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. Author manuscript; available in PMC: 2015 Apr 6.
Published in final edited form as: Cancer Gene Ther. 2014 Sep 19;21(10):434–440. doi: 10.1038/cgt.2014.47

Gene-directed enzyme prodrug therapy for localized chemotherapeutics in allograft and xenograft tumor models

KH Carruthers 1, G Metzger 2, MJ During 3, A Muravlev 4, C Wang 3, E Kocak 2,5
PMCID: PMC4387004  NIHMSID: NIHMS670301  PMID: 25236494

Abstract

Most chemotherapy regimens rely on systemic administration of drugs leading to a wide array of toxicities. Using viral-vector-mediated gene modification of muscle tissues, we have developed a method for gene-directed enzyme prodrug therapy that allows for localized drug administration. An inactive prodrug of geldanamycin was activated locally for inhibition of tumor growth without systemic toxicities. A recombinant adeno-associated virus (rAAV) was used to deliver β-galactosidase (LacZ) to the treatment group and green fluorescent protein to the control group. After 1 week, both groups received adenocarcinoma cells in the same location as the previous rAAV injection. The geldanamycin prodrug was administered 1 h later via intraperitoneal injection. Tumor growth was significantly suppressed in animals whose muscles were gene modified to express β-galactosidase compared with the control. Serum assay to access hepatotoxicity resulted in no significant differences between the animals treated with the inactive or activated form of geldanamycin, indicating minimal damage to non-target organs. Using gene-directed enzyme prodrug therapy, in combination with novel recombinant AAV vectors, we have developed a method for localized activation of chemotherapeutic agents that limits the toxicities seen with traditional systemic administration of these potent drugs.

INTRODUCTION

Most solid tumors, especially those found in the colon, have a high rate of recurrence even after complete surgical resection.1–4 The currently accepted treatment for these recurrences generally involves the use of systemically administered chemotherapeutic agents. As a result, these medications frequently cause constitutional symptoms that are a challenge for the patient, as well as toxicities at distant organs that can cause lifelong impairment. Therefore, it would be a significant improvement if patients undergoing surgical resection of a primary tumor could receive a plastic surgical reconstruction involving not just a space-occupying muscle flap, but actually a therapeutic flap that could deliver a chemotherapeutic agent in a localized manner to prevent future recurrence.

This idea of a therapeutic muscle flap is not new. It has, in fact, been described in several experimental publications where viral vectors were used for various therapeutic end points, including the production of cytokines to achieve local immune modulation with antitumor effects. 5–10 Outside of this work, there are no other reports of gene-modified tissues being used in this manner.

However, locally activated prodrugs are another option for eliciting targeted antitumor therapies. These metabolically inert compounds can be locally activated in a number of ways, and experiments using antibodies and viruses to target the expression of the prodrug-activating enzyme are widely known.11,12 These protocols, known as antibody-directed enzyme prodrug therapy (ADEPT) or virus-directed enzyme prodrug therapy (VDEPT), are specific forms of the more general gene-directed enzyme prodrug therapy (GDEPT) and are rapidly attracting attention from the scientific community as potentially viable techniques for localizing chemical compounds that are known to be systemically toxic.13

Therefore, the purpose of this study was to develop a simple method for localizing the effects of a toxic chemotherapeutic agent using GDEPT mediated by a novel adeno-associated virus (AAV). The chemotherapeutic drug chosen for analysis in this study was geldanamycin, a potent heat-shock protein 90 inhibitor that is known to be effective against many carcinogenic cell lines, but the usage of which has been limited because of its extreme hepatotoxicity.14,15 A prodrug version of this agent was created for the purpose of these GDEPT experiments to determine if local activation of the drug could be achieved in areas where an AAV-expressing β-galactosidase was expressed.15

MATERIALS AND METHODS

Viral vector construction and administration

A recombinant adeno-associated virus (rAAV) vector was used for gene delivery. This rAAV vector, known as serotype rAAVrec2, was derived in our laboratory using a PCR shuffling technique from human and novel nonhuman primate viral isolates and has been successfully used in other gene therapy protocols, including one study published by our laboratory.16–21 Specifically, an rAAV vector containing the gene for either green fluorescent protein (GFP) or β-galactosidase (LacZ) was constructed. The cDNA was cloned into the high expression pAM AAV cis-plasmid containing the hybrid CBA promoter and WPRE 3′ sequence. The subsequent pAAV-CBA-LacZWPRE was used to generate high titer rAAV vectors expressing either GFP or LacZ using transfection techniques with helper plasmids as previously described by our laboratory.16–21 The resulting rAAV-GFP was used as a marker for the control group, whereas the rAAV-LacZ, used in the treatment group, would locally express β-galactosidase to activate the inactive prodrug form of geldanamycin.

Allogeneic tumor model

Eight-week-old immunocompetent C57BL/6 mice (Charles River Laboratories, Wilmington, MA, USA) (approximate weight 22 g) were divided into treatment and control groups with three mice in each population. Treatment of mice was in accordance with the guidelines approved by the Institutional Animal Care and Use Committee. Animals were anesthetized via intraperitoneal injection of ketamine/xylazine (87/13 mg kg−1) and the left quadriceps femoris muscle was shaved and sterilized. Gene modification was accomplished using direct injection of the viral vector using a 50 μl Hamilton syringe with a 30-gauge needle. Previous experiments conducted in our laboratory have identified this as an effective method for localizing the viral vector gene products while limiting the operative time required for transduction. Viral vectors were titered using real-time PCR. Stock solutions of rAAV-GFP and rAAV-LacZ were each diluted to a concentration of 6.7 × 1011 vg ml−1. From that stock solution, treated mice received 1 × 1010 virions of rAAV-LacZ in 15 μl via intramuscular injection in the ventral portion of quadriceps femoris muscle, which expressed β-galactosidase as the gene product for subsequent activation of the chemotherapeutic prodrug. The control mice received 1 × 1010 virions of rAAV-GFP in 15 μl via the same protocol, which delivered GFP to the surrounding tissue.

Intramuscular tumor establishment

One week following administration of the viral vector, both the treated and control groups received allogeneic colon carcinoma tumor cells (MC38) via intramuscular injection. MC38 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY, USA) and the medium was supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin–streptomycin (Life Technologies). Cells were passaged until 70% confluence was achieved. On the day of injection, cells were pelleted and resuspended in serum-free medium. A total of 5 × 105 MC38 cells were injected to the ventral area of the quadriceps muscle of each immunocompetent C57BL/6 mouse.

Chemotherapeutic prodrug construction and administration

The commercially available drug geldanamycin (InvivoGen, San Diego, CA, USA) was modified to its prodrug form by adding a large polysaccharide molecule to the original chemical structure, resulting in Compound 25: C36H53N3O14 (17-demethoxy-17[(2-β-galactopyranosylethyl)amino]-geldanamycin) (Figure 1). This prodrug form, once administered, could be activated by β-galactosidase's cleaving action on the polysaccharide addition, reverting the geldanamycin to its original, active, form only in tissues where rAAV-LacZ was expressed.

Figure 1.

Figure 1

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Molecular structure of the geldanamycin prodrug, Compound 25: C36H53N3O14 (17-demethoxy-17[(2-β-galactopyranosylethyl) amino]-geldanamycin).

One hour after the tumor cells were injected, the geldanamycin prodrug was administered systemically, via intraperitoneal injection, at a dose of 50 mg kg−1 suspended in pure dimethyl sulfoxide. The mice were then retreated in a similar manner with the prodrug at a dose of 50 mg kg−1 on days 3 and 7 following tumor injection (Figure 2).

Figure 2.

Figure 2

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Recombinant adeno-associated viruses (rAAVs) were used to gene modify the quadriceps femoris muscles of mice by direct injection. In the treatment group, gene modification was accomplished with rAAV-LacZ to express β-galactosidase. In the control group, muscles were injected with rAAV-GFP to express GFP protein. After 1 week, colon cancer cells were implanted into the muscle. After 1 h, animals were treated with intraperitoneal injection of the geldanamycin prodrug. Mice were then retreated with the prodrug on days 3 and 7 following tumor injection. GFP, green fluorescent protein.

Tumor growth analysis

All mice were housed individually and monitored daily for tumor growth and signs of distress. Starting 14 days after cell injection, tumors were serially measured and average growth curves were generated for both the treated and control populations. At 26 days, it was determined that the tumor burden on a number of the mice had grown to a point of interference with normal gait. Based on this observation, it was determined that the experiment should be ended and all mice killed, in accordance with Institutional Animal Care and Use Committee standards. At necropsy, the tumor was surgically exposed and photographed for documentation of visual differences between the tumor volumes in each population. Tumors were subsequently excised, cleaned of excess uninvolved tissue and weighed using a calibrated gram scale. All tumor weights were measured and recorded to the nearest 0.1 g. Averages and standard deviations were calculated for the two populations. A Student's t-test was used to compare the groups and P ≤ 0.05 demonstrated a significant result.

Determination of systemic toxicity

Post-mortem blood samples were collected and sent for serum analysis. A complete hepatic function panel, including aspartate aminotransferase and alanine aminotransferase (ALT), was requested for each mouse and averages were calculated for each population. The average level of hepatic enzymes for the treated mice was then compared with that of the control mice and to previously published data using an analysis of variance to determine the extent of hepatotoxicity caused by the activated form of the prodrug.22

In vitro analysis of cellular proliferation

To further characterize the extent of cellular proliferation inhibition caused by the activated geldanamycin prodrug, an MTS assay was performed. A total of 1.5 × 103 MC38 cells were plated in 100 μl of media in duplicate into wells of a 96-well plate (four wells per plate: two control wells and two treated wells) using aseptic technique. The geldanamycin prodrug was added at a concentration of 10 μm to all wells and a 24 h incubation allowed the cells sufficient time to adhere to the bottom of the wells. After incubation, β-galactosidase was added at a 1 μm concentration to the treatment wells, whereas an equal volume of media was added to the control wells for consistency. The plates were incubated until confluence was achieved in the control well (~72 h). To assess the number of viable cells present in all wells at this time, an MTS assay was performed. Twenty microliters of MTS reagent (Promega, Madison, WI, USA) was pipetted into each of the wells and incubated for 1 h. Absorbance was measured at 490 nm wavelength using a standard plate reader. A Student's t-test was used to compare the treated and control groups and P ≤ 0.05 indicated a significant result.

Xenogeneic tumor model

All of the above-described experiments, both in vivo and in vitro, were repeated to determine xenogeneic tumor applicability for the model.

The in vivo experiment used to establish xenograft success used immunodeficient BALB/c nu/nu mice (Charles River Laboratories, Wilmington, MA, USA) and the human colon carcinoma cell line, SW620. SW620 cells were cultured in Leibovitz's L-15 medium (Life Technologies) and the medium was supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin–streptomycin (Life Technologies). Cells were cultured as previously described for the allogeneic model. A total of 2 × 105 SW620 cells were injected to the ventral area of the quadriceps muscle of each immunodeficient BALB/c nu/nu mouse.

Both experiments were otherwise conducted according to identical protocols, including the post-mortem tissue handling and serum analyses.

The in vitro MTS experiment to further quantify the in vivo results was also conducted using SW620 cells. A total of 1.0 × 103 SW620 cells were plated in 100 μl of media into wells of a 96-well plate using aseptic technique, as described in the MC38 experiment. The remainder of the experimental protocol was the same. Published data of the mean concentration of traditional geldanamycin producing 50% growth inhibition (GI50) and 50% apoptosis (LD50) in SW620 cells were used for comparison with the treated cells.23 All subsequent analyses were unchanged.

RESULTS

Allogeneic tumor model

Tissues treated with GDEPT have a slower tumor growth rate and result in smaller masses compared with non-treated control tumors

To assess the tumor growth over time, volumetric measurements were recorded serially and compared between populations. Even at the earliest time point, a slight difference was identified between the treated and non-treated mice, and this difference grew as the time progressed. However, it was not until the final time point that a statistically significant difference was achieved (P = 0.007) (Figure 3a). Once the mice were killed because of excessive tumor burden in the hind limb, surgical exposure of the tumor in the muscle allowed for clear visualization of the extent of tumor growth and appreciation of the muscle involvement (Figure 4). Tumors were then excised and weighed to calculate the final average tumor mass in each population. Results indicated that the mice that did not receive the rAAV-LacZ to activate the administered prodrug experienced over two times the MC38 tumor burden of those that were able to activate the prodrug (P = 0.003) (Figure 5).

Figure 3.

Figure 3

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At 2 weeks after administration of prodrug, all mice were examined and tumor volume was measured using topical calipers. Measurements were recorded and repeated every 3 days until the mice were killed at day 26. In both the (a) allograft and (b) xenograft models, tumor mass began to increase more quickly for the control group (rAAV-GFP and prodrug) than the treated group (rAAV-LacZ and prodrug) and a significant difference was achieved by 26 days. *P ≤ 0.05 demonstrated a significant result. Data are mean ± s.d.; n = 3 for each group; Student's t-test: Pallograft = 0.007, Pxenograft = 0.004. GFP, green fluorescent protein; rAAV, recombinant adeno-associated virus.

Figure 4.

Figure 4

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Mice were killed 26 days after administration of prodrug and tumors were surgically exposed. Mice treated with a combination of (a) green fluorescent protein (GFP) and prodrug (control) demonstrated a tumor that was visibly larger than the mice treated with a combination of (b) LacZ viral vector and prodrug (gene-directed enzyme prodrug therapy (GDEPT) treated). Images shown are from the allograft tumor model.

Figure 5.

Figure 5

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At 26 days postimplantation, the average tumor mass in the control group (rAAV-GFP and prodrug) was significantly greater than the average tumor mass for the treated group (rAAV-LacZ and prodrug) for both the allograft and xenograft tumor models. *P ≤ 0.05 demonstrated a significant result. Data are mean ± s.d.; n = 3 for each group. Student's t-test: Pallograft = 0.003, Pxenograft = 0.002. GFP, green fluorescent protein; rAAV, recombinant adeno-associated virus.

Gene modification and activation of the chemotherapeutic prodrug did not result in systemic toxicities

Determination of systemic toxicities was achieved by serum analysis of hepatic enzymes. A complete liver function panel was obtained and a analysis of variance was used to compare the results between the treated populations, the non-treated populations and the expected normal values, obtained from the existing lterature.22 Although slight fluctuations were identified, these were considered to be reasonable naturally occurring variations and not a function of drug-induced toxicity. This assumption was further supported by the previously published data by Supko et al.,23 who reported a 32- to 49-fold increase in alanine aminotransferase and aspartate aminotransferase values in animals treated with traditional geldanamycin. Thus, if the activated prodrugs were causing systemic toxicities, liver function tests would be expected to be significantly higher than baseline. In this study, no statistically significant differences were identified, indicating no systemic effect, despite the local activation of a potent hepatotoxic chemo-therapeutic agent (Table 1a).

Table 1.

Liver function serum assays: (a) C57BL/6+MC38 and (b) BALB/c+SW620

Measure Normala
 rAAV-GFP+prodrug
 rAAV-LaCZ+prodrug
 P-value
Mean (n = 60) s.d. Mean (n = 3) s.d. Mean (n = 3) s.d.
(a) C57BL/6+MC38
    ALT (IU l–1) 77.0 36.0 47.2 12.9 50.1 15.3 0.2
    AST (IU l–1) 255.0 160.0 329.9 162.9 322.9 63.7 0.6
    Albumin (g dl–1) 3.2 0.4 2.9 0.4 3.1 0.6 0.4
    Globulin (g dl–1) 1.2 0.3 1.1 0.8 0.9 0.4 0.3
    Total bilirubin (U l–1) 0.4 0.1 0.3 0.4 0.4 0.1 0.3
(b) BALB/C+SW620
    ALT (IU l–1) 77.0 36.0 53.2 22.6 46.0 20.4 0.2
    AST (IU l–1) 255.0 160.0 291.1 182.0 304.7 127.8 0.8
    Albumin (g dl–1) 3.2 0.4 2.8 0.4 2.8 0.3 0.1
    Globulin (g dl–1) 1.2 0.3 1.2 0.7 0.9 0.2 0.3
    Total bilirubin (U l–1) 0.4 0.1 0.5 0.3 0.5 0.1 0.1
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Abbreviations: ALT, alanine aminotransferase;AST, aspartate aminotransferase.

a

Schnell et al.22

In vitro assays confirmed in vivo results, indicating decreased cellular proliferation after treatment with the activated prodrug

MTS assay was used to verify the extent of proliferation inhibition caused by the treatment of activated geldanamycin prodrug. The non-treated MC38 cells had 124% growth, compared with the treated cells that had only 29% growth in the given 72 h period (Figure 6). This represented a significant difference in the cellular proliferation following drug treatment (P = 0.004).

Figure 6.

Figure 6

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In vitro cellular proliferation assay (MTS) was performed on both the MC38 and SW620 cell lines to quantify the rate of cellular doubling in the control (plain media and prodrug) and treated (β-galactosidase and prodrug) groups. The control cells proliferated at a significantly greater rate than the cells that were treated with the activated prodrug. *P ≤ 0.05 demonstrated a significant result. Data are mean ± s.d.; n = 2 for each group. Student's t-test: Pallograft = 0.004, Pxenograft = 0.003.

Xenogeneic tumor model

Analyses of the SW620 and immunodeficient BALB/c nu/nu experiments yielded results consistent with the MC38 and immunocompetent C57BL/6 experiments reported above (Figures 3b and 5, 6, 7 and Table 1b). Cellular growth inhibition of the activated prodrug, assessed via MTS analysis, was compared with previously published data on the effects of traditional geldanamycin on SW620 cells in vitro. These published experiments reported a GI50 of 1 × 10−7.8m and a LD50 of 1 × 10−4.9 m, indicating that the activated prodrug version of geldanamycin was able to limit cellular growth at a rate similar to that of the traditional drug.23 These results strongly indicate the xenograft applicability of the proposed model.

Figure 7.

Figure 7

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Tumors were excised, cleaned of excess uninvolved tissue and weighed using a calibrated gram scale. All tumor weights were measured and recorded to the nearest 0.1 g. Averages and standard deviations were calculated for (a) the control group (rAAV-GFP and prodrug) and (b) the treated group (rAAV-LacZ and prodrug). Images shown are from the xenograft tumor model. Note: Images do not represent the actual size of the specimens, only the relative difference in tumor mass between the two populations. GFP, green fluorescent protein; rAAV, recombinant adeno-associated virus.

DISCUSSION

Localizing the effects of chemotherapeutic agents has long been a goal for cancer treatment.24 Only recently, however, have scientists considered the possibility of combining gene therapy and plastic surgery to accomplish this goal.5–10,21 Gurtner and his co-workers6–10 performed the foundation work, which focused on using ex vivo viral perfusion of microvascular free flaps to deliver locally tumor-suppressing agents to the reconstructive bed. While the initial results were promising, the proposed methods needed to be modified to expand applicability to pedicled flaps and to reduce operative time necessary for viral vector perfusion. To this end, we have introduced a novel method that uses direct injection as the method of gene modification and can be applied directly to the area of tumor resection or to the pedicled muscle flap used for wound coverage.

Furthermore, this study indicates the possibility of resurrecting drugs previously discontinued from human use because of systemic toxicities. Many of these agents, including geldanamycin, were highly effective in the treatment of a wide range of cancers, but never reached clinical applicability because of non-target toxicities, especially in the liver.14,15 By creating a prodrug version of these agents, and using rAAVs to enzymatically target the activation to the area of local recurrence, these agents could, potentially, hold the potential to treat human disease. Additionally, the transition from ex vivo perfusion to direct intramuscular injection of the viral vector allows for minimal additional operative time and would allow a wider range of surgeons and institutions to offer therapeutic flaps in the future.

We believe that the success of our initial results was not only dependent on the protocol followed but also the novel recombinant AAV vector used. AAVs have consistently been the choice for targeted gene therapy regimens because they are not been proven to transmit any known human disease.25 However, adeno-associated viruses can be improved even further through the creation of recombinant AAV vectors that have enhanced tissue tropism. This technique was used by our current experimental protocol, as well as previously published in one of our laboratory's earlier studies that demonstrated the advantages of our unique recombinant viral vector over traditional viral sero-types.21 Using this unique rAAV, created by our laboratory, which consistently demonstrated an affinity for local muscle tissues, we were able to localize successfully the chemotherapeutic activation, which was an essential component of our goal to limit systemic toxicities.

Although success was achieved in our experiments using a small animal model, human applications of GDEPT for the localization of chemotherapeutic agents are still in infancy. Subsequent experiments to determine the applicability of these techniques in a larger animal model are needed, especially to see if reasonable concentrations of viral vector gene products can be achieved on a larger scale. We anticipate that larger animals, or humans, might possibly need multiple injections of the rAAV over a period of hours and this change would add to the operative time required. Additionally, further analysis of the systemic effects of the activated prodrug is needed. Although our past experience caused us to focus on the possible hepatic insult caused by prodrug activation, the effects on other distant targets should undoubtedly be explored. Finally, testing is needed to determine the duration of viral vector expression. We know from our preliminary experiments that viral gene product expression peaks at ~ 4 weeks after administration. However, how long a therapeutic dose is maintained after that time point is still unknown. Even in the event of an eventual loss of viral expression, subsequent injections could, theoretically, be a reasonable option for keeping the β-galactosidase active over an indefinite period of time.

Using gene-directed enzyme prodrug therapy, in combination with novel recombinant AAV vectors, we have developed a method for localized activation of chemotherapeutic agents that limits the toxicities seen with traditional systemic administration of these potent drugs. This allows for the use drugs that have known antitumor effects against many carcinogenic cells to not be limited by the systemic toxicities previously encountered and for patients to avoid the constitutional symptoms that frequently cause refusal of treatment. Additionally, this technology could be used to combat local recurrences immediately following solid tumor resection before tumorigenic proliferation is ever established, especially in cases where it could be indicated to augment traditional plastic surgical reconstruction methods by gene-modifying muscle flaps.

ACKNOWLEDGEMENTS

We would like to acknowledge Peng George Wang, PhD, of the Ohio State University Department of Chemistry, for his efforts constructing the geldanamycin prodrug (Compound 25). The project described was supported by Pilot Project No. 11876 Grant UL1TR001070 from the National Center For Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Advancing Translational Sciences or the National Institutes of Health. This project was supported by the National Center for Research Resources, the National Center for Advancing Translational Sciences and the Office of the Director, National Institutes of Health, through UCSF-CTSI Grant Number KL2 (K12) RR024130. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. This study supported through the Institutional Research Grant Number IRG-67-003-47 from the American Cancer Society, administered through the Comprehensive Cancer Center at The Ohio State University.

Footnotes

CONFLICT OF INTEREST

The authors KHC, GM, MJD, AM, CW and EK have no commercial associations or financial disclosures that might pose or create a conflict of interest with information presented in the attached manuscript.

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