Abstract 656: Histological and 3D morphological evaluation of mammary cancers and primary cells from genetically engineered mice with only one copy of Brca1 disrupted in combination with Trp53 haploinsufficiency
Authors: Sahar J. Alothman, Weisheng Wang, Bhaskar V. Kallakury, Priscilla Furth
Journal: Cancer Research (2016)

Abstract Background: Previously established genetically engineered mouse (GEM) models with spontaneous mammary cancer development have both Brca1 alleles disrupted withTrp53 haploinsufficiency (Brca1f11/f11/MMTV-Cre/Trp53+/-). Cell culture plates with nanoimprinted scaffolds (SCIVAX Life Sciences, Inc) purportedly increase concordance between in vitro/in vivo results but reports are limited to human cancer cell lines. Here we characterized hyperplasia and cancer development in Brca1f11/WT11/MMTV-Cre/Trp53+/- mice and established conditions for 3D culture of primary mammary epithelial cells (MEC) using the imprinted plates. Methods: Time-course of mammary hyperplasia and cancer development was defined in female Brca1f11/WT11/MMTV-Cre/Trp53+/- (n = 23) and Brca1f11/f11/MMTV-Cre/Trp53+/- (n = 13) mice euthanized at age 6 and 12 months (m) or when largest tumor reached 1 cm3. Mammary tissue was taken at necropsy for histology (age 6/12m) and primary MEC culture (age 6m) using EpiCult-B (StemCell Technologies) on nanoimprinted scaffold plates. Primary normal (wild-type, n = 2) and precancerous MEC (Brca1f11/f11/MMTV-Cre/Trp53+/-, n = 2; Brca1f11/WT11/MMTV-Cre/Trp53+/-; n = 2) were cultured. Optimal conditions for spheroid growth were established by comparison of spheroid formation from thoracic vs. inguinal mammary glands with different numbers of plated cells and percentage FBS after 7 days culture. Endpoints included sphere number, size and presence/absence of concurrent monolayer growth. Spheres were harvested for histological examination. Hematoxylin and eosin (H&amp;E) stained sections were read for histology and immunohistochemistry (IHC) for Pan-Cytokeratin (Pan-CK) and Ki67 performed for in vivo/in vitro comparisons. Results: Forty-two percent of Brca1f11/WT11/MMTV-Cre/Trp53+/- mice developed mammary cancer by age 12m compared to 100% of Brca1f11/f11/MMTV-Cre/Trp53+/- mice. Cancers appeared by age 6m only in Brca1f11/f11/MMTV-Cre/Trp53+/- mice. Mammary cancers in both models were carcinomas of either adeno, anaplastic or sarcomatoid types. All were Pan-CK positive and proliferation rates were similar between the two genetic models. Spheres developed from all primary MEC attempted. Optimal conditions for sphere formation were plating of 10,000 cells obtained from inguinal mammary gland per well in 5% FBS EpiCult-B. Conclusions: Here we report development of spontaneous mammary cancers in Brca1 insufficiency mice that are more representative of disease development in women who carry only one allele with a BRCA1-mutation and established conditions for use of cell culture plates with nanoimprinted scaffolds for 3D culture of normal and preneoplastic primary murine mammary epithelial cells. Supported by NCI, NIH 1RO1CA112176 (PAF). Citation Format: Sahar J. Alothman, Weisheng Wang, Bhaskar V. Kallakury, Priscilla Furth. Histological and 3D morphological evaluation of mammary cancers and primary cells from genetically engineered mice with only one copy of Brca1 disrupted in combination with Trp53 haploinsufficiency. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 656.