Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice

Abstract

The ability to genetically manipulate mice has led to rapid progress in our understanding of the roles of different gene products in human disease. Transgenic mice have often been created in the FVB/NJ (FVB) strain due to its high fecundity, while gene-targeted mice have been developed in the 129/SvJ-C57Bl/6J strains due to the capacity of 129/SvJ embryonic stem cells to facilitate germline transmission. Gene-targeted mice are commonly backcrossed into the C57Bl/6J (B6) background for comparison with existing data. Genetic modifiers have been shown to modulate mammary tumor latency in mouse models of breast cancer and it is commonly known that the FVB strain is susceptible to mammary tumors while the B6 strain is more resistant. Since gene-targeted mice in the B6 background are frequently bred into the polyomavirus middle T (PyMT) mouse model of breast cancer in the FVB strain, we have sought to understand the impact of the different genetic backgrounds on the resulting phenotype. We bred mice deficient in the inducible nitric oxide synthase (iNOS) until they were congenic in the PyMT model in the FVB and B6 strains. Our results reveal that the large difference in mean tumor latencies in the two backgrounds of 53 and 92 days respectively affect the ability to discern smaller differences in latency due to the Nos2 genetic mutation. Furthermore, the longer latency in the B6 strain enables a more detailed analysis of tumor formation indicating that individual tumor development is not stoichastic, but is initiated in the #1 glands and proceeds in early and late phases. NO production affects tumors that develop early suggesting an association of iNOS-induced NO with a more aggressive tumor phenotype, consistent with human clinical data positively correlating iNOS expression with breast cancer progression. An examination of lung metastases, which are significantly reduced in PyMT/iNOS-/- mice compared with PyMT/iNOS+/+ mice only in the B6 background, is concordant with these findings. Our data suggest that PyMT in the B6 background provides a useful model for the study of inflammation-induced breast cancer.

Fig. 1

PyMT tumorigenesis in the FVB and B6 strains. (A) The day at which the first mammary tumor was detected by palpation in each mouse was recorded and a Kaplan-Meier survival plot generated. Log rank analysis indicated a highly significant difference, P < 0.001 between the curves. FVB mice N = 44, B6 mice N = 46. (B) Whole mounts of mammary glands from 3 week old PyMT/iNOS^+/+ mice of the FVB (left panel) and B6 (right panel) strains

Fig. 2

Effects of iNOS deficiency on mammary tumor development. (A) Whole mounts of PyMT/iNOS^+/+ and PyMT/iNOS^−/− #4 mammary glands from 11 week FVB mice or 20 week B6 mice. LN = lymph node. (B) Kaplan Meier plots of the tumor latency of PyMT/iNOS^+/+ annd PyMT/iNOS^−/− mice of the 2 strains. Data were analyzed by the log rank test. PyMT/iNOS^+/+ FVB N = 44, PyMT/iNOS^−/− FVB N = 57, PyMT/iNOS^+/+ B6 N = 46, PyMT/iNOS^−/− B6 N = 26

Fig. 3

Mammary tumor growth kinetics. (A) Mean tumor volume per mouse. FVB N = 32, B6 N = 44. Data are means ± SEM. (B) Tumor latency for individual B6 PyMT/iNOS^+/+ and PyMT/iNOS^−/− tumors. Data were derived from examining 460 B6 PyMT/iNOS^+/+and 260 PyMT/iNOS^−/−tumors

Fig. 4

Distribution of B6 mammary gland tumor burden. (A) Total tumor burden was significantly lower in PyMT/iNOS^−/− mice, ***P < 0.001. (B) Examination of individual tumors indicated that a significant difference in genotypes was only found in the #3 and #4 mammary glands, although in each gland the mean tumor burden was lower in PyMT/iNOS^−/− mice. *P < 0.05 ANOVA followed by Bonferroni post tests. PyMT/iNOS^+/+ B6 N = 24 (black bars), PyMT/iNOS^−/− B6 N = 25 (grey bars)

Fig. 5

Tumor latency of individual B6 mammary glands. Tumor latency was lower in the #1 mammary gland in PyMT/iNOS^+/+ mice compared with all other mammary glands (upper panel). A similar result was observed in PyMT/iNOS^+/+ mice, with an increase in disparity between the latency of the #1 gland and all other glands (middle panel). When the #1 and #4 latencies for both genotypes were overlayed, a greater difference in mean tumor latency was observed between the #4 glands when compared with the #1 glands (lower panel)

Fig. 6

Tumor metastasis. (A) HE stained paraffin embedded sections of PyMT/iNOS^+/+ (left panel) and PyMT/iNOS^−/− (right panel) lungs. (B) Comparison of metastatic tumor burden in FVB and B6 mice *P < 0.05 Mann-Whitney U test. PyMT/iNOS^+/+ FVB N = 14, PyMT/iNOS^−/− FVB N = 13, PyMT/iNOS^+/+ B6 N = 17, PyMT/iNOS^−/− B6 N = 21