Title: Phenotypic dissection and therapeutic manipulation of cell differentiation programs in the salivary gland epithelium and human Adenoid Cystic Carcinomas
Description: Salivary glands (SGs) are important exocrine glands of the craniofacial region, whose main role is to produce and secrete saliva, a seromucous solution necessary for a diverse spectrum of critical functions, such as the preliminary digestion and swallowing of solid food, the articulation of speech, the maintenance of dental enamel and the prevention of oral infections. The production and secretion of saliva is orchestrated by a large and diverse collection of epithelial cell populations. Although many of the cell types that form the SG epithelium can be recognized morphologically and investigated using histological assays, it is currently impossible to achieve their differential purification from primary tissues as live cells, due to the lack of surface markers known to be either selectively or preferentially expressed by various cell subsets. This critical gap in knowledge limits our capacity to conduct functional studies in many areas of SG biology, including studies aimed at elucidating the developmental relationships that link different cell types (e.g. testing whether selected cell types can act as progenitors for the generation of others), studies elucidating the roles played by different cell types during regeneration of the SG epithelium following injury (e.g. radiotherapy), and studies investigating the biology of SG malignancies characterized by a heterogeneous cell composition, such as Adenoid Cystic Carcinomas (ACCs).

In this work, we aimed to advance our understanding of the cell composition of the salivary gland epithelium and to identify surface markers that enable the differential purification of its various cell types by fluorescence-activated cell sorting (FACS), in order to facilitate functional investigations of their individual capacity to act as stem/progenitor cells in prospective assays. In the first portion of our studies, we leveraged single-cell RNA sequencing (scRNA-seq) to dissect the transcriptional identities of various epithelial cell populations found in normal murine SGs, and discovered surface markers that allowed us to purify eight distinct cell types by FACS. We then used bulk RNA sequencing to generate high-resolution transcriptomic profiles of seven of these populations, and annotated their identity (e.g. acinar, ductal, basal, myoepithelial) in terms of anatomical location and differential expression of lineage-specific biomarkers. Furthermore, using a three-dimensional (3D) in vitro organoid tissue culture assay, we tested each of the newly identified SG populations for stem/progenitor properties, and demonstrated that organoid forming capacity is primarily restricted to only one of them, characterized by a basal phenotype, and able to function as a bipotent progenitor in vitro.

Finally, we used FACS to examine the effects of radiotherapy on the cell composition of the mouse SG epithelium, and demonstrated that, of the eight newly identified populations, at least four display preferential sensitivity to radiation injury. In the second portion of our studies, we tested whether the surface markers that we identified as differentially expressed between different subtypes of SG epithelial cells could also be leveraged to achieve the purification of the two subsets of malignant cells known to co-exist in Adenoid Cystic Carcinoma (ACC), one of the most common and lethal forms of human SG malignancy. A defining feature of ACC is the presence of two distinct cell populations, resembling myoepithelial and ductal cell types found in the normal salivary gland epithelium. However, little is known about the developmental relationship linking these two cell populations, their individual capacity to sustain the growth of malignant tissues upon xeno-transplantation, as well as their distinct behavior in terms of responses to therapeutic manipulations.

By utilizing cell surface markers identified as differentially expressed in the mouse SG epithelium, we developed a sorting strategy that enabled us to isolate the two major subtypes of malignant cells found in ACCs. By conducting prospective xeno-transplantation experiments in immunodeficient mice, we demonstrated that, contrary to common belief, myoepithelial-like cells are highly tumorigenic (i.e. do not represent an indolent component of the tumor) and can act as progenitors of ductal-like cells. Furthermore, by investigating differences in the transcriptional profiles of myoepithelial-like and ductal-like cells, we discovered that the two cell types differ in the expression of multiple components of the biochemical pathways that control retinoic acid (RA) signaling. We find that RA direct and inverse agonism have opposing effects on cell composition through distinct molecular mechanisms, whereby direct agonism facilitates differentiation of myoepithelial-like to ductal-like cells, and inverse agonism induces selective cell death of ductal-like cells.

Finally, we demonstrate that inhibition of RA signaling with inverse agonists is able to profoundly impair in vivo growth of human ACCs implanted in immunodeficient mice. Overall, the findings reported in this study advance our understanding of the cellular composition of both normal and malignant SG epithelia, establish novel and robust analytical assays for the purification of multiple subtypes of SG epithelial cells, and reveal novel strategies for the therapeutic manipulation of differentiation programs in human ACCs.