Breast cancer is one of the most commonly diagnosed cancers among American women, and while early detection and intervention have led to a reduction in the associated mortality rate over the past few decades, the development of targeted, patient-specific therapies promises greater success in treating the progressed disease. For example, estrogen receptor (ER), which is expressed by tumors in a subset of breast cancer patients, can facilitate expression of genes responsible for growth and proliferation of breast cancer cells, and thus represents an attractive therapeutic target.
Given the rapid pace and growing expense of therapeutic development, high-content in vitro screening approaches represent an attractive approach for assaying target-specific effects of novel drug candidates. However, since cell-extracellular matrix (ECM) interactions, which may not be well represented in traditional 2D cell culture models, are often crucial to the expression of drug targets, the application of 2D cell culture models is not always the most effective means to screening targeted therapeutic compounds. Specifically, in the context of breast cancer, several investigations have reported a loss of ER-expression throughout standard 2D culture.
In order to explore the effect of 2D versus 3D culture on both ER expression and the consequential response of each culture model to a variety of ER-targeting and non-targeting therapeutics, we implemented the commercially available Wood cell model (Cellaria Biosciences, which was derived from an ER (+), PR (weak+), grade 1, stage T2N0M0 invasive ductal and lobular carcinoma of the breast in a Caucasian female, age 65-69, with a BMI of 33.98. Spheroids cultured in Thermo Fisher multi-well tissue culture plates for 2D adherent culture or Corning® Ultra-low Attachment Spheroid Micro-plates for 3D spheroid culture were dosed with chemotherapeutic agents, labeled with Thermo Fisher’s LIVE/DEAD™ Fixable Red Dead Cell Stain and with Thermo Fisher antibodies against ER and Ki67 (to determine proliferation), and cleared with Visikol® HISTO-M™ for high-content imaging via the Thermo Fisher CX7 LZR. As a result, cell viability and proliferation could be quickly compared across a range of drug treatments in both formats. Herein, we present compelling findings suggesting a difference in expression of ER in 2D versus 3D spheroid culture and consequently a difference in therapeutic response.
Wood cell culture
Wood cells were maintained in Basal Renaissance Essential Tumor Medium (RETM) with RETM supplement, 25 µg/mL cholera toxin, 5% fetal bovine serum, 1x antibiotic-antimitotic in a humidified, 37°C, 5% CO2 incubator and passaged via light trypsinization with 1X TrypLE and 1 mM EDTA upon reaching 80% confluence. Trypsinized cells were resuspended in complete medium and 1 x 103 cells were plated in each of 384 wells of a Corning Round Bottom Ultra Low Attachment Spheroid Microplate or 2 x 103 cells were plated in each of 96 wells of a Thermo Fisher optically clear, tissue culture treated plate. Spheroids and 2D cell cultures were maintained under standard culture conditions for 2 days, half of the media volume was exchanged to dose with drugs listed in Table 1 in complete media, and cultures were maintained for an additional 2 days prior to labeling.