Exploring our in vitro model system for the factors that control it

We investigated possible epidemiological factors associated with how well primary cultures from non-diseased breast explants formed secondary breast ductal structures in culture. We found that African American women developed these structures, on average, in half the time of European white women. Based on this observation, we compared gene expression, stem cell compartment size and stem cell potency between breast explant cultres from African American and European White women

We found that African American reduction mammoplasty cell lines had increased proportions of stem cells, defined as CD24-/44+/49f+ cells relative to European White, and that these cells were also more capable of bi-potency than those from European White derived cell lines. Although preliminary, we also found that in samples from European White women stem cell compartments are more consistent with those seen in post-menopausal derived cell lines. However, this aspect of the study is limited by the lack of available post-menopausal cell lines. The present results rely on a very small number for post-menopause analysis, one African American cell line and two European White cell lines. Another limitation of this study is the that we have not determined the percent admixture in the self-declared African American and European-derived White

subjects. We have additional samples that may reflect patients that have more European White lineage than African American lineage, so all cell lines need to be validated for admixture.

We know that there are “windows of vulnerability,” for genetic mutations, in breast development that coincide with breast developmental phases (Martinson, Lyons, Giles, Borges, & Schedin, 2013). During these windows, women are more sensitive to genotoxic agents and this may affect their risk of developing breast cancer (Martinson et al., 2013; Russo & Russo, 2011). African American women’s stem cell characteristics are different then European-derived White women and they develop ductal structures in culture, differently, as well. African American women go through thelarche, one of the “vulnerability” phases, at much younger ages than European derived white woman. They may therefore respond at different times and differently to factors that influence breast cancer (Bodicoat et al., 2014; Cabrera et al., 2014; Martinson et al., 2013).

We have developed an in vitro model of thelarche where we can test various chemicals and how they affect breast architecture. The Latimer tissue engineering system is a model of thelarche when it spontaneously undergoes ductal development, in which breast stem cells play a role. Our first attempts at using this system as a model for the thelarche utilized the simple characteristic of formation of epispheres. Episphere formation is an active process that has been documented in time lapse movies in the laboratory.

We have shown that the development of JL BRL-14 epispheres is responsive to estrogen and BPA in a dose dependent manner. With further validation and use of other estrogenic and antiestrogenic compounds this early work lays the foundation for the development of a medium throughput assay. The use of high throughput instruments such as the Thermofisher CellinsightTM will allow us to turn this assay into a medium throughput assay. Additional work is already under way, and it is our hope that we can test additional chemicals for their putative role in hormone disruption of breast

differentiation. RNA sequencing will be used to determine the gene expression changes and potential mutation that are present as a result of these exposures. In addition, pre- disposed cultures that are already on the path to cancer can be used instead of reduction mammoplasties in order to see visible steps towards cancer.

Episphere formation from women of different ancestries could also be tested when the system is optimized. The two cell lines tested were both European White, but they had the capacity to form architecture over later passages. Only one was

reproducible and another approach to chemical testing might be to use only one for all chemicals in the tradition of the ESCREEN.

An interesting experiment would compare nucleotide excision repair function of normal stem cells in African American and European-derived White women to their respective non-stem cell populations with the unscheduled DNA synthesis assay. Then, using the Latimer tissue engineering system, we would expose the derived cells to

mutagenic chemicals. This would give us valuable information. First, can non-diseased stem cells be transformed in this system to malignancy? Then, do African American and European-derived White women explants respond differently to insult? If malignant cells are formed, do we see differences in the malignances of European White and African American women, i.e. the resultant breast cancer subtype? This can be taken further by testing the resulting transformed cells by again sorting and measuring the nucleotide excision repair capacity after malignancy, in the parent, cancer stem cells, and cancer non-stem cells. A major obstacle in this experiment would be maintaining the stem cell state during the transformation process. Historically, it has been shown that

transformation assays are extremely difficult to make work in vitro and the long period of time necessary for exposure to cause enough somatic mutations would cause the stem cells to differentiate (Tralau & Luch, 2012).