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Radiosensitization mediated by a transfected Anti-erbB-2 single-chain antibody in vitro and in vivo
Int. J. Radiation Oncology Biol. Phys., Vol. 42, No. 4, pp. 817– 822, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/98/$–see front matter
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Chemical Sensitizers and Protectors RADIOSENSITIZATION MEDIATED BY A TRANSFECTED ANTI-ERBB-2 SINGLE-CHAIN ANTIBODY IN VITRO AND IN VIVO MURRAY A. STACKHOUSE, DONALD J. BUCHSBAUM, WILLIAM E. GRIZZLE, SHEILA J. BRIGHT, CHRISTINE C. OLSEN, SREEKANTH KANCHARLA, MATTHEW S. MAYO, AND DAVID T. CURIEL Departments of Radiation Oncology, Pathology, Medicine and Gene Therapy Program, University of Alabama at Birmingham, Birmingham, AL Purpose: The erbB-2 receptor is overexpressed in several human cancers, including ovarian, prostate, and breast. We have developed plasmid and adenoviral vectors expressing an anti-erbB-2 single chain antibody (sFv), directed to the endoplasmic reticulum (ER) of target cells, that is cytotoxic to tumor cells overexpressing erbB-2 through induction of apoptosis. The anti-erbB-2 sFv also sensitizes erbB-2 overexpressing cells to the cytotoxic effects of cisplatin. On this basis, we hypothesized that human ovarian cancer cells expressing anti-erbB-2 sFv with downregulated erbB-2 product, p185erbB-2, also would be sensitized to ionizing radiation. Therefore, we designed experiments to test the ability of the anti-erbB-2 sFv to radiosensitize human ovarian cancer cells in vitro and in vivo. Methods and Materials: To test our hypothesis, we established subcutaneous (s.c.) tumors in the flanks of nude mice with SKOV3.ip1 human ovarian cancer cells and SKOV3 cells stably expressing the ER directed antierbB-2 sFv (SKOV3/pGT21). The tumors were treated with 10 Gy 60Co, or received no radiation. We then determined the regression rate, delay in regrowth, and time to tumor doubling of the tumors treated with radiation in the transfected group and controls. In addition, SKOV3.ip1 and SKOV3/pGT21 tumors were dissected from the irradiated animals and assayed for differences in p185erbB-2 expression at 12 weeks after irradiation by immunohistochemistry. Further, in vitro clonogenic survival assays were performed on the parental SKOV3.ip1 and SKOV3/pGT21 cell lines. Results: A statistical analysis of the combined data was done for two in vivo experiments. The analysis of the combined data showed that animals with irradiated tumor SKOV3/pGT21 had a significantly higher regression rate (p 5 0.0055), longer delay in regrowth (p 5 0.0001) and time to tumor doubling (p 5 0.0004), than those animals with tumor SKOV3.ip1 that received radiation. We observed a similar significant effect for the same parameters in the unirradiated tumor SKOV3/pGT21 compared to unirradiated tumor SKOV3.ip1. Immunohistochemical analysis of the SKOV3/pGT21 tumor cells demonstrated focal accumulation of p185erbB-2 in scattered clumps of cells and less p185erbB-2 membrane expression than cells of SKOV3.ip1 tumors. However, SKOV3.ip1 and SKOV3/pGT21 cells had similar in vitro sensitivity to radiation. Conclusions: These data support the hypothesis that tumors with reduced p185erbB-2 expression mediated by the anti-erbB-2 sFv are rendered more susceptible in vivo to the cytotoxic effects of ionizing radiation than tumors that maintain their normal expression of p185erbB-2. However, a similar effect was not observed with the same tumor cells in vitro. Thus, as has been described by others (1, 2), in vitro and in vivo results do not always correlate. Therefore, appropriate assays to assess clinical relevance need to be determined for each particular system studied. © 1998 Elsevier Science Inc. p185erbB-2, Gene therapy, Single-chain antibody, Radiation therapy, Radiosensitization.
INTRODUCTION
to be capable of malignant transformation of rodent fibroblasts in vitro (4). In addition, transgenic mice carrying either normal or mutant erbB-2 develop a variety of tumors, including neoplasms of mammary origin (5). Importantly, it has been shown that amplification and/or overexpression of the erbB-2 gene occurs in a variety of human epithelial carcinomas, including malignancies of the ovary, breast,
ErbB-2 is a 185 kDa transmembrane protein kinase receptor with extensive homology to the family of epidermal growth factor receptors (EGFR) (3). Several lines of evidence suggest that aberrant expression of the erbB-2 gene may play an important role in neoplastic transformation and progression. Specifically, ectopic expression of erbB-2 has been shown
Acknowledgments—We thank Robert Stockard for technical assistance and Sally Lagan for help with the preparation of this manuscript. This work was supported by American Cancer Society Grant RPG-98-059-01-CCE (MAS) and National Cancer Institute Grants RO1 CA68245 (DTC) and RO1 CA72532 (DTC). Accepted for publication 6 July 1998.
Presented at the 10th International Conference on Chemical Modifiers of Cancer Treatment, Clearwater, FL, Jan 28 –31, 1998. Reprint requests to: Murray A. Stackhouse, Ph.D., University of Alabama at Birmingham, Department of Radiation Oncology, 1824 6th Avenue South, WTI 624, Birmingham, AL 35233-6832. 817
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lung, prostate, and stomach (6 – 8). Further, a direct correlation has been noted between overexpression of erbB-2 and aggressive growth of specific tumors, as well as reduced overall survival of patients with adenocarcinomas of the breast and ovary (9, 10). Thus, these findings are consistent with the concept that erbB-2 overexpression may be a key event in malignant transformation and progression. A novel means to achieve erbB-2 down-regulation in cell lines overexpressing erbB-2 has been described (11, 12), in which intracellular expression of a single-chain antibody (sFv) that binds to erbB-2 targeted to the endoplasmic reticulum (ER) dramatically reduces the cell surface expression of p185erbB-2. Reduced p185erbB-2 expression mediated by the anti-erbB-2 sFv results in inhibition of cellular proliferation and cytotoxicity (11, 13, 14). Further studies documented that the intracellular expression of sFv anti-erbB-2 (ER) resulted in induction of apoptosis both in vitro and in vivo (15). These studies led to the isolation of clones of SKOV3 cells with partial reduction of erbB-2 expression via stable expression of sFv anti-erbB-2 (ER), designated SKOV3/pGT21, that were shown to have enhanced sensitivity to cisplatin (16, 17). This effect is presumably due to an altered balance in cellular repair of DNA damage vs. apoptosis. On this basis, we hypothesized that sFv-mediated downregulation of erbB-2 oncoprotein could lead to radiosensitization in an analogous manner to cisplatin. In this study, we demonstrate that erbB-2 downregulation leads to increased radiation response of xenografts of human ovarian adenocarcinomas stably expressing the antierbB-2 sFv. However, the same anti-erbB-2 sFv expressing cells of ovarian cancer were not radiosensitized in vitro. Thus, survival results based on in vitro studies may not always correlate with in vivo data, as has been reported with isogenic p211/1 and p212/2 HCT116 human colorectal cancer cells (1). METHODS AND MATERIALS Cell culture The human ovarian carcinoma cell line SKOV3.ip1, a SKOV3-derivative cell line, was kindly provided by Janet Price (University of Texas, M. D. Anderson Cancer Center, Houston, TX). This cell line was maintained in DME/F12 media supplemented with 10% FCS and L-glutamine (200 mg/ml) at 37°C in a 100% humidified 5% CO2 atmosphere. The SKOV3 cells (ATCC, Rockville, MD) were transfected, and cells stably expressing the anti-erbB-2 sFv were isolated and designated SKOV3/pGT21 (16). SKOV3/ pGT21 cells were maintained in DME/F12 media containing 800 mg/ml G418, an antibiotic, to maintain expression of the transfected anti-erbB-2 sFv (Life Technologies, Bethesda, MD). Cell survival assays in vitro Two hundred to 5,000 SKOV3.ip1 or SKOV3/pGT21 cells were plated 6 h prior to irradiation in T-25 flasks (Falcon, Franklin Lakes, NJ). The cells were then mock
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irradiated or irradiated using a 60Co therapy unit (Picker, Cleveland, OH) at a dose rate of 0.80 Gy/min and were returned to the incubator for colony formation. Fourteen days later, the colonies were fixed in ethanol and stained with 1% Crystal Violet. Colonies containing greater than 50 cells were counted. Survival was calculated as the average number of colonies counted divided by the number of cells plated times plating efficiency (PE); where PE was fraction of colonies counted divided by cells plated without radiation. The survival data was generated using the Fit v2.4 software (kindly provided by Dr. N. Albright, University of California at San Francisco). Animal studies For the first in vivo experiment, 3 groups of experimental animals were used: 1. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3.ip1 cells in the flank. 2. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3/ pGT21 cells in the flank. 3. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3/pGT21 cells in the flank. On Day 0, all animals had tumors of 4 – 8 mm size. Group 1 and 2 tumors received a single dose of radiation (10 Gy). In the second experiment, 4 groups of experimental animals were used: 1. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3.ip1 cells in the flank. 2. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3.ip1 cells in the flank. 3. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3/pGT21 cells in the flank. 4. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3/pGT21 cells in the flank. On Day 0, all animals had tumors of 4 – 8 mm size. Group 1 and 3 tumors received a single dose of radiation (10 Gy). The mice were shielded so as to allow irradiation of a single flank (6 mice at a time). Tumor growth was measured 3 times a week in 2 dimensions using a caliper, and the change in tumor size (product of the 2 dimensions) was calculated. The data from both experiments were pooled for analysis. Immunohistochemistry SKOV3.ip1 and SKOV3/pGT21 derived tumors growing in nude mice were removed 12 weeks after radiation and stained for expression of p185erbB-2. The method used has been described previously (8, 18). Briefly, the tumors were cut into multiple 2- to 3-mm fragments, fixed in neutral buffered formalin and processed to paraffin blocks. Paraffin sections (5 mm) were deparaffinized in xylene and were subsequently rehydrated before immunostaining. The slides were treated with 3.0% hydrogen peroxide for 5 min to quench endogenous peroxidase activity. In addition, the sections were incubated with preimmune goat serum (1.0%) for 1 h to decrease nonspecific staining. The sections were subsequently incubated with a monoclonal antibody (MAb) (clone 3B5, Oncogene Science, Uniondale, NY) to the erbB-2 gene product, p185erbB-2, for 1 h at room temperature. The remainder of the procedure was conducted using a commercial secondary detection system (Biogenex, San Ramon, CA), which contained a biotinylated link (anti-
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to assess the validity of pooling the data. There were no indications that pooling was inappropriate. RESULTS
Fig. 1. Clonogenic survival assay of SKOV3.ip1 and SKOV3/ pGT21 human ovarian cancer cells. Two hundred to 5,000 cells were plated 4 h prior to 60Co irradiation.
mouse immunoglobulin) antibody and an avidin horseradish peroxidase conjugate. Diaminobenzidine tetrahydrochloride was used as the substrate for visualization of antigen-antibody reactivity. The slides were lightly counterstained with hematoxylin. Statistical analysis Comparisons between groups on delay in regrowth and time to tumor size doubling were performed using the log rank test (19). Pairwise comparisons were made within a tumor type, between those animals with tumors that received radiation vs. those animals that did not, within animals who did not receive radiation between tumor types, and within animals who received radiation between tumor types. Tumor regression rates were compared between groups using Fisher’s exact test (20). Statistical comparisons were made on the combined data from both in vivo experiments. Comparisons were made between experiments
An in vitro assay of colony formation was used to determine the radiation sensitivity of human ovarian cancer cells expressing or not expressing anti-erbB-2 sFv. As shown in Fig. 1, there was no difference in survival between the parental SKOV3.ip1 cells and the anti-erbB-2 sFv expressing SKOV3/pGT21 cells following exposure to radiation varying between 2 and 8 Gy. Thus, no radiosensitization was observed by downregulation of erbB-2 in vitro. We also were interested in evaluating if the anti-erbB-2 sFv would produce radiosensitization in vivo. Xenografts of human ovarian cancer cells expressing or not expressing the anti-erbB-2 sFv were established. The data from the two experiments were combined and analyzed to assess the effect of radiation in human ovarian xenografts expressing an anti-erbB-2 sFv. Overall, there were four groups of animals in this analysis, 10 with tumor SKOV3/pGT21 that did not receive radiation, 10 with tumor SKOV3/pGT21 that did receive radiation, 5 with tumor SKOV3.ip1 that did not receive radiation, and 10 with tumor SKOV3.ip1 that received radiation. Animals with irradiated tumor SKOV3/ pGT21 did not have a significantly different regression rate ( p 5 0.582), but had a significantly longer delay in regrowth ( p 5 0.0088) and time to tumor doubling ( p 5 0.0042), than those animals that did not receive radiation (Table 1 and Fig. 2). Animals with irradiated tumor SKOV3.ip1 did not have a significantly different regression rate ( p 5 0.524), but had a significantly longer delay in regrowth ( p 5 0.0001) and time to tumor doubling ( p 5 0.0001), than those animals that did not receive radiation. Animals with unirradiated tumor SKOV3/pGT21 had a significantly higher regression rate ( p 5 0.026), longer delay in regrowth ( p 5 0.0001) and time to tumor doubling ( p 5 0.0001), than those animals with unirradiated tumor SKOV3.ip1. Animals with irradiated tumor SKOV3/ pGT21 had a significantly higher regression rate ( p 5 0.0055), longer delay in regrowth ( p 5 0.0001) and time to tumor doubling ( p 5 0.0004), than those animals with tumor SKOV3.ip1 that received radiation. Recurrent SKOV3.ip1 and SKOV3/pGT21 tumors from the second treatment experiment were tested for expression
Table 1. Comparisons between animals bearing SKOV3.ip1 and SKOV3/pGT21 subcutaneous tumors that were untreated or treated with 10-Gy single-dose 60Co radiation with respect to regression rate, growth retardation time, and time to tumor size doubling
SKOV3.ip1 SKOV3.ip1 SKOV3/pGT21 SKOV3/pGT21
Number of animals
Radiation dose (Gy)
Regression rate (%)
Growth retardation time (d)*
Time to tumor size doubling (d)*
5 10 10 10
0 10 0 10
0 20 70 90
7 (7–7) 43.5 (37–45) 53.5 (39–62) 106 (65–134)
25 (21–28) 55 (48–62) 60.5 (44–66) 111 (72–136)
* Median values with interquartile range in parentheses.
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Fig. 2. Effect of single-fraction 60Co external beam irradiation on the growth of established human ovarian tumors. The data are the median tumor size (bidimensional product) from the combination of two experiments, with 5 to 10 animals per group.
of p185erbB-2 by immunohistochemical techniques. In Fig. 3A–F, A to C are representative viable areas of the SKOV3.ip1-derived xenograft tumors at 12 weeks after radiation. There are extensive areas of continuing necrosis
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in these tumors (area “n” of A), probably secondary to local vascular insufficiency. Most of the cells in the viable areas of the tumor demonstrate strong membrane expression of p185erbB-2. In contrast, Fig. 3D to F demonstrates representative areas of viable xenograft tumors derived from SKOV3/pGT21. These tumors were transfected stably with the pGT21 anti-erbB-2 sFv (ER) plasmid prior to implantation into the nude mice. Tumors were then removed 12 weeks after radiation and, similar to the nontransfected SKOV3.ip1-derived tumors, these tumors continued to have large areas of necrosis. However, there is a marked difference in the cellular expression of p185erbB-2 in these transfected tumors, as well as differences in their overall histopathology. As can be noted from the curved arrows in Fig. 3E and F, there is focal accumulation of p185erbB-2 in scattered clumps of tumor cells; such patterns are not noted in nontransfected SKOV3.ip1 tumors. Also, the membrane pattern of staining for p185erbB-2 is less in the majority of cells. Note that the cells between the small straight arrows demonstrate a marked reduction in the membrane expression of p185erbB-2 compared to SKOV3.ip1 cells. Similarly, many cells show large cleared vacuoles (thick straight arrows) and many other cells have “cleared” cytoplasm with respect to erbB-2 immunostaining. Although scattered similar-appearing cells with vacuoles can be identified in the nontransfected SKOV3.ip1 tumors, such cells are much more numerous in the tumors derived from SKOV3/pGT21 that were transfected. DISCUSSION
Fig. 3. Immunohistochemistry of SKOV3.ip1 and SKOV3/pGT21irradiated tumors. A to C are representative viable areas of SKOV3.ip1-derived tumors growing in nude mice (X400) at 12 weeks after radiation. In contrast, D to F demonstrate representative areas of viable SKOV3/pGT21-derived tumors (X400) growing in nude mice and removed 12 weeks after irradiation. These tumor cells were stably transfected with the pGT21 anti-erbB-2 sFv (ER) plasmid prior to implantation into the nude mice. The tumors were removed 12 weeks after radiation.
We did not observe in vitro radiosensitization by downregulation of erbB-2 expression following transfection with anti-erbB-2 sFv (ER) by a standard colony formation assay. In contrast, human ovarian cancer cell xenografts were radiosensitized significantly by the expression of the antierbB-2 sFv. Unirradiated SKOV3/pGT21 xenografts regressed initially, followed by a period of growth delay and then regrowth. Immunohistochemical staining for p185erbB-2 confirmed that the expression level and the pattern of p185erbB-2 expression were altered in nodules of tumors stably transfected with anti-erbB-2 sFv (ER) and removed 12 weeks following radiation. Therefore, the alteration of erbB-2 expression in the SKOV3/pGT21 tumors may partially explain the observed enhanced radiation sensitivity in vivo. Another potential factor in the observed enhanced radiation sensitivity in the anti-erbB-2 sFv-expressing SKOV3/pGT21 tumors is apoptosis. The role of apoptosis was not determined in this study, but it has been shown previously that the expression of the anti-erbB-2 sFv leads to induction of apoptosis in the SKOV3 cell line in vitro and in vivo (15). Wouters et al. showed that, in isogenic p211/1 and p212/2 HCT116 human colon cancer cells, increased apoptosis in the p212/2 cells did not lead to increased radiosensitivity in vitro (2). It was also shown that apoptosis was not the basis for the observed radiosensitization observed in vivo (2). Although apoptosis is increased by
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the expression of the anti-erbB-2 sFv in human ovarian adenocarcinoma cells (15), it did not increase the sensitivity of the SKOV3/pGT21 cells in vitro. Apoptosis may also have played a role in the observed initial regression and growth delay of the unirradiated SKOV3/pGT21 xenografts. The role of apoptosis in the observed enhanced radiation effect in vivo is unknown at this time. The experiments described with a stably transfected cell line represent the ideal situation to test for radiosensitization mediated by the anti-erbB-2 sFv. The use of viral or other gene delivery vectors may not be as effective in actual gene therapy applications as obtained in these experiments. The development of selective and efficient gene delivery vectors remains a challenge for effective gene therapy. The association of overexpression of the erbB-2 gene product with neoplastic transformation has led to the development of methods to reduce erbB-2 available receptor levels in target tumor cells. MAbs have been developed that exhibit high-affinity binding to the extracellular domains of the erbB-2 protein (21, 22). A number of studies have demonstrated that a subset of these MAbs can elicit growth inhibition of overexpressing erbB-2 tumor cells both in vitro and in vivo (23, 24). In addition, these antibodies were shown to enhance chemosensitivity of tumor cells (25, 26). No correlation of increased sensitivity to ionizing radiation has been reported for erbB-2 downregulation. Nevertheless, there is a high degree of homology between erbB-2 and the epidermal growth factor receptor (EGFR), and MAb to EGFR has abrogated the radiation resistance observed when EGF binds to its receptor (27). Furthermore, MAb to EGFR
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also enhanced the cytotoxicity of radiation therapy in A431 (EGFR1) tumors in vivo (28). Therefore, it is not surprising that erbB-2 downregulation may lead to radiosensitization. We are not the first group to show a difference between in vitro and in vivo radiation sensitivity. Waldman and colleagues demonstrated a discrepancy between the in vitro and in vivo sensitivity to radiation of isogenic p211/1 and p212/2 HCT116 human colon cancer cells (1). This finding was confirmed by Wouters et al. (2). Two important contributions of these articles relate to 1. the importance of cell cycle control and the sensitivity of cancer cells to cytotoxic agents, and 2. the validity of the in vitro clonogenic cell survival assay for assessing the potential of therapeutic agents. Lamb and Friend (29) addressed the ramifications of the lack of correlation between in vitro and in vivo survival responses, and suggested that caution should be observed before rejecting clonogenic cell survival assays as a method to predict clinical treatment responses (29). As suggested by Lamb and Friend “no one test may predict clinical outcome in all situations.” We concur with this assessment and suggest that lack of correlation between our in vitro and in vivo results does not mean that the results obtained are invalid. Cell cycle control, apoptosis, and growth regulation are a common denominator in these studies, and the importance of selecting appropriate assays to demonstrate the efficacy of the experimental treatment is crucial. The potential clinical efficacy of experimental cancer treatments need to be viewed in the light of these studies where in vitro and in vivo results do not correlate.
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Chemical Sensitizers and Protectors RADIOSENSITIZATION MEDIATED BY A TRANSFECTED ANTI-ERBB-2 SINGLE-CHAIN ANTIBODY IN VITRO AND IN VIVO MURRAY A. STACKHOUSE, DONALD J. BUCHSBAUM, WILLIAM E. GRIZZLE, SHEILA J. BRIGHT, CHRISTINE C. OLSEN, SREEKANTH KANCHARLA, MATTHEW S. MAYO, AND DAVID T. CURIEL Departments of Radiation Oncology, Pathology, Medicine and Gene Therapy Program, University of Alabama at Birmingham, Birmingham, AL Purpose: The erbB-2 receptor is overexpressed in several human cancers, including ovarian, prostate, and breast. We have developed plasmid and adenoviral vectors expressing an anti-erbB-2 single chain antibody (sFv), directed to the endoplasmic reticulum (ER) of target cells, that is cytotoxic to tumor cells overexpressing erbB-2 through induction of apoptosis. The anti-erbB-2 sFv also sensitizes erbB-2 overexpressing cells to the cytotoxic effects of cisplatin. On this basis, we hypothesized that human ovarian cancer cells expressing anti-erbB-2 sFv with downregulated erbB-2 product, p185erbB-2, also would be sensitized to ionizing radiation. Therefore, we designed experiments to test the ability of the anti-erbB-2 sFv to radiosensitize human ovarian cancer cells in vitro and in vivo. Methods and Materials: To test our hypothesis, we established subcutaneous (s.c.) tumors in the flanks of nude mice with SKOV3.ip1 human ovarian cancer cells and SKOV3 cells stably expressing the ER directed antierbB-2 sFv (SKOV3/pGT21). The tumors were treated with 10 Gy 60Co, or received no radiation. We then determined the regression rate, delay in regrowth, and time to tumor doubling of the tumors treated with radiation in the transfected group and controls. In addition, SKOV3.ip1 and SKOV3/pGT21 tumors were dissected from the irradiated animals and assayed for differences in p185erbB-2 expression at 12 weeks after irradiation by immunohistochemistry. Further, in vitro clonogenic survival assays were performed on the parental SKOV3.ip1 and SKOV3/pGT21 cell lines. Results: A statistical analysis of the combined data was done for two in vivo experiments. The analysis of the combined data showed that animals with irradiated tumor SKOV3/pGT21 had a significantly higher regression rate (p 5 0.0055), longer delay in regrowth (p 5 0.0001) and time to tumor doubling (p 5 0.0004), than those animals with tumor SKOV3.ip1 that received radiation. We observed a similar significant effect for the same parameters in the unirradiated tumor SKOV3/pGT21 compared to unirradiated tumor SKOV3.ip1. Immunohistochemical analysis of the SKOV3/pGT21 tumor cells demonstrated focal accumulation of p185erbB-2 in scattered clumps of cells and less p185erbB-2 membrane expression than cells of SKOV3.ip1 tumors. However, SKOV3.ip1 and SKOV3/pGT21 cells had similar in vitro sensitivity to radiation. Conclusions: These data support the hypothesis that tumors with reduced p185erbB-2 expression mediated by the anti-erbB-2 sFv are rendered more susceptible in vivo to the cytotoxic effects of ionizing radiation than tumors that maintain their normal expression of p185erbB-2. However, a similar effect was not observed with the same tumor cells in vitro. Thus, as has been described by others (1, 2), in vitro and in vivo results do not always correlate. Therefore, appropriate assays to assess clinical relevance need to be determined for each particular system studied. © 1998 Elsevier Science Inc. p185erbB-2, Gene therapy, Single-chain antibody, Radiation therapy, Radiosensitization.
INTRODUCTION
to be capable of malignant transformation of rodent fibroblasts in vitro (4). In addition, transgenic mice carrying either normal or mutant erbB-2 develop a variety of tumors, including neoplasms of mammary origin (5). Importantly, it has been shown that amplification and/or overexpression of the erbB-2 gene occurs in a variety of human epithelial carcinomas, including malignancies of the ovary, breast,
ErbB-2 is a 185 kDa transmembrane protein kinase receptor with extensive homology to the family of epidermal growth factor receptors (EGFR) (3). Several lines of evidence suggest that aberrant expression of the erbB-2 gene may play an important role in neoplastic transformation and progression. Specifically, ectopic expression of erbB-2 has been shown
Acknowledgments—We thank Robert Stockard for technical assistance and Sally Lagan for help with the preparation of this manuscript. This work was supported by American Cancer Society Grant RPG-98-059-01-CCE (MAS) and National Cancer Institute Grants RO1 CA68245 (DTC) and RO1 CA72532 (DTC). Accepted for publication 6 July 1998.
Presented at the 10th International Conference on Chemical Modifiers of Cancer Treatment, Clearwater, FL, Jan 28 –31, 1998. Reprint requests to: Murray A. Stackhouse, Ph.D., University of Alabama at Birmingham, Department of Radiation Oncology, 1824 6th Avenue South, WTI 624, Birmingham, AL 35233-6832. 817
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lung, prostate, and stomach (6 – 8). Further, a direct correlation has been noted between overexpression of erbB-2 and aggressive growth of specific tumors, as well as reduced overall survival of patients with adenocarcinomas of the breast and ovary (9, 10). Thus, these findings are consistent with the concept that erbB-2 overexpression may be a key event in malignant transformation and progression. A novel means to achieve erbB-2 down-regulation in cell lines overexpressing erbB-2 has been described (11, 12), in which intracellular expression of a single-chain antibody (sFv) that binds to erbB-2 targeted to the endoplasmic reticulum (ER) dramatically reduces the cell surface expression of p185erbB-2. Reduced p185erbB-2 expression mediated by the anti-erbB-2 sFv results in inhibition of cellular proliferation and cytotoxicity (11, 13, 14). Further studies documented that the intracellular expression of sFv anti-erbB-2 (ER) resulted in induction of apoptosis both in vitro and in vivo (15). These studies led to the isolation of clones of SKOV3 cells with partial reduction of erbB-2 expression via stable expression of sFv anti-erbB-2 (ER), designated SKOV3/pGT21, that were shown to have enhanced sensitivity to cisplatin (16, 17). This effect is presumably due to an altered balance in cellular repair of DNA damage vs. apoptosis. On this basis, we hypothesized that sFv-mediated downregulation of erbB-2 oncoprotein could lead to radiosensitization in an analogous manner to cisplatin. In this study, we demonstrate that erbB-2 downregulation leads to increased radiation response of xenografts of human ovarian adenocarcinomas stably expressing the antierbB-2 sFv. However, the same anti-erbB-2 sFv expressing cells of ovarian cancer were not radiosensitized in vitro. Thus, survival results based on in vitro studies may not always correlate with in vivo data, as has been reported with isogenic p211/1 and p212/2 HCT116 human colorectal cancer cells (1). METHODS AND MATERIALS Cell culture The human ovarian carcinoma cell line SKOV3.ip1, a SKOV3-derivative cell line, was kindly provided by Janet Price (University of Texas, M. D. Anderson Cancer Center, Houston, TX). This cell line was maintained in DME/F12 media supplemented with 10% FCS and L-glutamine (200 mg/ml) at 37°C in a 100% humidified 5% CO2 atmosphere. The SKOV3 cells (ATCC, Rockville, MD) were transfected, and cells stably expressing the anti-erbB-2 sFv were isolated and designated SKOV3/pGT21 (16). SKOV3/ pGT21 cells were maintained in DME/F12 media containing 800 mg/ml G418, an antibiotic, to maintain expression of the transfected anti-erbB-2 sFv (Life Technologies, Bethesda, MD). Cell survival assays in vitro Two hundred to 5,000 SKOV3.ip1 or SKOV3/pGT21 cells were plated 6 h prior to irradiation in T-25 flasks (Falcon, Franklin Lakes, NJ). The cells were then mock
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irradiated or irradiated using a 60Co therapy unit (Picker, Cleveland, OH) at a dose rate of 0.80 Gy/min and were returned to the incubator for colony formation. Fourteen days later, the colonies were fixed in ethanol and stained with 1% Crystal Violet. Colonies containing greater than 50 cells were counted. Survival was calculated as the average number of colonies counted divided by the number of cells plated times plating efficiency (PE); where PE was fraction of colonies counted divided by cells plated without radiation. The survival data was generated using the Fit v2.4 software (kindly provided by Dr. N. Albright, University of California at San Francisco). Animal studies For the first in vivo experiment, 3 groups of experimental animals were used: 1. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3.ip1 cells in the flank. 2. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3/ pGT21 cells in the flank. 3. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3/pGT21 cells in the flank. On Day 0, all animals had tumors of 4 – 8 mm size. Group 1 and 2 tumors received a single dose of radiation (10 Gy). In the second experiment, 4 groups of experimental animals were used: 1. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3.ip1 cells in the flank. 2. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3.ip1 cells in the flank. 3. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3/pGT21 cells in the flank. 4. Day minus 6, 5 athymic nude mice received 1 3 107 SKOV3/pGT21 cells in the flank. On Day 0, all animals had tumors of 4 – 8 mm size. Group 1 and 3 tumors received a single dose of radiation (10 Gy). The mice were shielded so as to allow irradiation of a single flank (6 mice at a time). Tumor growth was measured 3 times a week in 2 dimensions using a caliper, and the change in tumor size (product of the 2 dimensions) was calculated. The data from both experiments were pooled for analysis. Immunohistochemistry SKOV3.ip1 and SKOV3/pGT21 derived tumors growing in nude mice were removed 12 weeks after radiation and stained for expression of p185erbB-2. The method used has been described previously (8, 18). Briefly, the tumors were cut into multiple 2- to 3-mm fragments, fixed in neutral buffered formalin and processed to paraffin blocks. Paraffin sections (5 mm) were deparaffinized in xylene and were subsequently rehydrated before immunostaining. The slides were treated with 3.0% hydrogen peroxide for 5 min to quench endogenous peroxidase activity. In addition, the sections were incubated with preimmune goat serum (1.0%) for 1 h to decrease nonspecific staining. The sections were subsequently incubated with a monoclonal antibody (MAb) (clone 3B5, Oncogene Science, Uniondale, NY) to the erbB-2 gene product, p185erbB-2, for 1 h at room temperature. The remainder of the procedure was conducted using a commercial secondary detection system (Biogenex, San Ramon, CA), which contained a biotinylated link (anti-
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to assess the validity of pooling the data. There were no indications that pooling was inappropriate. RESULTS
Fig. 1. Clonogenic survival assay of SKOV3.ip1 and SKOV3/ pGT21 human ovarian cancer cells. Two hundred to 5,000 cells were plated 4 h prior to 60Co irradiation.
mouse immunoglobulin) antibody and an avidin horseradish peroxidase conjugate. Diaminobenzidine tetrahydrochloride was used as the substrate for visualization of antigen-antibody reactivity. The slides were lightly counterstained with hematoxylin. Statistical analysis Comparisons between groups on delay in regrowth and time to tumor size doubling were performed using the log rank test (19). Pairwise comparisons were made within a tumor type, between those animals with tumors that received radiation vs. those animals that did not, within animals who did not receive radiation between tumor types, and within animals who received radiation between tumor types. Tumor regression rates were compared between groups using Fisher’s exact test (20). Statistical comparisons were made on the combined data from both in vivo experiments. Comparisons were made between experiments
An in vitro assay of colony formation was used to determine the radiation sensitivity of human ovarian cancer cells expressing or not expressing anti-erbB-2 sFv. As shown in Fig. 1, there was no difference in survival between the parental SKOV3.ip1 cells and the anti-erbB-2 sFv expressing SKOV3/pGT21 cells following exposure to radiation varying between 2 and 8 Gy. Thus, no radiosensitization was observed by downregulation of erbB-2 in vitro. We also were interested in evaluating if the anti-erbB-2 sFv would produce radiosensitization in vivo. Xenografts of human ovarian cancer cells expressing or not expressing the anti-erbB-2 sFv were established. The data from the two experiments were combined and analyzed to assess the effect of radiation in human ovarian xenografts expressing an anti-erbB-2 sFv. Overall, there were four groups of animals in this analysis, 10 with tumor SKOV3/pGT21 that did not receive radiation, 10 with tumor SKOV3/pGT21 that did receive radiation, 5 with tumor SKOV3.ip1 that did not receive radiation, and 10 with tumor SKOV3.ip1 that received radiation. Animals with irradiated tumor SKOV3/ pGT21 did not have a significantly different regression rate ( p 5 0.582), but had a significantly longer delay in regrowth ( p 5 0.0088) and time to tumor doubling ( p 5 0.0042), than those animals that did not receive radiation (Table 1 and Fig. 2). Animals with irradiated tumor SKOV3.ip1 did not have a significantly different regression rate ( p 5 0.524), but had a significantly longer delay in regrowth ( p 5 0.0001) and time to tumor doubling ( p 5 0.0001), than those animals that did not receive radiation. Animals with unirradiated tumor SKOV3/pGT21 had a significantly higher regression rate ( p 5 0.026), longer delay in regrowth ( p 5 0.0001) and time to tumor doubling ( p 5 0.0001), than those animals with unirradiated tumor SKOV3.ip1. Animals with irradiated tumor SKOV3/ pGT21 had a significantly higher regression rate ( p 5 0.0055), longer delay in regrowth ( p 5 0.0001) and time to tumor doubling ( p 5 0.0004), than those animals with tumor SKOV3.ip1 that received radiation. Recurrent SKOV3.ip1 and SKOV3/pGT21 tumors from the second treatment experiment were tested for expression
Table 1. Comparisons between animals bearing SKOV3.ip1 and SKOV3/pGT21 subcutaneous tumors that were untreated or treated with 10-Gy single-dose 60Co radiation with respect to regression rate, growth retardation time, and time to tumor size doubling
SKOV3.ip1 SKOV3.ip1 SKOV3/pGT21 SKOV3/pGT21
Number of animals
Radiation dose (Gy)
Regression rate (%)
Growth retardation time (d)*
Time to tumor size doubling (d)*
5 10 10 10
0 10 0 10
0 20 70 90
7 (7–7) 43.5 (37–45) 53.5 (39–62) 106 (65–134)
25 (21–28) 55 (48–62) 60.5 (44–66) 111 (72–136)
* Median values with interquartile range in parentheses.
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Fig. 2. Effect of single-fraction 60Co external beam irradiation on the growth of established human ovarian tumors. The data are the median tumor size (bidimensional product) from the combination of two experiments, with 5 to 10 animals per group.
of p185erbB-2 by immunohistochemical techniques. In Fig. 3A–F, A to C are representative viable areas of the SKOV3.ip1-derived xenograft tumors at 12 weeks after radiation. There are extensive areas of continuing necrosis
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in these tumors (area “n” of A), probably secondary to local vascular insufficiency. Most of the cells in the viable areas of the tumor demonstrate strong membrane expression of p185erbB-2. In contrast, Fig. 3D to F demonstrates representative areas of viable xenograft tumors derived from SKOV3/pGT21. These tumors were transfected stably with the pGT21 anti-erbB-2 sFv (ER) plasmid prior to implantation into the nude mice. Tumors were then removed 12 weeks after radiation and, similar to the nontransfected SKOV3.ip1-derived tumors, these tumors continued to have large areas of necrosis. However, there is a marked difference in the cellular expression of p185erbB-2 in these transfected tumors, as well as differences in their overall histopathology. As can be noted from the curved arrows in Fig. 3E and F, there is focal accumulation of p185erbB-2 in scattered clumps of tumor cells; such patterns are not noted in nontransfected SKOV3.ip1 tumors. Also, the membrane pattern of staining for p185erbB-2 is less in the majority of cells. Note that the cells between the small straight arrows demonstrate a marked reduction in the membrane expression of p185erbB-2 compared to SKOV3.ip1 cells. Similarly, many cells show large cleared vacuoles (thick straight arrows) and many other cells have “cleared” cytoplasm with respect to erbB-2 immunostaining. Although scattered similar-appearing cells with vacuoles can be identified in the nontransfected SKOV3.ip1 tumors, such cells are much more numerous in the tumors derived from SKOV3/pGT21 that were transfected. DISCUSSION
Fig. 3. Immunohistochemistry of SKOV3.ip1 and SKOV3/pGT21irradiated tumors. A to C are representative viable areas of SKOV3.ip1-derived tumors growing in nude mice (X400) at 12 weeks after radiation. In contrast, D to F demonstrate representative areas of viable SKOV3/pGT21-derived tumors (X400) growing in nude mice and removed 12 weeks after irradiation. These tumor cells were stably transfected with the pGT21 anti-erbB-2 sFv (ER) plasmid prior to implantation into the nude mice. The tumors were removed 12 weeks after radiation.
We did not observe in vitro radiosensitization by downregulation of erbB-2 expression following transfection with anti-erbB-2 sFv (ER) by a standard colony formation assay. In contrast, human ovarian cancer cell xenografts were radiosensitized significantly by the expression of the antierbB-2 sFv. Unirradiated SKOV3/pGT21 xenografts regressed initially, followed by a period of growth delay and then regrowth. Immunohistochemical staining for p185erbB-2 confirmed that the expression level and the pattern of p185erbB-2 expression were altered in nodules of tumors stably transfected with anti-erbB-2 sFv (ER) and removed 12 weeks following radiation. Therefore, the alteration of erbB-2 expression in the SKOV3/pGT21 tumors may partially explain the observed enhanced radiation sensitivity in vivo. Another potential factor in the observed enhanced radiation sensitivity in the anti-erbB-2 sFv-expressing SKOV3/pGT21 tumors is apoptosis. The role of apoptosis was not determined in this study, but it has been shown previously that the expression of the anti-erbB-2 sFv leads to induction of apoptosis in the SKOV3 cell line in vitro and in vivo (15). Wouters et al. showed that, in isogenic p211/1 and p212/2 HCT116 human colon cancer cells, increased apoptosis in the p212/2 cells did not lead to increased radiosensitivity in vitro (2). It was also shown that apoptosis was not the basis for the observed radiosensitization observed in vivo (2). Although apoptosis is increased by
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the expression of the anti-erbB-2 sFv in human ovarian adenocarcinoma cells (15), it did not increase the sensitivity of the SKOV3/pGT21 cells in vitro. Apoptosis may also have played a role in the observed initial regression and growth delay of the unirradiated SKOV3/pGT21 xenografts. The role of apoptosis in the observed enhanced radiation effect in vivo is unknown at this time. The experiments described with a stably transfected cell line represent the ideal situation to test for radiosensitization mediated by the anti-erbB-2 sFv. The use of viral or other gene delivery vectors may not be as effective in actual gene therapy applications as obtained in these experiments. The development of selective and efficient gene delivery vectors remains a challenge for effective gene therapy. The association of overexpression of the erbB-2 gene product with neoplastic transformation has led to the development of methods to reduce erbB-2 available receptor levels in target tumor cells. MAbs have been developed that exhibit high-affinity binding to the extracellular domains of the erbB-2 protein (21, 22). A number of studies have demonstrated that a subset of these MAbs can elicit growth inhibition of overexpressing erbB-2 tumor cells both in vitro and in vivo (23, 24). In addition, these antibodies were shown to enhance chemosensitivity of tumor cells (25, 26). No correlation of increased sensitivity to ionizing radiation has been reported for erbB-2 downregulation. Nevertheless, there is a high degree of homology between erbB-2 and the epidermal growth factor receptor (EGFR), and MAb to EGFR has abrogated the radiation resistance observed when EGF binds to its receptor (27). Furthermore, MAb to EGFR
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also enhanced the cytotoxicity of radiation therapy in A431 (EGFR1) tumors in vivo (28). Therefore, it is not surprising that erbB-2 downregulation may lead to radiosensitization. We are not the first group to show a difference between in vitro and in vivo radiation sensitivity. Waldman and colleagues demonstrated a discrepancy between the in vitro and in vivo sensitivity to radiation of isogenic p211/1 and p212/2 HCT116 human colon cancer cells (1). This finding was confirmed by Wouters et al. (2). Two important contributions of these articles relate to 1. the importance of cell cycle control and the sensitivity of cancer cells to cytotoxic agents, and 2. the validity of the in vitro clonogenic cell survival assay for assessing the potential of therapeutic agents. Lamb and Friend (29) addressed the ramifications of the lack of correlation between in vitro and in vivo survival responses, and suggested that caution should be observed before rejecting clonogenic cell survival assays as a method to predict clinical treatment responses (29). As suggested by Lamb and Friend “no one test may predict clinical outcome in all situations.” We concur with this assessment and suggest that lack of correlation between our in vitro and in vivo results does not mean that the results obtained are invalid. Cell cycle control, apoptosis, and growth regulation are a common denominator in these studies, and the importance of selecting appropriate assays to demonstrate the efficacy of the experimental treatment is crucial. The potential clinical efficacy of experimental cancer treatments need to be viewed in the light of these studies where in vitro and in vivo results do not correlate.
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