Patient-Derived Breast Cancer Tissue Cultures for Anti-Endocrine Drug Assays

Abstract

Breast cancer is a complex and heterogeneous pathology, characterized by a variety of histological and molecular phenotypes. The majority of the breast cancers express the estrogen receptor alpha (ER), which plays a pivotal role in the pathobiology of the disease and are therefore classified as ER-positive (ER⁺). In fact, targeting of the ER signaling pathway is the main therapeutic strategy for ER⁺ breast cancer. Despite the success of endocrine therapy, intrinsic and acquired resistance are reported in 30-50% of the ER⁺ breast cancers. However, the mechanisms underlying ER heterogeneity and therapeutic resistance are far from being fully disclosed, and efficacious clinical strategies to overcome resistance are still pending. One of the hurdles in studying ER⁺ breast cancer resistance is related with the scarcity of experimental models that can recapitulate ER heterogeneity and signaling. This is the case of ER⁺ breast cancer cell models, typically based on cells derived from metastasis, which also fail to recapitulate tumor complexity. Primary cultures of patient-derived breast cancer cells are difficult to establish, and generally characterized by stromal fibroblasts overgrowth and rapid loss of phenotypic and molecular traits of the tumor cells, including ER expression. Ex vivo cultures of breast cancer tissue have been reported to retain the tissue architecture, with preservation of the tumor microenvironment (TME) and ER expression for short periods of time.Given the cumulating evidence on the role of the TME in sustaining ER⁺ tumor cells, we hypothesized that TME preservation in culture would favor the long-term retention of ER expression and signaling. We employed alginate encapsulation to provide a supporting scaffold to breast cancer tissue microstructures, coupled to dynamic culture to improve the lifespan of the culture by avoiding diffusional limitations. In this chapter, we provide a detailed description of this culture methodology, which has been previously published by our group (Cartaxo et al., J Exp Clin Cancer Res 39:161, 2020), based on electrostatically driven breast cancer tissue encapsulation in alginate, coupled to culture under agitation in a defined culture medium. We also describe challenge of the ex vivo model with an ER activator and inhibitors (anti-endocrine drugs) and a gene expression endpoint of drug response using reverse transcription PCR-based analysis of three distinct genes downstream of ER.

Keywords: Alginate; Breast cancer; Estrogen receptor signaling; Ex vivo; Explants; Patient-derived cancer tissue culture; RT-qPCR; Tumor microenvironment.