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(The FASEB Journal. 2002;16:1749-1754.)
© 2002 FASEB

Prevention of spontaneous mammary adenocarcinoma in HER-2/neu transgenic mice by foreign DNA

LUCIA SFONDRINI*, DARIO BESUSSO*, CRISTIANO RUMIO{ddagger}, MONICA RODOLFO{dagger}, SYLVIE MÉNARD* and ANDREA BALSARI§1

* Molecular Targeting Unit,
{dagger} Melanoma Genetics Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy; and
{ddagger} Department of Human Anatomy,
§ Institute of Pathology, University of Milan, 20133 Milan, Italy

1Correspondence: Chair of Immunology c/o Molecular Targeting Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. E-mail: balsari{at}istitutotumori.mi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Unmethylated CpG-oligodeoxynucleotides (CpG-ODNs) are recognized as a ‘danger signal’ and are potent immunostimulators. To test whether tumors might be prevented by maintaining the innate immune system on continuous alert, proto-neu transgenic female mice, which develop spontaneous mammary tumors, were systemically treated with CpG-ODNs at 10-day intervals. Tumor incidence and number of tumors/mouse were significantly lower in treated mice compared with the control group. Moreover, CpG-ODN systemic treatment significantly reduced lung metastases induced by intravenous inoculation of N202.1A cells derived from a spontaneous mammary carcinoma. Growth of established tumors was modestly inhibited after CpG-ODN systemic treatment but strongly on peritumoral application. Our data indicate that systemic repeated injection of CpG-ODN to maintain the innate immune system on continuous alert prevents the onset of genetically determined tumors and confers tumor protection when the tumor load is low.—Sfondrini, L., Besusso, D., Rumio, C., Rodolfo, M., Ménard, S., Balsari, A. Prevention of spontaneous mammary adenocarcinoma in HER-2/neu transgenic mice by foreign DNA.


Key Words: murine tumors • CpG-oligodeoxynucleotides • danger signal • innate immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
STRATEGIES THAT STIMULATE the ability of the immune system to recognize and destroy cancer cells via selective killing mechanisms have shown promise in the treatment of cancer. However, the induction of active immunity against tumor antigens is difficult because most such antigens identified to date are normal or slightly modified (mutated) autoantigens with poor immunogenicity. An alternative strategy involves broad stimulation of the immune system using immunomodulatory agents. As proposed by Matzinger (1) , the immune system does not distinguish self from nonself, but rather responds to ‘danger’, that is, anything causing tissue distress or lytic cell death. According to the danger model, the immune system can fail to exert strong surveillance or to eradicate tumors because they are not recognized as ‘dangerous’. Studies have demonstrated that foreign DNA from bacteria, parasites, yeast, and insects is recognized as dangerous by the mammalian immune system and can potently stimulate it (2 3 4) . Unmethylated CpG-dinucleotide-containing sequences were found to be responsible for this immunostimulatory effect of bacterial DNA (5 6 7) . In vitro and in vivo analyses of rodents, primates and other animals indicate that unmethylated CpG motifs reproducibly stimulate innate immunity; indeed CpG-containing oligodeoxynucleotides (ODNs) enhance host resistance to a variety of infectious microorganisms and accelerate the development of long-lived pathogen-specific immunity (3 , 8 9 10) . Moreover, a single dose of CpG-ODN has been reported to provide protection for ~2 wk before pathogen exposure (10 , 11) . An antitumor effect of CpG-ODNs was recently evaluated in different experimental models (12 13 14 15 16) .

We asked whether tumor development might be prevented by maintaining the innate immune system on continuous alert through repeated CpG treatment of N202 proto-neu transgenic mice. In these mice, the presence of the rat neu proto-oncogene driven by the mammary tumor virus promoter/enhancer induces spontaneous focal mammary tumors that overexpress the neu-encoded p185 protein in all females (17) , although stochastic development of the tumors and the long latency period indicate a requirement for additional events in tumor formation. In HER-2/neu transgenic mice, immunological tolerance to HER-2/neu, analogous to observations in patients with HER2/neu-overexpressing breast cancer, has been documented (18) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and cells
FVB-NeuN (#N202) transgenic mice (17) , on the H-2q FVB inbred background and carrying the rat HER-2/neu proto-oncogene under the transcriptional control of the mouse mammary tumor virus promoter, were bred and treated in accordance with institutional guidelines.

Tumor cell line N202.1A, derived from a spontaneous mammary carcinoma in an FVB-neuN transgenic mouse, was obtained from Dr. P. Nanni (Istituto di Cancerologia, Bologna, Italy) and routinely maintained in RPMI medium supplemented with 10% FBS, glutamine, and antibiotics.

Oligonucleotides
Purified single-stranded phosphorothioated ODN 1668 (5'-TCCATGACGTTC CTGATGCT-3') containing a CpG motif (19) was synthesized under endotoxin-free conditions by M-Medical-GENENCO (Firenze, Italy). Phosphorothioate modification was used to reduce the susceptibility of the ODN to DNase digestion, thereby significantly prolonging its half-life in vivo.

Analysis of CpG-ODN effect on spontaneous tumors
Ten-week-old individually tagged virgin FVB-NeuN female mice were inoculated intraperitoneally (i.p.) with CpG-ODNs (20 µg dissolved in 200 µL of saline) every 10 days until 54 wk of age (11 mice). A control group (11 mice) was left untreated. Mammary glands were inspected twice each week and two perpendicular diameters of tumor masses were recorded. Progressively growing masses of >3 mm in mean diameter were considered tumors. Tumor volumes were calculated as {pi}/6 x length x width2 (2) .

In another set of experiments, different CpG-ODN injection schedules were evaluated in FVB-NeuN female mice displaying a palpable spontaneous tumor of ~4 mm (mean diameter). Groups of 4 to 10 mice were inoculated with CpG-ODN i.p. (20 µg in 200 µL of saline twice weekly for 4 wk or 40 µg daily for 5 consecutive days) or peritumorally (20 or 40 µg in 200 µL of saline daily for 5 days). Tumor growth was monitored twice weekly with two perpendicular diameters of tumor masses recorded and volumes calculated as above.

Analysis of CpG-ODN effect on inoculated tumors
To evaluate memory immune response, four FVB-NeuN female mice treated with CpG-ODNs and tumor-free at 54 wk of age were injected subcutaneously (s.c.) in the right flank with 3 x 105 N202.1A carcinoma cells and tumor growth monitored twice weekly as above. Four male mice of the same age were injected s.c. with N202.1A cells only and used as control.

In experiments to evaluate CpG-ODN activity on experimental lung metastases, groups of eight male FVB-NeuN mice injected intravenously (i.v.) with 3 x 105 N202.1A carcinoma cells were inoculated i.p. with 20 µg CpG-ODNs in 200 µL of saline 4 h before or 2 or 48 h after tumor cell injection, or with 40 µg CpG-ODNs in 200 µL of saline 4 h before tumor cell injection and in the ensuing 4 days. Control mice were injected with tumor cells only. All mice were killed 4 wk later and superficial lung metastases were counted after lung insufflation with 10% India ink and bleaching in Fekete solution.

Immunofluorescence and cytotoxicity assays
Mouse serum samples were obtained during the experiment for prevention of spontaneous tumors by bleeding from the retroorbital venus plexus. Flow cytometry was used as described previously (20) to detect reactivity of sera against the tumor cell line N202.1A.

The induction of CTL and NK responses was assessed by a standard 4 h 51Cr release assay. Three mice bearing a small s.c. tumor were inoculated i.p. with CpG-ODNs (20 µg in 200 µL of saline) four times every 10 days. The day after the last CpG-ODN treatment, mice were killed and splenic lymphocytes were cultured at effector/target ratio of 200:1, 100:1, 50:1, and 25:1 with 51Cr-labeled N202.1A or YAC target cells. Splenic lymphocytes from untreated mice were used as control. Percent cytotoxicity was calculated as follows: % cytotoxicity = [(sample cpm) - (spontaneous cpm)/(total cpm) - (spontaneous cpm)] x 100.

Morphological examination and histochemistry
Mammary gland tissue samples were obtained from six female transgenic mice treated with 20 µg CpG-ODNs every 10 days from 10 wk of age. At 200, 250, and 300 days of age, two animals were killed. Untreated female transgenic mice were used as controls. Samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E) for evaluation by light microscopy.

For histochemistry, sections were collected on silanizated slides, deparaffinized, and rinsed in TBS/BSA 0.3%. After retrieval of the antigen with microwave oven in Tris-HCL 0.01 M buffer pH 8 and the quenching of endogenous peroxidase in H2O2 0.3% in TBS/BSA 0.3% for 15 min, sections were incubated with primary antibody against F4/80 (Caltag Laboratories, Burlingame, CA) for 1 h at 4°C, followed by Vectastain detection kit (Vector Laboratories, Burlington, Canada). The sections were then counterstained with light H&E and mounted.

Statistics
Differences in tumor incidence were calculated by Fisher’s exact test; differences in mean number of tumors per mouse, mean tumor volume, and mean number of lung metastases were calculated by unpaired t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The effect of CpG-ODNs on the development of spontaneous mammary tumors was evaluated in 22 transgenic virgin female mice randomly divided into two groups and treated i.p. with CpG ODNs (20 µg/mouse) every 10 days starting at 10 wk of age or left untreated. Tumor incidence and number of tumors/mouse were significantly lower in treated mice compared with the control group. All untreated mice developed mammary tumors before 305 days of age, whereas 5 of 11 of CpG-ODNs-treated mice were tumor-free (P=0.03, Fisher’s exact test) (Fig. 1 A) at that time and four were still tumor-free at the end of the experiment (380 days). The number of mammary palpable tumors was also significantly lower in mice treated with CpG-ODNs vs. the control group (P=0.02, unpaired t test) (Fig. 1B ), with almost twice the number of tumors in controls compared with the treated mice (27 vs. 14) by the end of the experiment. Tumor growth rate did not differ significantly in the two groups (Fig. 1C ). The reduced tumor incidence in CpG-ODN-treated mice was not due to inhibition of mammary gland development since light microscopy analysis of mammary tissue architecture revealed no differences between the treated and control mice. Indeed, tissues from both groups revealed normal organization of ducts and alveoli and hyperplasia of ductal epithelium (Fig. 2 A, B). Immunohistochemical analysis of mammary tissue glands from CpG-ODN-treated mice, using the F4/80 antibody, revealed few macrophages as well as in control mice (Fig. 2C ). Small nodules of in situ carcinomas showed foci of macrophage infiltration in CpG-ODN-treated mice (Fig. 2E ) whereas large carcinomas obtained from treated or control mice were negative for F4/80 staining (Fig. 2F ).



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  		Figure 1. Spontaneous mammary carcinogenesis in FVB-NeuN transgenic female mice treated every 10 days from the age of 10 wk with CpG-ODNs ({blacksquare}) compared with untreated control mice ({square}). A) Tumor incidence; B) number of palpable mammary carcinomas per mouse (mean±SE) calculated as cumulative number of incident tumors/total number of mice. C) Tumor volume (mean±SE) in mice that developed tumors.



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  		Figure 2. Light microscopy images of H&E-stained mammary gland and carcinomas obtained from FVB-NeuN transgenic mice at 300 days of age. Mammary gland from mice untreated (A) or treated with CpG-ODNs (B). Tissue from both groups revealed normal organization of ducts and alveoli and hyperplasia of ductal epithelium (bar=100 µm). Immunohistochemical analysis for detection of F4/80-positive infiltrating cells: normal mammary gland showing focal macrophage infiltration (C), in situ carcinoma from untreated mice showing no macrophage infiltration (D), in situ carcinoma (E), and large carcinoma (F) from CpG ODN-treated mice showing a strong macrophage infiltration and no macrophage infiltration, respectively (bar=50 µm).

Because the profound tolerance of HER2/neu transgenic mice to the encoded oncoprotein can be partially overcome by vaccination (18) , we investigated whether extended CpG treatment had induced a memory immune response to HER2/neu. The four mice treated for 10 months with CpG-ODN and still tumor-free at the end of experiment (380 days) and four untreated, age-matched male mice as controls were injected s.c. with 3 x 105 N202.1A neu-positive carcinoma cells derived from a spontaneous mammary carcinoma in an FVB-NeuN transgenic mouse. No differences in tumor latency or in tumor growth rate were observed between CpG-ODN-treated and control mice (data not shown). Corresponding to the lack of memory response, serum samples obtained during the experiment investigating the prevention of spontaneous tumors showed the absence or very low titers of antibodies reacting with N202.1A neu-positive cells. In vitro assay using splenic lymphocytes obtained from three mice repeatedly i.p. treated with CpG-ODN showed no T cell cytotoxicity against N202.1A carcinoma cells (<2% of cytotoxicity at an effector/target ratio of 200:1). On the contrary, the same splenic cells showed 13–15% cytotoxicity on YAC target cells (at the same effector/target ratio) vs. 4–8% from untreated mice, indicating NK activation induced by CpG-ODN.

Because i.p. delivery of CpG-ODNs at 20 µg/mouse every 10 days did not significantly influence the growth rate of spontaneous tumors (Fig. 1C ), we tested other doses, routes, and schedules of treatment. In mice bearing small spontaneous mammary tumors, no significant tumor inhibition was observed using increased i.p. doses (up to 40 µg) and increased frequency of administration (up to every day for 5 days) (data not shown), whereas significant inhibition (P=0.01, unpaired t test) was observed when CpG-ODNs at 40 µg were injected directly at the tumor site for 5 days (Fig. 3 ).



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  		Figure 3. Inhibition of the growth of established mammary tumors in FVB-NeuN transgenic female mice treated at the tumor site with 40 µg of CpG-ODNs for 5 days (arrows) ({blacksquare}) compared with untreated control mice ({square}). Mean tumor volume (±SE).

Mice inoculated i.v. with N202.1A carcinoma cells formed significantly fewer lung metastases after 4 wk if treated with CpG-ODNs (20 µg/mouse) 4 h before or 2 h after tumor cell inoculation compared with control mice (P<0.05 unpaired t test) (Fig. 4 ). In mice injected with CpG-ODNs at a dose of 40 microgram, administered i.p. four hours before and in the 4 subsequent days, inhibition of experimental metastases was more extensive, although still incomplete. No inhibitory effect was observed when CpG-ODNs were administered 48 h after N202.1A cell injection (Fig. 4) .



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  		Figure 4. Number of superficial lung metastases (mean±SE) in pericardiac lobe at 4 wk after i.v. injection of 3 x 105 N202.1A tumor cells in mice inoculated i.p. with 20 µg CpG-ODNs 4 h before (B) or 2 h (C) or 48 h (E) after tumor cell injection or with 40 µg CpG-ODNs 4 h before tumor injection and for the next 4 days (D). Control mice received tumor cells only (A). *P < 0.05, unpaired t test.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Systemic administration of the CpG-ODN prevented the onset of mammary carcinoma in transgenic N202 mice, with 36% of mice remaining tumor-free after 380 days of observation. This extent of protection is comparable to or greater than that obtained with specific vaccines consisting of the extracellular domain of p185neu oncoprotein (21) or the neu NT DNA plasmid (22) or with vaccinations using irradiated whole cells transfected to express a mycobacterial protein (23) or infected with a rat neu recombinant vaccinia virus (18) . A higher protection was observed in a study using 10 repeated vaccinations with human HER2 cDNA ligated into the VR1012 expression vector (24) . These authors hypothesized that at least part of the protective effect of repeated human HER2 vaccinations might rest in the CpG sequences present in the VR1012 expression vector. Greater protection was also reported after treatment with exogenous interleukin-12 (IL-12) (25) or Tamoxifen (26) , in which the mechanism of prevention involves a reduction in the number of potential target cells for transformation, consistent with the clear loss in terminal alveoli cells in the mammary glands of treated mice (26 , 27) .

In therapy experiments, we observed significant growth inhibition of established tumors after peritumoral application of CpG-ODNs whereas systemic treatment induced only modest inhibition. On the other hand, systemic treatment led to a significant reduction in the number of lung metastases induced by i.v. inoculation of N202.1A tumor cells. It is possible that activation of the innate immunity is sufficient to prevent spontaneous mammary carcinomas and experimental metastases, when tumor load is low, whereas activation of both innate and acquired immunity is probably required for established tumors. Consistent with the notion of a predominant or exclusive role for innate immunity in the inhibitory effect of CpG-ODNs on spontaneous mammary tumors is the lack of protection against tumor challenge in mice receiving long-term treatment with CpG, as well as the absence of specific antitumor T lymphocytes and antibodies or of selection of HER2/neu-negative tumors in CpG-ODN-treated mice (data not shown).

Involvement of innate immunity in CpG-ODN-induced inhibition of experimental metastasis is in accord with results of a recent study using a lac-Z-transduced fibrosarcoma and mice analyzed 3 days after tumor cell inoculation (13) : the short time course of those experiments makes it unlikely that tumor-specific CTL development or that B-cell-derived, specific antitumor antibodies were involved in the CpG-ODN inhibitory effect.

The innate immune system can rapidly detect infections through cells such as dendritic cells, macrophages, monocytes, and neutrophils, which lack the sophisticated and exquisitely antigen-specific receptors of T and B cells, but instead have evolved a set of pattern recognition receptors (PRRs) that enable them to partially sense the environment. Some PRRs appear to be expressed only on particular cell subsets whereas others are more widely expressed (28) . Depending on the cell types activated and the molecular pathways triggered by particular PRRs, the immune system as a whole appears to integrate the resulting signals and respond in a fashion that has been evolutionarily selected for this pattern or combination of molecular patterns. Recently, a new family of PRRs, the Toll-like receptors, has been identified; the CpG-ODN 1668 used in our study has been shown to activate immune cells via the Toll-like receptor 9 (7) . In addition to the direct effects of CpG on NK cells (6) , B cells (5) , monocytes, macrophages (29) , and dendritic cells (30 , 31) , which play an important role in anti-tumor immunity, this motif induces enhanced production of several cytokines endowed with an antitumor activity, such as TNF-{alpha}, IL-12, and interferon-{gamma} (6 , 19) .

CpG peritumoral treatment of established tumors induced more potent inhibition of tumor growth than when administered at a distal site. This finding might rely on a greater local activation of innate immunity since inhibition was already observed in the first days of treatment. However, the possibility cannot be excluded that, as recently reported (32) , peritumoral CpG-ODN application activates antigen-presenting cells that have taken up tumor-derived material and enhances their migration to local lymph nodes where tumor-specific T cells are primed. In contrast to previous observations with s.c. induced tumors (12 , 32) , peritumoral treatment with CpG-ODN in our model inhibited but did not eradicate the tumors, possibly reflecting the low immunogenicity of spontaneous tumors. Indeed, the efficiency of CpG-ODN peritumoral therapy has been reported to correlate with the antigenicity of the tumors (32) .

In conclusion, our data show that systemic repeated injection of CpG-ODN to maintain the innate immune system on continuous alert prevents the onset of genetically determined tumors and confers tumor protection when the tumor load is low. A clear advantage of this immune intervention over vaccination with tumor antigens is the lack of requirement for selection and purification of tumor antigens. Assessment of clinical safety of these reagents is essential, especially since the lack of immunological memory implies a chronic treatment regimen.


   ACKNOWLEDGMENTS
 
Partially supported by AIRC and FIRST. L.S. was supported by a fellowship from FIRC. We thank Mrs. S. Celon for technical assistance and Mrs. L. Mameli for manuscript preparation.

Received for publication April 22, 2002. Accepted for publication July 15, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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