PDF(Pubmed)
Abstract：
UNASSIGNED: Primary luminal breast cancer cells lose their identity rapidly in standard tissue culture, which is problematic for testing hormone interventions and molecular pathways specific to the luminal subtype. Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge. Our goal was to adapt short-term organoids of luminal breast cancer for parallel testing of genetic and pharmacologic perturbations.
UNASSIGNED: We freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 μl replicates for increased throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these \"zero-passage\" organoids by withdrawing β-estradiol and adding 4-hydroxytamoxifen. We also examined sulforaphane as an electrophilic stress and commercial neutraceutical with reported anti-cancer properties. Downstream mechanisms were tested genetically 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, and organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times.
UNASSIGNED: We achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Success was mostly independent of clinical parameters, supporting general applicability of the approach. 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 tested. Combining sulforaphane with knockout of NQO1 (a phase II antioxidant response gene and downstream effector of sulforaphane) also yielded a breadth of organoid growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined.
UNASSIGNED: Zero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. This includes testing drug mechanisms of action in different clinical cohorts. A future goal is to relate inter-patient variability of zero-passage organoids to long-term outcomes.
UNASSIGNED: We freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 μl replicates for increased throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these \"zero-passage\" organoids by withdrawing β-estradiol and adding 4-hydroxytamoxifen. We also examined sulforaphane as an electrophilic stress and commercial neutraceutical with reported anti-cancer properties. Downstream mechanisms were tested genetically 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, and organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times.
UNASSIGNED: We achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Success was mostly independent of clinical parameters, supporting general applicability of the approach. 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 tested. Combining sulforaphane with knockout of NQO1 (a phase II antioxidant response gene and downstream effector of sulforaphane) also yielded a breadth of organoid growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined.
UNASSIGNED: Zero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. This includes testing drug mechanisms of action in different clinical cohorts. A future goal is to relate inter-patient variability of zero-passage organoids to long-term outcomes.
摘要：
背景原发性管腔乳腺癌细胞在标准组织培养中迅速失去身份，这对于测试激素干预和管腔亚型特有的分子途径是有问题的。乳腺癌类器官被认为可以更好地保留肿瘤特征，但管腔亚型病例的长期生存是一个持续的挑战。我们的目标是使管腔乳腺癌的短期类器官适应遗传和药理学扰动的平行测试。方法我们从管腔肿瘤刮片中新鲜分离出患者来源的细胞，将类器官形式小型化为5微升重复，以增加通量，并设置14天的终点以最小化漂移。通过撤回β-雌二醇并添加4-羟基他莫昔芬，可以在这些零代器官中模拟治疗性激素靶向。我们还研究了萝卜硫烷作为亲电应力和具有抗癌特性的商业营养药物。通过两个互补sgRNA的慢病毒转导和Cas9稳定在类器官培养的第一周进行遗传测试下游机制。通过RT-qPCR或RNA测序测量转录变化，和器官表型通过连续明场成像定量，数字图像分割,和细胞倍增时间的回归模型。结果我们在从肿瘤擦伤中启动管腔乳腺癌类器官并将其维持至14天零传代终点方面取得了>50%的成功率。成功大多与临床参数无关，支持该方法的一般适用性。在终点时，零过器官中ESR1和PGR的丰度始终保持在患者变异性的范围内。然而,在接受测试的管腔内乳腺癌病例中,对激素戒断和阻断的反应性差异很大.将萝卜硫烷与NQO1（一种II期抗氧化反应基因和萝卜硫烷的下游效应子）的敲除结合也产生了广泛的类器官生长表型，包括用萝卜硫素抑制生长，NQO1基因敲除促进生长，和生长拮抗作用结合在一起。结论零代器官是一种快速且可扩展的方式，可以从患者来源的材料中询问管腔乳腺癌细胞的特性。这包括测试不同临床队列中的药物作用机制。未来的目标是将零代器官的患者间变异性与长期结果相关联。
