Transcriptome analysis of mammary epithelial subpopulations ...

BMC Genomics 2008, 9:591http://www.biomedcentral.com/1471-2164/9/591DiscussionWe have described a comprehensive transcriptome analysisof three distinct mammary epithelial cell populations,basal/myoepithelial cells, estrogen receptor negativeluminal epithelial cells and estrogen receptor positiveluminal epithelial cells [5,8]. These data provide new supportfor the distinct identities of these three populationsand, in particular, justify distinguishing between twomajor subpopulations within the luminal epithelium. Wehave termed these luminal ER - and luminal ER + cells as itis expression of the estrogen receptor which makes themmost easily distinguishable in tissue sections (Figure 1).However, each of them expresses, in addition, a largenumber of unique genes which must be of relevance totheir in vivo functions. It is also clear that there are genesin common to the two luminal populations but which arenot expressed by the myoepithelial cells. Expression ofthese genes, for cytoskeletal proteins (e.g. keratin 18) ortight junction components (e.g. claudins), for instance,must reflect common aspects of luminal epithelial cellfunction. Most obviously, they are for proteins importantin maintaining the structure of the luminal cell layer andthe integrity of the lumen itself.Comparison of our data set with other published data setsfor separated myoepithelial and luminal cells[6,26,27,29] showed good concordance between genespreviously identified as enriched in mouse mammarymyoepithelial and luminal cells [6]. However, there wasless agreement with genes previously identified asenriched in human myoepithelial and luminal cells. Anumber of factors are likely to contribute to these differences.Clearly, species differences could be important. Forinstance, it is known that while K14 is a basal/myoepithelialcell marker and K18 a luminal epithelial cell markerthroughout the mouse mammary epithelium and in theducts of the human breast, in the Terminal Ductal LobuloalveolarUnits of the human breast, K14 can beexpressed by the luminal epithelial cells [63]. Furthermore,technical differences could influence the outcomeof the analyses. In particular, it should be noted that Stingland colleagues used the same Affymetrix platform as ourselvesfor their mouse analysis [6]. Finally, comparingthree distinct populations against each other, rather thanjust two, improves the contrasts between the populationsand enables more population specific genes to bedetected. For example, a gene which is present in thebasal/myoepithelial cells and one of the luminal populations,but not the other, may not be detected as being significantlydifferentially expressed when onlymyoepithelial and total luminal cells are compared. However,when all three populations are compared againsteach other, the contrast with the luminal population fromwhich the gene is absent enables the differential expressionof the gene to be detected.A novel functional cell type in the mammary epitheliumThe nature of the luminal ER - population as a discreteentity has been confirmed by its uniform staining profilewith the 33A10 antibody [8]. However, this populationappeared to contain within it further distinct functionalcell types. Use of network interaction mapping on thetranscriptomic profiles of the luminal ER - cells identifieda Toll-like receptor (TLR) signalling module includinggenes for the three components of the bacterial lipopolysaccharide(LPS) receptor, Tlr4, CD14 and Ly96, as well asdownstream transducers of Toll-like receptor signallingsuch as Irak2 and the pro-inflammatory cytokine Tnf,which is a TLR signalling target [38] (Figure 4 and 5). Flowcytometric analysis of the mammary epithelium usinganti-CD14 and anti-CD61 (a mammary progenitor cellmarker) [11] identified a small number of CD61 + cells inthe CD24 +/Low Sca-1 - basal population and four distinctsubpopulations within the luminal ER - cells, namelyCD61 + , CD61 + CD14 + , CD14 + and double negative cells.This raises the possibility of a differentiation hierarchy inwhich basal stem cells generate basal CD61 + progenitorswhich then become CD61 + progenitors with luminalcharacteristics. These then either lose CD61 + and becomethe double negative cells (although presumably expressingother markers as yet undefined) or first acquire CD14expression, becoming CD61 + CD14 + progenitors, andthen lose CD61 expression to become the CD14 + population.If this hypothesis is correct, it suggests that the luminalER - population contains two distinct functional celltypes, the double negative cells and the CD14 + cells.The function of the double negative cells remains unclear.However, given that the expression of all components ofthe LPS receptor can be found in the luminal ER - population,it is likely that the CD14 + cells have a distinct functionwithin the mammary epithelium as non-professionalimmune cells. Note that CD45 + cells were excluded fromthis analysis, so these are unlikely to be contaminatinghaematopoietic cells. Milk is an excellent growth mediumfor bacteria and it would be evolutionarily advantageousto have a cell type present in the breast capable of indicatingthe presence of bacterial contamination through Tolllikereceptor signalling pathways. However, it is also likelythat over-stimulation of this pathway in CD14 + mammaryepithelial cells would lead to serious inflammation andwould therefore be the cause of mastitis.The presence of the distinct subpopulations within theluminal ER - cells indicates that the gene expression profileof this population is derived from a mixture of differentcell types. However, as the luminal ER - cells do all share atleast one distinctive marker (high levels of expression ofthe epitope bound by the 33A10 antibody) [8] it is likelythat the luminal ER - profile includes genes whose expressionis common to all the cells of this subpopulation, asPage 18 of 28(page number not for citation purposes)