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. Author manuscript; available in PMC: 2006 Jul 1.
Published in final edited form as: Cancer Gene Ther. 2006 Jan 1;13(1):82–90. doi: 10.1038/sj.cgt.7700863

Combination Effect of Oncolytic Adenovirotherapy and TRAIL Gene Therapy in Syngeneic Murine Breast Cancer Models

Wei Guo 1, Hongbo Zhu 1,2, Lidong Zhang 1, John Davis 1,3, Fuminori Teraishi 1, Jack A Roth 1, Clifton Stephens 4, Juan Fueyo 5, Hong Jiang 5, Charles Conrad 5, Bingliang Fang 1,3,✉
PMCID: PMC1343537  NIHMSID: NIHMS2121  PMID: 16037823

Abstract

TRAIL gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.

Keywords: virotherapy, synergism, liver metastasis, TRAIL, murine tumor model

INTRODUCTION

With more than 40,000 American women dying of breast cancer every year1,2, new therapies are urgently needed. Two agents that have been extensively investigated are the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and various oncolytic viruses3,4,5,6. However, preclinical and clinical studies have revealed that the preclinical application of these two agents is hampered by either their weak anticancer activity or their systemic toxicity. Therefore, the development of strategies that maximize their anticancer activity and minimize their systemic toxicity is essential to the success of these agents in treating cancer.

In this study, we hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. An oncolytic adenovirus replicates in tumor cells and helps the replication-defective TRAIL virus replicate, thereby increasing the expression of the gene and the viral copy number. This increased expression of TRAIL leads to apoptosis, thereby facilitating the lysis of the tumor cells, the release of vector progenies, and the spread of the vector within the tumor cells.

However, on the basis of data from a study of a human adenovirus in a normal mouse lung model7, many investigators have assumed that human adenoviruses could not replicate in mouse cells. Very recently, though, Wang et al. reported that some mouse cancer cell lines were permissive to a human oncolytic adenovirus8, suggesting that murine syngeneic cancer models are valuable in assessing oncolytic adenovirus antitumor activity in vivo, especially in immunocompetent settings. Because an immune response to a viral infection is considered a potential limitation for oncolytic virotherapy, data based on immunocompetent animals should be more valuable than those based on immunocompromised animals.

Thus, in this study we evaluated the antitumor effects of an oncolytic adenovector, Delta243,4, in both human and murine breast cancer cell lines and the effects of TRAIL gene therapy combined with oncolytic adenovirotherapy in these cell lines. We found that the combined therapy produced enhanced antitumor activity over either agent alone in both the human and the murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. This finding of synergism between the two therapies has the potential to affect the development of oncolytic virotherapies.

MATERIALS AND METHODS

Cells Lines and Cell Culture

The human breast cancer cell lines MDA-MB-231 and MDA-MB-435 and the mouse breast cancer cell lines 4T1 and LM2 were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, antibiotics, and glutamine. The cells were cultured at 37°C in a humidified incubator containing 5% CO2.

Adenoviruses

The adenovectors Ad/TRAIL-F/RGD, Ad/CMV-GFP, and Delta24 5,9 were described previously. The expansion, purification, titration, and quality analyses of the three vectors were performed at the vector core facility of our institution, The University of Texas M. D. Anderson Cancer Center, as described previously10.

Cell Viability Assay

We treated the human breast cancer cell lines and the mouse breast cancer cell lines with the following adenovectors: 1) Ad/CMV-GFP, 2) Ad/TRAIL-F/RGD, 3) Delta24, 4) Ad/CMV-GFP plus Ad/TRAIL-F/RGD, 5) Ad/CMV-GFP plus Delta24, and 6) Ad/TRAIL-F/RGD plus Delta24. Cells treated with phosphate-buffered saline (PBS) were used as a mock control. Cell viability was determined by an XTT assay (cell proliferation kit II, Roche Molecular Biochemicals, Indianapolis, IN) as described previously6,11. Briefly, 5 × 103 cells/well were seeded in 96-well plates; one day later, the cells were treated with different adenovectors at multiplicities of infection (MOIs) ranging from 30 to 3,000 particles per cell. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Cell growth and viability were quantified by using the XTT assay over time after treatment. Each experiment was performed in quadruplicate, and each series was repeated at least twice.

Western Blot Analysis

The 4T1 cells were washed with cold PBS and lysed in Laemmli’s lysis buffer. Equal amounts of lysate were separated by electrophoresis on a 10% sodium dodecyl sulfate polyacrylamide gel and then transferred to Hybond-enhanced chemiluminescence membranes (Amersham Biosciences, Arlington Heights, IL). The membranes were blocked with a blocking buffer containing 5% low-fat milk and PBS containing 0.05% Tween-20 for at least for 1 h and sometimes overnight at 4°C, washed three times, again with PBS containing 0.05% Tween-20, and then incubated with primary antibodies for at least 1 h at room temperature. After being washed again with PBS containing 0.05% Tween-20, the membranes were incubated with peroxidase-conjugated secondary antibodies and developed with a chemiluminescence detection kit (ECL, Amersham Biosciences). (Mouse antihuman Rb, phosphoplus Rb [ser 780, ser 795, ser807], and rabbit antimouse Rb were obtained from Cell Signaling Technology, Inc., ( Beverly, MA) β-actin was used as the loading control.

Luciferase Assay

3 × 104/well cells were seeded in 24 well plates for overnight. They were then grown in serum free medium for 24 hours followed by transfection with 250 ng of a E2F-1-Luciferase reporter plasmid and 1ng of pRL-CMV (expressing Renilla luciferase, Promega Life Science, Madison, MI) for normalization of transfection efficiency12. FuGENE 6 (Roche Molecular Biochemicals, Indianapolis,IN) was used for plasmid transfection. The cells were kept in serum free medium for another 24 hours and then harvested for luciferase activity assay, using Dual-GloTM Luciferase Assay System (Promega Life Sceince) as instructed by the manufacturer. Cells transfected with pCNDA3.1 as control.

In Vitro Adenovirus Replication

The 4T1 cells (1 × 106) were seeded in a 60-mm culture dish. One day after the seeding, the cells were infected with different viruses at an MOI of 300 particles per cell. In the combined treatment groups, the total MOI was 300, with a 1:1 ratio for each vector. Four hours after being infected, the cells were washed with PBS three times to remove any free vectors in the medium. Four days later, both the supernatants and the cell lysates were collected, freeze-thawed three times, and titered on 293s cells by the limiting dilution method (determination of the 50% tissue-culture-infective dose) (TCID50)13,8. All cells infected with the different viruses and harvested at the same times were analyzed in the same assay. Each sample was tested twice with the TCID50 assay, and the data from two separate infection studies were averaged.

Animal Experiments

The animal experiments were carried out according to the “Guidelines for the Care and Use of Laboratory Animals” (NIH publication number 85-23) and the institutional guidelines of The University of Texas M. D. Anderson Cancer Center. Subcutaneous tumors were established in 6- to 8-week old Balb/c mice (Charles River Laboratories Inc., Wilmington, MA) by a single inoculation of 1.2 × 106 4T1 cells into the dorsal flank of each mouse. After the tumor diameters reached 3–5 mm, the mice were treated either with Delta24, Ad/TRAIL-F/RGD, or Ad/CMV-GFP alone or combinations of every pair of adenovectors. The adenovectors were administrated by intratumoral injection three times every 3 days. The tumor volumes were calculated with the formula a × b2 × 0.5, where a and b represented the largest and smallest diameters5,14, respectively. The mice were killed when their tumors reached 1.5 cm in diameter or became ulcerated.

The serum levels of alanine aminotransferase and asparate aminotransferase were measured, as previously described9, with blood samples collected from the tails of the mice on days 7 and 19 after the first treatment. Three mice from each treatment group were killed on day 24 after tumor cell inoculation. Liver, spleen, and kidney tissues were harvested for the pathologic studies. Liver micrometastases were counted in three mice from each treatment group, five sections per mouse, at 100X magnification. The histopathologic analysis was performed in the histology laboratory of the Department of Veterinary Medicine & Surgery at M. D Anderson Cancer Center.

Immunohistochemistry

For immunohistochemistry, 4 μm paraformaldehyde- fixed tumor sections were rehydrated and then incubated with a goat anti human adenovirus (Hexon) polyclonal antibody (Chemicon International Corp, Temecula, CA) (1: 200) in PBS containing 1% BSA for 60 min at room temperature. The slides were then stained with DakoCytomation LSAB+ Systems by using Dakoautostainer (DakoCytomation Corp, Glostrup, Denmark) to detect the expression of hexon. Counterstaining was performed with haematoxylin, and the slides were covered with Kaiser’s glycerin-gelatin.

Statistical Analysis

The differences among the treatment groups were assessed by using an analysis of variance (ANOVA). Synergisms in the combination treatments were analyzed with CalcuSyn software (Biosoft, Cambridge, UK). A P value of <0.05 was considered significant.

RESULTS

Combination of adenovirotherapy and TRAIL gene therapy enhanced cell killing in vitro

We evaluated the effects of the combined adenovirotherapy and TRAIL gene therapy. Two vectors were used with the total MOIs as the same as the total MOIs in the other single vector groups and with the ratio of the two vectors set to 1:1, both Delta 24 and Ad/TRAIL-F/RGD killed the MDA-MB-231, 4T1, and LM2 cells dose dependently. At the same total MOIs, Ad/TRAIL-F/RGD plus Delta24 was more effective than either Ad/TRAIL-F/RGD or Delta24 alone, suggesting that a combination of adenovirotherapy and TRAIL gene therapy is more effective than each therapy used alone in these human and murine breast cancer cells (Figure 1). There was no significant difference in cell killing when the ratio of Ad/TRAIL-F/RGD and Delta24 was changed to 3:1, 1:1, or 1:3. In contrast, combination of Delta24 with Ad/CMV-GFP resulted in either a comparable or a reduced cell killing when compared with Delta24 alone, suggesting that Ad/CMV-GFP did not contribute to detectable cell killing. A similar result was obtained when a newly constructed Ad/CMV-GFP-F/RGD was used as a control vector, suggesting that modification of F/RGD did not contribute to cell killing, either. In the MDA-MB-435 cells, however, only the treatment with Ad/TRAIL-F/RGD alone was effective in suppressing cell growth. Treatment with Delta24 did not result in substantial cell killing in this cell line compared with that treated with a control vector. Similarly, the results of the Ad/TRAIL-F/RGD plus Delta24 treatment were no better than those of the Ad/TRAIL-F/RGD treatment alone.

Fig. 1.

Fig. 1

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Cytotoxity effects of Ad/TRAIL-F/RGD combined with Delta24 in four breast cancer cell lines. Ad/CMV-GFP was used as the vector control. The cells were treated with viruses at various viral particles per cell. Cell viability was determined on day 4 after the treatment. The data presented are the means ± the standard deviation of two quadruplet assays.

To further evaluate the effect of the combined adenovirotherapy and TRAIL gene therapy, we determined the combination index (CI) at different MOIs in each cell line (Table 1) with CalcuSyn software (BioSoft, Cambridge, UK). We found that in the MDA-MB-231, 4T1, and LM2 cells, the combination of Delta24 with Ad/TRAIL-F/RGD led to a strong synergism (CI <0.3) in most doses when the total MOI was 100 or higher. In contrast, the Delta24 plus Ad/TRAIL-F/RGD treatment had an antagonistic effect on the MDA-MB-435 cells for all the doses tested (CI >1.1). This antagonistic effect might have been due to the fact that at a fixed total MOI, the effective dose for Ad/TRAIL-F/RGD in the combinations was reduced to half that of Ad/TRAIL-F/RGD alone.

Table 1.

Combination index (CI) of Ad/TRAIL-F/RGD and Delta24

MOI MDA-MB-231 MDA-MB-435 4T1 LM2
30 3.675 1.655 2.027 0.566
100 0.086 1.130 0.293 0.619
300 0.044 3.107 0.100 0.049
1000 0.078 6.694 0.181 0.088
3000 0.173 1.271 0.184 0.037
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Delta24, which has an eight-amino-acid-residue (24 base-pair) deletion in the viral E1A region responsible for binding the retinoblastoma (Rb) protein, reportedly replicates specifically in cells with inactivated Rb3,4,15. Because the MDA-MB-231 and MDA-MB-435 cells were dramatically different in their susceptibility, we tested Rb status in these two cell lines. The western blot analysis showed that the Rb proteins in the MDA-MB-231 cells were heavily phosphorylated (Figure 2A), suggesting that Rb was inactivated in this cell line. In contrast, although basal nonphosphorylated Rb was relatively weak in the MDA-MB-435 cells, phosphorylated Rb levels were either very low or nondetectable in these same cells, suggesting that the Rb protein is functional in this cell line. This result was consistent with the Rb-inactivation–dependent cytotoxicity of Delta24 and with the results of previous studies of Rb status in these two cell lines. To further evaluate Rb functions in these two cell lines, we tested the growth arrest mediated activation of an E2F-1 activating promoter by using luciferase gene as a reporter. Under starvation synchronized growth arrest conditions, endogenous E2F may be released and activated 16,17,18. The result showed that starvation dramatically induced E2F activity in MDA-MB-231 but not in MDA-MB-435 (Figure 2B), indicating that Rb protein in is functional and E2F-1 not released in MDA-MB-435 cells. In contrast, in MDA-MB-231 cells, Rb protein might be inactivated and release of E2F-1 permitted.

Fig. 2.

Fig. 2

Fig. 2

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A) Expression of Rb and phosphorylated Rb in MDA-MB-231 and MDA-MB-435 cells. Antibodies specific for the phosphorylated ser780, ser795, and ser807 sites were used to detect Rb protein phosphorylation. The result represents one of three different independent experiments with similar results. B) Growth arrest mediated activation of an E2F-1 promoter linked to a luciferase gene in MDA-MB-231 and MDA-MB-435 cells. The value represent mean + S.D of two assays. * P<0.01.

Delta24 helped E1-deleted adenovector replication in murine cancer cells

Delta24’s effective cell killing in the murine breast cancer cells suggested that this oncolytic adenovirus might replicate in such cells. Because the E1 proteins encoded in Delta24 can help the E1-deleted vectors in combination treatment groups, replication of Delta24 in murine cancer cells may help the replication of E1-deleted adenovectors in combination groups containing Delta24. To test the replication of Delta24 and the E1-deleted adenovirus co-infected with Delta24 in the murine cancer cell lines, we treated the 4T1 cells with Ad/CMV-GFP, Ad/TRAIL-F/RGD, Delta24, Ad/CMV-GFP plus Ad/TRAIL-F/RGD, Ad/CMV-GFP plus Delta24 and Ad/TRAIL-F/RGD plus Delta24 at MOIs of 300 particles per cell. Four hours after being infected, the cells were washed with PBS three times to remove any free vectors in the medium. The cells were then cultured with fresh medium and harvested on days 1 and 4 after the infection. The titers of Delta24 alone and Delta24 plus Ad/TRAIL-F/RGD or Ad/CMV-GFP dramatically increased 4 days after the cells were infected. In the cells treated with the E1-deleted vectors only, no viral replication was detected (Table 2).

Table 2.

In Vitro Replication Titer

Treatment 1 day 4 days
Ad/CMV-GFP ND ND
Ad/TRAIL-F/RGD ND ND
Delta24 1.8x103 5.9x105
Ad/CMV-GFP+Ad/TRAIL-F/RGD ND ND
Ad/CMV-GFP+Delta24 1.4x103 1.6x105
Ad/TRAIL-F/RGD+Delta24 1.9x103 2.7x105
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ND: Not detectable

We evaluated the replication of the E1-deleted adenovectors in the 4T1 cells co-infected with Delta24 by measuring green fluorescent protein (GFP) expression in fresh cells treated with cell lysates from the 4T1 cells treated with the various adenovectors. The treatment with cell lysates from the 4T1 cells treated with Delta24 plus Ad/CMV-GFP led to a dramatic increase in GFP expression (Figure 3). In comparison, treatment with cell lysates from the 4T1 cells treated with Ad/CMV-GFP only and Ad/CMV-GFP plus Ad/TRAIL-F/RGD did not produce any detectable GFP expression. This result demonstrated that Delta24 can replicate in murine breast cancer cells and that it can help E1-deleted adenovector replication.

Fig. 3.

Fig. 3

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Delta24-aided Ad/CMV-GFP replication in 4T1 cells. The 4T1 cells were infected by Delta24 plus Ad/CMV-GFP (total MOI of 300). Four days later, the supernatants and cell lysates were harvested. After being frozen and thawed three times and spun down, the supernatants were diluted to 1:1000 and then infected in 293s cells in a 96-well plate. Ad/CMV-GFP and Ad/TRAIL-F/RGD plus Ad/CMV-GFP were used as the control. The result represents one of three different independent experiments with similar results.

Oncolytic adenovirotherapy combined with TRAIL gene therapy suppressed tumor growth in syngeneic murine tumors in vivo

These promising in vitro results led us to test the effect of oncolytic adenovirotherapy combined with TRAIL gene therapy in vivo in syngeneic murine breast cancers. The treatments with Ad/TRAIL-F/RGD or Delta24 alone significantly retarded subcutaneous tumor growth when compared with the Ad/CMV-GFP treated group (P <0.05).

Nevertheless, the treatment with Ad/TRAIL-F/RGD plus Delta24 further suppressed the growth of the tumors derived from the 4T1 cells. Two mice in this group did not develop tumors whereas all mice in the other treatment groups developed tumors. The suppression of tumor growth in this treatment group was significant when compared with groups treated with Ad/TRAIL-F/RGD or Delta24 alone (P <0.05)(Figure 4). In comparison, combinations of Delta24 with Ad/CMV-GFP or Ad/TRAIL-F/RGD with Ad/CMV-GFP did not improve the tumor suppression when compared with groups treated by Delta24 or Ad/TRAIL-F/RGD alone (P >0.05). We also evaluated the possible liver toxicity of these treatments by measuring the serum liver enzymes alanine aminotransferase and asparate aminotransferase. Blood samples were collected before the treatment started and on days 7 and 19 after the first treatment. All of the values were within normal ranges, and no substantial differences were found among the groups (data not shown).

Fig. 4.

Fig. 4

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Suppression of 4T1 tumor growth in vivo. Subcutaneous tumors derived from the 4T1 cells were treated with various reagents as indicated. Tumor volumes were monitored over time after the treatments. The values represented the means ± the standard error of 12 mice per group. The mean tumor volume in the animals treated with Ad/TRAIL-F/RGD or Delta24 alone differed significantly from that of Ad/CMV-GFP treated group (P <0.05). Treatment with Ad/TRAIL-F/RGD plus Delta24 differed significantly from all other treatment groups (P <0.05).

We also evaluated the histologic changes that occurred in the tumor, liver, kidney, and spleen on days 7 and 19 after the first treatment with the virus. Lymphocyte infiltration in subcutaneous tumors from Ad/TRAIL-F/RGD plus Delta24 treated mice was more frequently than that of other treatments. Whether increased this lymphocyte infiltration may contribute to therapeutic benefit is not yet clear. Nevertheless, except for some mild myeloid hyperplasia in the spleen and liver and modest (1+) focal, subacute hepatitis in some of the mice, we noticed no other pathologic changes and found no substantial differences among the groups (data not shown), suggesting that the treatments were well tolerated.

To test whether Delta24 replicate in vivo in tumors, we evaluated the expression of adenoviral hexon in tumor sections 19 days after the first treatment by using immunohistologic staining with an anti-hexon antibody. Hexon positive cells were only detected in tumor treated with Delta24 or Delta 24 plus Ad/CMV-GFP or plus Ad/TRAIL-F/RGD but not in other tumor samples treated with Ad/CMV-GFP, Ad/TRAIL-F/RGD, or both (Figure 5A). Hexon, a capsid protein, is expressed after viral DNA replication. Massive expression of Hexon in syngeneic tumors treated with Delta24 but not in that treated with E1-deleted vectors only further demonstrated that Delta24 can replicate in syngeneic tumor in vivo.

Fig. 5.

Fig. 5

Fig. 5

Fig. 5

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A) Expression of hexon in the tumor on 19 days after the first treatment. The treatment is indicated at the top (up panel) or bottom (low panel). B) Effects of micrometastases in the liver H&E staining of the liver section. The treatment is indicated at the top. C) Number of tumor nodules in the liver section. The tumor nodules were evaluated in nine liver sections of three mice per group. The values represent the means (per field) ± the standard error of three mice per group.* P<0.05.

Adenovirotherapy combined with TRAIL gene therapy suppressed spontaneous liver metastases

When we evaluated the liver sections on day 19 after the first treatment, we found numerous micrometastases in the livers of the animals treated with Ad/CMV-GFP. The animals treated with Delta24 or Delta24 plus Ad/TRAIL-F/RGD had remarkably reduced micrometastases. We then counted the micrometastases in five liver sections that were located at least 5 mm apart from each other (three animals per group). The average number of micrometastases (per high-power field) in the animals treated with Delta24 or Delta24 plus Ad/TRAIL-F/RGD were significantly smaller than those in the other groups (P <0.05) (Figure 5B,C). This result suggested that oncolytic adenovirotherapy alone or combined with TRAIL gene therapy can suppress spontaneous liver metastases from 4T1 tumors.

DISCUSSION

Our results showed that in cells susceptible to adenovirotherapy and TRAIL gene therapy, cell killing is enhanced when the two therapies are combined. Moreover, the results of the combination studies showed that Delta24 can help the E1-deleted adenovector replicate in murine cancer cell lines, suggesting that at least to some degree, human adenoviruses can replicate in murine cancer cells. This result was consistent with results previously reported by Wang et al.13,8, who found that some mouse cancer cell lines can be permissive to human oncolytic adenoviruses. Our study of Delta24 in murine breast cancer cells confirmed their findings.

A major limitation in the field of replication-selective oncolytic adenoviruses, however, has been the lack of tumor models available for assessing antitumoral efficacy animals that are immunocompetent. Thus far, most of the preclinical studies on oncolytic adenoviruses have been performed in immunodeficient mice with human xenograft tumor models,19,20,21. These models may not be as predictive in clinical settings because a patient’s immune response to an oncolytic viral infection cannot be effectively evaluated. Thus, Dr. Kirn’s group’s finding of a human adenovirus replicating in mouse tumor cells is an important contribution to preclinical studies on oncolytic virotherapy.

Delta24 is a human Ad5 that contains a 24-bp deletion in its E1A region. This deletion removes the Rb binding site in E1 and causes viral replication specifically in Rb gene-defective cells. Rb regulates cell cycle progression through a mechanism that transcriptionally down-regulates the activity of genes required for cells to progress to the S phase. Inactivation of Rb occurs in various breast cancer cells lines. In our study, we found that Delta24 effectively killed three of the four cell lines we tested but not the human breast cancer cell line MDA-MB-435, which was resistant to Delta24. The western blot analysis showed that Rb was hyperphosphorylated in the MDA-MD-231 cells but hypophosphorylated in the MDA-MB-435 cells, suggesting that Rb is inactivated in MDA-MB-231 cells but not in MDA-MB-435 cells, a result consistent with the results of previous studies21,22. In the MDA-MB-435 cells, the intact Rb protein is expressed, whereas in the MDA-MB-231 cells, the protein functions abnormally because of a p16 deficiency15,24. E2F activity assay also supported this conclusion.

In this study, we found that although both adenovirotherapy with Delta 24 and TRAIL gene therapy with Ad/TRAIL-F/RGD killed murine breast cancer cells in vitro, their combination significantly increased cell killing in vitro and suppressed tumor growth in vivo more than either one alone, indicating that such combinations may have therapeutic advantages. Thus, as reported 19,20,21, oncolytic adenovirus-mediated TRAIL gene therapy can be a good strategy for cancer therapy. However, even though these syngeneic tumor models are useful for assessing a host’s immune response to oncolytic viruses, they may be limited in terms of their usefulness in toxicity studies and in the monitoring of virus spread within tumors. These limitations are due to the ineffectiveness of human adenoviruses in replicating in normal mouse tissues. Moreover, their ability to replicate may be much more attenuated in mouse tumor cells than in human tumor cells.

4T1 cells have often been used as a tumor model for studying metastases22,25,26. Yoneda and colleagues reported that metastases can be identified in the lungs and liver of syngeneic, immunocompetent mice around 2 weeks after tumor inoculation26. The pattern and histologic appearance of such metastases are similar to what is seen in humans. Our results showed that Delta24 alone or combined with TRAIL gene therapy can inhibit the micrometastases in the liver. However, whether this reduction in liver micrometastases was the result of a reduction in tumor size of the original subcutaneous tumor was not clear. Interestingly, treatment with Ad/TRAIL-F/RGD alone did not significantly reduce micrometastases in liver cells, although its effect on the primary subcutaneous tumor was similar to that of Delta24 alone, suggesting that factors other than a simple reduction in the primary tumor burden are involved in the antimetastasis effect of the oncolytic virus. It is possible that vector leakage from intratumoral injections may lead to a systemic infection in metastatic tumor cells. Because an oncolytic virus can replicate in these tumor cells, the virus may kill the cells before they are seeded in the liver to form metastasis nodules.

Acknowledgments

We thank Henry Peng and Li Wang for propagating the adenovirus and conducting the vector quality test, Alma J. Vega for helping prepare the manuscript, and Gayle Nesom for reviewing the manuscript. This article represents the partial fulfillment of the requirements for a Ph.D. for J. J. Davis.

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