Differential effects of high-dose gamma irradiation on the production of transforming growth factor-beta in fresh and established human ovarian cancer.

Tumor cells from five freshly isolated ovarian tumors and four established human ovarian carcinoma cell lines were analyzed for the production of the immunoinhibitory cytokine transforming growth factor-beta (TGF-beta) before and after exposure to gamma irradiation and/or the cytokines TNF-alpha plus IFN-gamma. All fresh tumors secreted high levels of TGF-beta when compared to the levels produced by the established ovarian carcinoma cell lines. TGF-beta produced by fresh tumors was significantly reduced after high doses of gamma irradiation (10,000 cGy). In contrast with the established cell lines, irradiation significantly increased TGF-beta secretion. Exposure of fresh tumor cells to cytokines followed by irradiation caused significant reduction of TGF-beta released when compared to the amount released after exposure to cytokines only. However, in the established cell lines, cytokines followed by irradiation again significantly increased TGF-beta production. These data indicate that high doses of irradiation in fresh ovarian tumors, unlike established ovarian carcinoma cell lines, can significantly reduce the local production of this potent immunoinhibitory cytokine. This effect could work to further amplify weak immunological responses within the tumor. In addition, these findings indicate major differences between fresh tumor samples and established cell lines and warn against the sole use of continuous cell lines as models for tumors growing in vivo.

tional protein endowed with a broad range of biological metastatic origin obtained at the time of surgery and four human epithelial ovarian carcinoma cell lines (UCI-101, activities on cells of different lineages [for review, 10]. Secretion of TGF-b has been detected from normal human UCI-107, SKOV-3, and T-222) were used for this study.
UCI-101 and UCI-107 were kindly provided by Dr. Alberto ovarian epithelium as well as from a wide variety of tumors [11][12][13][14]. The predominant effect of TGF-b on most normal Manetta, University of California, Irvine (UCI). SKOV-3 was purchased from American Type Culture Collection cells in vitro is reported to be inhibition of proliferation [15]. Because of this effect, it has been proposed that the (ATCC), while T-222 was kindly provided by Dr. Benjamin Bonavida, University of California, Los Angeles. All the unrestrained proliferation of some cancers may, in part, be due to the loss of normal growth inhibitory pathways such established tumor cell lines were maintained at 37ЊC, 5% as that caused by TGF-b [15]. However, it has recently been CO 2 in complete media (CM) containing RPMI 1640 (Gibco shown that TGF-b also markedly inhibits immune activity Life Technologies, Grand Island, NY), supplemented with including T, B, and NK cell effector functions [16,17]. 10% fetal bovine serum (FBS, Gemini Bioproducts, Calaba-These immune inhibitory effects could allow tumors secresas, CA). All of the fresh tumors (OVA-2, OVA-4, OVAting TGF-b to suppress and escape the immune response, 5, OVA-7, and OVA-13) were serous papillary adenocarcithus contributing to tumor growth. nomas and were maintained at 37ЊC, 5% CO 2 in CM con-An exciting recent approach to cancer immunotherapy taining RPMI 1640 supplemented with 5% FBS. Briefly, has been the use of genetically altered tumor cells (GATC) single-cell suspensions were obtained by processing solid engineered to secrete immune-enhancing cytokines capable tumor samples under sterile conditions at room temperature. of augmenting and stimulating antitumor immune responses Viable tumor tissue was mechanically minced in RPMI 1640 [for review , 18]. Transduction of cytokine genes into tumor to portions no larger than 1-3 mm 3 and washed twice with cells allows these proteins to function in their most physio-RPMI 1640. The portions of minced tumor were then placed logic way (i.e., paracrine) and to reach high local concentrainto 250-ml trypsinizing flasks containing 30 ml of enzyme tions within the tumor microenvironment at the immunizasolution [0.14% Collagenase Type I (Sigma) and 0.01% tion site. Moreover, the use of such GATC has been associ-DNAse (Sigma, 2000 kU/mg)] in RPMI 1640 and incubated ated with the induction of specific antitumor immunity in a on a magnetic stirring apparatus overnight at 4ЊC. Enzymativariety of tumor-cytokine combinations [for review , 18].
cally dissociated tumor was then filtered through 150-mm Recent studies investigating the immunizing effects of nylon mesh to generate a single-cell suspension. The resulcytokine-transduced tumor cells have suggested that the imtant cell suspension was then washed twice in RPMI 1640. munizing potential of a particular GATC is due to not only The percentage of tumor cells was determined by cytokeratin the specific cytokine transduced into the tumor but also the expression using immunohistochemical techniques. In this inhibitory cytokine(s) intrinsically secreted by such tumors regard, all the fresh tumor samples evaluated contained [19][20][21][22]. Such data support the concept that the balance ú99% tumor cells. between the quantity of immune-enhancing and suppressive Analysis of human TGF-b secretion. Tumor cell were cytokines produced within the tumor milieu can affect the seeded at a density of 1 1 10 5 cells/ml in CM in tissue ability of the immune system to respond to the tumor. culture flasks (Corning) and after a 48-hr incubation at 37ЊC, We have recently developed human ovarian carcinoma supernatants were aspirated, rendered cell-free by centrifutumor vaccines secreting different cytokines to be used as gation at 1500 rpm for 10 min, and stored at 020ЊC. TGFvaccines for women with advanced ovarian cancer [23,24]. b concentration was determined by ELISA, employing a We have proposed that such cytokine-secreting vaccines will commercially available kit (Research & Diagnostic Systems, be mixed with irradiated autologous tumor cells obtained at Minneapolis, MN) utilizing TGF-b standards ranging from the time of surgical debulking. Because of this, we have 31.2 to 2000 pg/ml. All samples were assayed in duplicate. directed our interest in evaluating the production of certain Standard regression lines were generated by plotting log 10 immune inhibitory cytokines, particularly TGF-b, in several concentration vs log 10 optical density, creating correlation fresh tumors and four established ovarian cell lines. The coefficients greater than 0.98 in all cases. The maximal alpurposes of our study were (1) to quantify the production lowed sample duplicate error was 10%. Duplicates falling of TGF-b by these various ovarian tumor cells and (2) to outside this error were reanalyzed. evaluate the effects of high doses of gamma irradiation, IFN-g and TNF-a preincubation. Cells from fresh tu-TNF-a / IFN-g exposure, and the combination of the two mors and established cell lines were cultured in CM conprocedures on the secretion of TGF-b.
taining a combination of IFN-g and TNF-a (500 units/ml MATERIALS AND METHODS each) for 3 days, washed, and reseeded at a density of 1 1 10 5 cells/ml in CM for subsequent studies. Recombinant Fresh tumors and tumor cell lines. Single-cell suspensions from five adenocarcinomas of the ovary of primary or human IFN-g (sp act 2.5 1 10 7 U/mg) was purchased from respectively. This difference in the levels of TGF-b produc-TGF-b Production in Fresh Ovarian Tumor Cells and Estabtion was shown to be significant (P õ 0.02).

lished Ovarian Carcinoma Cell Lines before and after High Doses
Secretion of TGF-b after high-dose gamma irradiation.

of Gamma Irradiation
TGF-b secretion was evaluated 48 hr after irradiation (10,000 cGy) and expressed as a normalized value as pg/ to pg/ml/10 5 viable cells/48 hr. Again, with the fresh tumors, a significant decrease in TGF-b production was noted in all the specimens when compared to the production of TGF-b Genzyme Corp. (Cambridge, MA) and recombinant human after cytokine exposure only (P õ 0.04). In contrast, for TNF-a (sp act 1.0 1 10 7 U/mg) was obtained from Genenthe established cell lines, cytokine exposure combined with tech, Inc. (San Francisco, CA).
irradiation consistently and significantly increased TGF-b IFN-g and TNF-a preincubation followed by high-dose production (P õ 0.02) ( Table 2). gamma irradiation. Tumor cells were irradiated with a total dose of 10,000 cGy using a Cs 137 source at a rate of 200 DISCUSSION cGy/min with or without prior preincubation with cytokines and then seeded at a density of 1 1 10 5 cells/ml in CM without cytokines. An aliquot of the cytokine-treated cells The production of inhibitory cytokines by a wide variety of tumors has been suggested as one of the major mecha-was also seeded in CM without additional cytokines to follow the natural course of TGF-b secretion but without the nisms by which these cells can evade destruction by the immune system [9-12]. The fact that ovarian cancer remains influence of irradiation. Media collected from flasks containing irradiated cells previously preincubated with cyto-confined to the peritoneal cavity, even at advanced stages of the disease, suggests that the growth of this malignancy kines and media collected from flasks containing irradiated control cells were collected after 48 hr for TGF-b evaluation. could be related to a local phenomenon of immunosuppression [4,5]. The local deficiency of antitumor immune ef-Statistical analysis. Significance analysis was performed fector mechanisms in the peritoneal cavity of patients with using a paired Student t test. Only P values õ0.05 were advanced stages of ovarian cancer [4,5] as well as the detecconsidered significant.
tion of high levels of inhibitory cytokines [5,6] in the ascites fluid of these patients supports this hypothesis.

RESULTS
TGF-b is a multifunctional cytokine with predominantly suppressive effects on immune system function [for review, Secretion of TGF-b by fresh tumors and established ovarian cell lines. Cell-free supernatants from freshly isolated 10]. Tumors that produce TGF-b secrete active TGF-b within the local environment [16] and it is therefore likely that this tumors and established ovarian cell lines were collected and analyzed for the levels of TGF-b by ELISA. The results in cytokine could inhibit the effector functions of T, B, and NK cells at the tumor site [17]. In this regard, it has been shown Table 1 show that all freshly isolated tumors as well as the established cell lines secreted TGF-b with a range of secre-that TGF-b is effective in blocking the differentiation and/ or the proliferation of naive CTLs and markedly inhibits the tion between 636 and 1793 pg/ml/10 5 cells/48 hr (mean 1158) and 415 to 480 pg/ml/10 5 cells/48 hr (mean 435), cytotoxic activity of NK effector cells [17]. expression of fresh ovarian tumors. We have shown that TGF-b Production in Fresh Ovarian Tumor Cells and cytokines markedly upregulate surface antigen expression Established Ovarian Carcinoma Cell Lines after Exposure to TNFand that irradiation causes persistent expression of this a / IFN-g and the Combination of 10,000 cGy Plus TNF-a / upregulated state (Santin et al., submitted). In this regard, cell lines (P õ 0.02). In our study we have confirmed the purity of the tumor cells in fresh tumor specimens by differ-Note. TGF-b levels were determined by ELISA from supernatants collected from tumor cells treated with TNF-a / IFN-g or with TNF-a / ential counts of Giemsa-stained cytospin slides as well as IFN-g followed by irradiation (i.e., 10,000 cGy). All samples were assayed by cytokeratin expression using immunohistochemical techin duplicate along with known standards as described under Materials and niques. In this regard, our fresh tumor samples contained Methods. P value for fresh tumor cells treated with TNF-a / IFN-g vs ú99% tumor cells.
those treated with TNF-a / IFN-g and irradiation was õ0.04. P value for The effects of gamma irradiation on TGF-b secretion indiestablished tumor cells treated with TNF-a / IFN-g vs those treated with TNF-a / IFN-g plus irradiation was õ0.02. Data from one experiment cated that lethal doses of irradiation were able to significantly are shown and are representative of two different studies with similar results. decrease the production of TGF-b in all fresh tumors tested. In contrast, however, all the established cell lines increased TGF-b secretion after lethal irradiation. One possible expla-The importance of TGF-b secretion in experimental animal models has been underscored by recent experiments of nation for these differences may be due to the differential capability of the fresh tumors and established cell lines to cytokine gene transduction that have found that the production of inhibitory cytokines from the transduced tumors can respond with reparative processes to the lethal damages caused by 10,000 cGy. Indeed, the much slower growth rate significantly affect the outcome of their interaction with host immune system cells [19][20][21][22]. While the data are not shown, exposure of tumor cells to have also found that TGF-b can dramatically inhibit the effects of IL-2 gene therapy with TGF-b-producing tumors IFN-g plus TNF-a caused an unpredictable response in the fresh tumors. Indeed, some tumors showed an increase in [21]. The failure to induce immunity against TGF-b-secreting tumors is in agreement with our unpublished data on a TGF-b secretion after cytokine exposure while others showed a decrease in its production. In contrast, in all four rat glioblastoma tumor (T9) genetically engineered to secrete IL-2 which was also shown to be a high secretor of TGF-established cell lines, cytokine preincubation induced a significant increase in TGF-b secretion (P õ 0.006) (data not b. In contrast, McAdam et al. have been able to reverse the in vivo inhibitory effects of TGF-b by immunizing animals shown).
When we evaluated the combined effects of cytokines and with tumor cells engineered to secrete high levels of IL-2 [22]. Thus, it appears that balances in the quantity of enhanc-irradiation on TGF-b secretion, again we found an opposite effect of irradiation on the fresh tumor samples and estab-ing or inhibitory cytokines within the tumor milieu may provide a possible explanation for the different outcomes lished cell lines. Indeed, in all fresh tumors a significant decrease in TGF-b production (P õ 0.04) after the combina-reported in these studies.
Our previous studies evaluated the effects of TNF-a / tion of the two procedures was always noted when compared to the levels of TGF-b secretion after exposure to cytokines IFN-g and high-dose gamma irradiation on surface antigen forming growth factor b as a suppressive factor, Cancer Immunol.