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"Suicide" of Breast Cells is Only Cytostatic In Vitro But Anti-tumoral In Vivo on Breast MCF7-ras Tumor

FRANCK GADAL, JORGE BASTIAS, MING X. WEI and MICHEL CRÉPIN

INSERM 553. Hôpital Saint-Louis and Université de Paris13, 1 avenue Claude Vellefaux, 75475 Paris Cédex 10, France

Abstract. Gene therapy with Herpes Simplex Virus thymidine Ganciclovir monophosphate and, subsequently, converted by kinase gene (HSV-tk) is effective in various tumor models in host cellular kinases to the di- and triphosphate forms (an vitro and in vivo. We compared the efficacy of the HSV-tk gene analog of the guanine triphosphate). The integration of therapy in vitro and in vivo in MCF-7 and MCF7-ras cells Ganciclovir triphosphate arrests DNA replication and causes which form tumor in athymic mice. After viral infection, cells (5). The present study compared the feasibility of a were treated with GCV (Ganciclovir) and live cells were HSV-tk "suicide" gene therapy in vitro and in vivo in the counted. The in vitro treatment significantly inhibited cell MCF-7 cell model characteristic of hormone-dependent growth but did not induce early and late , measured, human breast cancer (6). MCF7-ras cells, which overexpress respectively, by annexin or by propidium iodide staining and a Ras oncogene, are characteristic of hormone-sensitive breast significant cell death. The HSV-tk/GCV treatment of MCF7- cancer (7, 8) and form tumor in athymic mice without ras tumor in athymic mice showed a significant inhibition of estrogen treatment. The effect on the growth, viability and tumor development until 60 days post-treatment. Some mice apoptosis of HSV-tk/GCV-treated cells were compared to the showed a complete tumor eradication without tumor regrowth antitumor effect. after the end of treatment. In conclusion, we demonstrated that the HSV-tk/GCV system is not very efficient in vitro, but very Materials and Methods efficient in vivo in our animal breast cancer model. Cell cultures. The epithelial cell line MCF-7, established originally Breast carcinoma is one of the most frequent and from a pleural effusion of a breast adenocarcinoma, was obtained available treatments (surgery, and/or from ATCC (Rockville, MD, USA). MCF7-ras, derived from antiestrogen hormonotherapy with Tamoxifen) are not totally MCF7 by transfection of the H-ras oncogene, was kindly provided efficient. Chemotherapy and hormonotherapy have side- by Dr. F. Calvo (Hôpital St. Louis, Paris, France). M11 retrovirus- producing packaging cells (Laboratoire d’Histologie, Bobigny, effects and there is very often a tumor relapse under cancer France) were used to produce virus with a titer from 2.104 to 106 treatment. Furthermore, hormonotherapy with Tamoxifen is cfu/ml. NIH 3T3 tk- cells did not express endogenous thymidine not effective in all breast cancer because some breast cancers kinase and were obtained from (UFR Léonard de Vinci, are hormone-independent and disease recurrence is Laboratoire d’Histologie). All cell lines were cultured in commonly observed (1). Gene therapy with Herpes Simplex Dulbecco’s modified Eagle’s medium with glutamax I tm (Gibco Virus thymidine kinase gene (HSV-tk), which is effective in Invitrogen, Cergy Pontoise, France) supplemented with 10% Fetal various tumor models both in vitro and in vivo (2-4), involves Calf Serum (FCS) (Gibco Invitrogen), 50 Ìg/mL streptomycin (Gibco Invitrogen) and 50uI/ml penicillin (Gibco Invitrogen). transferring a chemosensitizing "suicide" gene to tumor cells. Tumor cells are exposed to a nontoxic pro-drug such as Virus and colony-forming assay. M11 cells were seeded in a T 75cm2 Ganciclovir (GCV), which is metabolized by HSV-tk into the (Falcon, VWR International, France) in DMEM 10% FCS. When they were at 70-80% of confluence, 5mL medium was replaced by 5mL of fresh medium for 24 hours. Viral supernatant was filtered through 0.45 Ìm and kept at –80ÆC. The titer of viral supernatant Correspondence to: Pr. Michel Crépin, SMBH Faculty of Medicine, was determined as follows. NIH 3T3 tk- cells were seeded for 2 days Université de Paris 13, 74 rue Marcel Cachin, 93017 Bobigny, in 6-well plate (Falcon) in DMEM 10% FCS at a density of France. Tel: 01-53-72-40-26, Fax: 01-53-72-40-27, e-mail: 105 cells/well, then they were infected for 3 hours with serial [email protected] dilutions of the viral supernatant supplemented with 8 Ìg of polybren (Sigma-Aldrich, Saint-Quentin Fallavier, France). One mL Key Words: Gene therapy, MCF7-ras tumor, HSV/tk-GCV. of fresh DMEM 10% FCS medium was added and the medium was

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Figure 1. Inhibition of MCF-7 and MCF7-ras cells proliferation by increasing concentrations of GCV. Exponentially growing MCF-7 and MCF7-ras cells were plated into 24-well plates (Falcon) at a density of 1.104 cells/well in triplicate. After 4 days, increasing concentrations of GCV were added to the cells. After 4 days of treatment, the number of viable cells for each well was determined by Malassez cell counting and trypan blue exclusion test. Data are shown as the mean value of triplicate experiments. Bar: SD, *: p<0.05.

changed the following day. Then 1mL of HAT medium (Gibco Invitrogen) was added and changed every 2 days until colony formation. Colonies were stained with giemsa’s solution for counting.

In vitro sensitivity of GCV. Cells were seeded for 2 days in DMEM supplemented with 10% FCS in 24-well plate (Falcon) at a density of 2.104 cells/well. Then cells were infected with 0.4mL/well of viral supernatant plus 8 Ìg/mL of polybren. After 3 hours, the cells were washed with 1x Phosphate Buffer Saline (PBS) (Gibco Invitrogen) and 1 mL of fresh DMEM 10% FCS was added to each well. Two days later, 1 mL/well of DMEM 10% FCS with GCV (Roche, Neuilly-sur Seine, France) 5 ÌM was added. After 4 days, the cells were harvested with 0.25% trypsin 0.2% EDTA (Gibco Invitrogen) Figure 2. Effect of retrovirus particles, with or without GCV, on the and counted by trypan blue exclusion assay. growth of MCF-7 and MCF7-ras cell lines. Exponentialy growing MCF-7 and MCF7-ras cells were plated into 24-well plates (Falcon) at a density Annexin V-FITC labelling. Cells were seeded in 6-well plate of 2.104 cells/well in triplicate. After 48 hours, cells were infected with (Falcon) in DMEM 10% FCS at a density of 1.105 cells/well. At the 0.4ml/well of viral supernatant (DMEM 10% FCS) and 8 Ìg/ml of indicated time after infection and treatment with GCV, the cells polybren for 2 hours. Then the cells were washed with 1x PBS and 1ml of were harvested, counted (plasma membrane integrity was fresh DMEM 10% FCS was added. Two days later, GCV at a estimated by trypan blue exclusion assay) and apoptosis was concentration of 5 ÌM was added to the cells. Viable cell number was detected and quantified by staining with annexin V-FITC determined by trypan blue exclusion assay after 4 days of treatment. Data (Clontech, CA, USA). Soon after the initiation of apoptosis, most are shown as the mean value of triplicate experiments. Bar: SD, *: p<0.01. cell types translocated phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface and PS could easily be detected by staining with the FITC conjugate of annexin V. The cells were incubated in the dark for 15 minutes at room before harvesting, then trypsinized, centrifuged at low speed, temperature with annexin V-FITC and/or propidium iodide as washed once with 1x PBS buffer, and then resuspended in DMEM described in the kit user manual. Five thousand cells were analyzed without serum at a density of 10.106 cells/ml. For subcutaneous by flow cytometry (FACScan®, Becton Dickinson, France S.A.). injection, 2.106 MCF7-ras cells in 200 Ìl were injected into the flanks of athymic mice (Harlan, France). When the tumors had Animal studies. For animal injections, culture cells in the reached a volume of about 200 mm3, 5.106 retrovirus-producing proliferative stage were changed with fresh medium 24 hours cells M11 (Laboratoire d’Histologie) in 200 Ìl were injected into

814 Gadal et al: In Vitro and In Vivo Gene Therapy of Breast Cancer Cells

Table I. Effect of HSV-tk-infected cell lines with or without GCV treatment on the viability of MCF-7 and MCF7-ras cells. The live cell number was determined by using Malassez cell and trypan blue exclusion assay 4 days after GCV treatment. Cell viability was calculated from the relationship: viability = ((alive-death)/alive) x 100. Data are shown as the mean value of triplicate experiments.

Viability MCF7 % MCF7-ras %

Control 96±4.0 95±4.0 HSVtk 96±0.5 97±5.0 HSV-tk/GCV 90±1.5 97±5.0

the tumor bed. Before GCV injection, the animals were randomly separated into 2 groups after tumor formation and M11 injection into the tumor bed. Mice in group 1 (n=6) received 1x PBS administration (in the volume corresponding to GCV administration) and mice in group 2 (n=6) received GCV Figure 3. Apoptosis induced by the "suicide" gene therapy of MCF-7 and treatment. Five days later, the animals started to be treated with MCF7-ras cells. Cells were seeded in 6-well plate (Falcon) in DMEM 10% GCV, which was administered twice a day for 14 days at a dose of FCS at a density of 1.105 cells/well. At the indicated time after infection 150 mg/Kg/day (15). Tumor volumes were regularly measured twice and treatment with GCV, cells were harvested and counted. Early and late a week with an external caliper. Tumor volumes were calculated as apoptosis were detected and quantified by staining cells with annexin V- 2 FITC and propidium iodide as described in Materials and Methods. follows: 4/3 x  x R1 x R2, where R1 and R2 are two diameters of Percentages of apoptotic cells are the measurements of annexin V-and/or the tumor and R1

In vitro treatment of HSV-tk-infected MCF7 and MCF7-ras cells with GCV-induced cell growth arrest but not a significant cell death (cytostatic effect). Before investigating the HSV-tk apoptosis phase indicated that the HSV-tk virus did not "suicide" gene effect with GCV prodrug, we tested the effect induce apoptosis for the two cell lines, 1, 2.5, 24 and 48 of GCV alone on MCF7 and MCF7-ras proliferations. hours after the beginning of the infection (data not shown). Figure 1 shows that, with 10 ÌM of GCV, the cell growths of Thus viral infection by HSV-tk does not affect the cell MCF7 and MCF7-ras lines slowed down in a dose- growth and viability of MCF7 and MCF7-ras cell lines. dependent manner. We obtained 65% and 70% of cell HSV-tk infected-cells in contact with GCV (5 ÌM) for 4 growth inhibition for MCF7 and MCF7-ras, respectively, days showed an important growth inhibition (Figure 3). with 50 ÌM of GCV. Since we observed that a high MCF7 and MCF7-ras growth were inhibited by 53%±1 and concentration of GCV (50 ÌM) increased the cell death rate 35%±1, respectively. When GCV was added, the death rate measured by trypan blue exclusion test (data not shown), we normally induced by GCV was not very important for the used a concentration of 5 ÌM of GCV in order to avoid a MCF7 cell line (Table I). The viability of MCF7-ras cells masking effect of the in vitro "suicide" gene therapy. was the same as the MCF7-ras control (Table I). We When the two cell lines were exposed to virus confirmed these results for the MCF7 and MCF7-ras cell supernatant obtained as described in the Materials and lines by annexin V-FITC labelling in vitro (Figure 3). This Methods section, we observed that the HSV-tk infection was experiment indicated, for the MCF7 cell line, that the not responsible for a significant decrease of growth and "suicide" gene therapy was able to induce significant, but not viability as compared to the control (Figure 2, Table I). In very important, apoptosis. One hour after GCV treatment, order to evaluate the effect of our HSV-tk virus infection we did not find significant apoptosis (annexin V and protocol on early and late apoptosis, as well as the death propidium iodide staining) induced by HSV-tk/GCV rate, we performed an annexin V-FITC and propidium therapy for both MCF7 and MCF7-ras cells. After 24 hours iodide labelling of infected and non-infected cells. The of incubation with GCV, a significant increase of the calculated percentages of cells in early (annexin V-FITC- infected MCF7 cell apoptosis-GCV mediated of 8%±1 as positive cells) or late (propidium iodide-positive cells) compared to the apoptotic MCF7 control cells was detected.

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Figure 4. "Suicide" gene therapy with GCV treatment of MCF7-ras tumors in athymic mice. When established tumors reached a volume of 200 mm3, after approximately 4 weeks, 5.106 retrovirus-producing cells M11 were injected into the tumor bed in 200 Ìl of DMEM (day 0). GCV was administered twice a day for 14 days (days 5 to 19), at a dose of 150 mg/Kg/day in the treated group (n=6). The control group (n=6) received 100 Ìl of 1x PBS. Tumor volumes were regularly measured twice a week with an external caliper as described in the Materials and Methods section. Bar: SD.

After 48 hours of treatment, almost the same difference Even after the end of GCV treatment at day 19, we did not (7%±1) between apoptotic MCF7 control cells and HSV- observe a relapse in GCV-treated mice and two mice showed tk/GCV MCF7 cells was found. a complete tumor regression. In contrast, in the control Results for the MCF7-ras cell line, indicated and group, all tumors were exponentially growing. On day 63, all confirmed that the "suicide" gene therapy was not able to control mice bearing huge tumors were sacrificed. Thus, induce cell death. GCV treatment could not induce HSV-tk/GCV treatment of MCF7-ras tumors blocked tumor significant apoptosis between control MCF7-ras cells and development and irreversibly induced a tumor regression. HSV-tk/GCV MCF7-ras cells after 1, 24 or 48 hours of GCV treatment (Figure 3). In conclusion, it appears that Discussion the MCF7 cell line, but not MCF7-ras, was slightly more sensitive in vitro to HSV-tk/GCV "suicide" gene therapy. The HSV-tk/GCV "suicide" gene therapy has been widely shown to be very efficient for the in vitro and in vivo HSV-tk/GCV therapy induced a strong MCF7-ras tumor treatment of various cells lines and transplanted tumors (2- regression in nude mice. Furthermore, we wanted to know if 4). In this study, we assessed the feasibility of a HSV-tk- MCF7-ras tumor-induced cells could be destroyed by the mediated gene therapy for the treatment of a hormone- HSV-tk/GCV "suicide" gene therapy in vivo in spite of lack of sensitive breast adenocarcinoma (6-8). In vitro MCF7 and sensitivity in vitro. So we used immune-depressed mice or MCF7-ras proliferations were compared for HSV-tk/GCV athymic mice for the development of the MCF7-ras tumor. "suicide" gene therapy. We also compared the effects of As shown in Figure 4, from day 0 to day 6, the MCF7-ras HSV-th/GCV therapy on the in vitro proliferation of MCF7- tumor volumes of treated and untreated groups were not ras to the MCF7-ras xenografted growth in nude mice. significantly different. However, these differences were We showed that direct injection into the MCF7-ras tumor significantly enlarged from day 6. In the GCV -treated group, bed with retroviral vector-producing cells followed by GCV tumors started to regress from day 6 and their growth was injections was efficient to cure MCF7-ras xenografted completely inhibited by day 14, until day 63 (data not shown). tumors. This efficacy was so important that in 2 mice of the

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GCV-treated group, tumors were completely eradicated destruction. These results suggested that the distant without tumor relapse. However, in the other 4 GCV- "bystander" effect is immune-mediated, but its precise treated mice, tumor growth was completely inhibited even mechanism remains to be elucidated. after the end of GCV injections (day 19). Tumor regression was not due to the retroviral vector-producing cells because Acknowledgements no tumor size variation was observed as compared to the control. This tumor regression under HSV-tk/GCV We are grateful to Professor J.L. Salzmann for help and providing treatment could be due to the "bystander" effect, first laboratory facilities for this work and we thank M.Etienne and demonstrated by Moolten et al. (9), which consists of the S.Beranger for their skillful technical assistance. transfer from cell to cell, infected or not, of phosphorylated GCV by gap junction (10) and/or apoptotic core (11). In our References study, due to the fact of complete tumor regression even after GCV treatment, HSV-tk/GCV therapy might have a 1 Litherland S and Jackson IM: Antiestrogens in the management "memory" effect to prevent tumor regrowth. of hormone-dependent breast cancer. Cancer Treat Res 14: In vitro results showed that the MCF7-ras cell line was not 183-194, 1988. sensitive to HSVtk/GCV therapy as far as toxicity and early 2 Kunishige I, Samejima Y, Shiki I, Moriyama A, Meruelo D, Saji F and Murata Y: Suicide gene therapy for human uterine apoptosis induced by phosphorylated GCV. The MCF7 cell adenocarcinoma cells using herpes simplex virus thymidine line was not very sensitive to this effect induced by GCV. kinase. Gynecol Oncol 72: 16-25, 1999. However, for these two cell lines, we found that HSV- 3 Lechanteur C, Princen F, Lo Bue S, Detroz B, Fillet G, Gielen tk/GCV "suicide" gene therapy could induce an important J, Bours V and Merville M-P: HSV-1 thymidine kinase gene antiproliferative cytostatic effect (12, 13). Treatment of therapy for colorectal adenocarcinoma-derived peritoneal MCF7 cells was more efficient than MCF7-ras cell carcinomatosis. Gene Ther 4: 1189-1194, 1997. treatment: 53% of growth inhibition with a weak toxicity and 4 Izquiero M, Cortes M, De Felipe P, Martin V, Diez-Guerra J, weak early and significant late apoptosis for MCF7 cells, and Talavera A and Perez-Higueras A: Long-term rat survival after malignant brain tumor regression by retroviral gene therapy. 36% of growth inhibition, no toxicity and early and late Gene Ther 2: 66-69, 1995. apoptosis for MCF7-ras cells. These differences can be 5 Loubiere L, Guettari N, Caruso M and Klatzmann D: explained by the fact that the MCF7 and MCF7-ras cell lines Nucleoside analogue metabolism by herpes simplex virus type I were not representative of the same cancer grade thymidine kinase and gene therapy for cancer and AIDS. progression: grade II for MCF7 which is not tumorigenic in International Antiviral News 4: 161-163, 1995. nude mice and grade III for MCF7-ras. Overexpression of 6 Thompson EW, Reich R, Shima TB, Albini A, Graf J, Martin the ras oncogene could also confer to MCF7-ras its GR, Dickson RB and Lippman ME: Differential regulation of resistance to in vitro "suicide" gene therapy. Perhaps the slow growth and invasiveness of MCF-7 breast cancer cells by antiestrogens. Cancer Res 48: 6764-6768, 1988. down in growth without cell death could be explained by too 7 Dickson RB, Kasid A, Huff KK, Bates SE, Knabbe C, Bronzert short an exposure time to GCV and/or to an insufficient D, Gelmann EP and Lippman ME: Activation of growth factor GCV dose. However, we know that this "suicide" gene secretion in tumorigenic states of breast cancer induced by 17‚- therapy induced a fast cell death and that it is not necessary estradiol or v-Ha-ras oncogene. Proc Natl Acad Sci USA 84: to use a high dose of GCV to obtain cell destruction (5). 837-841, 1987. Taking all these results together, we can conclude that 8 Sommers CL, Papageorge A, Wilding G and Gelmann EP: the immune system was absolutely necessary for the total Growth properties and tumorigenesis of MCF-7 cells transfected h destruction of MCF7-ras neoplastic cells (14, 15). In vitro with isogenic mutant of ras . Cancer Res 50: 67-71, 1990. 9 Moolten FL: Tumor chemosensitivity conferred by inserted cell growth was slowed down without cell death or herpes thymidine kinase genes: paradigm for prospective cancer apoptosis induced by HSV-tk/GCV, but in vivo we observed control stategy. Cancer Res 46: 5276-5281, 1986. a total destruction of these cells. Thus, we can postulate 10 Dilber MS, Abedi MR, Christensson B, Bjorkstrand B, Kidder that the "bystander" cell killing effect is mostly immune- G M, Naus C, Gahrton G and Smith E: Gap junctions promote mediated (16, 17). the bystander effect of herpes simplex virus thymidine kinase in In contrast to our in vitro results, a clear conclusion can be vivo. Cancer Res 57: 1523-1528, 1997. drawn that HSV-tk gene therapy was efficient in treating 11 Colombo BM, Benedetti S, Ottolenghi S, Mora M, Pollo B, Poli experimental breast cancer in athymic mice. These results G and Finocchiaro G: The "bystander effect": association of U-87 cell death with ganciclovir-mediated apoptosis of nearby cells and would suggest that the immune system might play an lack of effect in athymic mice. Human Gene Ther 6: 763-772, 1995. important role in tumor regression in vivo, explaining, in part, 12 Haberkorn U, Khazaie K, Morr I, Altmann A, Muller M and the discrepancy between the in vitro and in vivo results (18). Van Kaick G: Ganciclovir uptake in human mammary In conclusion, in this breast cancer model the immune carcinoma cells expressing herpes simplex virus thymidine system plays an important role for total tumor cell kinase. Nuclear Med Biol 25: 367-373, 1998.

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13 Vandier D, Rixe O, Besnard F, Kim M, Rikiyama T, Goldsmith 17 Kuriyama S, Mitoro A, Yamazaki M, Tsujinoue H, Nakatani T, M, Brenner M, Gouyette A and Cowan K H: Inhibition of Akahane T, Toyokawa Y, Kojima H, Okamoto S and Fukui H: glioma cells in vitro and in vivo using a recombinant adenoviral Comparison of gene therapy with the herpes simplex virus vector containing an astrocyte-specific promoter. Cancer Gene thymidine kinase gene and the bacterial cytosine deaminase Ther 7: 1120-1126, 2000. gene for the treatment of hepatocellular carcinoma. Scand J 14 Hall S, Sanford M-A, Atkinton G and Chen S: Induction of Gastroenterol 34: 1033-1041, 1999. potent antitumor natural killer cell activity by herpes simplex 18 Kuriyama S, Kikukawa M, Masui K, Okuda H, Nakatani T, virus-thymidine kinase and ganciclovir therapy in an Akahane T, Mitoro A, Tominaga K, Tsujinoue H, Yoshiji H, orthotopic mouse model of prostate cancer. Cancer Res 58: Okamoto S, Fukui H and Ikenaka K: Cancer gene therapy with 3221-3225, 1998. HSV-tk/GCV system depends on T-cell-mediated immune 15 Wei MW, Bougnoux P, Sacre-Salem B, Peyrat M-B, Lhuillery responses and causes apoptotic death of tumor cells in vivo. Int C, Salzmann J-L and Klatzmann D: Suicide gene therapy of J Cancer 83: 374-380, 1999. chemically induced mammary tumor in rat: efficacy and distant bystander effect. Cancer Res 58: 3529-3532, 1998. 16 Kruse CA, Lamb C, Hogan S, Smiley WR, Kleinschmidt- Demasters BK and Burrows FJ: Purified herpes simplex thymidine kinase retroviral particles. II. Influence of clinical parameters and bystander killing mechanisms. Cancer Gene Received June 21, 2004 Ther 7: 118-127, 2000. Accepted October 14, 2004

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