New C3H KitN824K/WT cancer mouse model develops late-onset malignant mammary tumors with high penetrance

Abstract

Gastro-intestinal stromal tumors and acute myeloid leukemia induced by activating stem cell factor receptor tyrosine kinase (KIT) mutations are highly malignant. Less clear is the role of KIT mutations in the context of breast cancer. Treatment success of KIT-induced cancers is still unsatisfactory because of primary or secondary resistance to therapy. Mouse models offer essential platforms for studies on molecular disease mechanisms in basic cancer research. In the course of the Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program a mouse line with inherited polycythemia was established. It carries a base-pair exchange in the Kit gene leading to an amino acid exchange at position 824 in the activation loop of KIT. This KIT variant corresponds to the N822K mutation found in human cancers, which is associated with imatinib-resistance. C3H Kit^N824K/WT mice develop hyperplasia of interstitial cells of Cajal and retention of ingesta in the cecum. In contrast to previous Kit-mutant models, we observe a benign course of gastrointestinal pathology associated with prolonged survival. Female mutants develop mammary carcinomas at late onset and subsequent lung metastasis. The disease model complements existing oncology research platforms. It allows for addressing the role of KIT mutations in breast cancer and identifying genetic and environmental modifiers of disease progression.

Figure 1

Hematological characterization of C3H Kit^N824K/WT mutant mice. Red blood cell counts (a), mean corpuscular volume (b) and mean corpuscular hemoglobin (c), platelet counts (d), total white blood cell counts (e) and percentage of granulocytic cells (f) of controls (WT) and heterozygous (Het) mutant animals (n = 9–11 per group, age 20-21 weeks), representative pictures of IHC for MPO of the spleen of a control (upper panel) and a mutant animal (lower panel) at age 6 months (g), Cell counts of femoral bone marrow derived from female control and heterozygous mutant mice collected at age 6 (n = 10 per group) or 12 months (n = 4 per group) (1 h), representative picture of bone marrow histology in the femoral epiphysis of 12 months old control (upper panel) and mutant (lower panel) animals (i). ****p < 0.0001; ***p > 0.001, **p < 0.01, *p < 0.05 (Wilcoxon Rank Sum Test).

Figure 2

Genetic characterization of C3H Kit^N824K/WT mutant mice. Sequence tracing of the mutation in exome 17 of the Kit gene (a) sequence obtained from a control animal (upper panel) and from heterozygous animal showing the mutant phenotype (lower panel), (b) exemplary depiction of genotyping results using PCR-RFLP method (upper panel) or heteroduplex detection using the lightscanner device (lower panel).

Figure 3

Pathology of the gastrointestinal tract of C3H Kit^N824K/WT mutant mice: Representative pictures of the forestomach wall of a control (a) and two mutant animals (b,c) showing a layer of hyperproliferating cells between the inner circular and outer longitudinal muscle layer (arrows), the glandular stomach wall of a control (d) and two mutant animals (e,f), higher magnification in g-i shows the thin layer of ICCs between the muscular layers (g, arrow), which is thickened due to ICC hyperplasia in mutant animals (h,i, arrows), IHC for KIT of the gastric wall in a control (j) and a mutant (k) animal—higher magnification in l—demonstrating KIT-positivity of ICCs in the wild-type and hyperproliferating cells (arrow) in the mutant animal, macroscopic (inserted picture) and histologic appearance of the cecum in a 6 months old wild-type animal (m) and a 12 months old control (p) compared to a 6 months old mutant with prominent Peyer´s patches (star) (n,q) and a 12 months old mutant animal with ingesta retention and extensive inflammation (star) (o,r).

Figure 4

Pathology of mammary tumors found in older female C3H Kit^N824K/WT mutant mice: Macroscopic (a) and histological (b,c) appearance of mammary tumors found in older female mutant mice. A high magnification of a region in the periphery of the tumor (c) shows normal mammary ducts (black arrows) and mammary intraepithelial neoplasia (MIN) lesions (bright pink arrows). Overview of immunohistochemistry staining results (negative = blue, positive = brown) and schematic depiction of observed staining patterns (membranous, cytoplasmic, nuclear) and staining intensities (dark brown = strong, light brown = weak) in 14 investigated tumors (d). Representative IHC pictures of KIT (e), ER (f,g), PR (h,i) and ERBB2 (j) in the mammary tumors. Examples of a negative (left) and a positive (right) staining for ER and PR are shown. Nuclear ER was also detected in some tumors (d). Mammary tumor Giemsa staining representative images for the identification of mast cells (k,l). H&E-stained representative picture of a lung metastasis found in an old mutant female with a mammary tumor (m).

Figure 5

Histological characterization of mammary tumors in C3H Kit^N824K/WT mutant mice: Tumors showed solid (a), glandular (b) or mixed (c) morphology, with strong Ki67 positivity observed in all types (a–c). Representative pictures of different mixed morphological tumor types (d–f), the most frequent type (d) with solid and glandular morphologies present in the same tumor (upper left), with necrotic areas (lower left), cysts (upper right) and fat droplet infiltration (lower right) were observed (d). A sclerotic/solid tumor is shown (e), with keratin deposition (upper right) and connective-tissue-rich sclerotic areas surrounding solid sheets (lower right), and an example of a papillary/solid tumor (f) with solid areas (upper right) and papillary (finger-like) tumor growth projections (lower right).

Figure 6

Phenotypic variability of the cecum in mutant N2-hybrid animals. Macroscopic pictures of the cecum derived from four 8 months old N2-hybrid littermates showing polycythemia (a–d), histopathology of the tumor depicted in (d) in 10-fold (e) and 40-fold (f) magnification.