Acute inflammation is essential for initiating and coordinating the body's response to injuries and infections. However, in acute inflammatory diseases, inflammation is not resolved but propagates further, which can ultimately lead to tissue damage such as in sepsis, acute respiratory distress syndrome and deep vein thrombosis. Currently, clinical protocols are limited to systemic steroidal treatments, fluids and antibiotics that focus on eradicating inflammation rather than modulating it. Strategies based on stem cell therapeutics and selective blocking of inflammatory molecules, despite showing great promise, still lack the scalability and specificity required to treat acute inflammation. By contrast, polymeric particle systems benefit from uniform manufacturing at large scales while preserving biocompatibility and versatility, thus providing an ideal platform for immune modulation. Here, we outline design aspects of polymeric particles including material, size, shape, deformability and surface modifications, providing a strategy for optimizing the targeting of acute inflammation.

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection post-stasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serum-free medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ.

Aging is associated with tissue-level changes in cellular composition that are correlated with increased susceptibility to disease. Aging human mammary tissue shows skewed progenitor cell potency, resulting in diminished tumor-suppressive cell types and the accumulation of defective epithelial progenitors. Quantitative characterization of these age-emergent human cell subpopulations is lacking, impeding our understanding of the relationship between age and cancer susceptibility. We conducted single-cell resolution proteomic phenotyping of healthy breast epithelia from 57 women, aged 16-91 years, using mass cytometry. Remarkable heterogeneity was quantified within the two mammary epithelial lineages. Population partitioning identified a subset of aberrant basal-like luminal cells that accumulate with age and originate from age-altered progenitors. Quantification of age-emergent phenotypes enabled robust classification of breast tissues by age in healthy women. This high-resolution mapping highlighted specific epithelial subpopulations that change with age in a manner consistent with increased susceptibility to breast cancer.