with ensuing quantitative cell killing. Moreover, we have confirmed that a cytotoxic bystander effect can be induced by genetically modified CD34+ cells in juxtaposition to tumor cells. Furthermore, we have demonstrated that this bystander effect is also achieved against proliferating human primary endothelial cells. More importantly, tumorigenicity was inhibited in vivo as a consequence of the bystander cytotoxicity. These augmented effects might thus potentially overcome the requirement to achieve near-universal quantitative tumor cell transduction in tumor foci, a major challenge in gene therapy for cancer. In effect, the establishment of a bystander effect has been an important factor contributing to the overall efficiency of molecular chemotherapy approaches in gene therapy (1) . In this regard, numerous studies have examined the biological basis of the bystander effect.
It appears that the presence of intercellular gap junction communications is an important determinant of the bystander phenomenon, although not a universal one (18) . In addition, apoptotic bodies generated during killing by toxin gene delivery may function as a vehicle for disbursement of toxin gene metabolites to nontransduced tumor cells (19) . In most cases, this effect has been sought between tumor cells or between tumor cells and surrounding endothelial cells (20) . To our knowledge, we have herein proposed for the first time the exploitation of a bystander effect induced from genetically modified cells with endothelial lineage.
The use of circulating cellular vehicles was first proposed a decade ago. Then, human TILs were used as vehicles for retroviral-mediated gene transfer based on a putative preferential localization at tumor sites (3 , 4) . It was also hypothesized that TILs could be an enriched source of natural killer cells and CTLs specific for tumor antigens. On this basis, technology for their expansion in culture was developed, and TILs were the first immune cells to be genetically modified and applied in a human gene therapy clinical trial against cancer (21) . It was soon observed that whereas TILs do include CTL- and natural killer-activated cells, only a few of these cells from these mixed cell populations are specific for the tumor from which they are isolated. Furthermore, reinfused TILs localize poorly into tumors, and their required expansion in vivo using interleukin-2 was rather toxic to patients. Although several strategies have been applied to improve treatments based on TILs and other lymphocytes, including an elegant reengineering of their tropism (22 , 23) , modest localization of TILs in tumors remains a limitation for the efficacy of this poorly tolerated and expensive therapy.
In contrast, the target of our proposed CD34+-based cellular vehicles is the nascent vascular endothelium of angiogenic tumors, which should be much more accessible to these circulating cells. This hypothesis is being tested in vivo. On the other hand, mature endothelial cells have also been shown to localize into areas of angiogenesis (24) and have been used effectively to deliver apolipoprotein E as a treatment for hypercholesterolemia in a murine apolipoprotein E knockout atherosclerosis model (25) and for delivery of the TK toxin (7) . In both cases, the major limitation to further development of the strategy is our inability to isolate and expand in vitro mature autologous endothelial cells. Furthermore, the alternative use of allogeneic cells established and expanded in vitro would introduce all of the encumbrances of an allogeneic cellular transplant. In marked contrast, we have proposed herein the use of autologous CD34+ cells, which are easily obtainable in large numbers from peripheral blood. Our use of these cells is in accord with the expanding use of progenitor and stem cells in a variety of therapeutic contexts (26 , 27) . The potential of CD34+ cell subsets as novel systemic vectors for toxin and other genes warrants further investigation. Our observations to date clearly establish the feasibility of cell-based gene delivery into disseminated areas of tumor angiogenesis as a rational strategy for cancer gene therapy.
ACKNOWLEDGMENTS
We thank Drs. Joanne T. Douglas, Masato Yamamoto, and Enrique Casado for critical reading of the manuscript and helpful suggestions.
FOOTNOTES
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.
1 To whom requests for reprints should be
addressed, at Division of Human Gene Therapy, 1824 6th Avenue South,
WTI 620, Birmingham, Al 35294. Phone: (205) 975-7594; Fax:
(205) 975-7949; E-mail: jesus.gomez{at}ccc.uab.edu 
2 The abbreviations used are: EPC, endothelial
progenitor cell; TK, thymidine kinase; GCV, ganciclovir; HUVEC, human
umbilical vein endothelial cell; HSV-TK, herpes simplex virus thymidine
kinase; MOI, multiplicity of infection; FDG, fluorescein
di-ß-D-galactopyranoside; FACS, fluorescence-activated
cell sorting; TIL, tumor-infiltrating lymphocyte; MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(sulfophenyl)-2H-tetrazolium,
inner salt.
Received 5/18/00; revised 8/29/00; accepted 8/29/00.
REFERENCES
This article has been cited by other articles:
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  J. Wels, R. N. Kaplan, S. Rafii, and D. Lyden Migratory neighbors and distant invaders: tumor-associated niche cells Genes & Dev., March 1, 2008; 22(5): 559 - 574. [Abstract] [Full Text] [PDF]
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 F. A. Scappaticci Mechanisms and Future Directions for Angiogenesis-Based Cancer Therapies J. Clin. Oncol., September 15, 2002; 20(18): 3906 - 3927. [Abstract] [Full Text] [PDF]
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). Five days later, the percentage of viable cells was measured
using a MTS assay. Bars, SD.
