Interleukin 12-mediated prevention of spontaneous mammary adenocarcinomas in two lines of Her-2/neu transgenic mice

Abstract

The ability of interleukin (IL)-12 to prevent tumors when administered to individuals with a genetic risk of cancer was studied in two lines of transgenic mice expressing rat HER-2/neu oncogene in the mammary gland. Female BALB/c (H-2(d)) mice carrying the activated HER-2/ neu oncogene show no morphological abnormalities of the mammary gland until 3 wk of age. They then progress through atypical hyperplasia to in situ lobular carcinoma and at 33 wk of age all 10 mammary glands display invasive carcinomas. Adult FVB mice (H-2(q)) carrying the HER-2/neu protooncogene develop mammary carcinomas with a longer latency (38-49 wk) and a lower multiplicity (mean of 2.6 tumors/mice). Treatment with IL-12 (5 daily intraperitoneal injections, 1 wk on, 3 wk off; the first course with 50 ng IL-12/day, the second with 100 ng IL-12/day) begun at 2 wk of age in BALB/c mice and at 21 wk of age in FVB mice markedly delayed tumor onset and reduced tumor multiplicity. Analogous results were obtained in immunocompetent and permanently CD8(+) T lymphocyte-depleted mice. In both transgenic lines, tumor inhibition was associated with mammary infiltration of reactive cells, production of cytokines and inducible nitric oxide synthase, and reduction in microvessel number, in combination with a high degree of hemorrhagic necrosis.

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.

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.

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.

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.

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.