Viral Vector Delivery in Solid-State Vehicles: Gene Expression in a Murine Prostate Cancer Model

D. Robert Siemens, J. Christopher Austin, Sean P. Hedican, James Tartaglia, Timothy L. Ratliff

Affiliations of authors: D. R. Siemens, J. C. Austin, S. P. Hedican, Department of Urology, The University of Iowa, Iowa City; J. Tartaglia, Virogenetics Corporation, Troy, NY; T. L. Ratliff, Department of Urology, The University of Iowa Cancer Center and The University of Iowa Prostate Cancer Research Group, Iowa City.

Correspondence to: Timothy L. Ratliff, Ph.D., Department of Urology, The University of Iowa, 200 Hawkins Dr., 3 RCP, Iowa City, IA 52242-1089 (e-mail: tim-ratliff{at}uiowa.edu).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: Although there are increasingly more clinical trials involving gene therapy, efficient gene transfer remains a major hurdle to success. To enhance the efficiency of delivery of viral vectors in gene therapy protocols, we evaluated the effect of various matrices to act as a vehicle for recombinant virus during intratumoral injection. METHODS: The ability of several vehicles (catgut spacer, polyglycolic acid, chromic catgut, and gelatin sponge matrix) to deliver the canarypox virus ALVAC to the cells of the murine prostate cancer cell line RM-1 was studied in vitro and in vivo. ALVAC recombinants encoding the murine cytokines interleukin 2 (IL-2), interleukin 12 (IL-12), and tumor necrosis factor-{alpha} (TNF-{alpha}) were used to assess enhancement of antitumor activity after intratumoral inoculation. Confirmatory experiments were conducted by use of another mouse prostate cancer cell line, RM-11, and a mouse bladder cancer cell line, MB-49. All statistical tests were two-sided. RESULTS: The gelatin sponge matrix proved to be the most effective solid-state vehicle for delivering viral vectors to cells in culture. In addition, this matrix statistically significantly enhanced expression of ALVAC-delivered reporter genes in tumor models when compared with fluid-phase delivery of virus (P = .037 for the RM-1 model and P = .03 for the MB-49 model). Statistically significant growth inhibition of established tumors was observed when a combination of the three recombinant ALVAC viruses expressing IL-2, IL-12, and TNF-{alpha} was delivered with the matrix in comparison with 1) fluid-phase intratumoral injection of the ALVAC recombinants, 2) no treatment, or 3) treatment with parental ALVAC (all P<.05). CONCLUSIONS: Viral vector delivery in a solid-state vehicle resulted in improved recombinant gene expression in vivo and translated to greater inhibition of tumor growth in an immunotherapy protocol for heterotopic tumor nodules. The efficient delivery of reporter genes described herein may prove useful in many solid tumor gene therapy protocols.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Gene therapy protocols for cancer are based on eradicating tumor cells either directly (e.g., toxic genes) or indirectly (e.g., genes that elicit antitumor immune responses). Alternatively, corrective gene therapy involves the replacement or inactivation of defective genes in neoplastic cells (e.g., p53 [also known as TP53]). There are increasingly more clinical gene therapy trials under way and, although many investigations have demonstrated a great promise in preclinical studies, the efficient and accurate delivery of therapeutic genes remains a formidable task in all solid-tumor oncology.

We have previously reported one approach to cancer immunotherapy involving the transfer of genes encoding the cytokines interleukin 2 (IL-2) and tumor necrosis factor-{alpha} (TNF-{alpha}) utilizing the canarypox viral vector ALVAC (1). The ALVAC virus was shown to efficiently infect murine prostate cancer cells, RM-1, and to produce high levels of extrinsic gene product. In addition, antitumor immunity was induced when tumor cells were infected by ALVAC cytokine recombinants and injected subcutaneously in the flanks of male C57BL/6 mice. The ALVAC virus is particularly well suited for the direct injection of tumors because the inability of the canarypox virus vectors to replicate in human cells greatly reduces the risk of systemic adverse events. The absence of replication also means that the virus will not propagate through the tumor mass; therefore, the expression of the delivered gene products is restricted to the sites of delivery. Our preliminary studies have shown a restricted pattern of expression after direct injection of fluid-phase ALVAC into the prostate or a tumor at a subcutaneous site. Moreover, any clinical gene therapy protocol for prostate cancer involving the intraprostatic delivery of therapeutic genes will be hampered by the multifocal and often cryptic nature of neoplastic and preneoplastic lesions in the prostate. Thus, methods to enhance the distribution and expression of recombinant genes are needed.

Numerous substances have been used as carriers to enhance and sustain the delivery of soluble products to both neoplastic and non-neoplastic tissue (2,3). In these studies, a gelatin sponge matrix was determined to be the most efficient in vitro and was shown to enhance delivery and, hence, reporter gene expression when injected intratumorally. The previously described tumor suppression in this model by recombinant virus encoding genes for IL-2, interleukin 12 (IL-12), and TNF-{alpha} was also markedly improved when the vector was delivered by the gelatin matrix as compared with fluid-phase injection.

In this study, we tested the ability of different matrices to act as a carrier vehicle for the canarypox virus (ALVAC) to improve the delivery of the vector in a heterotopic murine prostate cancer model.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Animals and Tumor Cells

The murine prostate cancer cell line RM-1 used for these studies mimics multistep carcinogenesis by activating the ras and myc oncogenes and is used to induce an aggressive prostate carcinoma in vivo. This cell line retains many features of prostate cancer, including androgen responsiveness early in culture, expression of androgen receptor, and progression to androgen independence with time (4). MB-49, a chemically induced mouse bladder tumor, was used in concert with RM-1 throughout the in vitro and in vivo gene expression experiments. Both RM-1 and MB-49 are syngeneic to C57BL/6 mice. The myc- and ras-transformed BALB/c RM-11 prostate cancer cell line was used for complementary in vivo tumor outgrowth studies. Cultured cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS). Mice (6-8 weeks old at the time of study initiation) were obtained through the National Cancer Institute, Bethesda, MD, and were allowed free access to food and water. All animal studies were approved by the Animal Review Board of the University of Iowa and were performed in accordance with institutional guidelines.

Gene Transfer Vectors

ALVAC is a canarypox virus that can infect mammalian cells but is restricted to avian species for replication (5). It has been shown to be a safe and effective vector in both humans and animals (6,7). The viral strain from which ALVAC was obtained was isolated from a pox lesion on an infected canary. Parental ALVAC, ALVAC vectors encoding murine IL-2, murine IL-12 and murine TNF-{alpha}, as well as the reporter gene constructs ß-galactosidase (ALVAC-lacZ), green fluorescent protein (ALVAC-GFP), and luciferase (ALVAC-luciferase) were developed at Virogenetics Corporation (Troy, NY).

Delivery Systems

Four solid-state delivery matrices were compared with fluid-phase delivery of viral vector to cells in vitro and in vivo: polyglycolic acid, chromic catgut, catgut spacer, and gelatin sponge. These substances were chosen because of their absorbable nature and relatively low tissue reaction. Polyglycolic acid (Davis and Geck, Inc., Wayne, NJ) and chromic catgut (Ethicon, Inc., Somerville, NJ) are both absorbable suture materials. Plain catgut spacer material (MDTech, Gainesville, FL) is a commercially available product used for prostate cancer brachytherapy protocols. An absorbable gelatin sponge (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) is prepared from purified pork skin gelatin granules and is used as a hemostatic agent. All delivery systems were prepared in 6-mm lengths and were tested in vitro and in vivo through an 18-gauge B-D spinal needle (Becton Dickinson and Co., Franklin, NJ) to better mimic intraprostatic injection in a clinical trial.

As previously described for the carrier delivery for insulin (2), the virion concentration to be delivered by an individual matrix was determined by weighing the matrices before and after viral absorption. The dry and wet weights of the matrices after 1 minute of viral absorption were recorded, and the subsequent volume delivered was calculated. Subsequently, to ensure that the gelatin sponge matrix was reliably delivering this weight-calculated number of viral particles, comparisons were made with the use of particle determination by measurement of the optical density (OD) at 260 nm after digestion of the gelatin matrix. A calculated particle concentration was delivered into solution by the gelatin sponge matrix and then digested by a combination of collagenase (0.16%), bovine serum albumin (2.5%), and deoxyribonuclease (0.001%) in phosphate-buffered saline (PBS) (pH 7.2); these three reagents were obtained from Sigma Chemical Co., St. Louis, MO.

Infection and Reporter Gene Assays

For in vitro analysis, RM-1 or MB-49 cells were harvested from tissue culture plates and replated with DMEM containing 10% FCS and 10 mM HEPES buffer (pH 7.0) on the day before infection. The medium was changed to DMEM with 2% FCS at the time of viral infection with either ALVAC-luciferase or ALVAC-lacZ delivered directly into the culture or via the delivery matrices. The viral vectors were added to the cells at the multiplicity of infection (MOI)-plaque-forming units (pfu) per cell shown in each experiment. The cells were then incubated for 6 hours at 37 °C in an atmosphere of 5% CO2 when the medium was changed back to DMEM with 10% FCS. Reporter assays were performed 48 hours after virus addition (unless otherwise stated). All in vitro experiments were performed in triplicate and repeated in at least two independent experiments.

For the in vivo gene expression studies, RM-1 or MB-49 cells were harvested from the tissue culture plates by treatment with 10 mM EDTA and were washed with PBS. The cells were then resuspended in DMEM in a concentration of 5 x 106 pfu/mL, and 0.1 mL was injected subcutaneously into the backs of mice. The ALVAC vectors recombinant for luciferase or ß-galactosidase were injected either directly (fluid phase) or via the delivery systems at a concentration of 3 x 106 pfu/mL approximately 10 days after tumor implantation. Tumors at that time were approximately 8 mm by 8 mm (approximately 200 mg wet weight). Tumors were harvested for reporter gene assays at various times after infection as described for individual experiments. Experiments were performed at least twice for the RM-1 and the MB-49 tumors in vivo.

We determined ß-galactosidase transgene expression in vitro after briefly fixing the cells with 0.5% glutaraldehyde for 10 minutes, washing them with PBS, then incubating them in X-gal (5-bromo-4-chloro-indolyl ß-D-galactopyranoside) at 37 °C for at least 4 hours. ß-Galactosidase cleaves this substrate into an indigo compound, such that cells producing the ß-galactosidase gene product are stained blue. We quantified ß-galactosidase expression by visualizing a representative area of the culture plate under high power (x40) and recording the percentage of blue cells. In vivo, after the tumor was harvested and weighed, sections were incubated in the X-gal solution containing the detergents sodium deoxycholate (0.01%) and Nonidet P-40 (0.02%). Representative tissue sections were then scored for percentage of blue-stained cells. Tumors infected with the parental ALVAC (not recombinant for the lacZ gene) were used as negative controls.

The luciferase assay from cell lysates was performed with the use of a commercial luciferase assay kit (Promega Corp., Madison, WI) following the manufacturer's recommendations. The Monolight 2010 Luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI) was used for the luciferase assay. Internal controls were performed by reading background luminescence for each assay as well as the periodic evaluation of the variation between replicates. Luciferase assays of infected subcutaneous tumors were performed after each tumor was homogenized with the Tissue Tearor (Biospeck Products, Inc., Fisher Scientific, Itasca, IL) in 0.5 mL of cell lysis buffer.

Tumor Inhibition Studies

RM-1 (5 x 105) or RM-11 (1 x 105) cells were injected subcutaneously in the backs of mice in a volume of 0.1 mL as described above. Approximately 10 days after tumor implantation, a total of 8.4 x 106 pfu of recombinant ALVAC vector was injected intratumorally. The ALVAC vectors used were the IL-2, IL-12, and TNF-{alpha} constructs in equal concentrations (2.8 x 106 pfu each). The vectors absorbed by the gelatin sponge matrix were injected in an 18-gauge needle and were compared with three separate 33-µL injections of the fluid-phase product (8.4 x 106 total pfu). Other controls included parental ALVAC absorbed by the gelatin sponge matrix, matrix only, and a no treatment group.

Tumor outgrowth, determined by tumor size as a function of time, was measured approximately three times a week. Survival of the tumor-bearing mice was also determined. Mice were killed for humane reasons if a single tumor was greater than 25 mm in any dimension or if the mice appeared to be ill from the tumor burden. Each experimental group contained four to six mice, and experiments were repeated at least twice.

Statistical Evaluation

Differences were analyzed by the Mann-Whitney rank sum test, including the nonparametric data for ß-galactosidase and luciferase reporter assays. The gene expression data are recorded as the means and the 95% confidence intervals (CIs). The rank sum test (Mann-Whitney) was also used to compare average tumor volume between matrix-delivered and fluid-phase-delivered treatment groups at individual time points. These data were also presented in the figures as the means and the 95% CIs. A one-way analysis of variance of log-transformed data was also used to compare all control groups with the gelatin sponge matrix-delivered treatment group at individual time points for the tumor outgrowth studies. Survival data were analyzed for significance with the use of the Cox proportional hazards regression model. For all statistical analyses, we used a computer software program, SAS (SAS Institute, Inc., Cary, NC) (8), or Statistix (Analytical Software, Seattle, WA; Version 1.0, 1996). All reported P values are two-sided, and statistical significance was determined as a P value of less than .05.


    RESULTS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Determination of Virion Concentration

The calculated volume delivered by the individual matrices was determined by the wet weight after absorption of the virus in the 18-guage delivery needle. The mean wet weight and dry weight of at least five samples were assessed for each delivery matrix, and the results of three separate determinations are presented in Table 1. The known volumes absorbed by the individual matrices allowed comparisons to fluid-phase delivery both in vitro and in vivo by calculation of the number of viral particles or pfu. Thus, pfu delivery via either matrix or fluid was equivalent, given the absorbable capacity of each matrix. Equivalency was verified in the gelatin sponge matrix system as determined by the particle count read as OD at 260 nm (data not shown).


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Table 1. Calculation of volume of vector absorbed by individual matrices

 
Delivery of Reporter Genes In Vitro

To examine the ability of each matrix to deliver the calculated virion concentration in cell culture, we determined ß-galactosidase and the firefly luciferase expression of cells 48 hours after infection with ALVAC-lacZ or ALVAC-luciferase vectors. The virus delivered by the matrices was compared with the addition of a known MOI (pfu) of fluid-phase virus in cell culture. As shown in Fig. 1, A, no significant differences were found with the percentage of RM-1 cells infected by the ß-galactosidase vector (MOI 10 : 1) in the gelatin sponge matrix-delivered group (mean = 48%; 95% CI = 41%-55%) versus the fluid-phase group (mean = 49%; 95% CI = 44%-55%). It is interesting that there was a trend to less ß-galactosidase expression for the other delivery systems, especially for the chromic catgut. These results were consistent over a wide range of MOI (10 : 1 to 200 : 1) for the RM-1 cell line, as well as for experiments using the MB-49 cell line.



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Fig. 1. Comparison of in vitro delivery by different matrices. A) Percentage (95% confidence interval) of ß-galactosidase-expressing RM-1 cells detected 48 hours after in vitro infection with ALVAC-ß-galactosidase. No statistically significant difference (P = .66, Mann-Whitney rank sum test) was seen between the gelatin sponge matrix delivery and direct infusion of the fluid-phase product. B) Luciferase activity (relative light units) of RM-1 cell lysates 48 hours after infection with ALVAC-luciferase. No statistically significant difference in luciferase activity was observed between the gelatin sponge matrix delivery and direct infusion of fluid-phase product (P = .08, Mann-Whitney rank sum test). Gene expression shown represents experiments performed in triplicate at a 10 : 1 multiplicity of infection (MOI). Experiments at different MOIs (10 : 1 to 200 : 1) in both the RM-1 and MB-49 cell lines demonstrated similar in vitro gene expression between the gelatin sponge matrix delivery and fluid-phase delivery.

 
Similarly, there was no significant difference between the luciferase activity (relative light units [RLU]) of RM-1 tumor cell lysates infected with ALVAC-luciferase (MOI 10 : 1) delivered by the gelatin sponge (mean = 1.8 x 107 RLU; 95% CI = 9.2 x 106-2.5 x 107 RLU) compared with delivery of the fluid-phase product in vitro (mean = 1.1 x 107 RLU; 95% CI = 7.7 x 106-1.5 x 107 RLU) (Fig. 1, B). No statistically significant difference was found between these two groups (P = .08, Mann-Whitney rank sum test). Similarly, no differences in gene transfer were observed between the gelatin sponge and fluid-phase groups in the MB-49 cell line (P = .46, Mann-Whitney rank sum test). Presumably, the entire viral load delivered into culture by this matrix was available to infect the tumor cells. Again, there was a trend to lower expression with the other delivery systems. The lower gene expression observed with the use of these systems most likely reflects either the inability to deliver the entire calculated volume or the inability of the matrix to release the absorbed virus in cell culture.

Transgene Expression in a Heterotopic Tumor Model

To determine the ability of these carrier systems to deliver the viral vectors in vivo, we first injected ALVAC-luciferase (3 x 106 pfu) into established subcutaneous RM-1, either in fluid-phase or via the various delivery systems. The chromic system was not tested, given its consistently poor transfer of virus to cells in culture. Forty-eight hours after infection, the tumors were harvested and the luciferase assay was performed. The gelatin sponge matrix delivery consistently resulted in significantly (P = .037, Mann-Whitney rank sum test) enhanced gene expression (mean = 34 226 RLU; 95% CI = 14 673-52 012 RLU) over fluid-phase delivery (mean = 2961 RLU; 95% CI = 29-7707 RLU) (Fig. 2, A). The other delivery systems (polyglycolic acid and catgut spacer) did not consistently enhance gene expression in injected tumor nodules. Similar experiments in the heterotopic MB-49 tumor model confirmed the ability of the gelatin sponge matrix to significantly improve (P = .03, Mann-Whitney rank sum test) the delivery of viral vectors compared with direct injection of the fluid-phase product. Fig. 2, B, shows the improved luciferase expression with gelatin sponge delivery in the RM-1 tumor model at different doses (pfu) of the ALVAC-luciferase vector (note log scale for RLU in Fig. 2, B).



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Fig. 2. Comparison of in vivo delivery by different matrices. A) Mean luciferase activity (95% confidence interval) of harvested subcutaneous RM-1 tumors (n = 5 per group) 48 hours after infection by 3 x 106 plaque-forming units of ALVAC-luciferase delivered by different matrices. Statistically significant differences (P = .037, Mann-Whitney rank sum test) in mean luciferase activity were observed only between the gelatin sponge matrix delivery and fluid-phase injection. Similar experiments in MB-49 subcutaneous tumors also resulted in statistically significant increases (P<.03, Mann-Whitney rank sum test) in luciferase gene expression with gelatin sponge delivery compared with fluid-phase delivery. B) Mean luciferase activity (95% confidence interval) in log scale of RM-1 subcutaneous tumors (n = 4 per group) 48 hours after infection with ALVAC-luciferase at different virion concentrations.

 
To determine if the improved in vivo gene expression consistently observed at 48 hours was conserved over time, we harvested subcutaneous RM-1 tumors infected with 3 x 106 ALVAC-luciferase for luciferase assays 24, 48, 72, and 96 hours after injection. Those tumors that were injected with virus in the gelatin sponge matrix had remarkably greater luciferase activity than intratumoral fluid-phase virus injection at each period of time (Fig. 3).



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Fig. 3. Mean luciferase activity (95% confidence interval) of heterotopic RM-1 tumor nodules after infection by 3 x 106 plaque-forming units of ALVAC-luciferase. Tumors (n = 5 per group) were harvested at various times after infection. Values represent combined data from two separate experiments. A statistically significant difference (all P values <.05, Mann-Whitney rank sum test) was observed at each time point between the gelatin sponge-delivered groups and the fluid-phase-delivered groups except at 96 hours (P= .058, Mann-Whitney rank sum test).

 
Enhanced gene expression was consistently demonstrated under various conditions (virion concentration, time) when the gelatin sponge matrix was used to deliver the vectors. To determine if improved biodistribution throughout the tumor was partly responsible for this improved expression, heterotopic tumors were infected by ALVAC encoding the reporter genes GFP or ß-galactosidase. Viewing tumors under the fluorescent microscope after ALVAC-GFP infection revealed much brighter and more widespread fluorescence when the vectors were delivered by the gelatin sponge matrix than when the vectors were delivered by fluid phase (Fig. 4, A and B). For comparisons, controls for background fluorescence were determined by infecting tumors with parental ALVAC (data not shown).







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Fig. 4. Histopathology sections of heterotopic RM-1 tumor nodule 24 hours after infection with 3 x 106 plaque-forming units of ALVAC-green fluorescent protein (GFP) or ALVAC-ß-galactosidase (lacZ). Parental ALVAC control infected RM-1 tumor under fluorescent microscopy showed no fluorescence (data not shown). A) Limited and localized GFP expression demonstrated under fluorescent microscopy when ALVAC-GFP delivered by fluid-phase injection. B) Greater gene expression and wider distribution seen when ALVAC-GFP vector delivered by the gelatin sponge matrix. C) RM-1 tumor infected with parental ALVAC control, stained with nuclear fast red (original magnification x63). D) ALVAC-lacZ-infected tumor stained with X-gal and counterstained with nuclear fast red. Limited ß-galactosidase expression was seen in needle tract after injection (arrows) of the fluid-phase product (original magnification x63). E) Lower power magnification of tumor infected with ALVAC-lacZ vector delivered by gelatin sponge matrix, demonstrating qualitatively higher gene expression with greater distribution (original magnification x25).

 
Similarly, fluid-phase injection of tumors by ALVAC-lacZ consistently revealed ß-galactosidase activity only within a relatively narrow distribution along the needle tract. Delivery by the gelatin sponge matrix, however, resulted in substantially more widespread distribution. These results were also controlled for endogenous ß-galactosidase activity by staining tumors infected with parental ALVAC vector (Fig. 4, C-E).

Tumor Outgrowth Studies

To determine if the enhanced gene expression found with the matrix delivery of vectors translated into improved biologic effect, we treated established subcutaneous RM-1 tumors (mean tumor volume = 110 mm3) with ALVAC vector expressing IL-2, IL-12, and TNF-{alpha} delivered in the gelatin sponge matrix or via fluid-phase injection. Control groups included 1) parental ALVAC delivered via the matrix, 2) a matrix only, and 3) a group with no treatment. Statistically significant (P values for all comparisons <.05, Mann-Whitney rank sum test) tumor inhibition, as determined by tumor volume over time, was seen in the treatment group only when delivered by the matrix (Fig. 5, A). This tumor inhibition was greatest within the first 6 or 7 days after infection with the recombinant virus, although the inhibitory effects remained significant through 13 days. Further comparison between groups was not possible because many control mice were killed after day 13. Several tumors (three of five) in the treatment group delivered by the gelatin matrix demonstrated substantial inhibition of growth for a period of 10 days after gene transfer, although all tumors did eventually grow out. Tumor volumes at days 4-13 in the gelatin sponge matrix-delivered group were statistically significantly smaller (P values for all comparisons <.05, Mann-Whitney rank sum test) than tumors in the fluid-phase injection group, as well as those in the control groups. Similarly, a statistically significant (P<.005; Cox proportional hazards regression model) increase in survival was seen for mice treated with the recombinant virus delivered by gelatin matrix as compared with those mice treated with the fluid-phase injection (Fig. 5, B). The differences observed between matrix and fluid-phase delivery have been confirmed in four separate tumor outgrowth experiments.




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Fig. 5. RM-1 tumor inhibition studies. A) Tumor growth of preestablished RM-1 subcutaneous nodules in C57BL/6 mice (n = 5 per group) infected by ALVAC virus encoding murine interleukin 2, interleukin 12, and tumor necrosis factor-{alpha} (8.4 x 106 total plaque-forming units) delivered either by the gelatin sponge matrix or by the fluid-phase product. Controls include matrix only, parental ALVAC virus, and a no treatment group. Data are presented as mean tumor volumes (95% confidence intervals) for the gelatin sponge-delivery group and the fluid-phase-delivery group. The average tumor volume in the matrix-delivered treatment group was significantly different (P values for all comparisons were <.05, Mann-Whitney rank sum test) from that in the fluid-phase-delivered group at each of the measurement days 4-13. A one-way analysis of variance of log-transformed data was also used to compare the average tumor volume of the matrix-delivered treatment group with all of the other experimental groups and was found to be significantly different at each time point (P values for all comparisons were <.001, one-way analysis of variance). B) Mice in tumor outgrowth studies were followed for survival and were killed if their tumor measured more than 25 mm in any dimension or if they became ill from the tumor burden. Improved survival was observed in the gelatin sponge matrix treatment group (P<.005, Cox proportional hazards regression model).

 
For confirmation of these results in a different prostate tumor model, subcutaneous RM-11 tumor nodules in BALB/c mice were infected with the recombinant ALVAC vectors as previously described. Impressive, statistically significant tumor inhibition and regression (P values for all comparisons <=.036, Mann-Whitney rank sum test) were demonstrated in this model when ALVAC recombinant for the IL-2, IL-12, and TNF-{alpha} cytokines was delivered with the gelatin sponge matrix compared with fluid-phase delivery (Fig. 6, A). Inhibition of tumor growth was statistically significantly greater in the matrix-delivered group (P<.045, Cox proportional hazards regression model) than in the fluid-phase and control groups, resulting in a substantial survival benefit for those mice (Fig. 6, B).



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Fig. 6. RM-11 tumor inhibition studies. A) Tumor growth of preestablished RM-11 subcutaneous nodules in BALB/c mice (n = 6 per group) infected with ALVAC virus encoding murine interleukin 2, interleukin 12, and tumor necrosis factor-{alpha} (8.4 x 106 total plaque-forming units) delivered either by the gelatin sponge matrix or by the fluid-phase product. Data are presented as mean tumor volumes (95% confidence intervals) for the gelatin sponge-delivery group and the fluid-phase delivery group. Again, a statistically significant difference in tumor volume was found between the matrix-delivered treatment group and the fluid-phase-delivered treatment group (P values for all comparisons were <=.036, Mann-Whitney rank sum test) at days 3-13. B) Survival of mice from the tumor outgrowth studies demonstrates a significant increase in the gelatin sponge matrix treatment group. Long-term survival (tumor-free) of approximately 20% has been observed in three separate tumor outgrowth studies.

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Prostate cancer is an important public health concern in the United States and represents the most common visceral cancer and the second leading cause of cancer deaths among men in this country. The American Cancer Society estimates that, in 1999, approximately 179 300 new cases of prostate cancer will be diagnosed and about 37 000 men could die of the disease (9). Despite this enormous prevalence, management of the disease remains controversial, and 5-year biochemical failure rates for radical prostatectomy range anywhere from 27% (10) to 57% (11). It is imperative to develop alternative or adjuvant treatment strategies, such as the introduction of therapeutic genes, to better manage both clinically localized disease and metastatic disease.

Several reports investigating prostate cancer immunotherapy have been encouraging. Early studies by Sanda et al. (12) showed that granulocyte-macrophage colony-stimulating factor (GM-CSF)-transfected rat prostatic adenocarcinomas grew more slowly than parental tumors. Subsequently, Vieweg et al. (13) showed that IL-2-transfected rat R3327-MatLyLu prostate cancer cells also induced antitumor activity. Existing tumors had a decreased rate of outgrowth, and protection was gained against subsequent tumor challenge. Using the parental R3327G tumor that exhibits hormone responsiveness, Yoshimura et al. (14) also observed antitumor activity; however, neither cytotoxic T-lymphocyte activity nor protection against subsequent tumor challenge was observed. In a previous study (1), we have shown that the ALVAC virus can efficiently infect prostate cancer cells, produce high levels of extrinsic gene product, and induce antitumor immunity. RM-1 tumor cells were infected by ALVAC cytokine recombinants and injected subcutaneously in the flanks of male C57BL/6 mice. As single agents, ALVAC-IL-2, ALVAC-IL-12, ALVAC-GM-CSF, and ALVAC-TNF-{alpha} were effective in partially inhibiting tumor outgrowth. As a combination therapy of ALVAC-TNF-{alpha} with ALVAC-IL-2, ALVAC-IL-12, or ALVAC-GM-CSF, the tumor outgrowth inhibition was optimized. Subsequent studies assessing the ability of the ALVAC cytokine vectors to induce regression of existing tumors showed only limited effects (data not shown). Although this system seems perfectly suited for intratumoral treatment of established tumors, given the inability of the ALVAC vector to replicate in mammalian species, we hypothesized that the fluid-phase injection of the viral vector resulted in limited expression and/or distribution of the gene product, as has been reported in other models (15).

Although numerous clinical gene therapy trials for prostate cancer have been initiated and many investigators have demonstrated great promise for both tumoricidal and corrective gene therapies, improving delivery and enhancing gene expression are imperative. Most preclinical investigations and ongoing phase I clinical protocols for localized prostate cancer rely on direct intraprostatic injection for the delivery of viral vectors (16,17). However, the relative inaccessibility of the prostate and the often isoechoic nature of cancer foci on transrectal ultrasound make detection and localization difficult. Moreover, examination of autopsy and radical prostatectomy specimens has revealed multiple and separate tumor sites, suggesting the possibility of a field change in the prostate (18,19) and necessitating even more efficient and accurate distribution of any therapeutic vector.

Unfortunately, the efficient delivery of genetic material to tumors remains a formidable task for all solid-tumor oncology. Asgari et al. (20) have found significant inhibition of preestablished subcutaneous tumor nodules of human prostate cancer by a single injection of the recombinant adenovirus p53 vector. It is interesting that they were unable to detect p53 overexpression in these adenovirus wild-type p53-infected tumors 48 hours after intratumoral injection. Moreover, multiple injections (1 week apart) did not improve the antitumor effect. These results most likely reflect the poor efficiency of transfer when direct intratumoral injections are employed. These difficulties with vector delivery are not isolated to the prostate. The effective use of gene therapy in glial tumors of the brain is hampered by the inefficient delivery of viral vectors. High titers of adenovirus are required for gene transfer to gliomas (15), and the resulting inflammation of the high inoculum decreases the efficiency of the gene transfer. To improve delivery, Beer et al. (21) found sustained release of recombinant adenovirus when coupled to biodegradable microspheres, allowing the administration of lower doses of viral vectors to glioma tissue.

Numerous methods have been investigated to better deliver soluble products to both neoplastic and non-neoplastic cells. A polymer-based paste has been found to enhance local delivery of chemotherapeutic agents and decrease recurrence rates at tumor resection sites (3). A fibrin- and gelatin-based drug-delivery system has been shown to more slowly release and to improve the therapeutic effect of antibiotics (22). Poloxamer 407 has been shown to improve the delivery of adenoviral vectors in vascular smooth muscle based on ß-gal reporter gene expression (23). The absorbable gelatin sponge employed in this study is primarily used as an intraoperative hemostatic agent, but it has also been used to deliver a number of different compounds, including insulin (2) and various cytokines and growth factors (24,25), in order to improve and sustain delivery.

In our study, the ability of a known quantity of recombinant ALVAC absorbed by the gelatin sponge matrix to infect tumor cells in vitro was similar to that seen with infusion of fluid-phase virus into the culture medium. The retained viral particles were able to freely efflux from the gelatin sponge matrix in this liquid environment over a 4- to 6-hour incubation time. The other matrices showed markedly less consistency as a delivery vehicle, most likely explained by some loss of viral particles in the transfer of the matrix to the culture dish or the inability of the retained virus to reenter the media. It is interesting that the gelatin matrix mediated enhanced gene expression and also enhanced biodistribution when compared with the fluid-phase injection of the viral vector in preestablished subcutaneous tumor nodules. The expression of the respective products of ALVAC-GFP or ALVAC-lacZ delivered in fluid phase was limited, i.e., restricted to the needle tract (Fig. 4) and around the tumor margin. Moriuchi et al. (26) have described a similar distribution of viral vector infection after intratumoral injection of an intracerebral tumor, resulting in poor gene expression and biologic response.

In contrast to delivery in the fluid phase, the vectors delivered by the gelatin matrix showed a broad biodistribution, which often would extend the full breadth of small tumors (approximately equal to 0.5 cm in diameter). This enhanced gene transfer was shown to translate into an improved biologic effect in the heterotopic RM-1 murine prostate cancer model with the use of a cytokine-based immunotherapy protocol. RM-1 tumors with a mean volume of 100-500 mm3 (depending on the experiment) were significantly inhibited when the gelatin matrix was used to deliver ALVAC-IL-2, ALVAC-IL-12, and ALVAC-TNF-{alpha}. The inhibitory effects in these treated mice also resulted in a significant survival advantage with a single injection. Despite this dramatic increase in gene expression observed in these heterotopic tumors when viral vectors were delivered with the gelatin sponge matrix, systemic distribution of vectors was not found to be increased over fluid-phase delivery. Selected organs (spleen, liver, and kidneys) harvested at the time of these experiments demonstrated no increased luciferase gene expression when compared with fluid-phase delivery. Similarly, in experiments involving orthotopic delivery of viral vectors in the ventral prostate of mice, gelatin sponge matrix delivery resulted in only minimal gene expression in surrounding organs, including the testes, bladder, and seminal vesicles, that was no greater and often less than that of fluid-phase delivery (data not shown).

Although gene therapy approaches to immunotherapy have enhanced immune activation and provided enhanced therapy results, the control of preestablished tumors has remained problematic (27-29). Control of the immunogenic RENCA kidney tumors was limited to tumors established for a maximum of 7 days [nonpalpable tumors; (29)]. Likewise, the control of tumor growth by herpes simplex virus thymidine kinase/gancyclovir has been limited to a reduction in tumor growth rate but not tumor regression (30). Our studies show the induction of substantial tumor growth inhibition with a single injection of 8.4 x 106 pfu of ALVAC cytokine vaccine (Fig. 5, A). While the control of tumor growth does not result in a cure, it does provide a significant extension of survival for treated mice. This result is accomplished in spite of the use of the poorly differentiated, highly aggressive RM-1 tumor model (approximate in vitro doubling time of 12 hours) in which treatment was initiated when tumors were large (approximately 215 mm3). The differences between the two means of vector delivery were even more dramatic in the RM-11 tumor model (Fig. 6) and have resulted in the long-term tumor-free survival in approximately 20% of mice over three separate experiments.

Improving the delivery of viral vectors is paramount in any clinical gene therapy trial, and this is especially true for prostate cancer. Delivery in a solid-state matrix, such as the one that we have described, may allow for more efficient and widespread gene transfer for all nonreplicative vectors. The increased efficiency of delivery may also result in circumventing antiviral host immune responses by decreasing the viral concentration needed to attain a desired effect.


    NOTES
 
Supported by grant 98-84 from the Carver Foundation.

We thank Jan Rodgers (Department of Pathology, University of Iowa) for her help with histopathology and Dr. Charles Davis (Department of Biostatistics, University of Iowa) for his assistance with statistical analysis.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 

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Manuscript received May 6, 1999; revised November 29, 1999; accepted December 8, 1999.


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