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doi:10.1006/mthe.2000.0240, available online at http://www.idealibrary.com on IDEAL
Development of a Syngenic Murine B16 Cell Line-Derived Melanoma Susceptible to Destruction by Neuroattenuated HSV-1 Cathie G. Miller,* Claude Krummenacher,†,‡ Rosalyn J. Eisenberg,†,§ Gary H. Cohen,†,‡ and Nigel W. Fraser*,1 *Department of Microbiology, School of Medicine, †Department of Microbiology and ‡Center for Oral Health Research, School of Dental Medicine, and §Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received for publication August 24, 2000; accepted in revised form December 4, 2000
HSV-1 ICP34.5 mutants can slow progression of preformed tumors in rodent models. However, the current models available for study are limited due to the lack of a syngenic, low-immunogenic tumor model susceptible to HSV-1. Thus we have developed a new model to determine the role of the immune response in viral-mediated tumor destruction. The human herpesvirus entry (Hve) receptors (HveA, HveB, and HveC) and a control plasmid were transfected into B78H1 murine melanoma cells. Transfection of HveA and HveC conferred sensitivity to HSV-1 to these cells. A10 (HveA), C10 (HveC), and control cells were able to form tumors reproducibly in vivo. The transfection of the receptors into B78H1 cells did not induce a detectable in vivo immunogenicity to the tumors. Finally, A10 and C10 tumor-bearing mice treated with HSV-1 1716 had significant prolongation of survival compared to mock-treated mice. These data suggest that A10 and C10 will be useful as in vivo models for studying the role of the immune response in viral-mediated tumor destruction. Key Words: HSV-1; tumor model; melanoma; gene therapy; Hve receptor.
INTRODUCTION Malignant brain tumors occur frequently and although many different treatment procedures are currently being studied, existing therapies are mainly palliative (1). One of the major types of malignant brain tumors is melanoma (2). The natural affinity of HSV-1 for neuronal tissue, along with the availability of nonvirulent mutants of HSV-1, led to the proposal to use neuroattenuated HSV-1 to specifically kill tumor cells in the brain (3). While many groups have shown that attenuated HSV-1 is able to kill various human tumor cells in vitro and in vivo (4 –7), we have recently shown that the immune response is important in prolonging the survival of mice bearing intracranial syngenic melanomas (8). However, our ability to understand the mechanism of tumor cell death and the potential contribution of the immune response has been difficult to determine due to the lack of a syngenic, low-immunogenic animal model that is susceptible to HSV-1 killing. In this paper, we describe a new model
1 To whom correspondence and reprint requests should be addressed at 3610 Hamilton Walk, 319 Johnson Pavilion, Philadelphia, PA 19104. Fax: (215) 898-3849. E-mail:
[email protected].
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based on the B16 murine melanoma cell line transfected with the human herpesvirus entry mediators (Hve) (9 – 12). This new model should facilitate the elucidation of the mechanism of tumor cell death and the contribution of the immune response to this cell death. The use of replication-competent HSV vectors for tumor therapy is gaining acceptance as a viable approach. Work from our lab and others has shown that a neuroattenuated HSV-1, HSV-1 1716, is able to prolong survival in syngenic animals bearing intracranial tumors and can even cure some animals (4, 5). Furthermore, despite the importance of the immune response in syngenic tumors, HSV-1 1716 is able to destroy xenogenic human tumors transplanted into immunodeficient mice (13, 14). Further studies with other replication-competent HSV strains have shown equal ability to destroy tumors in animal models, depending on tumor location and mode of therapy (15, 16). Clinical trials are currently under way to determine the safety of this therapeutic modality (17–20). Although HSV-1 was thought to infect many different cell types derived from many animals, including 26 human melanoma cell lines in vitro (4), early work in murine tumor models demonstrated that many of the accepted murine melanoma cell lines, such as B16, were resistant to MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy 1525-0016/01 $35.00
ARTICLE HSV-1 infection and killing (4). Two murine melanoma lines which are susceptible to HSV-1 killing have been used; unfortunately these lines either are highly immunogenic (S91 melanoma) or do not have a syngenic host available (Harding-Passey melanoma) (4). These drawbacks have significantly inhibited the ability to determine how the immune response contributes to the tumor destruction and the increased survival. Therefore, new cell lines, which are susceptible to HSV-1 killing and have a low immunogenicity, have been created, based on the accepted melanoma cell line B16 (21–24). These cells were created by transfection of the human HveA, HveB, and HveC entry molecules into an amelanotic B16-derived cell line, B78H1 (25). The recent identification of herpesvirus entry genes has led to a better understanding of the mechanism of HSV entry. First, binding of HSV to cells occurs through interactions of herpes simplex virus glycoprotein C (gC) and/or gB with cellular heparin sulfate. Fusion of the virion envelope with the cell surface requires interactions between gB, gD, gH, and gL (26). It is now known that coreceptors, termed herpesvirus entry mediators, are required to bind to gD to allow viral entry (9 –11, 27). Currently, four human members of the Hve family have been discovered, and these have been termed HveA, HveB, HveC, and HveD. HveA is a member of the tumor necrosis factor receptor family (9). It is expressed in many tissues of the body, including liver, lung, kidney, spleen, and leukocytes (9, 28 –30). HveA (HVEM/TNFSM10/ ATAR/TR2) allows entry into human lymphoid cells (9). HveB, the poliovirus receptor-related protein 2 (Prr2/Nectin-2) (10, 31, 32), is a receptor for entry of pseudorabies virus and certain strains of HSV-2 and allows entry of certain gD mutants of HSV-1 (10). HveC, the poliovirus receptor-related protein (Prr1/HigR/Nectin-1), is a member of the immunoglobulin superfamily (11, 27, 33, 34). HveC is expressed in epithelial cells and cells of neuronal origin and is a receptor for HSV-1 and HSV-2 entry into epithelial cells (11, 27). It is also proposed to be responsible for virus spread to cells of the nervous system (11, 27). HveD, the poliovirus receptor (CD255/PRV), does not allow for the entry of any HSV-1 or HSV-2 viruses tested (10) and has not been used in this study. We show here that the HveA- and HveC-transfected tumor cell lines (A10 and C10, respectively) reproducibly produce tumors in syngenic C57Bl/6 mice. Furthermore, we demonstrate that these cells are sensitive to HSV-1 infection and that HSV-1 1716 prolongs survival when intracranial tumors are produced. In contrast, the HveBtransfected tumor cell line (B5) did not reproducibly produce tumors in syngenic C57Bl/6 mice and HSV-1 1716 is unable to prolong survival of mice bearing control tumor cells. Furthermore, these tumor cell lines retain a low immunogenicity, as secondary tumors can grow successfully in mice bearing A10, C10, and control tumors. Additionally, no detectable levels of specific antibodies for the Hve receptors developed in any of the mice. We believe that both the A10 and the C10 tumor lines will be MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
useful tools to further delineate the role of the immune response in viral-mediated tumor destruction.
METHODS Animals and viruses. Female C57Bl/6 (4 – 6 weeks of age, weighing approximately 20 g) mice were obtained from Taconic (Germantown, NY). To produce virus stocks, subconfluent monolayers of African green monkey kidney (Vero) cells were infected with HSV strain 1716 (stock titer 1 ⫻ 108 pfu/ml). HSV strain 1716 has a 759-bp deletion, which deletes part of the genes encoding ICP34.5, LAT, and orfP (35). Virus was concentrated from the culture and titered by plaque assay as previously described (36). All viral stocks were stored frozen in viral culture medium (DMEM containing penicillin and streptomycin) at ⫺70°C and thawed rapidly just prior to use. Serum-free medium was used for control (mock) inoculation studies as negative controls. Tumor cell lines. B78H1 cells (25) were obtained from Meenhard Herlyn (Wistar Institute, Philadelphia, PA). Generation and characterization of Hve-transfected cells are described elsewhere (Pat Spear, unpublished data). Briefly, B78H1 cells were transfected with the HveA (pBec10)- (9), HveB (pMW20)- (10), or HveC (pBG38)- (11) containing vectors or empty vector (pcDNA3) (Invitrogen). The plasmids used for the transfections were the same as those used for the generation of the stable receptorexpressing CHO cell lines previously reported (9 –11). Stable transfectants were selected after 14 days of antibiotic selection and then cloned for single-cell colonies by limiting dilution methods. FACS analysis was performed to further select for homogeneous receptor-expressing populations. For preparation of tumor implantation, cells were grown using DMEM containing 0.05% penicillin, 0.05% streptomycin, and 10% calf serum. When originally obtained, cells were grown and then frozen in 95% calf serum/5% DMSO so that all experiments could be initiated with cells of a similar passage number. On the day of intracranial injection, cells in subconfluent monolayer culture were passaged with 0.25% trypsin solution in EDTA, washed 1⫻ in cell culture medium, resuspended at the appropriate concentration in medium without serum, and held on ice. In vitro titration and determination of cytopathic effect. For viral titrations, subconfluent monolayers of A10, B5, C10, and control cells were infected at an m.o.i. of 0.1 with HSV-1 strain 17⫹ and 1716. Cells were incubated for 90 min at 37°C and overlaid with complete medium and incubated at 34°C until cytopathic effect (CPE) was observed (48 –72 h for A10 and C10 cells). As no CPE was observed with B5 or control cells, these flasks were incubated at 34°C for 10 days and then incubated at 37°C for an additional 7 days. Virus was harvested and titrations were preformed per standard lab techniques on Vero cells. For determination of CPE, cells were infected at multiple m.o.i. (0.01, 0.1, and 1.0) for 90 min at 37°C. Cells were then overlaid and incubated at 37°C and CPE was observed at 24-, 48-, and 72-h time points. CPE was assayed as the percentage of cells detached or rounded. Subcutaneous tumor production. Mice were anesthetized by interperitoneal injections of ketamine/xylazine (87 mg/kg ketamine/13 mg/kg xylazine). A patch of hair was removed from the flank to be injected using a chemical depilatory agent (Magic Shaving Powder, Carson Products Co., Savannah, GA). Subcutaneous injection of 1 ⫻ 106 A10, B5, C10, or control cells in a total volume of 50 l was performed using a Hamilton syringe and a 28-gauge needle. Intracranial tumor production. Mice were anesthetized by interperitoneal injections of ketamine/xylazine (87 mg/kg ketamine/13 mg/kg xylazine). The head was cleansed with 70% EtOH and betadine. A small midline incision was made in the skin of the head exposing the skull. Stereotactic injection of tumor cell suspensions was performed using a small-animal stereotactic apparatus (Kopf Instruments, Tujunga, CA). Injections were done with a Hamilton syringe through a 28-gauge needle. The needle was positioned at a point 2 mm caudal of the bregma and 1 mm left of the midline. Using a separate 27-gauge needle the skull was breached at the appropriate coordinates. The injection needle was advanced through the hole in the skull to a depth of 2 mm from the skull surface and then extracted 0.5 mm to create a potential space. Cells (5 ⫻ 104) in a total volume of 10 l were injected over 2 min. Following the injection, the
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needle was left in place for 2 min and then slowly withdrawn. The skin was sutured closed. Viral inoculation. Mice were anesthetized by interperitoneal injections of ketamine/xylazine (87 mg/kg ketamine/13 mg/kg xylazine) and the head was cleansed with 70% EtOH. Using a Hamilton syringe with a 28-gauge needle, the appropriate amount of virus was injected, in a volume of 10 l, through a midline incision at the same stereotactic coordinates used for tumor cell injection. The injection was performed over 2 min, and following the injection the needle was left in place for 2 min and then slowly withdrawn. The amount of HSV-1 1716 used in all experiments (5 ⫻ 104 pfu/mouse) was the lowest pfu that gave the longest survival in previous experiments (4). Enzyme-linked immunosorbent assay. Soluble receptor proteins, HveA (200t) (37), HveB (361t), or HveC (346t) (38), at 10 g/ml in PBS were bound to a 96-well plate overnight at 4°C. Plates were washed with 0.1% Tween 20 in PBS (PBS-T) and incubated in PBS with 5% milk and 0.2% Tween 20 (blocking solution) for 30 min at room temperature (RT). Plates were washed with PBS-T and incubated in blocking solution containing the appropriate mouse serum dilutions for 2 h at RT. After being washed with PBS-T the plates were incubated with HRP-conjugated goat antimouse antibody diluted 1000⫻ in blocking solution for 1 h at RT. Plates were then washed with PBS-T and with 20 mM citrated buffer, pH 4.5. The HRP substrate (ABTS; Moss, Inc., Hanover, MD) in citrate buffer, pH 4.5, was added and the absorbance at 405 nm was read with a microtiter plate reader (Perkin–Elmer, Wellesley, MA). Statistics. Data analysis, including calculations of means and standard deviations and repeated-measures analysis of variance, was performed using StatView statistical software (Abacus Concepts, Berkeley CA) on an Apple Macintosh computer (Cupertino, CA).
RESULTS Cells Transfected with HveA and HveC, but Not HveB, Are Sensitive to HSV-1 Strains 17⫹ and 1716 Murine melanoma cells that are sensitive to HSV infection were constructed by transfection of B78H1 cells with human herpesvirus entry mediators HveA (A10), HveB (B5), and HveC (C10) (Pat Spear, unpublished data). A control cell line transfected with plasmid alone was also constructed (Pat Spear, unpublished data). To ensure that these proteins were functional for viral entry, all four cell lines were infected with HSV-1 17⫹ and 1716, a neuroattenuated ICP 34.5 mutant (35). HveB allows entry of HSV-1 with mutant gD but is unable to allow entry of HSV-1 viruses with a wild-type gD; therefore the B5 cells should be resistant to both HSV-1 17⫹ and 1716 infection. As expected, both A10 and C10 cells were susceptible to both HSV-1 17⫹ and 1716, but neither B5 nor control cells were sensitive to infection (Table 1). HSV-1 17⫹ and 1716 were able to grow to high titers on both A10 and C10 cell lines. Neither B5 nor control cells supported the growth of HSV-1 17⫹ or 1716, as the low-level pfu may be residual virus from the input pfu of 2 ⫻ 106 (Table 1). Additionally, cultures of each cell line were infected with HSV-1 17⫹ at multiplicities of infection of 0.01, 0.1, and 1.0. CPE was monitored at 24, 48, and 72 h after viral infection. A10 cells were most sensitive to HSV-1 17⫹, with complete CPE by 48 h at an m.o.i. of 0.01 (Fig. 1B). C10 cells were also sensitive to HSV-1 17⫹, although complete CPE was seen by 48 h only at an m.o.i. of 1.0 (Fig. 1C). B5 and control cells show little or no sensitivity
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In Vitro Growth of HSV-1 17⫹ and HSV-1 1716 on Hve-Transfected B78H1 Cell Lines Hve-transfected cell line Virus
A10a
B5b
C10a
Controlb
17⫹c 1716c
3.0 ⫻ 108 3.33 ⫻ 108
5.0 ⫻ 104 1.0 ⫻ 102
3.33 ⫻ 107 3.33 ⫻ 106
8.0 ⫻ 104 2.0 ⫻ 102
a
Virus was harvested after complete CPE for A10 and C10 (72 h). Virus was harvested after 17 days for B5 and control. c 2 ⫻ 108 cells were infected at an m.o.i. of 0.1. b
to HSV-1 17⫹ at any time point. As HSV-1 1716 is the virus used in cancer therapy experiments in our lab (4, 8, 13, 14), the ability of HSV-1 1716 to lyse these cells was determined as above. After 72 h of viral infection, all of the C10 cells were lysed at an m.o.i. of 0.1 (Fig. 2B). After 48 h of viral infection, all of the A10 cells were lysed at an m.o.i. of 1.0 (Fig. 2C). These results confirm the ability of these proteins to mediate viral entry into and viral infection and lysis of these cells. Therefore, A10 and C10 cells may be useful to determine the ability of neuroattenuated HSV to mediate tumor destruction. Additionally, the availability of the control cells, which are resistant to HSV killing in vitro, may allow us to determine whether a protective immune response develops against tumor cells. This immune response to the tumor cells would then be able to potentially destroy distant metastases.
A10, C10, and Control Cells Produce Tumors in Vivo The expression of a human protein on the murine cell surface of these cells could alter the ability of the cells to produce tumors. Therefore, a subcutaneous tumor model was used to determine whether the transfected cell lines still formed tumors efficiently. Mice were implanted with 5 ⫻ 106 tumor cells on the left flank and monitored for tumor growth. As can be seen in Fig. 3, A10, C10, and control cells were able to produce tumors in all mice implanted (n ⫽ 10 for A10 and control, n ⫽ 15 for C10). Control tumors developed by week 3, while A10 and C10 tumors developed by week 4. However, only 33% of the B5 implanted mice grew tumors (n ⫽ 10; Fig. 3A), and these arose after week 7. Mice in which tumors did not grow were monitored for at least 240 days. To determine whether these tumor cells expressing human receptors for HSV infection maintained their lowimmunogenicity, the tumors were removed from the flanks of mice and the animals rested for 1 week. The same animals were then implanted with 5 ⫻ 106 tumor cells on the opposite flank and again monitored for tumor growth. Only those mice that grew primary tumors were used for the production of secondary tumors. All mice implanted with A10, C10, and control cells developed secondary tumors (Fig. 3B). Additionally, sera from these mice were collected and tested for the presence of antiMOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
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FIG. 1. HSV-1 17⫹ efficiently replicates in and kills A10 and C10 cells, but not B5 or control cells. Subconfluent cell cultures were infected with wild-type HSV-1 17⫹ with different multiplicities of infection (m.o.i.) as described under Methods and percentages of surviving cells were recorded at 24, 48, and 72 h. These data are representative of two separate experiments. (A) m.o.i. 0.01, (B) m.o.i. 0.1, (C) m.o.i. 1.0.
bodies toward the three receptors. No significant detectable antibody response was seen in any animals (data not shown). Only one of the mice implanted with B5 cells developed a secondary tumor. The ability of the control cell line to produce primary and secondary tumors in all mice implanted will make it suitable for future studies to determine the protective effects against distant metastases induced by HSV-1 therapy of tumors. The ability of A10 and C10 cell lines to produce primary and secondary tumors in all mice injected supports their suitability to produce syngenic tumors susceptible to infection by HSV.
HSV-1 1716 Prolongs Survival of A10 and C10 Tumor-Bearing Mice but Not That of B5 and Control Tumor-Bearing Mice To determine whether HSV-1 1716 is able to cause Hvetransfected tumor destruction, all four cell lines were implanted intracranially into syngenic C57Bl/6 mice. Mean MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
survival rates of mock- (culture sera) treated mice bearing A10, C10, and control tumors were similar (22.8 ⫾ 1.2, 19.1 ⫾ 0.4, and 15.4 ⫾ 3.5 days, respectively, Fig. 4), although the control tumors did grow more aggressively both intracranially and subcutaneously (data not shown). One of the mice implanted with B5 that was mock treated developed a tumor and survived for 49 days. Ten days following tumor implantation, a second group of tumor-bearing mice was treated with HSV-1 1716. A10 tumor-bearing mice had a mean survival of 27.9 ⫾ 0.9 days, whereas C10 tumor-bearing mice had a mean survival of 29.4 ⫾ 1.2 days (Fig. 4). Although survival for C10 was higher than for A10 tumor-bearing mice, the difference was not significant. Both A10 and C10 tumor-bearing mice treated with HSV-1 1716 had significant increases in survival over mock-treated mice (P ⫽ 0.002 and P ⬍ 0.0001, respectively). There was no significant difference in the mean survival rates of control tumor-bearing mice treated with HSV-1 1716 (19.8 ⫾ 2.1) compared to those mock treated (P ⫽
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FIG. 2. HSV-1 1716 efficiently replicates in and kills A10 and C10 cells, but not B5 or control cells. Subconfluent cell cultures were infected with HSV-1 1716 with different multiplicities of infection (m.o.i.) as described under Methods and percentages of surviving cells were recorded at 24, 48, and 72 h. These data are representative of two separate experiments. (A) m.o.i. 0.01, (B) m.o.i. 0.1, (C) m.o.i. 1.0.
0.3103) (Fig. 4). Two mice bearing B5 tumors, which were treated with HSV-1 1716, produced tumors and the survival rate was 51 days. These results demonstrate that HSV-1 1716 is able to prolong survival in both A10 and C10 tumor-bearing animals. Control cells may be used to further delineate any protective immune response conferred toward distant metastases by HSV-1 therapy.
DISCUSSION The use of neuroattenuated HSV for the therapy of tumors is currently being assessed in clinical trials (19, 20). HSV is uniquely suited for therapy of malignant brain tumors because it is able to lytically replicate in nondifferentiated tumor cells but not in terminally differentiated neuronal tissue. Our laboratory and others have shown that neuroattenuated HSV-1 is able to prolong survival in a murine intracranial model (4 –7, 15, 39 – 43). In order to better understand the mechanism of HSV-mediated tumor lysis, we have recently shown that in an immunodeficient syngenic intracranial murine model, lysis is not sufficient to prolong survival. Furthermore, using an immunocompe-
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tent model, there is an increase in the immune cell infiltration into tumors following viral therapy (8). However, a clear understanding of the contribution of the immune response to viral-mediated tumor lysis is difficult to determine with the animal models that currently exist. Toda and co-workers have shown that a neuroattenuated HSV-1 (G207) is able to induce a highly specific systemic anti-tumor immune response in two different models, a colorectal carcinoma model (CT26) in Balb/c mice and the Cloudman M3 S91 melanoma model (S91) (15). However, the CT26 cell line expresses the MuLV env gene product (44) and the S91 cell line is highly immunogenic in syngenic mice. Thus it is difficult to determine whether the immune response is induced from virus administration or is occurring due to immunogenic properties of the tumor cell line. Additionally, studies utilizing various other tumor models have been used to determine the ability of neuroattenuated HSV-1 to destroy tumors in vivo. Unfortunately, many of these are rat models, which are refractory to HSV-1 infection. Although neuroattenuated HSV-1 is able to provide beneficial results in these animals, it is generally due to either the bystander effect or the expresMOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
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FIG. 3. Production of tumors in vivo after subcutaneous injection of 5 ⫻ 106 Hve-transfected cells. (A) Primary tumor growth. All mice injected with A10, C10, and control cells grew tumors. Only 33% of the mice injected with B5 cells grew tumors (N ⫽ 10 for A10, B5, and control cells; N ⫽ 15 for C10 cells). (B) Secondary tumor growth. After allowing growth of primary tumors in mice for 6 weeks, tumor cells were injected into opposite flanks of mice that grew primary tumors. All A10, C10, and control cells injected into mice grew secondary tumors. Only 33% of the B5-injected mice grew secondary tumors (N ⫽ 10 for A10 and control cells, N ⫽ 15 for C10, and N ⫽ 3 for B5).
sion of a suicide gene (45– 49). Also, many of the studies being undertaken are using human tumors transplanted into immunodeficient mice (6, 14, 39, 50 –56), which demonstrate that HSV-1 is able to destroy xenogenic tumors without the aid of the immune response. However, human tumor transplants are generally slower growing in animals and thus the virus has more time to spread throughout the tumor. We have recently demonstrated that mice bearing syngenic tumors have an accelerated tumor growth intracranially compared to those growing xenographic tumors, and the virus is not able to spread throughout the tumor with subsequent destruction of the whole tumor (8). To further delineate the role of the immune response in viral-mediated tumor cell destruction and to better determine whether a new tumor-specific immune response develops following viral therapy, a new tumor model was needed, one that (1) was susceptible to HSV-1 infection, (2) had low immunogenicity, and (3) had a syngenic host. Although cell lines that are resistant to HSV-1 infection are considered rare, many murine melanoma cells are resistant to HSV-1 infection (27, 57). Therefore, new tumor cell lines were created using the low-immunogenic B78H1 melanoma cell line. This cell line, an amelanotic subclone of B16 melanoma cells, is resistant to HSV-1 infection and neuroattenuated HSV-1 is unable to prolong survival in mice bearing B16 or B78H1 intracranial tumors (57). Four different cell lines were created by transfecting the human herpesvirus entry proteins, HveA, HveB, and HveC, and the control plasmid into B78H1 cells. In this paper, we determined the ability of these cells MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
to serve as a new model to determine the contribution of the immune response to viral-mediated tumor lysis. Transfection of HveA and HveC into B78H1 cells rendered these cells susceptible to HSV-1 17⫹ and 1716 infection and lysis. As expected, transfection of neither HveB nor the control plasmid renders cells susceptible to HSV-1. For the transfected cells to be useful as a tumor model, the expression of the human receptors on the cell surface must not affect the ability of the cells to form tumors in vivo. A10, C10, and control cells were able to consistently form tumors in animals, but B5 were not. We also determined that these cells retained the low immunogenicity of the parental cell line as secondary tumors were able to grow and no detectable humoral immune response to the receptors is seen after tumor implantation. Therefore, the expression of the receptors at the surface of these cells does not in itself seem to alter the immunogenicity of the cells. Finally, we determined that HSV-1 1716 is able to prolong survival of animals bearing intracranial A10 and C10 tumors, but is unable to prolong survival of animals bearing intracranial B5 or control tumors. HveA is the principal receptor for entry of HSV-1 into human lymphoid cells (9). Overexpression of HveA, a member of the TNF/NGF receptor family (9, 28 –30), results in activation of NFB/AP-1 (28), suggesting that HveA’s natural function following ligand binding is to interact with and modulate the immune response (28). Additional studies have shown that antibodies toward HveA are able to block T cell proliferation and that HveA is involved in the control of T cell activation (58). Binding of LIGHT/HveM-L, the ligands for Hve A (59), also activated NK-B and stimulated T cell proliferation (60). Additionally, binding of LIGHT/HveM-L to adenocarcinoma HT-29 cells inhibited growth of the cells. However, whether this will affect the determination of the role of the immune response in tumor destruction is questionable, as B78H1 cells may not possess the downstream pathways that human HveA normally interacts with. HveC and HIgR, an alternative splice variant (27), mediate the entry of HSV-1 and HSV-2 strains, PRV, and BHV-1 (11). HveC, a member of the immunoglobulin superfamily, is widely expressed in human tissue, including epidermal, neuronal, myeloid, and lymphoid lineages (11, 27), and HveC is considered the main receptor for infection of mucosal surfaces and for spread from infection site to the nervous system (11). HveC is involved in cell– cell interactions (33, 34, 61) and has recently been renamed Nectin-1 since it is a cell– cell adhesion molecule, found at zonula adherens in epithelia cells and in adherens junctions in nonepithelial cells (33). Murine homologues of HveA, HveB, and HveC, which mediate entry of human alphaherpesviruses but with slight differences in specificity (62, 63), have been discovered, as reviewed by Spear et al. (64). In this study we used the human homologues as more is known about the function of these proteins. It is interesting that transfection of HveB inhibited tumor formation in animals implanted with B5 tumor cells. The potential reasons for this inabil-
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FIG. 4. Survival of C57Bl/6 mice after intracranial injection of 5 ⫻ 104 Hve-transfected cells followed by treatment injection of 5 ⫻ 104 pfu HSV-1 1716 on day 10. (A) A10. There is a significant increase in survival of mice receiving viral treatment compared to those receiving mock treatment, P ⫽ 0.002. N ⫽ 10 for HSV-1 1716 and mock treated. (B) B5. There is no significant increase in survival of mice receiving viral treatment compared to those receiving mock treatment. N ⫽ 5 for HSV-1 1716 and mock treated. (C) C10. There is a significant increase in survival of mice receiving viral treatment compared to those receiving mock treatment, P ⬍ 0.0001. (D) Control cells. There is no significant increase in survival of mice receiving viral treatment compared to those receiving mock treatment, P ⫽ 0.313. N ⫽ 10 for HSV-1 1716 and mock treated.
ity to form tumors may be due to the level of expression on the surface of the cells or may be due to the natural function of this protein in vivo. It is also of interest that expression of the HveA and HveC receptors on the surface of these cells does not appear to alter the low immunogenicity of these tumor cells, as they are able to form secondary tumors in vivo. However, studies into the immune response following viral therapy will have to account for the expression of these human receptors on these cells and for the fact that an immune response may
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be directed to these proteins as well as other viral and tumor antigens. The availability of the control cell line will aid in determining the specifics of the immune response. In conclusion, we have developed new syngenic, lowimmunogenic tumor cell lines to study the contribution of the immune response to viral-mediated tumor destruction. We have created two tumor cell lines, A10 and C10, which are susceptible to HSV-1 infection and lysis. Furthermore, the control cell line can be used to determine if MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
ARTICLE protection against syngenic distant metastasis arises, allowing for the determination of tumor-specific responses that develop after viral therapy. Additionally, many of the transgenic and knockout mice which are currently available are in the same background as these tumor cells, allowing for the determination of potentially specific immune regulators and pathways which may be important in anti-tumor therapy. ACKNOWLEDGMENTS The authors thank the following for contributions to this paper: Vikram Suri for technical assistance and Jennifer Driscoll for secretarial assistance. Additionally we thank Pat Spear for the Hve receptor-transfected cell lines. This work was supported by a grant from the NIH (NS 37516). C.G.M. was supported in part by an institutional training grant (NS 07180) and an individual training grant (CA 77903) from the NIH. C.K. was supported by a fellowship (823A-053464) from the Swiss National Science Foundation.
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