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.Significance: 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. Cancer Res; 78(21); 6209-22. ©2018 AACR.

Figure 1.. Characteristics of the human ILC and IDC cell lines used in the study.

A. Western blotting using the indicated antibodies on whole cell lysates from ILC (left; red) and IDC (right; blue) cell lines. β-Actin was used as a loading control. B-C. Time course (top) and relative ER target gene expression (bottom) after estrogen and anti-estrogen treatment in hormone-deprived (B) MDA-MB-330 and (C) BCK4 cells. Graphs show representative data (n=6) from two-three experiments. Control: vehicle (Ethanol). E2: Estradiol (100 pM); 4-OHT: 4-hydroxytamoxifen (100 nM); Fulv: Fulvestrant (ICI 182, 780, 100 nM). p-values are from two-way ANOVA with Dunnett’s multiple comparison test to Control. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. D. Merged images and higher magnification insets of co-immunofluorescence staining for E-cadherin (red) and p120 (green) in ILC (top) and IDC (bottom) cell lines. DAPI (blue) was used for counterstaining to mark nuclei. Scale bar: 10 μm.

Figure 2.. Human ILC cell lines exhibit superior growth in ULA culture than human IDC cell lines.

A. Phase contrast light microscopy images of ILC (red; top) and IDC (blue; bottom) cell lines in 6-well 2D and ULA plates 4 days post plating. Scale bar: 100 μm. B. Relative growth curves showing fold growth normalized to day 0 at each time point over 6 days for ILC (red; left) and IDC (blue; right). Graphs show representative data from three experiments (n=6). p-values are two-way ANOVA comparison of 2D and ULA. * p ≤ 0.05; **** p ≤ 0.0001.

Figure 3.. ILC and IDC cell lines exhibit varying morphologies in 3D culture.

Phase contrast light microscopy images of (A) ILC (red) and (B) IDC (blue) cell lines in soft agar (4 weeks post plating), Collagen I, Matrigel embedded and Matrigel on-top culture (ILC: 3 weeks post plating; IDC: 1 week post plating). Scale bar: 100 μm.

Figure 4.. Human ILC cell lines have differing preferences for adhesion to ECM proteins.

A. Phase contrast light microscopy images of ILC (red; left) and IDC (blue; right) cell lines in uncoated, Collagen I or BSA-coated plates 16 hours post-plating. Scale bar: 100 μm. B. Fold adhesion (normalized to BSA) of ILC (red) and IDC (blue) cell lines 16 hours post-plating. Graphs show representative data from two experiments (n=4). p-values are from ordinary one-way ANOVA with Dunnett’s multiple comparison test to BSA for each cell line. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.

Figure 5.. Human ILC cell exhibit limited migration ability in wound-scratch assays.

A. Snapshots of the scratch wounds in ILC (red; left) and IDC (blue; right) cell lines at the indicated time points. Scale bar: 100 μm. B. Snapshots of the scratch wounds in BCK4 (red) and MCF7 (blue) cell lines treated with 100 nM PMA. Arrows indicate migratory protrusions at the wound edge. Scale bar: 300 μm. C. Relative wound densities over hour 0 in ILC (red; left) and IDC (blue; right) cell lines over time with and without PMA treatment. Graphs shows representative data from two-three experiments (n=6–8). p-values are from two-way ANOVA comparison of -PMA vs +PMA. * p ≤ 0.05; **** p ≤ 0.0001.

Figure 6.. Human ILC cell lines exhibit limited migration and invasion towards FBS but SUM44 and MDA-MB-330 exhibit haptotaxis to Collagen I in transwell Boyden chamber assays.

A-D. Images (top) and quantification (bottom) of crystal violet-stained inserts with ILC (red; left) and IDC (blue; right) cell lines from (A) Chemotaxis (B) Haptotaxis (C) Collagen I invasion and (D) Matrigel invasion assays towards the indicated attractants after 72 hours. Graphs show representative data from two independent experiments (n=3 biological replicates). p-values are from unpaired t-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 7.. Transcriptional differences between human ILC and IDC cell lines and tumors.

A. Supervised clustering heat map of ILC (red) and IDC (blue) cell lines using differentially expressed genes. B. PCA clustering plot of ILC and IDC cell lines and tumors using top 1,000 differentially expressed genes. C. Venn diagrams of commonly upregulated (top) and downregulated (bottom) genes between ILC and IDC cell lines, tumors and organoids. D. Disease-specific survival curves for PPFIBP2 (top) and PLOD2 (bottom) in ER+, Luminal A ILC (red; left) and IDC (blue; right) patients in the METABRIC dataset. Patients were divided into two groups by PPFIBP2 (q3: third quadrant) and PLOD2 (q1: first quadrant) expression. p-values are from log-rank test. q-values were calculated using the Benjamini-Hochberg method to correct p-values for multiple comparisons testing within histology. The small ILC sample size (n=60) allows for limited statistical power in detecting survival differences after multiple testing correction.