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Annals of Surgical Oncology 9:500-504 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Combined Gene Therapy and Ionizing Radiation Is a Novel Approach To Treat Human Esophageal Adenocarcinoma

Vinay K. Gupta, MD, James O. Park, MD, Nora T. Jaskowiak, MD, Helena J. Mauceri, PhD, Saraswathy Seetharam, PhD, Ralph R. Weichselbaum, MD and Mitchell C. Posner, MD

From the Departments of Surgery (VKG, JOP, NTJ, MCP) and Radiation & Cellular Oncology (HJM, SS, RRW), The University of Chicago, Chicago, Illinois.

Correspondence: Address correspondence and reprint requests to: Mitchell C. Posner, MD, Department of Surgery, University of Chicago, 5841 South Maryland Ave., MC 5031, Chicago, IL 60637; Fax: 773-702-4444; E-mail: mposner@ surgery.bsd.uchicago.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The ability to infect tumor cells limits the antitumor effects of gene therapy. The addition of radiotherapy to treatment with Ad.Egr.TNF.11D, a replication-deficient adenovirus containing a radiation-inducible promoter, early growth response-1, and the tumor necrosis factor-{alpha} (TNF{alpha}) complementary DNA may enhance the therapeutic ratio.

Methods: Seg-1 human esophageal adenocarcinoma cells were treated with Ad.Egr.TNF.11D with or without radiation. TNF{alpha} levels were quantified with enzyme-linked immunosorbent assay. Athymic nude mice bearing Seg-1 tumors were randomized to buffer, ionizing radiation, Ad.Egr.TNF.11D, and combination therapy. Tumor growth delay was used to compare treatment regimens. TNF{alpha} levels were measured in tumor homogenates and plasma.

Results: Seg-1 cells treated with Ad.Egr.TNF.11D and ionizing radiation demonstrated increased TNF{alpha} levels at 72 hours compared with cells exposed to vector alone (124 ± 0 pg/mL vs. 31.11 ± 22 pg/mL; P = .008). In vivo, Ad.Egr.TNF.11D-treated tumors expressed low TNF{alpha} levels (151.5 ± 107.11 pg/mg protein) compared with tumors receiving combined treatment (793.92 ± 489.13 pg/mg protein; P = .067). Increased TNF{alpha} levels were associated with increased tumor growth delay after combined treatment (P < .05).

Conclusions: Radiotherapy enables focal stimulation of TNF{alpha} expression in Ad.Egr.TNF.11D-infected cells and thus improves local tumor control.

Key Words: Tumor necrosis factor-{alpha} • Esophageal cancer • Ionizing radiation • Gene therapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of esophageal adenocarcinoma continues to increase in the United States.1– 3 Despite the recent availability of a wide array of chemotherapeutic agents, the application of multimodality therapy, and the reduction in surgical mortality, the prognosis remains poor.4–6 Clinical investigations using combined induction chemotherapy and radiation in operable esophageal cancer have resulted in improved local tumor control and have produced a greater number of clinical responses than either preoperative chemotherapy or radiotherapy alone.7,8 Unfortunately, the improved objective response rates have not translated into a survival advantage in prospective randomized clinical trials.9,10 However, the subset of patients who achieve a complete pathologic response after combined-modality therapy have an improved disease-free and overall survival compared with nonresponders.7 The use of innovative antitumor strategies that address both the gross primary and occult systemic disease in patients with esophageal cancer should increase the number of patients who derive a survival benefit.

Tumor necrosis factor-{alpha} (TNF{alpha}) is a cytokine with direct cytotoxic and radiosensitizing antitumor effects in a variety of gastrointestinal neoplasms.11–14 The combination of TNF{alpha} with ionizing radiation (IR) in phase I clinical trials has resulted in complete tumor regression in a subset of patients; however, significant toxicities, including fever, nausea, anorexia, and hypotension, have prevented its continued investigation as a systemically administered therapeutic antitumor agent.15,16 Therefore, if TNF{alpha} delivery can be locally contained, a therapeutic benefit may be achieved in the absence of significant systemic toxicity.17

Gene therapy has demonstrated utility in enhancing local tumor control via delivery of DNA constructs that encode genes for a variety of cytotoxic lymphokines, such as TNF{alpha}.18 Gene therapy targeted by IR is a concept in which the complementary DNA for a cytotoxic gene is ligated downstream of a radiation-inducible promoter.19,20 The early growth response-1 gene (Egr-1) encodes a nuclear phosphoprotein21 involved in the transition of cells from G0 to G1 after growth stimulation.22 We reported that Egr-1 is transcriptionally induced after exposure to IR at doses of 3, 10, and 20 Gy.23 In previously published studies, we used an adenoviral vector containing the promoter of the Egr-1 gene linked to the complementary DNA for TNF{alpha} (Ad.Egr-TNF). When xenografts received combined treatment with Ad.Egr-TNF and IR, significant tumor regression was achieved. It is important to note that no loss of hind limb motility or skin desquamation was observed. These initial studies suggested an increase in the therapeutic ratio with little increase in acute effects.24 In this study, we used a new clinical grade adenoviral vector (Ad.Egr.TNF.11D), presently in human trials, to examine local and systemic effects with a xenograft model of human esophageal adenocarcinoma. We report that selective TNF{alpha} induction by IR in cells infected with Ad.Egr.TNF.11D enhances intratumoral TNF{alpha} concentrations and demonstrates greater growth delay than tumors treated with either vector or IR alone.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Seg-1 tumor cells (a gift of Dr. David Beers, University of Michigan) were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum, L-glutamine, 100 µg/mL of streptomycin, and 100 U/mL of penicillin. The cell line was incubated at 37°C in 7% CO2.

Radiation Inducibility of Ad.Egr.TNF.11D in Seg-1 Cells
A total of 2 x 105 Seg-1 tumor cells in 2 mL of growth medium were plated in six-well tissue culture plates overnight. The medium was removed, and cells were infected with Ad.Egr.TNF.11D (a gift of GenVec, Inc., Gaithersburg, MD) in serum-free medium at 0, .1, 1, and 10 multiplicities of infection (MOI) for 2 hours. After incubation, wells were rinsed with serum-free medium, and 2 mL of fresh growth medium was added. Irradiated cultures were exposed to 5 Gy with the Pantak PMC 1000TM x-ray generator (East Haven, CT) operating at rate of 192 cGy/minute, 250 kV, and 15 mA. Conditioned medium was harvested at 0, 6, 24, 48, and 72 hours and stored at -80°C. Human TNF{alpha} concentrations were determined by QuantikineTM enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Seg-1 Xenografts
All animal work was performed under protocols approved by the University of Chicago Animal Research Committee. A total of 3 x 106 Seg-1 tumor cells in 100 µl of serum-free medium were injected subcutaneously into the right hind limb of female athymic nude mice (Frederick Cancer Research Institute, Frederick, MD). Tumor volumes were measured three times weekly, and the volume was determined as previously described.24 Data are reported as a mean fractional tumor volume for each group ± SEM.

Seg-1 Xenograft Regrowth Studies
Nude mice bearing Seg-1 xenografts (mean volume, 261 ± 21.6 µl; n = 30) were randomized to one of four treatment groups: 10 µl of viral buffer (n = 5); 4 x 109 particle units x five doses of Ad.Egr.TNF.11D (n = 5); 10 µl of viral buffer and 5 Gy x five doses (n = 8); or combined Ad.Egr.TNF.11D and IR x five doses (n = 12). Three hours before IR, tumors were injected with either viral buffer or vector. Tumors were injected in five separate sites by using a 10-µl Hamilton syringe. Tumor beds were irradiated with a Pantak PMC 1000 x-ray generator operating at 150 kV and 25 mA at a dose rate of 192 cGy/minute. Animals were shielded with lead except for the tumor-bearing hind limb. Tumors were measured and recorded as described. Data are shown as the mean fractional tumor volume ± SEM for each treatment group. Statistical significance was determined by analysis of variance (Sigma StatTM 2.0; Jandel Scientific, Costa Madre, CA).

TNF{alpha} Induction in Tumor Xenograft and Plasma
Eighteen athymic nude mice bearing Seg-1 xenografts were randomized to four treatment groups: viral buffer (n = 3), Ad.Egr.TNF.11D (n = 3), viral buffer and IR (n = 6), or Ad.Egr.TNF.11D and IR (n = 6). Animals were injected intratumorally with viral buffer or Ad.Egr.TNF.11D on days 0 and 1. Tumors from the buffer plus IR and virus plus IR groups were exposed to 5 Gy on days 0, 1, 4, 5, and 6. Mice were killed by metaphane inhalation on day 7. Tumors were excised, snap frozen in liquid nitrogen, and stored at -80°C. Tumor samples were then thawed and homogenized in 500 µl of protein extraction buffer (150 mM of NaCl; 10 mM of Tris-HCl, pH 7.5; 5 mM of EDTA, pH 7.5; 10 µg/mL of aprotinin; 5 µg/mL of leupeptin; and 100 mM of phenylmethyl sulfonyl fluoride) by using the Brinkman PolytronTM homogenizer (Kinematica AG, Littau, Switzerland) for 30 seconds on ice. After four freeze/thaw cycles, tumor homogenates were centrifuged at 4°C at 10,000 rpm for 10 minutes by use of the RC5CTM refrigerated centrifuge (Sorvall Instruments, Inc., Newton, CT) with the SS-34 rotor. BioRad macroassay protein analysis (BioRad Laboratories, Hercules, CA) was performed on the tumor homogenate supernatants according to the manufacturer’s instructions. Blood was also obtained from corresponding animals by aspirating the inferior vena cava with a 1-mL syringe and a 23-gauge needle. After adding blood samples to 100 µl of .105 M of buffered sodium citrate on ice, the samples were centrifuged at 4°C at 3000 x g for 10 minutes. Plasma supernatant was extracted and stored at -80°C. Enzyme-linked immunosorbent assay was performed on the tumor homogenates and plasma supernatants to quantify human TNF{alpha} concentrations. Data represent mean human tumor TNF{alpha} concentration normalized to the protein concentration ± SEM and mean human plasma TNF{alpha} concentration ± SEM. Statistical analysis was performed with one-way analysis of variance (Sigma Stat 2.0).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IR Enhances TNF{alpha} Expression in Seg-1 Cells
Seg-1 cells incubated with Ad.Egr.TNF.11D at 10 MOI demonstrated peak TNF{alpha} secretion at 48 hours with a concentration of 38 ± 22 pg/mL in conditioned medium. In comparison, Seg-1 cells treated with 10 MOI Ad.Egr.TNF.11D and exposed to 5 Gy of IR demonstrated a TNF{alpha} concentration of 58 ± 22 pg/mL at 48 hours that was amplified to 124 ± 0 pg/mL at 72 hours (Fig. 1). At 72 hours, TNF{alpha} levels were significantly enhanced after exposure to IR (124 ± 0 pg/mL) compared with vector alone (26 ± 10 pg/mL; P = .008).



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  		FIG. 1. Radiation amplifies tumor necrosis factor-{alpha} (TNF{alpha}) expression in Ad.Egr.TNF.11D-infected tumor cells. A total of 2 x 105 Seg-1 cells were exposed to 10 multiplicities of infection of Ad.Egr.TNF.11D for 2 hours followed by either 5 Gy ({blacksquare}) or no radiation ({blacktriangleup}). At 72 hours, TNF{alpha} levels increased in the conditioned media from cells treated with Ad.Egr.TNF.11D plus ionizing radiation (IR) compared with vector alone (P = .008).

 
IR Enhances TNF{alpha} Expression in Seg-1 Xenografts
Tumors were harvested on day 7 to quantify intratumoral TNF{alpha} levels. No human TNF{alpha} was detected in tumors injected with viral buffer or exposed to IR alone. Tumors treated with Ad.Egr.TNF.11D alone expressed lower levels of TNF{alpha} (151.48 ± 107.11 pg/mg protein) compared with tumors treated with Ad.Egr.TNF.11D and IR (793.92 ± 489.13 pg/mg protein; P = .067; Fig. 2). IR exposure produced a 5.24-fold increase in intratumoral TNF{alpha} concentration.



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  		FIG. 2. Radiation amplifies tumor necrosis factor-{alpha} (TNF{alpha}) expression in Ad.Egr.TNF.11D-treated Seg-1 xenografts. Athymic nude mice bearing Seg-1 xenografts were randomized to one of four treatment groups: buffer alone (n = 3), ionizing radiation (IR; 5 Gy x 5 fractions; n = 3), intratumoral Ad.Egr.TNF.11D (4 x 109 particle units x two doses; n = 3), and combined Ad.Egr.TNF.11D and IR (n = 3). Tumors were harvested at day 7. TNF{alpha} was not detectable in buffer- and IR-treated xenografts. TNF{alpha} levels in tumor homogenates were increased in the combined Ad.Egr.TNF.11D plus IR treatment group compared with Ad.Egr.TNF.11D alone (P = .067).

 
IR Enhances Plasma TNF{alpha} Expression Without Systemic Toxicity
Tumors and plasma were collected on day 7 to quantify intratumoral and circulating human TNF{alpha} levels. Human TNF{alpha} was not detectable in plasma from mice treated with either viral buffer alone or IR alone. Plasma from mice receiving the combination of Ad.Egr.TNF.11D and IR demonstrated an increase in human TNF{alpha} compared with the plasma from Ad.Egr.TNF.11D-treated mice (.23 ± .13 pg/mL vs. .7 ± .1 pg/mg protein; P = .017). Intratumoral TNF{alpha} levels in the Ad.Egr.TNF.11D plus IR group (793.92 ± 6.67 pg/mg protein) were also increased compared with those in the vector-alone group (122.16 ± 107.12 pg/mg protein; Fig. 3). It is noteworthy that TNF{alpha} levels within the tumor after combined treatment were more than 3 log-fold higher than those detected in the plasma of corresponding animals. No evidence of local or systemic toxicity was observed in any experimental animal from any treatment group.



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  		FIG. 3. Radiation amplifies tumor necrosis factor-{alpha} (TNF{alpha}) expression in Seg-1 xenografts and mouse plasma after treatment with Ad.Egr.TNF.11D. Plasma was collected from mice in all four treatment groups at day 7. TNF{alpha} was not detectable in the plasma from buffer-treated (n = 3) or buffer- and ionizing radiation (IR)–treated (n = 6) animals. Plasma TNF{alpha} levels were greater in the combined Ad.Egr.TNF.11D plus IR treatment group (n = 6) compared with the Ad.Egr.TNF.11D-alone group (n = 3; P = .017). No local or systemic toxicity was observed in animals from any treatment group. Normalized for protein, intratumoral TNF{alpha} levels were 3 log-fold greater than plasma TNF{alpha} levels.

 
Combined Treatment With Ad.Egr.TNF.11D and IR Increases Tumor Growth Delay
Seg-1 xenografts treated with viral buffer alone demonstrated a high rate of growth, reaching a tumor volume six times its original volume by day 9. Five doses of 4 x 109 particle units of Ad.Egr.TNF.11D had little effect on slowing tumor growth. Tumors treated with vector alone reached a fractional tumor volume of 6 at day 14. Xenografts exposed to five 5-Gy fractions demonstrated regression to original volume, with regrowth beginning at day 12. Combined treatment with Ad.Egr.TNF.11D and IR produced significant regression to original volume, with regrowth beginning at day 18 (P < .05; Fig. 4). Compared with control tumors that tripled in volume, xenografts treated with Ad.Egr.TNF.11D alone demonstrated a 4-day delay in tumor growth, IR-treated tumors a 17-day delay, and combined vector- and IR-treated tumors a 25-day delay to reach the same mean volume.



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  		FIG. 4. Radiation increases growth delay in Ad.Egr.TNF.11D-treated Seg-1 xenografts. Athymic nude bearing Seg-1 xenografts (n = 30) were randomized to buffer alone ({blacktriangleup}, n = 5), buffer plus ionizing radiation (IR; 5 Gy x 5 fractions; total dose, 25 Gy; •, n = 8), intratumoral Ad.Egr.TNF.11D (4 x 109 particle units x 5 doses; {triangleup}, n = 5), and combined Ad.Egr.TNF.11D and IR ({blacktriangleup}, n = 12). Mean fractional tumor volumes in the IR group were greater than those in the combined-treatment group (Ad.Egr.TNF.11D plus IR, P < .05). Compared with buffer-treated controls, Ad.Egr.TNF.11D-treated xenografts took 4 days, IR-treated xenografts took 17 days, and Ad.Egr.TNF.11D- and IR-treated xenografts took 25 days to reach a mean fractional tumor volume of 3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These studies investigated the effects of a novel local treatment for human esophageal adenocarcinoma by using an adenoviral TNF{alpha} vector. It has been demonstrated that TNF{alpha} exerts significant antitumor activity in a wide variety of malignancies because of its inherent cytotoxicity and its radiosensitizing properties. However, significant systemic toxicities associated with intravenous TNF{alpha} administration have prompted investigations to identify other routes of TNF{alpha} delivery that are nontoxic but that still achieve antitumor activity. We demonstrate that transduction of the TNFA gene into target tumor cells by using a replication-deficient adenovirus containing a radiation-inducible promoter, Egr-1, produces TNF{alpha} expression by infected Seg-1 human esophageal adenocarcinoma cells in vitro. To study the effect of IR on TNF{alpha} expression, we treated Ad.Egr.TNF.11D-infected Seg-1 cells with 5 Gy and demonstrated a 4.8-fold increase in TNF{alpha} expression. These findings confirm that IR enhances cytokine expression by Ad.Egr.TNF.11D-infected tumor cells.

To study the antitumor interaction between TNF{alpha} and IR in vivo, we treated Seg-1 xenografts with Ad.Egr.TNF.11D and IR. A growth delay of 21 days was observed after combined treatment with Ad.Egr.TNF.11D plus IR compared with Ad.Egr.TNF.11D alone, and a delay of 8 days was observed compared with IR alone. Induction studies demonstrated a 5.25-fold increase in TNF{alpha} expression after combined treatment (Ad.Egr.TNF.11D and IR) compared with vector alone at day 7. These findings are in agreement with our previously published studies in which we demonstrated induction of TNF{alpha} protein for 21 days after a single intratumoral injection of vector with continued fractionation.24 Although plasma TNF{alpha} was also increased in Ad.Egr.TNF.11D- and IR-treated animals, plasma TNF{alpha} levels were more than 3 log-fold lower than intratumoral TNF{alpha} levels. No animal in any group exhibited local or systemic toxicity. These findings taken together suggest that TNF{alpha} expression can be spatially and temporally controlled by IR, leading to improved local tumor control without toxicity.

The use of viral vectors has had limited success in many in vivo animal tumor models because of poor infection rates. However, adenoviral vectors, such as the one used in this study, are capable of infecting both dividing and nondividing cells. Insertion of promoters, such as Egr-1, into adenoviral vectors enables stimulation of transgene expression by IR. Unlike other viral vectors, incorporation of the adenovirus vector into the genome is not required for gene expression by the infected tumor cell. Previous studies conducted in our laboratory to analyze the distribution of TNF{alpha} protein revealed granular intracytoplasmic staining of TNF{alpha} within infected tumor cells and on the surface on noninfected tumor cells. These findings suggested that TNF{alpha} mediates tumor cell killing by a bystander effect and provided evidence that infection of a large proportion of tumor cells is not required for an antitumor effect.24

Induction of a host T-cell immune response clears the adenoviral vector from the tumor microenvironment and prevents long-term TNF{alpha} expression. The fact that in vivo studies were performed in athymic nude mice suggests that the observed antitumor effects are in fact a result of the TNF{alpha} and IR interaction and are not due to a T cell–mediated inflammatory response. These preclinical studies using combined treatment with Ad.Egr.TNF.11D and radiation demonstrate enhanced antitumor efficacy in a human esophageal adenocarcinoma model. Accessibility of human esophageal tumors endoscopically enables intratumoral administration of the vector under direct vision, and therapeutic dosing can be administered as required. The esophagus can be effectively exposed to IR with external beam or intraluminal sources, or both, to enhance local TNF{alpha} expression and boost local tumor control. Increased TNF{alpha} levels may elicit systemic mediators, such as cytokines and matrix proteinases, that target occult distant disease and thereby further enhance the therapeutic ratio. Taken together, the local and systemic antitumor effects after combined Ad.Egr.TNF.11D and radiotherapy may increase the number of patients who achieve a complete pathologic response and subsequently may translate to a survival benefit in this patient population. The absence of local and systemic toxicities with intratumoral administration of Ad.Egr.TNF.11D supports the safe addition of TNF{alpha} genetic radiotherapy to current investigational antitumor protocols of esophageal cancer.


   Acknowledgments
 
Supported by gifts from the GenVec Corporation.

Received for publication November 5, 2001. Accepted for publication March 9, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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