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Annals of Surgical Oncology 8:32-37 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

Antisense TGF-ß2 Immunotherapy for Hepatocellular Carcinoma: Treatment in a Rat Tumor Model

Melinda Maggard, MD, Lingzhong Meng, MD, Bibo Ke, MD, Rene Allen, BS, Lara Devgan, BS and David K. Imagawa, MD, PhD

From the Department of Surgery (MM, BK, LD, DKI), UCLA Medical Center, UCLA School of Medicine, Los Angeles, California, and the Department of Surgery, Division of Transplantation (LM, RA, DKI), UC Irvine Medical Center, Orange, California.

Correspondence: Address correspondence and reprint requests to: Dr. David K. Imagawa, UC Irvine Medical Center, Dept. of Surgery, Div. of Transplantation, Bldg 26, Room 1001, Route 81, 101 The City Drive, Orange, CA 92868-3298; Fax: 714-456-8796; E-mail: dkimagaw{at}uci.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: The overexpression of transforming growth factor-beta (TGF-ß) in hepatocellular carcinoma (HCC) appears to induce immunosuppression toward the tumor cells.

METHODS: A rat HCC cell line, Morris hepatoma rat cell line (MRH)-7777 (MRH), was transfected with antisense TGF-ß2 in pCEP-4 vector and used as immunotherapy against the development of wild-type tumors. An enzyme-linked immunosorbent assay (ELISA) confirmed that TGF-ß2 production was markedly lower for antisense modified cells as compared to wild-type tumor cells. Tumors were initiated by injecting MRH cells into the flanks of Buffalo rats. This was followed by biweekly vaccinations with irradiated MRH cells (unmodified, pCEP-4 alone, or antisense TGF-ß2 modified).

RESULTS: In the group that received irradiated MRH unmodified cells, 55% of rats died from tumor burden, and 36% developed tumor regression. In the group that received irradiated MRH cells modified with pCEP-4 vector alone, 50% died from tumors and 33% had spontaneous regression. In animals treated with pCEP-4/TGF-ß antisense modified cells, none developed tumors. Cell-mediated cytotoxicity assays demonstrated a twofold increase in lytic activity in the effector cells of the animals treated with antisense modified cells.

CONCLUSIONS: These results demonstrate the successful treatment of HCC tumors in rats by a HCC vaccine genetically altered with antisense TGF-ß2. Decreased production of TGF-ß in HCC vaccine enhances immunogenicity against wild-type HCC tumor cells.

Key Words: TGF-ß— • Hepatocellular carcinoma— • Antisense— • Gene therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, affecting over 1 million people per year.1 The risk is highest in Southeast Asia and sub-Saharan Africa with an incidence of 30 per 100,000 people per year, typically occurring where hepatitis B virus infection is endemic. Curative treatment can be offered only in a minority of cases and is limited to surgical resection or transplant. Even under stringent criteria, 5-year survival approaches 50%.2 Alternative options such as chemoembolization, percutaneous alcohol injection, cryotherapy, and chemotherapy are reserved for palliation. The development of an effective immunotherapy using genetically altered tumor cells could have enormous implications in the treatment of HCC. We used a novel nonviral gene therapy approach to develop a vaccine that blocks the tumor-derived immunosuppressant, transforming growth factor-beta 2 (TGF-ß2) and prevents the progression of HCC in a rat tumor model.

TGF-ß2 was originally purified and cloned from a human glioblastoma cell line based on its immunosuppressive effects.3,4 First termed the glioblastoma-derived T-cell suppressive factor, it inhibits the production of cytotoxic T-lymphocytes, T-cell growth induced by interleukin-2 (IL-2), antigen-induced growth of MHC class II restricted T-cells, natural killer cell activation, macrophage activation, and IL-2 receptor production.5–12 TGF-ß1 also has immunosuppressive effects, including playing a role in tolerance in immunologically privileged sites, and downregulates expression of adhesion molecules. Furthermore, the creation of TGF-ß1 knock-out mice has demonstrated the critical role TGF-ß plays in regulating the immune system.13 These knock-out mice had diffuse infiltrates of lymphocytes in their organs and had marked immune dysfunction. This immune system disruption leads to death of the mice in about 30 days. TGF-ß1 shares 71% homology in the amino acid sequence with TGF-ß2, and is interchangeable in bioassays.9,14,15 Furthermore, it appears that the immunosuppressive function of TGF-ß, when overexpressed by tumor cells, generates a self-protective environment by blocking endogenous tumor immunosurveillance.

Previous work by Fakhrai et al.16 demonstrated the successful treatment of glioma tumors using inoculations of tumor cells modified with antisense TGF-ß2. Cells genetically transformed with antisense TGF-ß2 vectors produced less TGF-ß2 as well as less TGF-ß1. Injections with the modified antisense tumor cells induced complete regression of established wild-type intracranial tumors. Enhanced tumor cytotoxicity was demonstrated by cell-mediated cytotoxicity assays, suggesting that inhibition of TGF-ß production may release the immune blockade and allow for activation of T cells directed against the tumors. Independent work by Yuan et al.17 found that bleomycin-induced inhibition of TGF-ß production in hepatomas was associated with upregulation of IL-2 receptors, suggesting a possible link of TGF-ß to the HCC immunosuppressive effects through decreasing IL-2 responsiveness.

Since its inception in 1984, antisense RNA therapy has proved to be a powerful tool in the regulation of cellular growth. Low ratio of antisense molecules relative to endogenous sense molecules has been one obstacle limiting application of this technique. Antisense gene transcription must be driven by a strong promoter to produce the high levels required to generate an effect. Many HCC animal cell lines, as well as HCC tumors resected from patients, overexpress TGF-ß.18,19 Antisense oligomer treatment of hepatoma cells has been successful in suppressing growth of both human and rat tumor cell lines in vitro.20,21 We proposed to treat rat hepatocellular carcinomas using antisense TGF-ß2 modified tumor cells to determine if reduction of TGF-ß2 can enhance the in vivo immune response.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Cell Line
A rat HCC cell line, MRH-7777 (MRH) was obtained from the American Type Culture Collection (Rockville, MD). MRH cells were derived from Buffalo rats by induction with N-2 fluorenylphthalamic acid and are transplantable into Buffalo rats. Buffalo rats (6–8 weeks old) were obtained from Harlan-Sprague-Daley (Indianapolis, IN). All animal studies were conducted in accordance with the animal care policy of the University of California at Irvine and the Animal Research Committee. Cells were maintained in standard DMEM media with 20% horse serum and 5% fetal calf serum.

TGF-B2 Vector
TGF-ß2 antisense vector was obtained from H. Fakhrai, UCLA Medical Center, Los Angeles, CA.16 This vector was formed previously by using the simian TGF-ß2 cDNA fragment containing bases 1–870, which was ligated in reverse orientation in the HindIII-XhoI sites of the pCEP-4 vector (InVitrogen, San Diego, CA). Mouse and simian TGF-ß2 share 81% homology. Expression of the antisense molecule is driven by the cytomegalovirus promoter of the vector. The pCEP-4 vector also contains the hygromycin resistance gene driven by the herpes simplex virus thymidine kinase promoter, the Epstein-Barr virus origin of replication, and the gene for the Epstein-Barr virus nuclear-associated antigen protein 1. Genetic modification of the MRH cells was performed by using the lipofectamine transfection kit (Gibco BRL, Life Technologies, Rockville, MD) following the manufacturer’s instructions. MRH cells that had been successfully transfected with the vectors were selected with hygromycin at 200 µg/ml (Sigma, St. Louis, MO). MRH cells were grown back to confluency and used for experiments during their logarithmic phase of growth.

TGF-ß2 Bioassay
Standard ELISA rat TGF-ß2 kit (Genzyme, Cambridge, MA) was used to measure production of secreted TGF-ß by the tumor cells. Following transfection, when the cells reached logarithmic phase, the amount of TGF-ß2 secreted into the supernatant fluid over a 24-hour period was determined.

MRH Rat Tumor Model and Vaccination
Wild-type MRH tumors were established by injecting 1 x 106 unmodified MRH cells subcutaneously into the flanks of Buffalo rats. Subcutaneous tumors typically are palpable at 10 to 12 days following tumor cell injection. Biweekly immunizations with genetically altered and control tumor cells were performed. Cells were harvested during the logarithmic phase of growth. Inoculations were performed using tumor cells that had been irradiated with 6000 cGy. Irradiated cells were suspended in 200 µl of saline and were injected (shortly after being irradiated) into the flank of the rats opposite the one where the wild-type tumor cells had been injected. Previous studies have shown that irradiation does not inhibit antisense production.16,21 Inoculations were completed on days 5, 10, 15, and 20 following the initial injection with the wild-type tumor cells. Four treatment groups were established. Group 1 animals (n = 16) received injections of saline only. Group 2 (n = 11) received biweekly inoculations with 1 x 106 MRH cells that had been irradiated. Group 3 (n = 6) was vaccinated with 1 x 106 irradiated MRH cells modified with pCEP-4 vector alone. Group 4 (n = 10) received 1 x 106 irradiated MRH cells transfected with pCEP-4/TGF-ß2 antisense modified vector. Mean survival time and tumor volume were recorded for each experimental rat.

Cell-mediated Cytotoxic Assays
Standard chromium-release assays were used to assess the effects of immunization with TGF-ß2 antisense vectors. Harvested lymph node effector cells from immunized animals were stimulated in vitro with irradiated unmodifed MRH stimulator cells at 30:1 effector-to-stimulator ratio for 5 days in the presence of recombinant IL-2 and 50 units/ml. Target MRH cells (1 x 106 cells) were labeled with 400 uCi Na 51Cr by incubating overnight at 37°C. Effector cells were then mixed with labeled target cells at ratios of 3:1, 10:1, 30:1, and 100:1. A 4-hour 51Cr release assay was completed. Percent lysis was calculated (cpm exp–cpm bkgd)/(cpm max–cpm bkgd) x 100, where exp = experimental and bkgd = background.

Two different cytotoxicity experiments were performed. In the first set of assays, effector cells were collected from the animals that had received the various vaccines (saline, unmodified MRH cells, MRH pCEP-4 modified cells, and MRH pCEP-4/TGF-ß2 antisense modified cells). These lymphocytes were stimulated in vitro with irradiated unmodified MRH cells for 5 days. Their ability to kill unmodified MRH target cells was then determined using the chromium-release assay.

The second experiment tested the in vitro stimulation of cytotoxic effector cells. Effector lymphocytes were taken from naïve Buffalo rats. These cells were then stimulated in vitro with the various cell preparations (unmodified MRH cells, MRH pCEP-4 modified cells, or MRH pCEP-4/TGF-ß2 antisense modified cells). These effector cells were then tested for their ability to kill unmodified MRH cells using the chromium-release assay.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subcutaneous immunizations of modified MRH HCC cell line were used to treat Buffalo rats that had been given wild-type MRH tumor cells 5 days before inoculations were initiated. Results of tumor incidence for the treatment groups are detailed in Table 1. Three possible outcomes for tumor development were observed: tumor growth was completely blocked (BT); tumors grew for a time and then regressed (RT); or tumors progressively continued to grow (PT). Group 1 animals received saline injections but no additional cell vaccines. In this group, 15 of 16 rats (94%) developed tumors and died, and none showed spontaneous regression of tumors. Group 2 animals received biweekly inoculations with irradiated unmodified MRH cells. Six of 11 (55%) rats in Group 2 died from tumor burden, and 4 of 11 (36%) had spontaneous regression of their tumors. Group 3 animals were inoculated with irradiated MRH cells that had been modified with pCEP-4 vector alone. In Group 3, 3 of 6 rats (50%) died from tumors, and 2 of 6 (33%) had spontaneous regression of tumors. Group 4 animals received irradiated cells transfected with pCEP-4 antisense TGF-ß2 vector, and all animals in this group remained free of tumors (n = 10). These data are graphed in Fig. 1.


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  		Table 1. Effect on the development of wildtype tumor growth by antisense TGF-ß2 vaccinea
 


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  		FIG. 1. Incidence of tumor development in response to pCEP-4/TGF-ß2 antisense MRH cell inoculations. Wild-type MRH cells were injected subcutaneously into all animals on day 0. Biweekly injections of vaccine preparations were given for total of four treatments. Tumor development was completely blocked in the animals treated with irradiated MRH pCEP-4/TGF-ß2 antisense modified cells (Group 4). Inoculations were as follows: saline, Group 1 ({Delta}); irradiated unmodified MRH cells, Group 2 ({blacktriangleup}); irradiated MRH pCEP-4 modified cells, Group 3 ({square}); and irradiated MRH pCEP-4/TGF-ß2 antisense modified cells, Group 4 ({blacksquare}).

 
Supernatant produced by the tumor cells in tissue culture was collected and tested in an ELISA kit for quantity of TGF-ß2 secreted. Antisense pCEP-4/TGF-ß2 transfected MRH cells produced 92 pg/ml TGF-ß2 per 106 cells over a 24-hour period, a quantity markedly less than the unmodified MRH cells (1571 pg/ml). The biological activity of the secreted TGF-ß was confirmed by use of TF-1 cell bioassay as described previously.22

Cell-mediated cytotoxicity assays were performed by using lymphocyte effector cells collected from the immunized rats and these cells were stimulated in vitro with unmodified MRH cells. Effector cells from animals immunized with antisense TGF-ß2 modified MRH cells demonstrated a two- to threefold increase in lytic activity toward unmodified MRH cells as compared to the other groups ( Fig. 2). In a second set of experiments, lymphocyte effector cells were collected from naïve Buffalo rats. These cells were then stimulated in vitro with unmodified MRH, pCEP-4 modified MRH, or pCEP-4/TGF-ß2 antisense modified cells and were tested for their ability to kill unmodified MRH cells ( Fig. 3). Effector cells stimulated with pCEP-4/TGF-ß2 antisense modified cells had double the lytic response compared to cells stimulated with unmodified MRH cells or pCEP-4 modified MRH cells.



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  		FIG. 2. Production of cytotoxic effector cells by vaccination with MRH pCEP-4/TGF-ß2 antisense modified cells. Lymphocytes taken from animals immunized with the various cell preparations were stimulated with irradiated, unmodified MRH cells. These effector cells were then tested for their ability to kill target unmodified MRH cells using chromium-release assay. Effector cells from animals vaccinated with irradiated MRH pCEP-4/TGF-ß2 antisense modified cells had a twofold greater ability to kill the target cells compared to the other immunizations. Effector cells were collected from: animals given injections of saline, Group 1 ({Delta}); irradiated unmodified MRH cells, Group 2 ({blacktriangleup}); irradiated MRH pCEP-4 modified cells, Group 3 ({square}); irradiated MRH pCEP-4/TGF-ß2 antisense modified cells, Group 4 ({blacksquare}).

 


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  		FIG. 3. Generation of cytotoxic effector cells by in vitro stimulation with MRH pCEP-4/TGF-ß2 antisense modified cells. Lymphocytes taken from naïve Buffalo rats were stimulated in vitro with the various cell vaccine preparations—irradiated unmodified MRH cells ({blacktriangleup}), irradiated MRH pCEP-4 modified cells ({square}), and irradiated MRH pCEP-4/TGF-ß2 antisense modified cells ({blacksquare}). These effector cells were then tested for their ability to kill target, unmodified MRH cells using chromium-release assay. Effector cells stimulated with irradiated MRH pCEP-4/TGF-ß2 antisense modified cells had a twofold higher ability to kill target unmodified MRH cells than did the other stimulator cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These results demonstrate the successful treatment of MRH HCC tumors in rats using a vaccine created by genetically altering tumor cells with antisense TGF-ß2. Decreased production of the immunosuppressive TGF-ß in the tumor vaccine should release the inhibition of the immune system and allow for specific, tumor-directed immune reaction. Enhancement of the natural immune response was demonstrated by in vitro cell-mediated cytotoxicity assays. Interestingly, the use of irradiated unmodified MRH cells and cells modified with pCEP-4 vector alone was able to cause tumor regression in about one third of the animals. Cytotoxic assays failed to show a significant tumor-specific augmentation of immune lymphocytes in these cases, suggesting that this effect may not be specific for HCC tumors. However, animals treated with the pCEP-4/TGF-ß2 antisense modified cells were all protected from the development of tumors. These results suggest that antisense TGF-ß2 modification enhances immunogenicity against MRH HCC tumor cells and supports its potential application in the clinical setting.

This novel method of gene therapy was previously used by Fakhrai et al. for treatment of intracranial glioblastoma tumors in rats.16 They determined that a vaccine using tumor cells modified with antisense TGF-ß2 was able to eradicate established intracranial tumors completely. Our results point out that this type of vaccine treatment is not unique to central nervous system tumors and may be directed toward other tumors which overexpress TGF-ß2. In fact, similar vaccines could be designed to treat tumors that express other immunosuppressive growth factors. Additionally, the use of combination treatments could prove to be even more effective. For example, current immunotherapy directed to augment the body’s immune system, such as IL-2 treatment, may have an additive benefit when used in conjunction with this type of antisense treatment. Use of antisense modified vaccines may allow treatments like IL-2 immunotherapy to be given in lower, less toxic doses. It is important to note that no untoward effects, e.g., weight loss or increased incidence of infections, were noted in the animals treated with the antisense TGF-ß2 modified vaccine.

TGF-ßs are multifunctional polypeptides that have a vast array of functions affecting regulation of cellular growth. Three isoforms, TGF-ß1, TGF-ß2, and TGF-ß3, have been identified. As mentioned earlier, they participate in regulation of cell growth, cell differentiation, angiogenesis, and formation of extracellular matrix. Signal transduction works by binding specific serine/threonine kinase cell surface receptors, the serine/threonine kinases. Their immediate substrates have been identified as the SMAD proteins, which, once activated, move into the nucleus, where they initiate gene transcription in association with DNA binding partners.23 To date, eight members of the SMAD family have been identified. The wide range of effects generated by the TGF-ß proteins is a function of the SMADs. Further investigation, looking at the control of this signal transduction pathway and how it is altered by antisense TGF-ß2 therapy, may lead to insight into the mechanism of the immunosuppressive effects TGF-ß2 has on tumors.

We have confirmed that tumor immunity can be augmented by creating a tumor vaccine that blocks a tumor-derived immunosuppressant, thereby enhancing the host’s immune response against the wild-type tumor. Future studies will be performed to investigate the ability of TFG-ß2 antisense vaccines to treat established HCC tumors. More detailed analysis needs to be completed to detail the effects on the immune lymphocytes, i.e., to determine the subsets of lymphocytes that were augmented. We hypothesize that this method could be applied clinically as follows: Tumor cells would be collected at the time of surgical resection. Once cells were harvested, they could be modified with antisense treatment and then used to treat a recurrence or as a preventative vaccine. Although this may not be practical in the current clinical setting, work to elucidate the mechanism through which TGF-ß leads to tumor immunosuppression may lead to the development of a simpler form of vaccine. Further work also may lead to the creation of a preventative tumor vaccine for use in populations in which HCC is prevalent.


   Acknowledgments
 
This work was funded in part by a grant from the Chao Family Cancer Center at the University of California at Irvine. The authors would like to James Economou for his expert advice on this project. We are indebted to Dr. Fakhrai, Department of Neurosurgery, UCLA Medical Center, Los Angeles, CA, for his generosity in giving us the antisense TGF-ß2 vector.


   Footnotes
 
Presented at the 53rd Annual Meeting of the Society of Surgical Oncology, New Orleans, Louisiana, March 16-19, 2000.

Received for publication September 13, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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