Spatially correlated phenotyping reveals K5-positive luminal progenitor cells and p63-K5/14-positive stem cell-like cells in human breast epithelium

Abstract

Understanding the mechanisms regulating human mammary epithelium requires knowledge of the cellular constituents of this tissue. Different and partially contradictory definitions and concepts describing the cellular hierarchy of mammary epithelium have been proposed, including our studies of keratins K5 and/or K14 as markers of progenitor cells. Furthermore, we and others have suggested that the p53 homolog p63 is a marker of human breast epithelial stem cells. In this investigation, we expand our previous studies by testing whether immunohistochemical staining with monospecific anti-keratin antibodies in combination with an antibody against the stem cell marker p63 might help refine the different morphologic phenotypes in normal breast epithelium. We used in situ multilabel staining for p63, different keratins, the myoepithelial marker smooth muscle actin (SMA), the estrogen receptor (ER), and Ki67 to dissect and quantify the cellular components of 16 normal pre- and postmenopausal human breast epithelial tissue samples at the single-cell level. Importantly, we confirm the existence of K5+ only cells and suggest that they, in contrast to the current view, are key luminal precursor cells from which K8/18+ progeny cells evolve. These cells are further modified by the expression of ER and Ki67. We have also identified a population of p63+K5+ cells that are only found in nipple ducts. Based on our findings, we propose a new concept of the cellular hierarchy of human breast epithelium, including K5 luminal lineage progenitors throughout the ductal-lobular axis and p63+K5+ progenitors confined to the nipple ducts.

Integrating single-cell RNA-sequencing and functional assays to decipher mammary cell states and lineage hierarchies

“…Indeed, K5/K18 and K14/K18 double-positive cells are not uncommon in human TDLUs 61 . More recently, Boecker et al 65 identified K5 + K18/19 − and K5 + K18/K19 + populations in the luminal layer of ductal and TDLU breast tissue in situ 65 , while in human breast epithelial populations isolated by flow cytometry, the progenitor populations (Lin − CD49f + EpCAM hi ) include cells double-positive for K5/6 and K14 — and notably are also c-KIT + 40 . To add to the complexity of these marker patterns, K19 has been described both as a marker of progenitors 66 – 68 and highly expressed in differentiated luminal ER + cells 6 , 69 .…”

“…Boecker et al 65 termed the populations they identified as progenitors and intermediary cells, respectively, but it is difficult to definitively assign such functions purely on the basis of marker expression, or indeed ex vivo assays. Of course, human breast tissue cannot be lineage-traced through transgene activation as one can in the mouse, but use of cytochrome C oxidase (CCO) mutations in the mitochondrial genome has proven feasible as an approach.…”

Cytokeratin 5 determines maturation of the mammary myoepithelium

“…The basal cell layer is able to regenerate the whole mammary gland epithelial tree ( Böcker et al., 2002 ; Boecker and Buerger, 2003 ; Van Keymeulen et al., 2011 ). Development of LE and ME cells occurs in a complex hierarchical manner from the basal progenitors that can differentiate into both epithelial cell types depending on numerous signaling pathways and hormonal stimuli ( Arendt and Kuperwasser, 2015 ; Böcker et al., 2002 ; Boecker and Buerger, 2003 ; Boecker et al., 2018 ; Van Keymeulen et al., 2011 ). This epithelial differentiation process can be followed as changes in cell-type specific protein expression patterns, including expression of distinct cytokeratin (CK) family members ( Böcker et al., 2002 ; Boecker and Buerger, 2003 ; Boecker et al., 2018 ).…”

“…Development of LE and ME cells occurs in a complex hierarchical manner from the basal progenitors that can differentiate into both epithelial cell types depending on numerous signaling pathways and hormonal stimuli ( Arendt and Kuperwasser, 2015 ; Böcker et al., 2002 ; Boecker and Buerger, 2003 ; Boecker et al., 2018 ; Van Keymeulen et al., 2011 ). This epithelial differentiation process can be followed as changes in cell-type specific protein expression patterns, including expression of distinct cytokeratin (CK) family members ( Böcker et al., 2002 ; Boecker and Buerger, 2003 ; Boecker et al., 2018 ). Less than 5% of the mammary basal cells represent mammary stem cells that express CK5 without luminal epithelial (LE) markers CK8/18/19 or myoepithelial (ME) marker α-SMA ( Böcker et al., 2002 ; Fu et al., 2020 ).…”

“…Less than 5% of the mammary basal cells represent mammary stem cells that express CK5 without luminal epithelial (LE) markers CK8/18/19 or myoepithelial (ME) marker α-SMA ( Böcker et al., 2002 ; Fu et al., 2020 ). Expression of CK5 is also detected in the progenitor cells that can differentiate into mature luminal or ME cells, lacking the expression of CK5 ( Böcker et al., 2002 ; Boecker and Buerger, 2003 ; Boecker et al., 2018 ). Additionally, progenitor cell activity has been attributed to cells expressing CK14 simultaneous with LE or ME markers ( Arendt et al., 2014 ; Boecker et al., 2018 ; Fridriksdottir et al., 2017 ; Villadsen et al., 2007 ).…”