Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids

Abstract

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone Reprints and permissions information is available at www.nature.com/reprints.

Experimental models of endocrine responsive breast cancer: strengths, limitations, and use

“…However, EO771 cells were highly responsive to the isoflavone genistein, a compound that activates both ESR1 and ESR2. These results are consistent with ESR2 being reported to be expressed in EO771 cells and activation of ESR2 inhibiting breast cancer cell growth [145] .…”

“…EO771 mouse mammary tumor cells originated from a spontaneous ER+ mammary tumor in a C57BL/6 mouse [143] have been used as allografts for luminal B disease given their expression of ESR2 (ERβ), although these cells also have been classified as basal-like [144,145] . Since the stability and/or functionality of ER expression in EO771 tumors is inconsistently reported and basal-like tumors arise from allografted cells, EO771 has yet to be proven as a reliable and robustly responsive model in which to study ER+ breast cancers.…”

“…Other ER+ mouse mammary tumor cell lines have also been described, such as 67NR, but whether the resulting allografted tumors are responsive to antiestrogens has not been fully established [145] .…”

“…In addition to 3D growth of established cell lines, the generation of organoids from normal and neoplastic tissue from human subjects also offers novel opportunities to study key hypotheses in a more physiologically relevant context than simple 2D culture. Such organoids can be maintained and expanded in vitro and can be transfected with nucleic acids, enabling several lines of mechanism-based inquiry [ 146 ] . Patient-derived organoids can naturally exhibit some cellular heterogeneity.…”

Generation and functional characterization of murine mammary organoids

“… CRITICAL: After thawing, Matrigel must be held on ice until the time of plating. Alternatives: This protocol has been optimized using the Corning® Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix; however, alternative basement membrane matrices have been generated and used to grow mouse and human organoids ( Dekkers et al., 2021 ; Sachs et al., 2018 ). Matrigel is a naturally derived gel extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma and, as such, batch-to-batch variability in proteins content can occur.…”