~JOURNAL OF SURGICAL RESEARCH 70, 7-11 (1997)ARTICLE NO. JR975113 /--"'"

Adenoviral Thymidine Kinase Prodrug Gene TherapyInhibits Sarcoma Growth in Vivo

Howard M. Ross, M.D.,* Edward A. Hirschowitz, M.D.,t Thomas J. Russi,M.D.,tRonald G. Crystal, M.D., t Sumihiko Nawata, M.D.,* Michael E. Burt, M.D., Ph.D.,*

Murray F. Brennan, M.D.,* and Jonathan J. Lewis, M.D., Ph.D.*,l*Department of Surgery, Memorial Sloan-Kettering Cancer Center, and tDepartment of Pulmonary Critical Care Medicine,

Cornell University Medical Center, New York, New York 10021

Submitted for publication December 23, 1996

Local recurrence of sarcoma is due to residual tumorcells remaining alter surgical resection and is associ-ated with decreased survival. We implemented adenovi-ral-mediated transfer of the herpes simplex thymidinekinase (HSTK) gene with subsequent ganciclovir (GCV)administration to treat a model of residual sarcoma.[3H]Thymidine uptake in MCA sarcoma cells was deter-mined alter infection with replication incompetent ade-novirus of the AdMLP .HSTK construct in the presenceof GCV. In vivo efficacy was evaluated in a model ofresidual sarcoma when 9 mg of MCA tumor was im-planted into the latissimus muscle of Fischer 344 rats.Three days alter implantation, animals were random-ized to receive AdMLP .HSTK, AdCMV .Null, or viral sus-pension buffer intratumorally. From Day 4, animalswere administered b.i.d. GCV (50 mgikg) or saline ip.Tumors were excised on Day 14 and weighed. Statisticalanalysis was by Mann-Whitney U test. In vitro: [3HJ-thymidine incorporation was significantly decreased inMCA sarcoma cells infected with AdMLP .HSTK in thepresence of GCV (P < 0.05). In vivo: Growth of MCAsarcoma treated with AdMLP.HSTK and GCV was sig-nificantly inhibited. Final tumor weights in theAdMLP .HSTR/GCV group were lower than all controlgroups (P < 0.05). A significant antitumor growth effecton MCA sarcoma was seen with adenoviral-mediatedtransfer of the HSTK gene and GCV administration,both in vitro and in an in vivo model ofresidual disease.This prodrug gene therapy strategy warrants investiga-tion as an adjuvant modality in the management of sar-coma. @ 1997 Academic Press

INTRODUCTION

Local recurrente of soft tissue sarcoma poses a sig-nificant clinical problem and is associated with de-creased survival [1, 2]. Multimodality therapy has re-

duced the incidence of local recurrence but rates re-main high [3]. Patients with retroperitoneal sarcomatreated with resectional surgery can have a 40% rate oflocal recurrence [4]. Local recurrence in patients withextremity sarcoma treated with limb sparing surgeryand adjuvant brachytherapy ranges from 10 to 35% [3].Local recurrence in both the abdomen and extremityis due to residual tumor cells remaining after surgicalresection and adjuvant treatment. This study exploredthe local application of prodrug gene therapy to thegrowth ofMCA sarcoma in a model ofresidual disease.

Prodrug gene therapy for cancer involves the activa-tionofan anticancer agent via the expression ora for-eign transferred gene. We utilized an adenovirus vectorto transfer the herpes simplex viTal thymidine kinase(HSTK) gene. Adenovirus has been used to transferforeign genes into mammalian cells to affect tumorgrowth in multiple animal models [5-7]. Adenoviruscan be genetically engineered to be replication incom-petent and to contain up to a 7.5-kb exogenous gene.Adenoviral infection generates a strong inflammatoryresponse and this may be helpful in eliciting an im-mune response toward tumor cells [8].

Transfer of the HSTK gene with subsequent gan-ciclovir (GCV) administration yields cell death via amechanism described in 1986 [9]. Expression of theHSTK gene produces the viTal enzyme (HSTK), whichmonophosphorylates the nucleoside analog prodrugGCV. Mammalian cellular enzymes then triphosphory-late the monophosphorylated GCV. The incorporationof triphosphorylated GCV into DNA results in chaintermination and ultimately causes cell death [10]. Anobserved phenomenon associated with the HSTK/ GCVparadigm is the "bystander effect" [11]. This phenome-non involves the death of non-HSTK-transfected cellsadjacent to HSTK-transfected cells, and allows regres-sion of an entire tumor when only a fraction of the cellscomprising the tumor are genetically altered. .

In this study we examined the effect of adenoviral-mediated HSTK gene transfer with subsequent GCVadministration on MCA sarcoma tumor growth in vitroand in an in vivo model Qf residual disease.

Presented at the Annual Meeting of the Association for AcademicSurgery, Dearborn, Michigan, November 8-11, 1995.1 To whom correspondence should be addressed at Department of

Surgery, Box 126, 1275 York Avenue New York, NY 10021.

0022-4804/97 $25.00Copyright 10 1997 by Academic Press

AlI rights of reproduction in any forro reserved.

7

8 JOURNAL OF SURGICAL RESEARCH: VOL. 70, NO. 1, JUNE 1997

MATERIALS AND METHODS

Adenoviral VectorsConstruction of AdMLP.HSTK. A recombinant replication-defi-

cient adenovirus (AdMLP .HSTK) was constructed with the HSV tkgene downstream ofthe adenovirus majar late promoter. The 1.8-kbcDNA encoding HSV tk was first ligated into an adenovirus shuttleplasmid. The shuttle plasmid based on pBluescript II SK containeda multiple cloning Bite flanked on the 5' end by the left invertedterminal repeat of adenovirus, the E lA enhancer, along with theadenovirus majar late promoter and tripartite leader of adenovirustype 2. On the 3' end of the multiple cloning Bite there was a 3-kbregion homologous to adenovirus type 5. A linearized fragment ofthis recombinant ofthis recombinant plasmid was cotransfected into293 cells (American Type Culture Collection) along with ClaI cutadenovirus DNA from the E3 deletion mutant Ad-dI327. Singleplaques were amplified in 293 celIs. Recombinant virus was screenedby PCR amplification for the presence of HSV tk and the absenceof the El region to demonstrate successful homologous recombina-tion. A single PCR-positive plaque, AdMLP.HSTK, was amplified in293 cells and subsequently purified on cesium gradients. Viralstocks in excess of 1010 plaque forming units (pfu) per milliliter ofAd.MLP.HSTK were prepared and titered in 293 celIs [12].

Construction of AdCMV.Null. A control vector of replication-in-competent adenovirus containing an expression cassette with a CMVpromoter without the cDNA insert for HSTK was constructed usingthe same methods.

Construction of AdCMV.LacZ. A replication incompetent ade-noviral vector containing the marker gene, {3-galactosidase, undercontrol of the CMV promoter was constructed as previously de-scribed [13].

Tritiated Thymidine Uptake StudiesTritiated thyrnidine uptake was utilized as a measure of cellular

proliferation. The ability of recombinant adenovirus containing theHSTK gene to affect the viability ofmethylcholanthrene-induced ratrhabdomyosarcoma cells in the presence of GCV (Cytovene, Gan-ciclovir sodium, Syntex Laboratories, Inc., Palo Alto, CA) was as-sessed by (3H]thyrnidine uptake studies. The first in viera experimentevaluated the impact of increasing multiplicity of viral infection(m.oi.) with subsequent GCV administration on MCA sarcoma cellDNA synthesis. In triplicate plates, 105 MCA sarcoma cells wereincubated in RPMI, 2% fetal calf serum in the presence ofAdMLP.HSTK or AdCMV.Null at O, 10,50, and 100 m.oi. for 24 hr.GCV was then added to the infected cells to a concentration of 10p.g/ml for 48 hr. Cells were incubated for 6 hr with (3H]thyrnidine(0.5 p.Ci/ well; DuPont New England Nuclear, Boston, MA). The me-dium was aspirated, cells were washed three times with phosphate-buffered saline followed three times with ice-cold 5% trichloroaceticacid. Two hundred proof ethyl alcohol was added to the cells andthey were then aspirated. The cells were allowed to air-dry for 15min and then lysed with 500 p.l 0.1 N NaOH. pH was neutralizedwith 0.1 N HCl and the amount of (3H]-thyrnidine was quantified ina scintillation counter.

The second in viera experiment determined the effect on MCA sar-coma cells of increasing doses of GCV at an m.oi. of 100 and on MCAcells without prior viral exposure. This experiment was conducted toevaluate GCV toxicity in viera. In triplicate wells, 105 MCA sarcomacells were infected with either AdMLP .HSTK or AdCMV .Null at anm.oi. of 100 for 24 hr. One group of wells was not exposed to virus.GCV was then added to the wells at concentrations of O, 5, and 10p.g/ml for 48 hr. (3H]Thyrnidine uptake was evaluated as above.

ally. After betadine skin preparation, a 1-cm skin incision was madeayer the left latissimus muscle. Forceps and scissors were used tocreate a small cavity in the muscle. A 9-mg MCA sarcoma tumorspecimen, excised from a passage animal, was placed into the cavity.A 5-0 silk suture was placed around the tumor to facilitate lateridentification. The skin was then stapled closed and animals wereallowed to recover from anesthesia. Three days were allowed to passto enable the tumor to implanto

On Day 3, animals were anesthetized with pentobarbital sodium,their prior incision was opened with gentle traction, and the incorpo-rated tumor was identified. Under direct vision intratumoral injec-tion of 109 pfu of AdCMV.LacZ in 50 III of viral suspension bufferwas performed. The skin was closed and animals were allowed torecover from anesthesia. On Day 5, 48 hr post-AdCMV.LacZ infec-tion, animals were sacrificed via CO2 inhalation and their tumorswere excised with al-cm margin of surrounding muscle. The excisedflank was fixed in 10% Formalin on ice for 1 hr. The tissue was thenplaced in X Gal reagent for 4 hr at 37°C. The presence of expressedf3-galactosidase protein was revealed in the development of a bluechromophore. The presence of tumor expressing the f3-galactosidaseprotein was confirmed visually.

In Vivo Residual Tumor ModelIn vivo experiments assessed MCA sarcoma tumor growth in a

model of residual disease. Consistent tumor establishment was seenin 170/171 rats implanted with tumor in experiments conducted inour laboratory. This model attempted to approximate clinical postre-section sarcoma. On Day O, 41 Fischer rats underwent tumor implan-tation on Day O as described in the f3-galactosidase expression sec-tion. On Day 3, animals were anesthetized with pentobarbital so-dium, their prior incision was opened with gentle traction, and theincorporated tumor was identified. Animals were randomized to re-ceive intratumoral injection of AdMLP.HSTK 109 pfu, AdCMV.Null109 pfu, or viral suspension buffer in 50 III total volume through a28-gauge needle. Starting on Day 4 and continuing through Day 14,the animals were administered GCV (50 mg/kg) b.i.d. or saline ip.Tumors were excised and weighed on Day 14. The randomization ofanimals to both intratumoral treatlI1ent and subsequent ip adminis-tration of GCV or saline resulted in the creation of five experimentalgroups. The treatment group consisted of 12 animals that receivedintratumoral AdMLP.HSTK and then GCV ip. Control groups weredesigned to evaluate the individual effects of AdMLP .HSTK intratu-moral injection, intratumoral viral suspension buffer injection, andGCV ip administration. The effect of combined AdCMV .Null intratu-moral injection with subsequent GCV ip administration and the im-pact of intratumoral viral suspension buffer injection followed by ipsaline were algo evaluated.

Animal CareAnimals (male Fischer 344 rats (F344), 200-250 g, Charles River,

Kingston, NY) were treated in accordance with the "PrincipIes ofLaboratory Animal Care" formulated by the National Society ofMed-ical Research and the NIH "Guide for the Care and U se of LaboratoryAnimals" NIH Publication 86-23, revised 1985). Experiments wereapproved by the Institutional Animal Care and use Committee (lA-CUC), Memorial Sloan-Kettering Cancer Center. AlI rats were al-lowedaccess to standard laboratory rat chow (Purina Rat Chow, StoLouis, MO).

Statistical AnalysisComparison of counts per minute of incorporated tritiated thymi-

dine into MCA sarcoma cells under various conditions and Day 14tumor weights of animals in different groups was by the nonparamet-ric test to evaluate means, Mann-Whitney U. Significance was de-fined as P < 0.05.

RESULTSIn Vivo Foreign Gene Expression

To evaluate the ability tQ transfer a foreign gene vía recombi-nant adenovirus infection into MCA tumor growing in the flankof the Fischer rat, [3-galactosidase express ion was investigated.Two Fischer 344 rats were anesthetized with pentobarbital sodium(Wyeth Laboratories Inc., Philadelphia, FA) 50 mg/kg intraperitone-

In VitroIn vitro assessment of an antiproliferative effect of

AdMLP .HSTK and GCV revealed a significant de-

~

ROSS ET AL.: ADENOVlRAL PRODRUG GENE THERAPY INHIBITS MCA SARCOMA 9

crease in [3H]thymidine incorporation as the multiplic-ity ofAdMLP.HSTK infectionincreased from O to 100in the presence of GCV (P < 0.05). There was not asignificant decrease in [3H]thymidine incorporation asthe m.o.i. of AdCMV.Null increased from O to 100 inthe presence of GCV (Fig. 1).

Increasing GCV concentrations had a significantantiproliferative effect on MCA sarcoma cells infected,with AdMLP .HSTK. Ganciclovir administration toMCA sarcoma cells without prior viral infection did notresult in a significant change in [3H]thymidine incorpo-ration. There was no decrease in [3H]thymidine incor-poration of MCA sarcoma cells as GCV was increasedfrom O to 5 ¡.¡,g/ml. A small but significant decrease in[3H]thymidine incorporation was evident in cells in-fected with AdCMV.Null without GCV and with thosetreated with 5 and 10 ¡.¡,g/ml GCV (Fig. 2).

o 1 5 10Ganciclovir (pg/mI)

FIG.2. The efTect ofincreasing GCV dose on MCA sarcoma cellsinfected with AdMLP .HSTK, AdCMV .Null, or without prior adenovi-ral infection. (3H]Thymidine incorporation decreased as GCV dosewas increased in cells infected with AdMLP .HSTK (P < 0.05 for eachincrease in GCV). Error bars reflect standard deviation.

sisted of animals who received intratumoral AdMLP.HSTK and then ip saline (AdMLP .HSTK/saline), Ad-CMV.Null intratumorally with subsequent GCV (Ad-CMV.Null/GCV), intratumoral viral suspension buffer,and then subsequent GCV (buffer/GCV), and animalswho received intratumoral viral suspension buffer andthen ip saline (buffer/saline).

In Vivo

MCA sarcoma tumor growing in the flanks of theFisher rat expressed the ,B-galactosidase protein afterinfection with AdCMV .LacZ. The blue chromophore, re-sultant from expressed ,B-galactosidase protein and ex-posure to X-gal reagent, was evident in the tumor nod-ule. The percentage transfection based on visual esti-mation from cross sections of tumor after B-Galstaining was between 30 and 50%. This quantificationis only an approximation. (Fig. 3).

The mean tumor weight after excision on Day 14 isillustrated in Table 1. Tumors in the group of animalsthat received intratumoral AdMLP .HSTK with subse-quent b.i.d. ip GCV (AdMLP .HSTR/GCV) weighed 3.8g (:t 0.6 g). The mean tumor weight of animals in thisgroup (AdMLP .HSTR/GCV) was significantly smallerthan the mean tumor weights of animals in each of the.::ther control groups (P < 0.05). Control groups con-

DISCUSSION

140000 ,

O-'",* ?~-~~~~~~.

Despite optimal multimodality therapy, the problemof soft tissue sarcoma recurrence persists. This studydemonstrated the ability oí adenoviral-mediated pro-drug gene therapy to diminish the growth oí sarcomain vitro and in an in vivo model of residual disease. Theability to treat in vivo sarcoma with a herpes simplexthymidine kinase/ganciclovir prodrug gene therapystrategy is novel.

A scenario where the HSTR/GCV paradigm could beemployed clinically is to treat the tumor bed remainingafter excision of a soft tissue sarcoma. Local recurrenceof sarcoma must occur because of residual tumor ce11sremaining after operation. The tumor bed oí a resectedsarcoma has features that may enhance the success ofprodrug gene therapy. These characteristics are the míni-mal amount of residual disease that can exist and thesuperficial nature ofthese residual ce11s. A small amountof residual disease augments the likelihood oí successfultherapy. Superficial, nonbulky disease íacilitates adeno-virus access to tumor ce11s. The "bystander effect'; mayprevent the need for every tumor ce11 to be infected withvirus. The induced death of ce11s adjacent to adenovirus-infected ce11s maximizes potential antitumor efficacy.

Evaluating tumor treatment in a model which ap-proximated clinical postresection sarcoma was im-portant. Our model of residual sarcoma was created onthe principIe that a tumor must have an established

QI 105000

='.6S'"'c; 70000o...!!J

§OU 35000

AdMLP .HSTK*

o 10 100so

Multiplicity of Infection

FIG. 1. The effect of increasing multiplicity of viral infection(m.o.i.) and constant GCV dose on MCA sarcoma cell (3H]thymidineincorporation. Error bars reflect standard deviation. A significantantiproliferative effect was revealea as m.o.i. increased from O to100 (P < 0.05 for each increase in m.o.i.) of cells infected withAdMLP.HSTK prior to GCV.

10 JOURNAL OF SURGICAL RESEARCH: VOL. 70, NO. 1, JUNE 1997

FIG. 3. Photograph of MCA sarcoma tumor expressing the blue chromophore, indicating ¡J-galactosidase production. The metal pinentering the tumor indicates the path of the initial intratumoral injection.

blood supply to achieve rapid growth [14]. In this modelof residual disease, MCA sarcoma tumor implanted inthe latissimus muscle exhibited rapid consistent growthfrom Day 3 after implantation. In orde¡ to affect tumorgrowth and not tumor implantation, treatment with in-tratumoral adenovirus injection was started on Day 3.

Adenoviral infection and GCV administration exhib-ited independent antitumor growth effects in our study.Adenovirus infection is known to be cytopathic to cellsin vivo. Adenoviral infection elicits a strong infiamma-tory response ofpredominantly cytotoxic T (CD8+) lym-phocytes [8]. Tumor growth inhibition by GCV has beenseen in other studies [6]. MCA sarcoma cells were sen-sitive to elevated doses of GCV in vitro as revealedby decreased [3H]thymidine incorporation. The weight

gain of animal s ín the GCV alone group was les s ayerthe study length than animals not receiving GCV butnot significantly. We chose to treat animals with GCVfor 10 days to maximize antitumor effects ofthe combi-nation of GCV and TK, understanding that transgeneexpression was probably less than 10 days. GCV hadan antianabolic effect on the rats that could be relatedto both daily and total clases r~ceived. We do not havean explanation for the antitumor growth effect.

Clinically, the dimensions of a postresection fieldafter sarcoma excision can measure 20 X 20 cm. Theability to treat residual tumor cells ayer a large fieldwith adenoviral mediated prodrug gene therapy pres-ents a challenge. Further, although treated tumorswere smaller at the end of the treatment period, theywere not eradicated. We believe this indicates that thetreatment was able to kill a proportion of the initialtumor cells but cells remaining after transgene expres-sion stopped and cells not infected with the TK genegrow normally. Optimization of both vector and pro-drug are necessary and are currently under study inour laboratory. Strategies include more specifically tar-geting tumor cells through promoter manipulation anddiscovering means to enhance immune recognition oftumor. The demonstration ofadenovirus-mediated h:er-pes simplex thymidine kinase prodrug gene therapy toinhibit in vivo tumor growth has encouraged us to pur-sue further study ofthis modality as an adjuvant treat-ment of human soft tissue sarcoma.

TABLE 1

Day 14 Tumor Weights alter Excision

Tumor weight(mean :!: SEM in g)Group

3.8:t 0.6*6.9:t 1.28.3:t 1.87.2:t 1.2

19.7:t 2.2

AdMLP.HSTR/GCV (n = 12)AdMLP.HSTR/saline (n = 9)AdCMV.NuIVGCV (n = 9)Buffer/GCV (n = 7)Bufferlsaline (n = 4)

Note. SEM, standard error of the mean.* p < 0.05 vs all control groups.

ROSS ET AL.: ADENOVIRAL PRODRUG GENE THERAPY INHIBITS MCA SARCOMA 11

REFEREN CES

1. Jaques, D. P., Coit,D. G., Hadju, S. l., and Brennan, M. F. Man-agement of primary and recurrent soft-tissue sarcoma of theretroperitoneum. Ann. Surg. 212: 51, 1990.

2. Lewis, J. J., Leung, D., Casper, E. S., Woodruff,J. M., andBren-nan, M. F. The Association of Local Recurrence with SubsequentSurvival in Extremity Soft Tissue Sarcoma. Presented at theSociety of Surgical Oncology, 1996.

3. Harrison, L. B., Franzese, F., Gaynor, J. J., and Brennan, M. F.Long-term results of a prospective randomized trial of adjuvantbrachytherapy in the management of completely resected softtissue sarcomas of the extremity and superficial trunk. Int J.Radiat. Biol. Phys. 27: 259, 1993.

4. Sindelar, W. F., Kinsella, T. J., Chen, P. W., DeLaney, T. F.,Tepper, J. E., Rosenberg, S. A., and Glatstein, E. Intraoperativeradiotherapy in retroperitoneal sarcomas. Arch. Surg. 128: 402,1993.

5. Hwang, H. C., Smythe, W. R., Elshami, A. A., Kucharczuk,J. C., Amin, K. M., Williams, J. P., Litsky, L. A., Kaiser, L. R.,and Albeda, S. M. Gene therapy using adenovirus carrying theherpes simplex-thymidine kinase gene to treat in vivo modelsof human malignant mesothelioma and lung cancer. Am. J.Respir. Cell Mol. Biol. 13: 7, 1995.

6. Chen, S., Shine, H. D., Goodman, J. C., Grossman, R. G., andWoo, S. L. C. Gene therapy for brain tumors: Regression of ex-perimental gliomas by adenovirus-mediated gene transfer invivo. Frac. Natl. Acad. Sci. USA 91: 3054, 1994.

7. Clayman, G. L., EI-Naggar, A. K., Roth, J. A., Zhang, W., Goep-fert, H., Taylor, D. L., and Liu, T. In vivo molecular therapywith p53 adenovirus for microscopic residual head and necksquamous carcinoma. Cancer Res. 55: 1, 1995.

8. Yang, Y., Li, Q., Ertl, H. C., and Wilson, J. M. Cellular andhumoral immune responses to viTal antigens create barriers tolung-directed gene therapy with recombinant adenoviruses. J.Virol. 69: 2004, 1995.

9. Moolten, F. L. Tumor chemosensitivity conferred by insertedherpes thymidine kinase genes: Paradigm for a prospective can-cer control strategy. Cancer Res. 46: 5276, 1986.

10. Matthews, T., and Boehme, R. Antiviral activity and mecha-nism ofaction of ganciclovir. Rev. Infect. Dis. 10: 490, 1988.

11. Freeman, S. M., Abboud, C. N., Whartenby, K. A., Packman,C. H., Koeplin, D. S., Moolten, F. L., and Abraham, G. N. The"bystander effect": Tumor regression when a fraction ofthe tu-mor mass is genetically modified. Cancer Res. 53: 5274, 1993.

12. Guzman, R. J., Hirschowitz, E. A., Brody, S. L., Crystal, R. G.,Epstein, S. E., and Finkel, T. In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adeno-virus-mediated transfer of the herpes simplex virus thymidinekinase gene. Frac. Natl. Acad. Sci. USA 91: 10732, 1994.

13. Hirschowitz, E. A., Ohwada, A., Pascal, W. R., Russi, T. J.,Crystal, R. G. In vivo adenovirus-mediated gene transfer oftheEscherichia cali cytosine deaminase gene to human colon carci-noma-derived tumors induces chemosensitivity to 5-fluorocy-tosine. Hum. Gene Ther. 6(8):1055, 1995.

14. Folkman, J. The Tale of angiogenesis in tumor growth. Semin.Cancer Bial. 3: 65, 1992.