Wnt-activating human skin organoid model of atopic dermatitis induced by Staphylococcus aureus and its protective effects by Cutibacterium acnes.

A recently developed human PSC-derived skin organoid model has opened up new avenues for studying skin development, diseases, and regeneration. The current model has limitations since the generated organoids are enclosed, circular aggregates with an inside-out morphology with unintended off-target development of cartilage. Here, we first demonstrated that Wnt signaling activation resulted in larger organoids without off-target cartilage. We optimized further using an air-liquid interface (ALI) culture method to recapitulate structural features representative of human skin tissue. Finally, we used the ALI-skin organoid platform to model atopic dermatitis by Staphylococcus aureus (SA) colonization and infection. SA infection led to a disrupted skin barrier and increased production of epidermal- and dermal-derived inflammatory cytokines. Additionally, we found that pre-treatment with Cutibacterium acnes had a protective effect on SA-infected organoids. Thus, this ALI-skin organoid platform may be a useful tool for modeling human skin diseases and evaluating the efficacy of novel therapeutics.

The KRAS-variant and its impact on normal breast epithelial cell biology.

MicroRNA (miRNA)-binding site variants in 3' untranslated regions (3'UTRs) are a novel class of germ-line, functional mutations, which are now recognized as powerful biomarkers of human cancer risk and biology. The first mutation discovered in this class is the KRAS-variant, a let-7-binding site mutation in the 3'UTR of the KRAS oncogene. The KRAS-variant predicts increased cancer risk for certain populations, is a predictive biomarker of cancer treatment response across cancer types, leads to conserved tumor biology and elevated AKT signaling in KRAS-variant patient tumors, and was recently found to predict elevated TGF-ß and immunosuppression in cancer patients. Based on the functional biology of the KRAS-variant in cancer patients, here we chose to investigate altered normal cellular biology in the presence of the KRAS-variant, through interrogation of an isogenic normal breast epithelial cell line model with and without the KRAS-variant. We find that KRAS-variant normal breast epithelial cells exhibit a mesenchymal phenotype, which appears to be due to numerous molecular changes, including miRNA dysregulation and autocrine pathway alterations, including elevated TGF-ß, resulting in ZEB and SNAIL upregulation. Our findings support the hypothesis that the KRAS-variant has a fundamental biological impact on normal cellular biology, that is conserved in these patients when they develop cancer.

Estrogen withdrawal, increased breast cancer risk and the KRAS-variant.

The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42-37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer.

Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro.

BACKGROUND: The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. METHODS: We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 µg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. RESULTS: The level of luciferase activity of follicles with 3 µg pGL4 was significantly (P < 0.05) greater than the 1 µg and 2 µg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 µg of D-luciferin. CONCLUSIONS: The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a new bioluminescence imaging model. This study is the first to demonstrate that reporter genes can be transferred to intact granulosa cells with a lipid-mediated gene transfer method within intact follicles in vitro, and the level of transgene expression can be assessed by bioluminescence imaging in living intact antral follicles.