Comprehensive Phenotypic Characterization of Human Invasive Lobular Carcinoma Cell Lines in 2D and 3D Cultures

Abstract

Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin-mediated adherens junctions. Despite displaying unique histologic and clinical features, ILC still remains a chronically understudied disease, with limited knowledge gleaned from available laboratory research models. Here we report a comprehensive 2D and 3D phenotypic characterization of four estrogen receptor-positive human ILC cell lines: MDA-MB-134, SUM44, MDA-MB-330, and BCK4. Compared with the IDC cell lines MCF7, T47D, and MDA-MB-231, ultra-low attachment culture conditions revealed remarkable anchorage independence unique to ILC cells, a feature not evident in soft-agar gels. Three-dimensional Collagen I and Matrigel culture indicated a generally loose morphology for ILC cell lines, which exhibited differing preferences for adhesion to extracellular matrix proteins in 2D. Furthermore, ILC cells were limited in their ability to migrate and invade in wound-scratch and transwell assays, with the exception of haptotaxis to Collagen I. Transcriptional comparison of these cell lines confirmed the decreased cell proliferation and E-cadherin-mediated intercellular junctions in ILC while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism, and alternative cell adhesion molecules such as N-cadherin, some of which were differentially regulated in ILC versus IDC tumors. Altogether, these studies provide an invaluable resource for the breast cancer research community and facilitate further functional discoveries toward understanding ILC, identifying novel drug targets, and ultimately improving the outcome of patients with ILC. These findings provide the breast cancer research community with a comprehensive assessment of human invasive lobular carcinoma (ILC) cell line signaling and behavior in various culture conditions, aiding future endeavors to develop therapies and to ultimately improve survival in patients with ILC. .

Estrogen controls mTOR signaling and mitochondrial function via WNT4 in lobular carcinoma cells

“…Consistent with clinical observations, we identified tamoxifen-resistance in ER-positive ILC models, including de novo ER partial agonism by anti-estrogens [13,14]. These data raise the question of whether ER function and signaling is unique in the context of ILC.…”

Proteomic and Transcriptomic Profiling Identifies Mediators of Anchorage-Independent Growth and Roles of Inhibitor of Differentiation Proteins in Invasive Lobular Breast Cancer

“…SUM44PE (SUM44) cells were purchased from Asterand and BCK4 cells were kindly provided by Britta Jacobsen, University of Colorado Anschutz, CO. Cell lines were maintained as previously described [23] in the following media (Life Technologies) with 10% FBS: MDA-MB-134 and MDA-MB-330 in 1:1 DMEM:L-15, MCF7 and MDA-MB-231 in DMEM, T47D in RPMI, BCK4 in MEM with non-essential amino acids (Life Technologies) and insulin (Sigma-Aldrich). SUM44 cells were maintained as described [25] in DMEM-F12 with 2% charcoal stripped serum and supplements.…”

“…2D and ULA growth assays were performed as previously described [23]. ILC (15,000/96-well; 300,000/6-well) and IDC (5,000/96-well; 100,000/6-well) cells were seeded in regular (Thermo Fisher Scientific) or ULA (Corning Life Sciences) 96-well plates.…”

“…Immunoblots were performed as previously described [23] using 5% milk powder for blocking and developed using ECL (Sigma-Aldrich). Details of the antibodies used in Additional file 1: Table S1 .…”

“…We have recently published a comprehensive phenotypic characterization of human ER-positive ILC cell lines in 2D and 3D cultures, where we reported that ILC cells exhibit a remarkably unique ability to efficiently grow in ultra-low attachment (ULA) culture as compared to IDC cells [23]. Given the importance of anchorage-independence in tumor cell dissemination and metastasis [2, 13, 15, 24], herein we characterized the cellular and molecular mechanisms underlying the ability of human ILC cells to survive in detached conditions.…”