Establishment and Culture of Patient-Derived Breast Organoids

Abstract

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

Figure 1:. Establishing patient-derived breast organoids.

(A) Schematic representation of the major steps in establishing patient-derived breast tumors or normal organoids. (B) Representative images showing growth of DS117T patient-derived breast tumor organoids from establishment (passage 0) to long-term culture (passage 12). Blue arrows = fibrous material, yellow arrows = debris/dead cells, and red arrows = supporting cell types from the microenvironment (mainly fibroblasts). Scale bar = 100 μm.

Figure 2:. Patient-derived breast organoids representing different subtypes.

Top panel: morphological differences between organoid lines derived from patient breast tumors (B–E) of different histopathological and molecular subtypes. (A) The figure represents organoids derived from normal human breast tissue obtained post-reductive mammoplasty surgery. Scale bar = 50 μm. Bottom panel: H&E stained images of the same organoid lines. Scale bar = 100 μm. Abbreviation: TNBC = triple negative breast cancer.

Figure 3:. Growth in culture of different patient-derived breast tumor organoids.

Representative bright-field images and cell viability curves (A and B), as measured via a luminescent cell viability assay showing the growth in culture of different patient-derived breast tumor organoid lines over 12 days. Scale bar = 50 μm. Error bars represent SEM for n = 6.