hsv-1 therapy of primary tumors reduces the number of metastases in an immune-competent model of...

HSV-1 Therapy of Primary Tumors Reduces the Numberof Metastases in an Immune-Competent Model

of Metastatic Breast Cancer

Darby L. Thomas and Nigel W. Fraser*

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076

*To whom correspondence and reprint requests should be addressed at the Department of Microbiology, University of Pennsylvania School of Medicine,319a Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6067. Fax: (215) 898-3849. E-mail: [email protected].

The HSV-1 1716 mutant virus and similar oncolytic herpesviruses deficient in the �34.5 neuro-virulence gene are able to reduce the growth of tumors in mice. Here we demonstrate that HSV-11716 therapy moderately reduced the growth of tumors of the highly malignant, spontaneouslymetastasizing 4T1 mouse mammary carcinoma model. This moderate effect on 4T1 tumor growthwas likely due to poor replication kinetics of HSV-1 1716 in 4T1 cells. Interestingly, HSV-1 therapyof the primary tumor increased the survival time of mice. Coincident with this increase was areduction in metastases as determined by quantification of the number of metastatic cells in thelungs. HSV-1 therapy of the primary tumor was also able to reduce the establishment of a secondchallenge of 4T1 tumors. Moreover, infiltrates of both CD4� and CD8� T cells were detected inHSV-1 1716-treated tumors. An important role for the T cell infiltrates was confirmed when HSV-1therapy did not reduce the growth of 4T1 tumors in SCID mice. Collectively, these resultsdemonstrate that an HSV-dependent anti-tumor immune response is required for the reductionin primary 4T1 tumor growth and for the reduction in the establishment of metastases in thistumor model.

Key Words: HSV-1, gene therapy, 4T1 mammary tumor, metastasis, immune response

INTRODUCTION

Herpes simplex viruses with mutations in the �34.5 neu-rovirulence gene-encoding infected cell protein (ICP34.5)do not replicate in terminally differentiated cells, such ascells of the central nervous system, but can replicate individing cells, such as cancer cells [1–3]. ICP34.5 func-tions to inactivate the dsRNA-induced PKR pathway, toprevent host protein synthesis shutoff, by complexingwith protein phosphatase 1�, which results in the de-phosphorylation of eIF-2� [4]. The selective replication of�34.5-mutant viruses has been exploited to design repli-cating HSV-1 vectors for cancer therapy. HSV-1 therapycan increase the survival of mice harboring establishedbrain tumors and reduce the growth (sometimes com-pletely) of established subcutaneous tumors in mice afterintraneoplastic injection. Herpes vectors like G207,R3616, and 1716 have been shown to be effective in therapyof human tumors in immune-deficient mouse models, suchas colon, ovarian, glioma, breast, prostate, and melanoma,as well as in therapy of immune-competent mouse mod-els of glioma, colorectal, and melanoma [5–9 and re-

viewed in 10]. Consequently, results of completed phase Iclinical trials for both G207 and 1716 demonstrated notoxicity at doses up to 109 and 105, respectively, in pa-tients with malignant glioma and melanoma [11–13].

It is now well established that an important compo-nent of HSV-1 cancer therapy is an immune responseelicited by the virus. For example, Toda et al. [14] dem-onstrated that G207 therapy of a colon carcinoma modelacts as an “in situ vaccine” by eliciting a tumor-specificimmune response. Similarly, HSV-1 1716 therapy of abrain tumor model of metastatic melanoma requires mul-tiple components of the immune system [15]. In addition,preexisting immunity to HSV-1 can enhance HSV-1 1716therapy of brain tumors [16]. Considering these studies,we selected an immune-competent cancer model—the4T1 mouse mammary carcinoma model [17–19]—andasked whether oncolytic HSV-1 therapy would be effec-tive in this model of an aggressive metastatic cancer.

4T1 tumors established in the mouse mammary glandclosely resemble human breast cancer in their immuno-genicity and their growth and metastatic properties [20].Derived from Balb/c mice, 4T1 cells are considered very

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543MOLECULAR THERAPY Vol. 8, No. 4, October 2003Copyright © The American Society of Gene Therapy1525-0016/03 $30.00

weakly immunogenic with a relative antigenic strength ofless than 0.01 (with 9.9 being the most immunogenic)[21,22]. Tumors derived from 4T1 cells spontaneouslymetastasize primarily to the lungs (�95%), but also spreadto the liver (�75%) and brain (40%) as well as the bloodand lymph nodes [20]. Because 4T1 cells are 6-thiogua-nine resistant, micrometastases to different organs areeasily amplified and the number of metastatic cells iseasily quantified [17,20].

Here we demonstrated that HSV-1 1716 therapy ofestablished 4T1 mammary tumors moderately reducedthe growth rate of these primary tumors compared tothose treated with culture medium (mock). However, thetherapy of the primary tumor significantly reduced thenumber of clonogenic lung metastases and the mean sur-vival time of the mice compared to mock therapy. Inaddition, HSV-1 1716 therapy of primary 4T1 tumorsinhibited the establishment of a second challenge of 4T1tumor cells, implicating HSV stimulation of an anti-tu-mor immune response. This implication was supportedby the presence of infiltrating CD4� and CD8� T cells.Furthermore, HSV-1 1716 therapy did not affect thegrowth of primary tumors in immune-deficient SCIDmice, verifying that an immune response is required foreffective HSV-1 therapy of the primary 4T1 tumors as wellas for the reduction in the establishment of metastases.

RESULTS

HSV-1 1716 Replicates Poorly in 4T1 MammaryTumor CellsAlthough Balb/c mice are susceptible to HSV-1 infection,not all murine cell lines derived from susceptible mice can

be efficiently infected with HSV-1 [23,24]. Therefore, weexamined the ability of wild-type HSV-1 17� and the�34.5-mutant HSV-1 1716 to replicate in 4T1 cells com-pared to HSV-susceptible Vero cells. We infected Vero and4T1 cells with HSV-1 17� and 1716 at a multiplicity ofinfection (m.o.i.) of 0.1. Infected monolayers were har-vested over time and the number of plaque-forming units(pfu) was determined by plaque assay. HSV-1 17� repli-cated to high levels within 3 days in both Vero and 4T1cells (Fig. 1A). In contrast, HSV-1 1716 yields were threelogs lower in 4T1 cells than in Vero cells in the same timeperiod (Fig. 1B), suggesting that, unlike Vero cells, 4T1cells cannot complement the �34.5 mutation to supportreplication of this virus. Although 4T1 cells are suscepti-ble to killing by HSV-1 1716 at a high m.o.i. (data notshown), HSV-1 1716 replication is not as efficient in 4T1cells as in Vero cells.

HSV-1 1716 Only Moderately Reduces the Growth ofPrimary Mammary TumorsThough HSV-1 1716 does not replicate well in 4T1 cells,we still wanted to examine the potential of HSV-1 1716 toreduce the growth of established 4T1 tumors. A singleinjection of HSV-1 1716 had little effect on tumor growth(data not shown); therefore we chose to analyze the out-come of two and three separate injections of HSV-1 1716.Similarly, Chahlavi et al. [25] demonstrated that multipleinjections of lower quantities of virus are more effectivethan a single injection of a high dose of virus. Tumorsestablished subcutaneously in the abdominal mammarygland received intratumor injections of 5.4 � 105 pfu ofHSV-1 1716 virus on days 9 and 13 (double therapy), oron days 9, 13, and 16 (triple therapy), post-injection of

FIG. 1. Replication of HSV-1 17� and HSV-1 1716 in 4T1 and Vero cells. 4T1 and Vero cells were infected at a m.o.i. of 0.1 for 1 h with (A) HSV-1 17� or (B)HSV-1 1716 as described under Materials and Methods. The “zero” time point was taken after washing of the cell monolayers immediately following the 1-hinfection. Time points were taken in duplicate and were titered on Vero cells in triplicate. The mean pfu at each time point is presented in log scale � standarderror.

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tumor cells. We determined the mean tumor diameter(calculated as the square root of the product of two per-pendicular diameters) for each mouse every 3 to 4 daysuntil day 27 post-injection of tumor cells. Between days13 and 16, we found only a small increase in the meandiameter of tumors receiving double HSV-1 1716 therapy(Fig. 2A), and no increase in the mean diameter of tumorsreceiving triple HSV-1 1716 therapy (Fig. 2B), in com-parison to tumors treated with culture medium (mocktreated). Beginning at day 16, the difference in the meandiameters between HSV-1 1716- and mock-treated tumorswas significant each day until the endpoint for both dou-ble (Fig. 2A, P � 0.025) and triple therapy (Fig. 2B, P �0.002). Despite this significance, the mean diameter ofHSV-1 1716-treated tumors continued to increase afterday 16—at either a similar rate (Fig. 2A, double therapy)or a reduced rate (Fig. 2B, triple therapy) compared tomock-treated tumors, suggesting that HSV-1 therapy in-creased in effectiveness with increased inoculations. How-ever, therapy was not totally effective.

HSV-1 1716 Replicates Poorly in 4T1 MammaryTumorsHSV-1 1716 therapy has been demonstrated to treat braintumors in mice effectively, and replication of HSV-1 1716

in these tumors is characterized by an increase in virusdetection (by plaque assay) in the days following injec-tion [6,9]. Because HSV-1 1716 therapy was not very ef-fective in reducing the growth of primary 4T1 tumors, wenext wanted to determine whether HSV-1 1716 was rep-licating within the tumor. We repeated triple therapy ofprimary 4T1 tumors and harvested the tumors at the timepoints indicated in Fig. 3. On the days of therapy, weharvested the tumors immediately following injection ofvirus to determine the amount of input virus detectableby plaque assay. We found that, on the days followingeach injection of HSV-1 1716, virus was detected byplaque assay (Fig. 3). However, the quantity of virus de-creased instead of increased over time, though infectiousvirus was still detectable for up to 3 days. This result issimilar to results seen when HSV-1 1716 is injected di-rectly into the brain [6,26]. In these studies, Kesari et al.[26] found that, when injected into the ventricles whereHSV-1 1716 replicates in the ependymal cells, infectiousvirus can be detected by titration for up to 2 days. Wheninjected into the brain parenchyma, where HSV-1 1716cannot replicate, infectious virus is not detected at anytime postinjection. Considering these studies in the brain,our results suggest that inefficient viral replication is oc-curring in the 4T1 tumors.

FIG. 2. HSV-1 1716 therapy of 4T1 mammary tumors. Balb/c mice were injected subcutaneously in the abdominal mammary gland with 1 � 104 viable 4T1 cells.Mean tumor diameters were determined as described under Materials and Methods. Palpable tumors were injected with 20 �l containing either 5.4 � 105 pfuof HSV-1 1716 or DMEM (mock-infected) on (A) days 9 and 13 (double therapy; mock, n 24; 1716, n 23) or (B) days 9, 13, and 16 (triple therapy; mock,n 22; 1716, n 19). Arrows indicate the days when therapy was administered. *Significant differences between mock- and 1716-treated tumors. Error barsrepresent �standard error.

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HSV-1 1716 Therapy of Primary Mammary TumorsIncreases the Survival of MiceA recent study demonstrated that therapy of primary 4T1tumors with IL-12, delivered by gene gun, did not reducethe growth of primary tumors, yet increased the survivaltime of mice [27]—presumably due to a decrease in me-tastasis. Therefore, we reasoned that it was still possiblethat HSV-1 therapy, though only moderately effective onprimary 4T1 tumors, could prolong the survival time oftreated mice. To address this possibility, we divided themice described in Fig. 2 with primary tumors treated witheither two or three rounds of HSV-1 1716 therapy intotwo groups. We monitored the first group for survivalfollowing surgical removal of the primary tumor at day 27post-injection of tumor cells. Mice with primary tumors

that received double mock therapy (Fig. 4A) or triplemock therapy (Fig. 4B) survived a mean of 37.6 � 5.2 daysor 48.9 � 10.7 days, respectively. The difference we ob-served between the two therapies, which is not statisti-cally significant, is likely due to experimental variability,though we cannot formally rule out the effects of manip-ulating the tumor. One double-mock-therapy mouse sur-vived past day 100 post-injection of tumor cells (Fig. 4A).We presume that this mouse did not get metastases,which is in accordance with previous observations thatmetastasis (e.g., to the lungs) is less than 100% [20,28].Mice with primary tumors that received either double(Fig. 4A) or triple (Fig. 4B) HSV-1 1716 therapy survivedsignificantly longer, with a mean of 48.4 � 12.3 (P 0.014) or 58.1 � 7.7 days (P 0.005), respectively.

HSV-1 1716 Therapy of Primary Mammary TumorsReduces the Number of Metastatic Cells in the LungsEstablished 4T1 tumors form disseminated metastasesthroughout the body of the mouse [17,20]. The increasein survival time of mice with HSV-1 1716-treated primary4T1 tumors suggests that therapy reduced the extent ofmetastases. We therefore utilized the resistance of 4T1cells to 6-thioguanine to quantify the number of meta-static cells in the lungs. We chose to look exclusively atthe lungs since previous studies have demonstrated thatmetastasis to the lungs best represents the extent of 4T1cell metastasis throughout the mouse [20]. We sacrificedthe second group of mice from the primary tumor exper-iments described in Fig. 2 on day 27 and removed, disso-ciated, and cultured their lungs in 6-thioguanine to assaythe number of 4T1 colonies representing the number ofmetastatic cells. We determined the natural log (ln) of thenumber of metastatic colonies from each mouse to givenormality for statistical evaluation (data not shown).Mice receiving double mock therapy had a mean of69,665 � 66,434 metastatic colonies and mice receivingtriple mock therapy had a mean of 58,051 � 94,962metastatic colonies (Table 1). Significantly, mice receivingdouble or triple HSV-1 1716 therapy had a mean of 6781� 12,353 (P 0.004) or 13,033 � 32,597 (P 0.033)

FIG. 3. HSV-1 1716 replication in 4T1 tumors. Balb/c mice with established4T1 tumors were given triple therapy as described for Fig. 2. Two tumors pertime point were harvested on the days indicated post-injection of virus andprepared as described under Materials and Methods. Tumor lysates wereassayed for HSV-1 1716 levels by plaque assay on Vero cells in triplicate. Themean pfu at each time point is presented in log scale � standard error. Arrowsindicate the days when therapy was administered.

FIG. 4. Survival of mice with HSV-1 1716-treated 4T1 mammary tumors. On day 27 post-injection of tumor cells, tumors were surgically removed from micedescribed in Fig. 2 as described under Materials and Methods and the mice were monitored for survival. Kaplan–Meier survival curves are presented. (A) Doubletherapy: mock, n 12; 1716, n 12; P 0.014. (B) Triple therapy: mock, n 12; 1716, n 10; P 0.005.

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metastatic colonies, respectively (Table 1). These resultsdemonstrate that HSV-1 1716 therapy of the primary tu-mor can reduce the burden of metastases in the lungs. Inaddition, we see a strong correlation between tumor sizeand the number of metastatic colonies for each mouse(data not shown), consistent with other studies using thistumor model [20].

HSV-1 1716 Therapy of 4T1 Tumors Partially Protectsagainst a Second Challenge of 4T1 CellsA possible explanation as to how HSV-1 1716 therapy ofthe primary 4T1 tumor could reduce the number of me-tastases is that viral therapy stimulated an immune re-sponse to the tumor cells. We addressed this possibility byasking whether treating established primary 4T1 tumorswith HSV-1 1716 inhibited the growth of a second chal-lenge of 4T1 tumor cells. Specifically, established 4T1tumors were given triple therapy and then surgically re-moved. We then injected additional 4T1 tumor cells onthe contralateral side of the abdomen and monitoredthem for growth. Because 4T1 cells are very weakly im-munogenic, secondary tumors were able to grow in the 10mice given triple mock therapy and 6 of these 10 micedied before the endpoint at 38 days after the first injectionof tumor cells, presumably due to metastases (Fig. 5).Interestingly, 3 of 10 mice receiving triple HSV-1 1716therapy did not develop secondary tumors. Although thetumors that did form grew at a rate similar to that ofmock-treated tumors, only 1 of 10 mice receiving tripleHSV-1 1716 therapy did not survive until the endpoint ofthe experiment (Fig. 5). These results suggest that HSV-1therapy of primary 4T1 tumors can stimulate an anti-tumor immune response that reduces the establishmentof a second challenge of tumor cells.

CD4� and CD8� T Cells Are Present in HSV-1 1716-Treated TumorsIt is well established that both CD4� and CD8� T cells areessential for immune-mediated tumor rejection [reviewed

in 29]. To examine further a role for an anti-tumor im-mune response in HSV-1 1716 therapy of 4T1 tumors, weperformed immunohistochemical analyses on sections ofHSV-1 1716- and mock-treated tumors. Mice with estab-lished 4T1 tumors received triple HSV-1 1716 and mocktherapy as described above and we harvested and frozetumors for sectioning during and after the course of ther-apy. Tumor sections were then analyzed for histology andfor the presence of CD4� and CD8� T cells. As early as day12 post-injection of tumor cells, inflammatory cells, suchas neutrophils, could be detected throughout the mass ofHSV-1 1716-treated tumors (Fig. 6). CD4� and CD8� T

TABLE 1: Quantitation of clonogenic lung metastases from mice with HSV-1 1716-treated 4T1 mammary tumors

Therapyb No. of injectionsc nd

No. of mice with clonogenic lung metastasesa

Mean (ln)e p-valuef0–1000 1001–20,000 20,001–50,000 �50,000

Mock 2 11 2 2 1 6 69,665 � 66,434 (10.10)0.004

HSV-1 1716 2 12 5 5 2 0 6,781 � 12,353 (7.05)

Mock 3 9 2 4 0 3 58,051 � 94,962 (9.06)0.033

HSV-1 1716 3 9 6 2 0 1 13,033 � 32,577 (5.75)aMice described in Figure 2 were sacrificed on day 27 post-injection of 4T1 cells and the lungs were removed, processed, and cultured as described in Materials and Methods. The numberof 6-thioguanine-resistant 4T1 colonies stained with methelene blue were counted for each mouse. The number of mice with lung 4T1 colony numbers within each range is presented.bMock-treated mice received culture media and HSV-1 1716-treated mice received 5.4 � 105 pfu per injection.cEstablished 4T1 tumors received injections of either HSV-1 1716 or culture media (mock) either two (days 9 and 13 post-injection of 4T1 cells) or three times (days 9, 13, and 16post-injection of 4T1 cells) as described in Figure 2.dTotal number of mice per group.eMean number of 6-thioguanine-resistant 4T1 colonies � standard deviation. (ln) is the natural log of the mean number of metastases.fThe natural log (ln) of the number of metastases in the lungs of each mouse was used to determine the p-value for either two or three injections as described in Materials and Methods.

FIG. 5. Growth of a second challenge of 4T1 tumor cells after HSV-1 1716therapy of primary 4T1 tumors. 4T1 tumors were established, received HSV-11716 or mock therapy on days 9, 13, and 16, and were measured periodicallyas described for Fig. 2. On day 18 post-injection of 4T1 cells, the primarytumors were surgically removed, and another 1 � 104 4T1 cells were injectedinto the contralateral side. Seven days following the second injection, growthof tumors was again measured periodically until the endpoint of 38 days. n 10 each group until days 32, 35, and 38 when two mock-treated mice eachday and day 38 when one HSV-1 1716-treated mouse was euthanized becauseof illness. Arrows indicate the days when therapy was administered. *Signifi-cant differences between mock- and 1716-treated tumors. Error bars represent�standard error.

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cells could also be detected throughout the HSV-1 1716-treated tumors. Peak staining for CD4� and CD8� T cellsappeared between day 17 (shown in Fig. 6) and day 20post-injection of tumor cells and began to subside by day23 (data not shown). Very few inflammatory cells couldbe detected in mock-treated tumors (data not shown).

HSV-1 1716 Therapy Has No Effect on 4T1 TumorGrowth and Metastases in SCID MiceWe wanted to further confirm the importance of theimmune system in the reduction of metastases by exam-ining HSV-1 therapy of primary 4T1 tumors in immune-incompetent SCID mice, which are deficient in both Tcells and B cells. In addition, we wanted to address thepossibility that, without an adaptive immune response,HSV-1 1716 would grow uninhibited throughout the tu-mor and, therefore, more efficiently destroy the tumor. Asabove, we injected SCID mice with 1 � 104 viable 4T1cells subcutaneously in the abdominal mammary gland.Mice received intratumoral injections of either HSV-11716 or mock therapy on days 9, 13, and 16 post-injectionof cells, and we monitored the mean tumor diameter overtime. Interestingly, there was no difference in the growthbetween HSV-1 1716-treated and mock-treated primary4T1 tumors (Fig. 7). Consequently, there was also nosignificant difference in the number of metastatic cells inthe lung (data not shown). These results indicate that anintact immune response is needed for HSV-1 therapy ofthe primary tumor, as well as for the reduction of metas-tases.

DISCUSSION

The use of replication-restricted HSV-1 vectors has gainedacceptance as plausible for cancer therapy. This accep-tance is due to its efficacy in numerous rodent models ofcancer and its lack of toxicity in early clinical trials forglioma and melanoma [11–13]. Here we wanted to deter-mine whether oncolytic HSV-1 therapy of an aggressive,spontaneously metastasizing mammary carcinoma modelwould be effective. It was found that HSV-1 therapy hadonly a moderate effect on growth of the primary tumor,yet primary tumor therapy led to an increase in survivaltimes and a reduction of metastatic cells in the lungs.

Tumor therapy using �34.5-mutant viruses in onlysome models leads to complete regression of the tumor,demonstrating that the efficacy of HSV-1 therapy willlikely be tumor-type dependent. In the 4T1 mouse mam-mary carcinoma model, HSV-1 1716 only temporarily

FIG. 6. Presence of immune infiltrates in HSV-1 1716-treated 4T1 tumors. 4T1tumors were established and received HSV-1 1716 or mock therapy on days 9,13, and 16 as described for Fig. 2. During and after the course of therapy, micewere sacrificed and tumors harvested, frozen, and sectioned as describedunder Materials and Methods. Sections of HSV-1 1716-treated 4T1 tumorswere either stained with hematoxylin and eosin (H&E) or reacted with anti-CD4 or anti-CD8 antibodies and counterstained with methyl green to visualize

nuclei. Representative sections of tumors from days 12 and 17 are shown.Some of the lymphocytes and plasma cells in the day 17 section are indicatedwith white arrowheads. The day 17 photos are serial sections of the sametumor area. Cells positive for CD4 and CD8 are stained purple. Magnificationequals 200.

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reduced the growth of primary tumors. This lack of effi-cacy is likely due to the reduced replication kinetics ofHSV-1 1716 in these tumor cells and the poor replicationin 4T1 tumors as determined by plaque assay. Interest-ingly, wild-type 17� was able to replicate efficiently in4T1 cells, indicating that 4T1 cells are not able to com-plement the replication defect of HSV-1 1716.

In human breast cancer models, inefficient HSV-1 rep-lication in vitro also correlated with the poor oncolysis oftumors in vivo. In these studies, Toda et al. [30] found thatthe MDA-MB-435 and MCF-7 breast cancer cell lines aremore susceptible to HSV-1 mutant G207 infection at am.o.i. of 0.1 than T47D cells and that MDA-MB-231 cellswere not very susceptible to G207 infection at the samem.o.i. This differing susceptibility correlated with theability of G207 to reduce the growth of MDA-MB-435tumors but not of MDA-MB-231 tumors. Also similar to4T1 cells, mouse CT-26 colorectal tumor cells have a re-duced ability to support G207 replication at a low m.o.i.(0.1), but can be efficiently killed at a higher m.o.i. (1.0)[14,31]. Why HSV-1 has a reduced ability to replicate inthese different cell types has not been determined, yet theblock is not at the point of viral entry, like in B16 mela-noma cells [8], since 4T1 and CT-26 cells are capable ofbeing infected and lysed by HSV-1. Studies by Farassati etal. [32] have shown that efficient replication of �34.5-

mutant viruses is dependent on an activated Ras signalingpathway and they propose that components of the Rassignaling pathway can inactivate PKR, thus complement-ing the lack of anti-PKR activity in the �34.5-mutantviruses. We speculate that the oncogenic nature of 4T1cells is not due to an activated Ras pathway. In this regard,it is possible that other selectively oncolytic herpesvirusesthat do not have mutations in �34.5, such as hrR3 [33,34]and the recently described HF10 [35], might be moreefficacious in these tumor models and in other tumortypes that show resistance to replication and killing by�34.5-mutant viruses.

Interestingly, therapy of primary 4T1 mammary tu-mors in the absence of T and B cells is completely inef-fective, indicating that viral oncolysis alone is not suffi-cient for the reduction in tumor diameters observed withHSV-1 1716 therapy of 4T1 tumors in immune-competentmice. Similar observations have been made in other mod-els. We previously demonstrated that HSV-1 1716 therapydid not prolong survival of SCID mice with brain tumorsin an experimental model of melanoma [16]. In addition,therapy of CT-26 colorectal tumors, both flank and intra-cranial, with the HSV-1 mutant G207 was also ineffectivein athymic Balb/c mice [14,36]. However, in immune-competent mice, HSV therapy of CT-26 tumors acts as anin situ vaccine by inhibiting the growth of contralateraltumors or of a second challenge of tumor cells [14,31,36].In the 4T1 model, we found that HSV-1 1716 therapy ofthe primary tumor increased the mean survival time ofmice and reduced the metastatic burden in the lungs.Moreover, primary tumor therapy also can inhibit theestablishment of a second challenge of 4T1 tumors. It ispossible that the growth of secondary tumors could alsohave been inhibited if more time had been allowed be-tween removal of the primary tumor and the secondinjection of 4T1 cells, which would have potentially al-lowed more time to develop a proper anti-tumor immuneresponse. We were also able to detect an infiltration ofboth CD4� and CD8� T cells in HSV-1 1716-treated tu-mors. Collectively, these results, confirmed by the SCIDmice results, demonstrate that the immune system is es-sential for HSV-1 therapy of 4T1 mammary tumors andthat HSV-1 replication can act as a “flag” for the develop-ment of an anti-tumor immune response that can inhibitthe growth of primary tumors and the establishment ofmetastases in this model system.

The use of rodent models for the study of breast cancerhas significantly contributed to the understanding of thebiology of cancer and to the development of cancer ther-apeutics. Although there are limitations to the use ofrodent models, there are many similarities in structureand function between the mammary glands of humansand rodents [37], further stressing the importance of anal-yses of these models. For the studies presented here, weexamined oncolytic HSV-1 therapy on tumors of the 4T1mouse mammary carcinoma model [17–19]. 4T1 tumors

FIG. 7. HSV-1 1716 therapy of 4T1 mammary tumors in SCID mice. 4T1mammary tumors were established in SCID mice and received either HSV-11716 or mock therapy on days 9, 13, and 16 post-injection of tumor cells asdescribed for Fig. 2. n 10 each. Arrows indicate the days when therapy wasadministered. No significant difference was found at any time point. Error barsrepresent �standard error.

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established in the mammary gland of syngeneic Balb/cmice form disseminated metastases throughout the body,most frequently in the lungs (92–95%), as well as in theliver (�75%), brain (40%), blood, and lymph nodes[20,28]. This metastatic pattern shares some commonalitywith that of human breast cancer with metastases to thelungs (24–77%), liver (22–62%), and brain (30%)[20,38,39]. The 4T1 model has been used extensively inimmunotherapy studies [20,28,40,41] as well as antian-giogenic therapy studies [42,43]. Although HSV-1 1716therapy on primary tumors was only moderately effective,we propose that mouse 4T1 mammary tumors are a goodmodel for HSV-1 therapy studies. Accordingly, the studiesherein with this model should serve as a therapeutic base-line from which HSV-1 1716 therapy can be improved. Itis likely that other oncolytic HSV-1 vectors that expressimmune response-stimulating molecules [44,45] or angio-genesis inhibitors would be more effective in this model.

MATERIALS AND METHODSCell culture, viruses, and virus titrations. The 4T1 tumor cells were a kindgift from Dr. Fred Miller, Karmanos Cancer Center (Detroit, MI). 4T1 cellsand Vero cells were cultured in Dulbecco’s modified Eagle medium(DMEM) (GIBCO) supplemented with 5% fetal calf serum and were main-tained in 0.05% penicillin/streptomycin, at 37°C, in a humidified cham-ber, and in an atmosphere of 5% CO2. For the studies described here, weused the HSV-1 1716 virus, which contains a 759-bp deletion within bothcopies of ICP34.5, located in the terminal 1 kb of the long repeat region ofthe wild-type HSV-1 strain 17� [2]. Wild-type HSV-1 17� and the HSV-11716 mutant were propagated in Vero cells, purified, concentrated byhigh-speed centrifugation, and titered by black plaque assay as describedelsewhere using standard methods [46].

Replication of HSV-1 in cells. The efficiency of replication of HSV-1 17�

and HSV-1 1716 in cells was as previously described [9]. Briefly, both 4T1and Vero cells at 80–85% confluency were infected at a m.o.i. of 0.1 withHSV-1 17� and HSV-1 1716. Cells were infected for 1 h at 37°C. Theinoculum was removed and the cell monolayers were washed twice withDMEM and overlaid with 2 ml of culture medium. The “zero” time pointwas taken after washing of the cell monolayers immediately followinginfection. At the appropriate time points, the cells were scraped into themedium and frozen at 70°C. Samples were thawed and refrozen twiceprior to titering on Vero cells as described above.

Establishment and therapy of 4T1 mouse mammary tumors. FemaleBalb/c mice (Taconic, Germantown, NY) or SCID mice (Wistar InstituteAnimal Facility, Philadelphia, PA) at 6–10 weeks of age were injectedsubcutaneously in the abdominal mammary gland with 1 � 104 viable 4T1cells. Tumors were palpable by day 7–8 post-injection of 4T1 cells and hada mean tumor diameter of 3 to 5 mm. The mean tumor diameter wascalculated as the square root of the product of two perpendicular diameters[20]. On days 9 and 13 (double therapy) or days 9, 13, and 16 (tripletherapy), each tumor was injected using a 27.5-gauge needle in two orthree places with a total volume of 20 �l containing either 5.4 � 105 pfuof HSV-1 1716 or DMEM (mock-infected). The mean tumor diameter wasmeasured every 2–4 days using a caliper. Tumor diameters of each mousewere normalized by subtracting the mean tumor diameter measured onday 8 post-injection of tumor cells from the mean tumor diameter mea-sured on each subsequent day. On day 27 post-injection of 4T1 cells, micewere divided into two groups. The first group was assayed for survival andthe second group assayed for lung metastases as described below. SCIDmice were sacrificed and assayed for lung metastases as described below onday 26 post-injection of 4T1 cells.

For the second challenge of tumor cells, 4T1 tumors were established

and received triple therapy as described above. On day 18 post-injection of4T1 cells, mice were anesthetized using 70 mg/kg ketamine/xylazine andthe primary tumors were surgically removed. The wounds were suturedusing wound clips. Another 1 � 104 viable 4T1 cells were then injectedinto the contralateral side of the abdomen. The second challenge of cellswas injected on the same day as the primary tumor was removed to allowtime to monitor growth of the secondary tumors before the mock-treatedmice began to succumb to metastases starting approximately 38 dayspost-injection of primary tumor cells. Seven days following the secondinjection, the size of the tumors was again measured on the days indicated.Mice that became moribund were euthanized according to IACUC guide-lines of both the University of Pennsylvania and the Wistar Institute.

Replication of HSV-1 1716 in 4T1 tumors. Balb/c mice with established4T1 tumors were given triple therapy as described above. Tumors wereharvested on different days post-injection of virus and snap frozen. On thedays of viral therapy, tumors were harvested immediately after injection ofvirus. Tumors were thawed at 37°C in 3 ml of DMEM and homogenizedusing a tissue homogenizer. Tumor-cell suspensions were then refrozenand thawed twice. Following centrifugation, the tumor supernatant wasassayed for HSV-1 1716 levels by plaque assay as described above.

Survival analyses. On day 27 post-injection of tumor cells, mice wereanesthetized using 70 mg/kg ketamine/xylazine and the tumors weresurgically removed such that the tumor would not regrow. The woundswere sutured using wound clips. The mice were then monitored regularlyfor any signs of illness and were euthanized when moribund according toIACUC guidelines of both the University of Pennsylvania and the WistarInstitute.

Quantitation of clonogenic lung metastases. Metastases of 4T1 cells to thelungs were quantified as described [20]. Briefly, mice were sacrificed toremove the lungs, which were then treated with elastase and collagenaseand filtered through a 70-�m nylon cell strainer to isolate cells. Dilutionsof lung cells were plated in tissue culture medium in the presence of 60 �M6-thioguanine to allow for clonogenic growth of 4T1 cell colonies. Thio-guanine-resistant cell colonies, which appear in 10–12 days, were fixed inmethanol and stained with methylene blue for counting.

Immunohistochemistry. 4T1 tumors were established and received eitherHSV-1 1716 or mock injections on days 9, 13, and 16 post-injection oftumor cells. Two mice for each group were sacrificed and their tumorsremoved on days 10, 12, 14, 15, 17, 18, 20, 22, and 23 post-injection oftumor cells. The harvested tumors were embedded in Tissue-Tek OCTCompound (Fisher Scientific), snap frozen in 2-methylbutane, and sec-tioned at 6 �m. Sections were stained with standard hematoxylin andeosin or used in immunohistochemical staining. For immunohistochem-ical staining, briefly, sections were fixed in a descending series of ethanolwashes, quenched with 0.3% peroxide in PBS, and blocked in 5% goatserum. Primary antibody, either rat anti-mouse CD4 (L3T4) or CD8 (Ly-2)(1:100, 1:80, respectively; BD Biosciences, San Diego, CA) diluted in PBS,was incubated on sections at room temperature for 1 h. Secondary anti-body, biotinylated anti-rat made in goat (1:1500; Vector Laboratories,Burlingame, CA), diluted in PBS, was incubated on sections at roomtemperature for 45 min. Positive antibody reactivity was detected using anindirect avidin–biotin immunoperoxidase method (Vectastain ABC kit)with VectorVIP as the chromogen, and nuclei were then counterstainedusing methyl green, all used per the manufacturer’s instructions (VectorLaboratories). Sections were washed in between each step with PBS andwere blocked before each step except the chromogen and counterstainingsteps using 5% goat serum in PBS. As a negative control for specificity, 5%goat serum was used in place of primary antibody.

Statistical analyses. The unpaired t test was used to analyze differences inmean tumor diameters, and Kaplan–Meier curves, using the Wilcoxon testfor significance, were used for analyses of survival experiments. Statisticalanalyses of lung metastases were performed on the ln of the number ofmetastases to give a valid P value since the normal distribution of thenumber of metastases is very broad. All analyses were performed usingStatview, and a P value less than 0.05 was considered significant.

ARTICLE doi:10.1016/S1525-0016(03)00236-3


ACKNOWLEDGMENTS

We thank Kevin O’Brien at East Carolina University (Greenville, NC) for guid-ance with statistical analyses, Srikanth Yellayi for help with histological anal-yses, Daniel Ruge for technical assistance, and the members of the Fraserlaboratory for helpful discussions. This work was supported by a grant from theNational Institutes of Health (NS37516) to N.W.F. D.L.T. was supported by aNRSA (CA93034) from the National Cancer Institute.

RECEIVED FOR PUBLICATION JANUARY 10; ACCEPTED JULY 5, 2003.

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