Using organoids to understand mechanisms and consequences of aging in breast.

Aging is overwhelmingly the main risk factor for carcinomas in humans, indeed, nearly 80% of all breast cancers are diagnosed in women over 50 years of age. Invertebrates lack mammary glands, and the relationship between age and mammary tumor incidence in mice differs significantly from humans. Examination of breast tissue in situ and in primary culture has revealed the luminal epithelia as a site of significant phenotypic changes with age that merits additional scrutiny because that is the region where breast cancer cells of origin reside. We utilize human mammary epithelial organoids that are reconstituted from a diverse bank of primary human mammary epithelial cells (HMEC) to examine the mechanisms and consequences of aging that lead to increased breast cancer susceptibility. Our 3D culture system enables highly parallel phenotypic analysis of thousands of reconstituted organoids for long periods of time, up to 200 days, in Matrigel-free conditions. Maintaining the luminal lineage in standard culture has been a persistent challenge for more than three decades, however, reconstituted multilineage organoids and bilayer cultures provided the microenvironment cues to maintain luminal cells for long-term. Heterochronus luminal-myoepithelial organoids revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. Age-driven changes in luminal-myoepithelial cell gap- and tight-junctions are implicated as key facilitators of the age-related states. We provide evidence that the luminal lineage is exquisitely sensitive to microenvironment conditions, and that states of aging are cell-non-autonomously communicated through microenvironment cues over at least one cell diameter.