Patient-derived response estimates from zero-passage organoids of luminal breast cancer
Background: Primary luminal breast cancers lose hormone receptors rapidly in standard tissue culture.  Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge.   Methods: We freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 µl replicates to increase throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these “zero–passage” organoids by withdrawing ß-estradiol and adding tamoxifen. We tested sulforaphane as an electrophilic stress and commercial neutraceutical. Genetic perturbations were introduced by lentiviral transduction of two complementary sgRNAs and Cas9 stabilization for the first week of organoid culture. Transcriptional changes were measured by RT-qPCR or RNA sequencing; organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times.   Results: We achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Few clinical parameters reliably impacted success. Abundance of ESR1 and PGR in zero-passage organoids consistently remained within the range of patient variability at the endpoint. However, responsiveness to hormone withdrawal and blockade was highly variable among luminal breast cancer cases. Combining sulforaphane with knockout of NQO1 (a phase II enzyme) also yielded a breadth of growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined.   Conclusions: Zero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. Overall design: RNA-seq was performed on paired tumor scrapes, organoids, and 2D cultured cells from 6 different patients (considered as batches). 6 different luminal A breast cancer cell lines were used as additional controls. For 5 of the patient-derived organoid cultures, we also treated with 0.1% DMSO or 200 nM or 3 µM 4-hydroxytamoxifen from day 5 to day 14 of culture.