Abstract

Materials and Methods

Results

At week 33, lobular carcinomas were palpable in all 10 mammary glands of BALB–NeuT females carrying the activated HER-2/neu oncogene (Fig. ​(Fig.11 A). FVB–NeuN females carrying the HER-2/neu protooncogene display one or more mammary glands with tumor at 49 wk, and the mean number affected at 61 wk is still very low (Fig. ​(Fig.11 B). This rapid onset and total gland involvement suggests that expression of activated HER-2/neu in BALB–NeuT mice requires few, if any, additional genetic events to transform the mammary epithelial cell (7), whereas the delayed onset and asynchronous progression observed in the other line indicates that overexpression of HER-2/neu protooncogene results in stochastic tumor development.

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Figure 1

Progression of HER-2/neu carcinogenesis in untreated BALB–NeuT and FVB–NeuN female mice. (A) Successive alterations of the mammary gland in untreated BALB–NeuT mice and indications of the time of 5-d courses of rIL-12 administration (□, 50 ng/day; , 100 ng/day). Histology shows that ductular atypical hyperplasia (a) is already evident and widely distributed in all mammary glands at 3 wk of age. This hyperplasia progresses to carcinoma in situ (b) between wk 13 and 17, and then to an invading lobular carcinoma (c). (B) Successive alterations of the mammary gland in FVB– NeuN mice during the 61-wk follow-up. Squares (□, , as in A). Histology shows that the mammary glands are normal (a) until at least wk 29 of age. Thereafter the neoplastic process begins in one mammary gland with focal hyperplasia and carcinoma in situ (b) and progresses by giving rise to an invasive lobular carcinoma (c) between wk 39 and 48. Original magnification: ×630.

These kinetic patterns provide two distinct models of HER-2/neu mammary oncogenesis with which to test the effect of prolonged administration of low doses of rIL-12. Since histologic examination revealed that widespread atypical hyperplasia was already evident in the mammary glands of 3-wk-old BALB–NeuT mice, and HER-2/neu mammary oncogenesis quickly progressed to overt carcinoma, these mice first received a 5-d course of 50 ng of rIL-12 plus MSA, injected intraperitoneally at 2 wk of age. The dose was then increased to 100 ng, and 5-d courses followed by 3 wk off were run from wk 5 to 29 (Fig. ​(Fig.11 A). MSA control mice received similar courses of MSA only. Mice were inspected weekly. Those displaying progressively growing masses in all 10 mammary glands and those that reached 33 wk of age were killed and morphologically examined.

A significant delay in the onset of the first mammary tumor was evident in the treated mice as compared with the MSA controls. Moreover, at wk 33 the number of mammary glands with a palpable tumor was much lower (Fig. ​(Fig.2,2, top). Histologic examination of the mammary glands at progressive time points in a fashion blind to the treatment revealed a general delay in the progression of the HER-2/ neu oncogenesis in the treated mice. Atypical hyperplasia was less vigorous and less widely distributed than in the MSA controls, and no carcinoma in situ was found before wk 15 (data not shown). This delay in tumor progression was associated with deficient neovascularization and a marked increase in infiltrating CD8⁺, and to a lesser extent CD4⁺, lymphocytes (Table ​(Table1).1). Lymphocyte infiltration was concomitant with enhanced vascular cell adhesion molecule (VCAM)-1 expression (Fig. ​(Fig.3),3), as well as expression of mRNA for proinflammatory cytokines and their production (Table ​(Table1).1). Extensive expression of iNOS on tumors from treated mice was shown immunohistochemically (Fig. ​(Fig.3).3). This suggests that IL-12-primed NO exerted a cytotoxic and antitumor activity directly or through its reactive products (11, 12). Moreover, strong expression of IP-10 and MIG mRNA was revealed by reverse transcriptase PCR in the tumor area from treated mice (Table ​(Table1).1). These two chemokines mediate the antiangiogenic activity of IL-12, and, in combination, chemoattract activated T cells (13– 15). Their modulation is probably a consequence of IL-12– induced expression of IFN-γ and TNF-α in NK and T cells. IFN-γ and TNF-α also amplify the production of proinflammatory cytokines, angiogenic inhibitors, and NO by reactive cells (15). This multifactorial scenario offers an explanation for both the inhibition of tumor neoangiogenesis and vascular damage leading to the impressive ischemic and hemorrhagic necrosis of advanced carcinomas in rIL-12–treated BALB–NeuT mice (Fig. ​(Fig.4).4).

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Figure 2

Inhibition of mammary carcinogenesis in HER-2/neu transgenic female mice treated with rIL-12. Percentage of tumor-free mice and mean number of palpable mammary carcinomas per mouse calculated as cumulative number of incident tumors/total number of BALB–NeuT mice, BALB–NeuT mice CD8⁺ T lymphocyte depleted by neonatal thymectomy and anti-CD8⁺ mAb treatment, and FVB–NeuN mice treated with rIL-12 () or MSA only (□). Stars () denote time points at which tumor multiplicity was significantly different (P <0.05 at least) between MSA and rIL-12 treatment groups. Group sizes were: BALB– NeuT: 38 MSA control mice and 43 rIL-12–treated mice; thymectomized and anti-CD8⁺ mAb treated BALB–NeuT mice: 10 MSA control mice, 6 rIL-12–treated mice; FVB–NeuN: 8 mice each group.

Table 1

Effects of rIL-12 Treatment on Neovascularization, the Expression of Adhesion Molecules, the Secretion of Mediators, and the Infiltration by Reactive Cells in 7-mm Mean Diameter Tumors Growing in BALB–NeuT and FVB–NeuN Female Mice

		BALB–NeuT mice				FVB–NeuN mice
		MSA		MSA + IL-12		MSA		MSA + IL-12
Pathologic findings (immunohistochemistry)
Microvessel count		15.9 ± 2.1		9.5 ± 1.3 *		19.7 ± 3.7		10.5 ± 2.4*
Endothelial adhesion molecules
ICAM-1		+		+		+		+
VCAM-1		−		++		−		++
ELAM-1		−		+/−		−		−
Infiltrating cells
Macrophages		12.5 ± 4.1		13.4 ± 3.8		16.1 ± 4.2		14.2 ± 5.2
Granulocytes		5.2 ± 1.9		7.6 ± 2.3		5.0 ± 2.8		6.9 ± 3.0
CD8+ lymphocytes		2.9 ± 0.8		42.0 ± 8.3 *		4.8 ± 2.1		14.6 ± 6.1*
CD4+ lymphocytes		3.1 ± 1.7		15.2 ± 5.1*		3.6 ± 1.9		5.7 ± 2.6
Cytokines and mediators
IL-1β		−		+/−		−		+/−
TNF-α		+/−		+		+/−		+
IFN-γ		−		+		−		+
IL-6		+/−		+		+/−		+
iNOS		−		++		−		++
Reverse transcriptase PCR (mRNA)
Cytokines and chemokines
IL-1β		++		+++		NT‡		NT
TNF-α		−		+		NT		NT
IFN-γ		−		+/−		NT		NT
IL-6		++		++		NT		NT
GM-CSF		+/−		+		NT		NT
MIG		+		++		NT		NT
IP-10		+		++		NT		NT

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Microvessel counts were performed on cryostat sections tested with an antiendothelial (CD31) mAb as described in Materials and Methods. The expression of adhesion molecules, cytokines, and mediators on cryostat sections was probed with the corresponding mAb. From mice with multiple tumors, two or more samples (one sample per tumor) were evaluated. At least 10 fields were counted per sample. Values are expressed as mean ± SD of five Balb–NeuT and four FVB–NeuN mice. An arbitrary score (from − to +++) was used to indicate the amount of detected signal in a semiquantitative reverse transcriptase PCR analysis, as described in Materials and Methods. At least three independent tumors from Balb–NeuT mice were evaluated, and a representative expression pattern is shown.  

^*Value significantly different (P <0.001) than that in MSA-treated mice.  

^‡NT, not tested.  

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Figure 3

Cryostat sections of invading lobular carcinomas in MSA control (a and c) and IL-12–treated (b and d) BALB–NeuT mice. VCAM-1 is absent in tumor blood vessels of MSA control mice (a), whereas it is expressed in those of rIL-12–treated mice (b) as revealed by anti–VCAM-1 mAb. In the MSA control tumors, iNOS expression is almost undetectable in macrophages (c), whereas in rIL-12–treated mice it is clearly evident in macrophages intermingled among tumor cells (d) as shown by anti-iNOS mAb. Observations made in 7-mm mean diameter tumors. Similar patterns are evident in all established tumors. Original magnification: ×630.

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Figure 4

Histologic features of invading lobular carcinomas in BALB–NeuT (a) and FVB–NeuN (d) MSA control mice and in rIL-12–treated BALB– NeuT (b and c) and FVB–NeuN (e and f) mice. The impressive ischemic and hemorrhagic necrosis disaggregating the neoplastic masses becomes evident from wk 27 in BALB–NeuT (b) and wk 53 in FVB–NeuN (e) mice. In both cases it eventually results in extensive tumor destruction (c and f). Original magnification: ×630.

Our previous studies have shown that CD8⁺ lymphocytes play a pivotal role in the antitumor reaction elicited by IL-12, whereas CD4⁺ cells play an inhibitory role (8, 16, 17). To evaluate the weight of CD8⁺ cells in rIL-12-delayed oncogenesis, BALB–NeuT mice treated with rIL-12 or MSA only were thymectomized at 4 wk and received 200 μg of anti-CD8 mAb intraperitoneally. Both the progression of mammary oncogenesis and the protective effect of IL-12 were unaffected by this permanent CD8⁺ depletion (Fig. ​(Fig.2,2, middle).

The protective effect of rIL-12 was also evident in FVB– NeuN mice. Here, because of the slower tumor progression and the virtual absence of atypical mammary hyperplasia foci before wk 29, the initial 5-d course was administered to 21-wk-old mice (Fig. ​(Fig.11 B), followed the by same schedule as for the BALB–NeuT mice until wk 57. Mice displaying a tumor mass >2 cm mean diameter and those that reached the wk 61 of age were killed: 50% of the treated mice were tumor-free, whereas all of the MSA controls displayed palpable tumors. The mean number of mammary glands with a palpable tumor was 2.6 per mouse in the MSA controls compared with only 0.9 in the IL-12–treated mice (Fig. ​(Fig.2,2, bottom). Deficient peri- and intratumoral vascularization, enhanced expression of adhesion molecules, enhanced CD8⁺ infiltration, cytokine production, and iNOS activation (Table ​(Table11 and Fig. ​Fig.5),5), and large areas of hemorrhagic necrosis in established tumors (Fig. ​(Fig.4)4) were evident, as in the IL-12–treated BALB–NeuT mice.

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Figure 5

Cryostat sections of invading lobular carcinomas in MSA control (a and c) and IL-12–treated (b and d) FVB–NeuN mice. TNF-α (b) and IFN-γ (d) are evident in the tumor growth area in IL-12–treated mice, whereas both are absent (a and c) in the MSA control, as revealed by staining with anti–TNF-α and –IFN-γ mAb in tumor masses of 49-wk-old mice. Original magnification: ×630.

Increased spleen and liver extramedullary hematopoiesis and a slight exacerbation of hepatic periportal mononuclear infiltration were the only signs of chronic rIL-12–related lesions in both lines of mice, showing that this cytokine is effective at well-tolerated doses.

Discussion

rIL-12 administration in young BALB–NeuT mice carrying the activated Her-2/neu oncogene delays the appearance of tumor and reduces the number of mammary glands involved. In adult FVB–NeuN mice carrying the HER-2/ neu protooncogene, neoplastic progression is less impetuous and 50% of IL-12–treated mice are tumor-free at 1 yr. Since pathologic examinations suggest that rIL-12 does not lead to an appreciable failure in normal mammary tissue development, the increased percentage of FVB–NeuN–protected mice may mean that progression was delayed by rIL-12 beyond the 61 wk of the experiment.

The protection offered by rIL-12 could, of course, be ascribable to its triggered killing of neoplastic cells, thus off-setting the continuous generation of new transformed cells by the transgenic mammary tissue. However, in contrast with the rIL-12–induced immune reaction against established carcinomas (8), the results obtained with thymectomized BALB–NeuT mice suggest that CD8⁺ lymphocytes do not play a major role here, even if they are the dominant population of reactive cells infiltrating the tumor area in rIL-12–treated mice. It is possible that NK cells are the leading actors in this IL-12–provoked delay in oncogenesis (18). Since the NK1.1 mAb recognizes an allelic form of an NK cell antigen expressed by C57BL/6 but not BALB/c mice, the type and function of lymphocytes first involved are currently under investigation in (BALB/c × C57BL/6) NeuT⁺ F1 mice.

The nonessential role of cytotoxic CD8⁺ lymphocytes in vivo fits in well with both the marginal cytotoxic activity and the lymphokine release observed when T lymphocytes from rIL-12–treated mice were restimulated in vitro with APCs pulsed with the product of the rat HER-2/neu oncogene (p185), a self-protein in these transgenic mice (data not shown). These findings, along with morphologic data, rather suggest that rIL-12 essentially protects by halting angiogenesis (19) through the induction of several secondary cytokines and mediators, thus slowing down the transition from hyperplasia to overt carcinoma (20, 21). Moreover, the secondary cytokines and the tertiary chemokines and monokines elicited by IL-12 are apparently responsible for later vessel endothelial wall injury and hemorrhagic necrosis of established tumors (12, 13, 22–24). The endothelial vessel alteration and hemorrhagic necrosis observed in both lines of rIL-12–treated mice are reminiscent of those observed during rejection of an established transplantable mammary adenocarcinoma in BALB/c mice treated with systemic rIL-12 or adenocarcinoma cells engineered to release IL-12 (8).

Whether immunological maneuvers in healthy individuals at risk prevent the development of cancer is a question that has rarely been examined. Most studies have evaluated immunization before transplantation with frank tumor cells. Immunoprevention of cancer has been attempted in a few models of chemical carcinogenesis. Interpretation of these experiments may be confounded by the fact that carcinogens may be immunosuppressive.

Noguchi et al. have shown that similar doses of IL-12 inhibit 3-methylcholanthrene carcinogenesis in mice through the release of cytokines and mediators and nonspecific immune mechanisms (25). Our findings extend these observations by underscoring the potential of nonspecific immune mechanisms and their cross-talk with the endothelial cells of tumor vessels in the inhibition of HER-2/neu oncogenesis.

The HER-2/neu oncogene is expressed in a substantial proportion of human mammary carcinomas and its product seems to be a promising target for specific immune intervention (26). However, its expression in humans is not the same as the expression of a xenogenic oncogene in transgenic mice, and this issue may somewhat impact specific immunization rather than nonspecific immune mechanisms. The close resemblance of the progression of mammary carcinogenesis in HER-2/neu transgenic mice to that in women suggests that chronic administration of nontoxic rIL-12 regimens may be a significant prophylactic strategy. The direct proportionality between the length of carcinogenesis progression and the efficacy of rIL-12 suggest that a “soft” immunologic alternative to controversial and distasteful mastectomy may be envisaged.

Acknowledgments

Abbreviations used in this paper

Footnotes

References