[CANCERRESEARCH57, 4205-4209, October 1, 19971

Advances in Brief

Virally Directed Cytosine Deaminase/5-Fluorocytosine Gene Therapy Enhances

Radiation Response in Human Cancer Xenografts'

Nader N. Hanna, Helena J. Mauceri, Jeffery D. Wayne, Dennis E. Hallahan, Donald W. Kufe, andRalph R. Weichselbaum2Department ofSurgery (N. N. H., J. D. W.J and Department ofRadiation and Cellular Oncology (H. J. M., D. E. H., R. R. WI, Pritzker School ofMedicine, University of Chicago,Chicago, Illinois 60637, and Division of Cancer Pharmacology, Dana-Farber Cancer institute ID. W. K.], Boston, Massachusetts 02115

Abstract

Gene therapy combined with radiation therapy to enhance selectivelyradiation cytotoxicity in malignant cells represents a new approach forcancer treatment. We investigated the efficacy of adenoviral (AdS)directed cytosine deaminase/5-fluorocytosine (CD/5-FC) enzyme/prodrug

gene therapy to enhance selectively the tumoricidal action of ionizingradiation in human cancer xenografts derived from a human squamouscarcinoma cell line (SQ-20B). Tumor xenografts grown in hindlimbs ofnude mice were transfected with an adenoviral vector (Ad.CMV.CD)containing the cytosine deaminase (CD) gene under the control of a cytomegalovirus (CMV) promoter. Mice were Injected i.p. with 800 mg/kg ofS-FC for 12 days, and tumors were treated with fractionated radiation ata dose of 5 Gy/day to a total dose of 50 Gy. In larger tumors with a meanvolume of 1069 mm3, marked tumor regression to 11% of the originaltumor volume was observed at day 21 (P 0.01). The volumetric regres.sion of smaller tumors with a mean volume of 199 mini', which receivedthe same combined treatment protocol, was significant at day 12(P = 0.014). However, unlike large tumors, regression of the smallertumors continued until day 36 (P = 0.01), with 43% cured at day 26. Nocures or significant volumetric reduction in size was observed in tumorstreated with radiation alone; AdCMV.CD with or without radiation; or

with Ad.CMV.CD and 5-FC. These results suggest that the CD/S-FCgenetherapy approach is an effective radiosensitizing strategy and may lead tosubstantial improvement in local tumor control that would translate into

improved cure rates and better survival.

Introduction

Radiation therapy is frequently used as a primary or adjuvanttreatment for many human cancers with curative intent or to improvelocal tumor control. For many large epithelial and mesenchymaltumors or tumors of certain histological types, such as glioblastoma,complete tumor eradication and local cure following radiotherapy arerare. Potential mechanisms of radiation resistance include a largepopulation of clonogenic tumor cells, repopulation of tumor cellsbetween doses, cellular repair of radiation damage, and inherent tumor

radioresistance (1). Also microenvironmental factors such as tumorhypoxia may play an important role in clinical radioresistance.

Attempts to improve the therapeutic efficacy of radiation includethe concomitant administration of chemotherapeutic agents. However,the lack of differential effects on tumors and some normal tissueslimits the potential therapeutic efficacy of combining chemotherapeutic agents and radiotherapy due to associated toxicities. One of the

most widely used antineoplastic agent, as well as a clinical radiationsensitizer, is 5-FU.@In addition to its antitumor effects, 5-FU has beenshown in several prospective clinical trials to enhance radiation response and local tumor control in several types of human malignancies (2—6). The enhancement of radiation by 5-FU may involve

inhibition of the mechanisms of cell repair as a result of depletion ofcellular nucleotides and eradication of radioresistant cells in the Sphase of the cell cycle and additive cytotoxic tumor killing effects.

Gene therapy combined with radiotherapy represents a new approach for cancer treatment by selectively radiosensitizing malignantcells. We have previously described the concept of gene therapytargeted by ionizing radiation, whereby a cDNA encoding tumor

necrosis factor-a, is ligated downstream from a radiation-induciblepromoter (7—9).In this way, radiation acts as a “molecularswitch―toactivate transcription of tumor necrosis factor-a, which modifies thecellular response to radiation. Another gene therapy approach with thepotential to enhance the radiation killing of malignant cells is VDEPT(10—21).VDEPT incorporates a viral vector system to deliver a geneencoding a prodrug-activating enzyme to malignant cells. Followingintracellular expression of the enzyme, a nontoxic prodrug is converted to a toxic compound to sensitize selectively tumor cells toradiation. Normal cells not expressing the therapeutic gene are notaffected. Also, because the toxic drug is only produced intratumorally,systemic toxicity is avoided. Although several enzyme/prodrug sys

tems have been investigated for gene therapy (22, 23), VDEPT hasconcentrated mainly on two enzyme/prodrug combinations for clinicalapplication. The first is the HSVtk/GCV combination. The HSVtkenzyme phosphorylates GCV to GCV triphosphate, which acts as achain terminator in DNA synthesis, resulting in the death of dividingcells. The second is the CD/5-FC combination. CD converts nontoxic5-FC to toxic 5-FU. 5-FU inhibits thymidylate synthetase, blockingthe methylation reaction of dUMP to TMP. In this manner, it disruptsDNA synthesis and 5-fluorUTP formation, which is incorporated intoRNA, resulting in cell deaths in both dividing and nondividing cells.In addition to its antineoplastic effects, 5-Ri also sensitize the tumorcells to irradiation. Furthermore, this approach is reported to result intumor-specific immunity (24—26).The present in vivo experimentswere undertaken to investigate the efficacy of virally directed CD/5-FC combination to enhance the tumoricidal action of ionizingradiation in a human cancer xenograft model.

Received 7/15/97; accepted 8/14/97.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was tupported by NIH Grant T32CA09516, The Daniel F. and Ada L. Rice

Foundation, The Chicago Tumor Institute, The Center for Radiation Therapy, and National Cancer Institute Grant CA41068.

2 To whom requests for reprints should be addressed, at Department of Radiation and

Cellular Oncology, Box MC 9030, 5758 South Maryland Avenue, University ofChicago, Chicago, IL 60637. Phone: (773) 702-0817; Fax: (773) 834-7233; E-mail:rrw@rover.uchicago.edu.

Materials and Methods

Cell Lines. Studies were performedusing SQ-20B cells derived from apatient with squamous cell cancer of the larynx after recurrence followingprimary radiotherapy (27). The SQ-20B cell line forms undifferentiated squa

3 The abbreviations used are: 5-FU, 5-fluorouracil; VDEPT, virus-directed enzyme/

pro-drug therapy; HSVtk, herpes simplex virus thymidine kinase; GCV, ganciclovir; CD,cytosine deaminase; 5-FC, 5-fluorocytosine; CMV, cytomegalovirus.

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GENETIC RADIOTHERAPY WITH ENZYME/PRODRUGCOMBINATION

In larger tumors with a mean volume of 1096 mm' (Fig. la),radiation alone produced no volumetric regression compared to antreated controls. Animals in the control group and the radiation alonegroup were sacrificed at day 14 due to large tumor burden. TheAd.CMV.CDvectoraloneor combinedwith5-FCproducedvolumetric regression to a mean of 37% (day 14) of the original tumor volume.This volumetric regression was followed by tumor regrowth anddoubling by day 35. In tumors transfected with Ad.CMV.CD andtreated with 5-FC and 50 Gy, significant volumetric regression to 11%

was observed at day 21 (P = 0.01). This regression was statisticallysignificant beginning at day 11 (P = 0.0004) and continued throughday 28 (P = 0.049). No cures were observed in these tumors receivingthe combined treatment.

In smaller tumors with a mean volume of 199 mm@(Fig. lb),neither radiation alone nor Ad.CMV.CD with or without radiationproduced volumetric regression below the original tumor volume.Furthermore, no significant tumor control was observed in tumorstreated with Ad.CMV.CD and 5-FC injection. Small tumors traitsfected with Ad.CMV.CD and treated with 5-FC and 50 Gy demonstrated significant volumetric regression to 40% of original tumor

volume at day 33 (P = 0.0005), with no regrowth. This volumetricregression was significant beginning at day 12 (P = 0.014) andcontinued through day 36 (P = 0.01). Three of seven tumors in thatcombined group were cured at day 26 with no recurrence.

No treatment-related deaths occurred in treatment groups transfected with the vector and treated with 5-FC and radiation. Mild localedema and skin desquamation were observed in treatment groupsreceiving radiation therapy.

Discussion

Previous experiments using VDEPT has shown that the CD/5-FC

system was not effective when xenograft volume was large. Thepresent experiment was designed to study if the CD/5-FC combinationinteracts with irradiation to improve tumor control in large tumors.

This study clearly demonstrates that 5-FC selectively enhances thetumoricidal action of ionizing radiation in xenografts expressing theCD gene.

The interaction between 5-FU and irradiation has been experimentally studied in a variety of dosages, schedules, and modes of administration without any clear demonstration of therapeutic advantage ofone particular regimen (29—35).In this experiment, we administeredthe drug daily concomitant with radiation therapy to simulate clinicalprotocols. The i.p. route was chosen based on pharmacokinetic studiesof 5-FC in nude mice (13). Previous studies have shown that 5-FC isnot toxic to nude mice or other mouse strains (11, 24, 36). By contrast,5-FU was relatively toxic to nude mice when given via i.p. or oral

route at a dose of 25 and 32 pjg/kg, respectively. The half life of 5-FCis approximately 40 mm in nude mice, and plasma levels of 25 p@gor

greater could be maintained for approximately 4 h following a singlei_p. injection of 500 mg of 5-FCIkg of body weight (13). Huber et a!.(13) also demonstrated that the i.p. route was more efficacious thancontinuous tail infusion. Pharmacokinetics studies (14, 37) of 5-Rigenerated intratumorally from targeted CD/5-FC gene therapy demonstrated that intratumoral 5-Ri reached a maximal level at approximately 45 mm after 5-FC administration, with peak levels seventimes greater than those following systemic 5-Ri administration.Furthermore, 5-Ri levels remained elevated for a longer time, and

there was a 17-fold increase in the area under the curve of intratumoral 5-Ri compared to systemic drug administration.

The efficacy of VDEPT is dependent not only on the efficiency ofgene transfer but also upon the enzyme/prodrug system. The expression of CD enzyme in colon tumor cells has resulted in a 600-fold

mous cell cancer xenografts in nude mice and is relatively radioresistant (Di,,239 cGy; Ref. 28) when compared with other tumor cell lines.

Growth of Human Tumor Xenografts in Vivo. Xenografts were grown inthe hindlimbs of 6—8-week-oldathymic female nude mice (Frederick CancerResearch Institute, Frederick, MD) by s.c. inoculation of 5 X 106 cellssuspended in 100 ml of PBS. All animal procedures followed establishedguidelines for humane treatment of animals. Tumors were permitted to growfor 10—14days prior to treatment. At day 0, initial tumor volume wasdetermined by direct measurement with calipers and calculated by the formula(a X b x c/2), which is derived from the formula for an ellipsoid (ird3/6).

Tumor volumes were measured twice/week until animal death or for at least 45days from vector inoculation. Based on the day 0 tumor volume, mice wererandomly assigned to treatment groups (n = 7 in each group) such that themean volume of each treatment group was approximately equal. The effect oftreatment was studied in small (mean volume, 199 mm3) and large (meanvolume, 1069 mm3) xenografts.

Gene Transfer. SQ-20B xenograftswere injectedwith the Ad.CMV.CDvector (GenVec, Inc., Rockville, MD) via direct intratumoral injection using aHamilton syringe. The adenoviral vector (AdS) is a recombinant replicationdeficient type 5 adenovirus with El and E3 deletion. It contains the CD genedownstream of the 5‘long tenninal repeat sequence under the control of aconstitutive CMV promoter. Tumors were injected (twice/week for 2 weeks)with a dose of lO@plaque-forming units/injection on days 0, 4, 7, and 11 (Table1). The replication competent Adenovirus-(RCA) for the vector was 1 X l0@.

Chemicals and Dnig Treatment. The prodrug 5-FC was purchased fromSigma Chemical Co. (St. Louis, MO.). The prodrug was reconstituted in sterilewater to a concentration of 250 mg/l2.5 ml of 5-FC at a maximum dose of 800

mg/kg body weight (as limited by the drug solubility), injected i.p. daily, 5days/week for a total of 12 days (Table 1).

Xenograft Irradiation. Irradiated mice were immobilized in Lucite chambers, and the entire body was shielded with lead except for the tumor bed.Tumors were irradiated, 5 Gy/day, 4 days/week, beginning at day 3 (Table 1)to a total dose of 50 Gy using a Maxitron generator (1.88 Gy/min). Irradiationwas performed within 2 h after i.p. administration of 5-FC.

Experimental Design. To study the potential interaction between the vector Ad.CMV.CD, 5-FC, and X-irradiation, SQ-20B xenografts were grown toa mean volume of 1069 mm@.Animals were assigned to one of five treaunentgroups (n = 7 in each group): a combined treatment group receiving Ad.CMV.CD, 5-FC, and 50 Gy; and four control groups receiving no treatment, 50Gy alone, Ad.CMV.CD alone, or Ad.CMV.CD plus 5-FC. To compare theeffect of combined treaunent in small tumors, the same experiment wasconducted in xenografts with a mean volume of 199 mm@. An additionalcontrol group receiving Ad.CMV.CD and 50 Gy was included. The scheduleofdelivery ofthe vector, 5-FC, and irradiation, outlined in Table 1,was chosento simulate clinical radiotherapy. 5-FC administration was not begun until day3 to allow for stable transfection of tumor cells with the viral vector.

Statistical Analysis. Statistical significance was determined by variousstatistical tests including ANOVA, Kruskal/Wallis test, and Two-Sample

Wilcoxon Rank-Sum test. Data are reported as the percentage of original (day0) tumorvolume and graphedas the fractionaltumorvolume ±SE.

Results

No spontaneous tumor regression was observed in untreated controlgroups in both large and small tumors. The initial tumor size independently affected the rate of tumor growth across different treatmentgroups. Tumor doubling time was shorter in small tumors and wasstatistically significant compared to large tumors, starting at day 4(P = 0.0001) until day 36 (P = 0.0007).

Table I Schedule ofdelivery of viral vector (Ad.CMV.CD) at a dose of 10' plaqueforming units (PFU)/injection, 5-FC at a dose of 800mg/kg body weight, and

X-irradiation (XRT) at a dose of 5 Gy/day

Day

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0 5 10 15 20 25 30 35 40 45

DAY

.-.-XRT (50 Gy)

a10

0

0.1

b

‘1

-.I&-.Ad.QIIV.CD

—*-Ad.CMV.CD+ 5-PC

—E,-.AdXMV.CD+ 5-PC +XRT

Fig. 1. Fractional tumor volume of SQ-20B xcnografts assigned to different treatment groups(a 7 mice in each group): untreated controls,

500y; Ad.CMV.CD; Ad.CMV.CD with 5-FC; Ad.CMV.CD with 50 Gy; and Ad.CMV.CD with 5-FCand 50 Gy (XR1). a, large xenografts with a meanvolume of 1069 mm3; bars, SE. b, small xenograftswith a mean volume of 199 mm3; bars, SE.

100

@Control

-4--XRT(50 03v)

—&-A@CMV.CD

—--M.CMV.CD+XRT

-*-M.CMV.CD+5-PC

-6--M.CMV.CD+5-PC +XRT

1

0.10 5 10 15 20 25 30 35 40 45

DAY

increase in sensitivity to the prodrug 5-FC (13). Huber et aL (14) havealso reported that when only 2% of colorectal tumor mass containsCD-expressing cells, significant regression in all tumors was observedwhen mice were treated with nontoxic levels of 5-FC. Similar resultswere reported by Trinh et a!. (38). This bystander effect due to thereadily diffusible toxic catabolite 5-Ri (14, 39) appears to overcomelow gene transfer efficiency and accounts for the significant therapeutic effect. In the present experiment, a significant bystander therapeutic effect is evident by the tumor regression observed. A similarbystander effect has also been described with the HSVtk/GCV system,both in culture and in vivo (40—53).No cell contact is needed with theCD/5-FC system, as opposed to the HSVtk/GCV combination, whichrequires metabolic cooperation between cells through gap junctions(40, 43—45)and/or transfer of apoptotic vesicles (46, 47). The in vivomechanism of this bystander effect may require an intact immunological system (50—53).Trinh et a!. (38) has shown recently that theCD/5-FC combination is more efficacious compared to HSVtk/GCV

in controlling WiDr colorectal xenografts when only a small percentage of the tumor cells expressed the therapeutic gene due to a greaterbystander effect.

Khil et a!. (54) have reported that tumor cells transfected with the

CD gene are sensitized to radiation killing in vitro when exposed to5-FC for 72 h prior to radiation. This radiation enhancement effectwas evident only in preirradiated cells and was dependent on the drugconcentration and preirradiation exposure time. Similar radiation enhancement effects have been reported with various enzyme/pro-drugcombinations other than CD/5-FC. Recent reports have demonstratedthat the HSVtk/bromovinyl deoxyuridine enzyme/prodrug combination enhances radiation cytotoxicity in human glioma cells (19, 55—57). U-251 human glioma cells transduced with HSVtk and exposedto 40 @g/mlof bromovinyl deoxyuridine for 24 h prior to irradiationwere more sensitive to radiation compared to control cells. Kim et a!.(55)reportedrecentlythattheantiviralagentsbromovinyldeoxyuridine and acyclovir, administered systemically, selectively enhanced

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radiation cytotoxicity in rat 9L gliosarcoma tumor cells transfectedwith the HSVtk gene both in vitro and in vivo.

The CD/5-FC enzyme/pro-drug system is of particular clinicalinterest because the enzyme CD is not found in mammalian cells, andthe prodrug 5-FC is relatively nontoxic in humans (58, 59). 5-Ri canreadily diffuse into and out of cells, crossing biological membranes bynonfacilitated diffusion, accounting for a significant therapeutic bystander effect, which overcomes the need for targeted gene expressionin every tumor cell. Through its mechanisms of action involving DNAand RNA pathways, 5-Ri results in deaths of both dividing andnondividing cells. 5-Ri is also the radiosensitizing agent of choice

used presently in the treatment of several human tumors.The finding that no significant volumetric regression occurred in

tumors treated with ACLCMV.CD and 5-FC is explained by ourprevious finding that SQ-20B tumors are not sensitive to 5-Ri (28).

An unexpected and interesting finding was the observation thatlarge tumors injected with Ad.CMV.CD grew more slowly thanuninjected control tumors. The effect of the vector alone on tumorgrowth could be due to a direct cytotoxic effect of the adenovirus or

to an immunological effect as a result of: more effective antigenpresentation from dead CD+ cells; inflammatory response to CD+

dying cells with recruitment and activation of antigen presenting cells;or immunogenicity of the CD protein itself. A direct viral cytotoxiceffect seems unlikely because we have demonstrated previously thattreatment with adenoviral vector with or without radiation producedno significant tumor control in SQ-20B xenografts (8). This findingled us to add an additional control group, receiving Ad.CMV.CD plusirradiation in the second experiment with smaller tumors, to determinewhether the presence of the Ad.CMV.CD plays a role in tumorregression when combined with radiation. No tumor regression belowthe original tumor volume was observed in this group, confirming ourprevious finding (8). Taken collectively, these data suggest thatCD/5-FC combination is effective as a radiosensitizing strategy invivo, especially in tumors that cannot be controlled effectively withradiation alone or tumors not sensitive to 5-Ri.

Conclusion. VDEPT combined with radiotherapy for the treatmentof cancer offers a new potential to improve the therapeutic efficacy ofradiation through selective tumor cell radiosensitization. Radiosensitization via local conversion of 5-FC to 5-Ri can potentially lead tomore tolerable treatment without the local or systemic toxicity ofconventionally delivered 5-Ri. This approach would be particularlyuseful for the treatment of large unresectable tumors or tumors with ahigh rate of local failure after curative resection. Additional studiesare needed to determine the optimal radiosensitizing dose, as well asthe duration and sequence of delivery of 5-FC in relation to radiationtherapy.

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1997;57:4205-4209. Cancer Res   Nader N. Hanna, Helena J. Mauceri, Jeffery D. Wayne, et al.   XenograftsTherapy Enhances Radiation Response in Human Cancer Virally Directed Cytosine Deaminase/5-Fluorocytosine Gene

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