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Experimental Therapeutics |
Mogam Biotechnology Research Institute, Kyonggi-Do, 449–910, Korea [J. W., H. K., E. J. P., Y. H., Y. Y.]; and Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892 [S.-J. K.]
ABSTRACT
Many types of tumor cells overexpress transforming growth factor ß (TGF-ß), which is believed to promote tumor progression. We hypothesized that overexpression of the extracellular region of the type II TGF-ß receptor (soluble TßRII) would compete for or block TGF-ß binding to TßRs on immune cells, preventing TGF-ß-mediated immunosuppression and consequently resulting in the eradication of tumor cells. We tested this in the mouse thymoma cell line EL4, which has been reported to suppress cellular immunity by secreting a large amount of TGF-ß. Transduction of EL4 with recombinant retrovirus encoding soluble TßRII resulted in the secretion of heterogeneously glycosylated, 25 to 35 kDa truncated TßRII. Inoculation of 1 x 104 to 5 x 104 soluble TßRII-modified EL4 cells (EL4/Ts, EL4 cells transduced with recombinant retrovirus encoding soluble TßRII and neomycin resistance gene) s.c. to mice showed reduced tumorigenicity, as indicated by lower overall tumor incidence (7%, 1 of 14; P < 0.001) compared with unmodified EL4 (100%, 9 of 9) or vector-modified EL4 cells (EL4/neo, EL4 cells transduced with recombinant retrovirus encoding neomycin resistance gene; 100%, 4 of 4). Administration of mitomycin C-treated EL4/Ts cells (1 x 106) after EL4 inoculation (1 x 104) reduced tumor incidence from 100% (5 of 5 in mice inoculated with mitomycin C-treated EL4/neo) to 40% (4 of 10, P < 0.05), indicating that supply of soluble TßRII could actually block TGF-ß-mediated tumorigenesis. In vitro tumor cytotoxicity assays revealed 3–5-fold higher cytotoxic activity with lymphocytes from EL4/Ts-bearing mice compared with those from EL4- or EL4/neo-bearing mice, indicating that the observed tumor rejection was mediated by restoration of the tumor-specific cellular immunity. These data suggest that expression of soluble TßRII is an effective strategy for treating highly progressive tumors secreting TGF-ß.
INTRODUCTION
TGF-ß3 belongs to the TGF-ß superfamily, which regulates various physiological functions such as proliferation, differentiation, development, bone morphogenesis, and production of the extracellular matrix (1) . There are three different isoforms of TGF-ß in mammalian cells, i.e., TGF-ß1, TGF-ß2, and TGF-ß3,which have similar but different activities, cellular targets, and binding affinities to various receptors (2) .TGF-ß was first reported as a stimulator of phenotypic transformation of rat kidney fibroblasts (1) , but later it has been more frequently reported as an inhibitor of proliferation in a broad range of cell types including epithelial cells, endothelial cells, and hematopoietic cells (1) . The role of TGF-ß in tumorigenesis is somewhat paradoxical. Although TGF-ß acts as a potent growth inhibitor of some cancer cells (3, 4, 5) , at the same time it can act as a selective growth promoter when tumor cells somehow acquire resistance to TGF-ß-mediated growth inhibition (6, 7, 8, 9, 10, 11, 12, 13, 14) . Deletion (7, 8, 9) , reduced expression (10 , 11) , and mutation (12) of the TßR were suggested to be the underlying mechanisms for the loss of TGF-ß sensitivity in highly progressive tumors such as glioma, cutaneous T-cell lymphomas, and carcinomas from breast, stomach, prostate, and colon. Loss of heterozygosity is frequently detected in various human cancers on chromosome 18q21, where several candidate tumor suppressor genes such as the TGF-ß signaling mediators deleted in pancreatic cancer 4 (DPC4) and MADR2 exist (13 , 14) .
Many tumor cells secrete TGF-ß, which promotes their growth by acting as a paracrine modulator on stromal cells as well as paralyzing the immune surveillance system (15, 16, 17) . TGF-ß induces angiogenesis and regulates the production of the extracellular matrix and proteolytic enzymes as well as the expression of adhesion molecules, making tumor cells more invasive and metastatic (18, 19, 20) . Although the mechanism of transition from hyperplastic, oncogene-transformed cells to metastatic carcinoma is not clear, it has been demonstrated that epithelial-stromal interaction is important for phenotypic conversion and that one of the principal mediators of that process is TGF-ß (18) . TGF-ß is also a strong immunosuppressor (21, 22, 23, 24, 25, 26, 27) and acts by inhibiting the proliferation of lymphocytes (21 , 22) , the differentiation of lymphokine-activated killer cells (22) , the development of Th1 cells (23) , cytokine/cytokine receptor expression (24 , 25) , and cytotoxic activity of lymphocytes (26) . Suppressed immune function has been frequently observed in animals with TGF-ß-secreting tumors (24 , 25) . In addition, genetic modification of highly immunogenic tumor cells to express TGF-ß allowed escape from the immune surveillance system, resulting in increased tumor incidence (27) .
There are three types of TßR (28, 29, 30, 31) . TGF-ß signaling is mediated through a heterooligomeric complex of type I and type II receptors (28) . Although TßRII (3) can bind to TGF-ß in the absence of TßRI, TßRI always requires TßRII to bind TGF-ß. A primary TGF-ß-TßRII complex recruits TßRI, which is then phosphorylated in the GS domain by constitutively active TßRII (29) . Activated TßRI then phosphorylates Smad molecules, which detach from TßRI upon phosphorylation, form heterooligomeric complexes, translocate into the nucleus, and activate several genes in concert with other transcriptional factors (30) . TßRIII is a proteoglycan that has a high binding affinity for all of the isoforms of TGF-ß and recruits TGF-ß to type I and II receptors (31) . TßRIII does not have a known signaling motif in its cytoplasmic domain and seems to act as an adapter of TGF-ß for other TßRs (31) .
In this study, we applied the ability of TßRII to bind to TGF-ß in the absence of type I or type III receptors (28) . A previous study indicated that the extracellular region of TßRII is processed and secreted from cells and binds to TGF-ß (32) . We hypothesized that overexpression of the extracellular region of TßRII in tumor cells would prevent TGF-ß-mediated immunosuppression and subsequent tumorigenesis by directly binding to TGF-ß and blocking its action on the surrounding stroma and the immune system. We tested this possibility in the mouse thymoma cell line EL4 that has been reported to secrete TGF-ß and down-regulate the immune system of its host (33 , 34) . Here, we demonstrate that the expression of the extracellular region of TßRII in EL4 cells elicited tumor rejection and prevented the tumor progression.
MATERIALS AND METHODS
Vector Construction and Genetic Modification of Mouse Thymoma EL4 Cells.
MFG vector (35)
with internal ribosomal entry site and a neomycin resistance gene within the BamHI site (MFG/i.neo) was used as a backbone. To generate an MFG vector encoding soluble TßRII (MFG/Ts), the extracellular domain (amino acids 1–159) of human TßRII was amplified from a full-length human TßRII (36)
and cloned into the NcoI-BamHI site of MFG/i.neo (Fig. 1)
. As a control vector, an MFG vector with the neomycin resistance gene only (MFG/neo) was used. Genetic modification of EL4 cells was performed by recombinant retrovirus encoding MFG/Ts and MFG/neo. Briefly, 1 x 105 to 5 x 105 BOSC23 cells, a packaging cell line of kidney cell origin (37)
, were seeded the day before transfection. Each retroviral vector was transfected into BOSC23 using Lipofectamine as indicated by the manufacturer (Life Technologies, Inc., Gaithersburg, MD). Culture supernatants containing recombinant retrovirus were harvested 48–72 h after transfection and filtered through a 0.45 µm cellulose acetate filter (Micro Filtration Systems, Dublin, CA). Actively growing EL4 cells (2 x 105) were incubated for 4 h with 1 ml of viral supernatant diluted 1:2 in RPMI 1640/10% FBS (RPMI10) containing 8 µg/ml of Polybrene (Sigma Chemical Co., St. Louis, MO). EL4 cells were kept incubated with viral supernatant, which were diluted further with RPMI10 so that the final concentration of Polybrene reached 2 µg/ml. After overnight incubation, cells were washed with RPMI10 and cultured for another 24 h. The next day, EL4 cells were diluted to 1 x 105 cells/ml in RPMI10 containing 800 µg/ml of geneticin (G418 sulfate; Sigma) and selected for 10–14 days. Transduced EL4 cells were drug selected for 2–5 more passages by growing in RPMI10 containing G418.
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Western Blot.
To determine the expression and secretion of soluble TßRII, transduced EL4 cells were cultured in serum-free and protein-free hybridoma medium (Sigma) for 2 days. Culture supernatants of EL4, EL4/neo, and EL4/Ts cells were precipitated with 5 volumes of acetone at -70°C for 30 min and centrifuged. The pellet was analyzed by Western blotting with affinity-purified polyclonal rabbit antibody (1:2000) to human TßRII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and goat anti-rabbit antibody (1:5000; Kirkegaard & Perry Laboratory, Inc., Gaithersburg, MD) as primary and secondary antibodies, respectively. The blots were processed with ECL Western blot detection reagents as indicated by the manufacturer (Amersham). The amount of soluble TßRII secreted into the culture supernatant was determined by densitometric analysis with the known staining intensity of commercially available recombinant human TßRII (R&D System, Minneapolis, MN) as a control.
Tumorigenicity and Tumor Regression Study.
For the tumorigenicity study, 1 x 104 or 5 x 104 cells of either unmodified, vector-modified (EL4/neo), or soluble TßRII-modified (EL4/Ts) EL4 cells were inoculated s.c. into the backs of C57BL/6 mice, 4–6 weeks of age. After 7–10 days, mean tumor diameter was measured every 3 or 4 days until day 36 or until the mice died. For the tumor regression study, 4–6-week-old C57BL/6 mice were injected s.c. with 1 x 104 EL4 cells and subsequently with 1 x 106 mitomycin C-treated EL4/neo or EL4/Ts cells. Mitomycin C-treated cells were injected twice a week for 2 weeks, beginning on the day of tumor inoculation, so that a total of four injections were made. For mitomycin C treatment, cells (1 x 107 cells/ml) were incubated with mitomycin C (50 µg/ml) for 30 min in the 37°C CO2 incubator, after which they were thoroughly washed twice with RPMI10, once with PBS (pH 7.4), and then used for inoculation.
Tumor Cytotoxicity Assays.
Splenocytes were harvested 14 days after inoculation of 5 x 104 EL4, EL4/neo, or EL4/Ts cells. They were then stimulated in vitro with mitomycin C-treated EL4 cells (1 x 104 cells/well) at a 5:1 responder:stimulator ratio for 5–7 days in the presence of 100 units/ml of recombinant mouse IL-2 (Calbiochem-Novabiochem International, La Jolla, CA). In vitro-stimulated lymphocytes were mixed with Na 51Cr-labeled EL4 (1 x 104) at E:T ratios of 100:1, 75:1, 50:1, and 10:1 and incubated overnight in the 37°C CO2 incubator. Target EL4 cells (5 x 106) were labeled with 300 µCi (1 Ci = 37 GBq) of Na 51Cr for 90 min at 37°C. The percentage of specific lysis was calculated by the following formula: [(cpmexp - cpmmin)/(cpmmax - cpmmin)] x 100, where exp is experimental, min is spontaneous release, and max is maximum release.
RESULTS
Genetic Modification of EL4 Mouse Thymoma Cells.
We hypothesized that overexpression of the extracellular domain of TßRII (soluble TßRII) in EL4 would interrupt interaction between tumor-secreted TGF-ß and TßRs on the immune or stromal cells, resulting in prevention or alleviation of TGF-ß-mediated tumorigenesis. To test this hypothesis, a retroviral vector encoding the entire extracellular domain (amino acids 1–159) of type II human TßR was constructed (MFG/Ts; Fig. 1A
). As a control vector, MFG vector with the neomycin resistance gene only (MFG/neo) was also constructed. These vectors were transfected to BOSC23 packaging cells, and culture supernatants containing recombinant retrovirus were used to transduce EL4 mouse thymoma cells. Integration of retroviral vectors into chromosomal DNA was confirmed by genomic PCR. An 
0.48-kb fragment encoding soluble TßRII was amplified from the genomic DNA of soluble TßRII-modified EL4 cells (EL4/Ts; Fig. 1B
). Integration of neomycin resistance gene was also confirmed by the presence of an amplified 0.8-kb DNA fragment in both EL4/neo and EL4/Ts cells (data not shown). A Western blot indicated that EL4/Ts cells secreted 10–20 ng/ml of soluble TßRII of 25 to 35 kDa in size into the culture supernatant (Fig. 1C)
. This is consistent with a previous report by Lin et al. (36)
in which the extracellular domain of TßRII was detected as multiple bands of 25 to 35 kDa in COS cells due to heterogeneous glycosylation.
Tumorigenicity and Tumor Regression Study.
We studied the effect of soluble TßRII expression on the tumorigenicity of EL4 cells. The experiments were performed twice with minor modifications. According to our preliminary tumor induction test, inoculation of 1 x 104 to 1 x 105 EL4 cells induced tumors between days 10 and 14, and mice died on days 28–38 (data not shown). In the first tumorigenicity study, we injected s.c. 5 x 104 EL4 or EL4/Ts cells into the backs of C57BL/6 mice and observed tumor incidence for 36 days. All mice inoculated with EL4 developed tumors 28–32 mm in diameter, whereas in the group of mice inoculated with EL4/Ts, only 1 mouse of 10 mice developed a tumor (Table 1)
. The EL4/Ts-bearing mice with tumors showed delayed onset and progression about a week compared with EL4-bearing mice. In the second tumorigenicity study, mice were inoculated with 1 x 104 EL4, EL4/neo, or EL4/Ts cells and then observed for tumor progression until day 36. All of the mice inoculated with EL4 or EL4/neo developed tumors, whereas none of the mice inoculated with EL4/Ts did (Table 1
; Fig. 2
). Tumors 2 mm in diameter disappeared and reappeared several times in one mouse with EL4/Ts but disappeared ultimately (Fig. 2)
. All in all, only one of the 14 animals inoculated with EL4/Ts developed a tumor in contrast to 100% tumor incidence with EL4 or EL4/neo-bearing animals.
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. This indicates that the reduced tumorigenicity of EL4/Ts is not due to reduced growth rate caused by spontaneous genetic mutations.
In the tumor regression study, we tested the antitumorigenic effect of the secreted soluble TßRII on unmodified parental EL4 cells. We assumed that repeated injections of nondividing EL4/Ts cells near the primary tumor inoculation site would result in a transient elevation of the local concentration of soluble TßRII, which would neutralize TGF-ß-mediated immune suppression and tumorigenesis. Mice were subjected to inoculation of 1 x 104 unmodified EL4 cells, followed by four injections of mitomycin C-treated EL4/neo or EL4/Ts cells during 2 weeks. Repeated injections of nondividing EL4/Ts reduced tumor incidence from 100 to 20% (P < 0.01) and 40% (P < 0.05) at day 20 and day 34, respectively (Table 2)
. The increase in tumor incidence from 20 to 40% during the 14 days after day 20 suggests that a more intensive injection schedule might be required to completely block tumor growth.
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. Spleen cells were harvested at day 14 after tumor inoculation, stimulated in vitro with unmodified EL4 for 5–7 days, and evaluated for their ability to lyse unmodified EL4. As shown in Fig. 3
, lymphocytes from mice inoculated with EL4/Ts displayed 3–5-fold greater cytolytic ability at E:T ratios of 100:1 and 75:1 compared with those from mice injected with EL4 or EL4/neo.
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In this study, we demonstrated that expression of the soluble extracellular domain of the TßRII in tumor cells is an effective method for tumor therapy. EL4 cells modified to express soluble TßRII secreted heterogeneously glycosylated TßRII with a molecular mass between 25 and 35 kDa and elicited tumor rejection in almost 100% of the inoculated animals. Administration of nondividing EL4/Ts cells reduced tumor incidence induced by EL4 cells, indicating possible therapeutic use of soluble TßRII for preventing relapse after tumor removal.
Standard chromium release assays using lymphocytes from mice inoculated with EL4/Ts showed 3–5-fold higher cytolytic activity compared with those from EL4- or EL4/neo-bearing mice. This suggests that secreted soluble TßRII from EL4/Ts blocked TGF-ß-mediated immunosuppression, and this could be a major contributor of the active tumor eradication observed in mice with EL4/Ts inoculation.
Suppression of cell-mediated immunity has been observed previously in EL4-bearing mice and TGF-ß-mediated macrophage dysfunction, abnormal ratios of the Th1:Th2 population, and weakened Th1 response were proposed as the underlying mechanisms of suppressed immune function in these mice (33
, 34)
. Administration of neutralizing anti-TGF-ß antibody into EL4-bearing mice restored the ability of macrophages to secrete nitric oxide and TNF-
on LPS stimulation (33)
. Production of Th1-type cytokines by lymphocytes stimulated with anti-CD3 or PMA/A23187 was also restored by anti-TGF-ß antibody administration (34)
. Because active involvement of macrophages and Th1 cells is a prerequisite for full CTL development, it is possible that both of the previously proposed mechanisms underlie the enhanced cellular immune responses we observed in this study upon soluble TßRII therapy.
In this study, we did not compare invasive or metastatic activity between EL4 and EL4/Ts cells. However, considering the enhanced invasiveness in many TGF-ß-secreting tumors (18, 19, 20) , soluble TßRII-mediated segregation of TGF-ß from stromal cells or tumor cells themselves might also have played a role in preventing tissue invasion and consequent tumor regression. Oft et al. (18) indicated that TGF-ß is not only a major player converting oncogene-transformed, hyperplastic cells to invasive phenotype but also is important for maintaining its invasive feature by acting autocrine. It may well be possible that TGF-ß enhances tumorigenesis by maintaining the invasive state of EL4 in an autocrine manner besides acting paracrine to stromal and immune cells.
Several studies have indicated that blocking of TGF-ß expression in tumor cells alleviates tumorigenesis. Blockage of TGF-ß2 expression by antisense therapy in rat glioma cells eradicated established rat gliomas and protected rats from further tumor challenges (38) . This antitumor effect was suggested to be due to restored cellular immunity as can be seen from the higher cytotoxic activities of the lymphocytes from the animals inoculated with TGF-ß antisense-modified gliomas. Retention of TGF-ß around breast cancer cells by expressing TßRII having high affinities to both TGF-ß1 and TGF-ß2 also reduced tumor incidence (39) . Expression of IL-2 in a TGF-ß-secreting tumor, the murine mammary sarcoma EMT6, also reduced tumor incidence, indicating that the antagonistic effect of interleukin 2 on TGF-ß could alleviate tumorigenesis (40) .
Lin et al. have reported previously that the affinity of soluble TßRII to TGF-ß is about 5–10-fold lower than that of whole membrane-associated TßRII (32) . This would restrict the efficiency of soluble TßRII therapy and consequently would require a large amount of soluble TßRII expression to completely block the interaction between TGF-ß and membrane-associated TßRII. In addition, because soluble TßRII binds very weakly or not at all to TGF-ß2, its antitumor effect on TGF-ß2-secreting tumors could be minimal. For clinical application, the present approach needs modifications, such as the usage of a soluble TßRII with improved affinity to TGF-ß and the combination therapy using B7 or granulocyte-macrophage colony-stimulating factor to enhance tumor-specific immunity.
ACKNOWLEDGMENTS
We are grateful to Seongyoo Cho for help in the cytotoxicity assay, Dr. Hee-Yong Chung for comments on the retrovirus system, Jihyun Lee for help in the animal experiments, and Hyunsun Kang for critical reading of the manuscript.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Korea Green Cross Co. and the Ministry of Science and Technology of Korea. 
2 To whom requests for reprints should be addressed, at Mogam Biotechnology Research Institute, 341 Pojung-Ri, Koosung-Myun, Yongin-City, Kyonggi-Do, 449-910, Korea. Phone: 82-331-262-3206; Fax: 82-331-262-6622; E-mail: yydyun{at}kgcc.co.kr
3 The abbreviations used are: TGF-ß, transforming growth factor-ß; TßR, TGF-ß receptor.
Received 9/21/98. Accepted 1/18/99.
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