Exploring the role of BCL11A in the oesophagus and mammary gland using in vivo lineage tracing and 3D organoid models.

In the pursuit of effective cancer therapy, extensive disease heterogeneity remains one of the largest problems facing researchers. NGS sequencing has helped to identify specific markers of disease to deconvolute some of this heterogeneity, indeed it has also enabled the similarities of some epithelial tumours to be identified1. BCL11A has been identified as a triple negative breast cancer gene2. However, little is known about its function in the physiological mammary gland. In addition to its role in breast cancer, previous work in the lab has identified BCL11A as a selectively upregulated gene lung squamous carcinoma (LUSC)3 and some preliminary data suggest that it is also upregulated in oesophageal squamous carcinoma (OSCC). With proven roles in malignancy and little knowledge about its physiological function, this work reports the combined use of in vivo lineage tracing techniques and in vitro 3D organoids to explore the expression and gene function of Bcl11a in the oesophageal epithelium and mammary gland. Gene function studies were undertaken using mouse models of BCL11A overexpression: RosaCreERT2; Rosa-CAGG-LSL-BCL11A (BCL11AOE) and Bcl11a knock out: RosaCreERT2; Bcl11a flox/flox (Bcl11aKO). Bcl11a lineage tracing was achieved using a Bcl11aCreERT2; Rosa-CAG-LSL-tdTomato mouse model (Bcl11aCre). Within the murine mammary epithelium previous work in the lab revealed the localisation of Bcl11a expressing cells to the luminal progenitor compartment of the ductal network. Expansion of labelled cells in response to pregnancy was observed. To explore this physiological role further, mammary organoids were derived from single cells of the luminal progenitor compartment from both BCL11AOE and Bcl11aKO models. Organoids exhibiting these gene perturbations did not demonstrate phenotypic alterations. Further studies will be required to fully characterise the role of Bcl11a in the mammary gland. Oesophageal organoids cultivated from the BCL11AOE model demonstrated a lack of differentiation and a superior colony forming ability after passaging. Bcl11aKO organoids demonstrated an inhibition in growth and differentiation, inducing a loss of integrity in the organoid structures. Cells displayed a lower colony forming capacity after passage, suggesting Bcl11a expression was critical to organoid growth and differentiation. These findings were validated in vivo using lineage tracing. Bcl11a lineage tracing revealed the persistence and expansion of labelled cells in each layer of the squamous epithelium, for a period of up to 12 weeks post tamoxifen exposure. Labelling at 24 hours revealed cells present in both the basal layer (which houses progenitors) and in cells of the differentiated suprabasal layers. FACS sorted tdTomato-positive cells 24 hours post tamoxifen exposure demonstrated superior colony forming capacity compared to the tdTomato negative population. Taken together these results suggest that Bcl11a has a role in progenitor cells of the oesophageal epithelium with a potential function in maintaining normal growth and differentiation. Taken together these results suggest that Bcl11a is a valuable candidate for further study in the murine oesophageal epithelium.National centre for the refinement reduction and replacement of animals in research. Cancer Research U