Lgr5+ stem cells and their progeny in mouse epidermis under regimens of exogenous skin carcinogenesis, and their absence in ensuing skin tumors

Abstract

Actively proliferating Lgr5+ skin stem cells are found deep in the hair follicle (HF). These cells renew the HF and drive its expansion in anagen phase. Their long residence and continuous mitotic activity make them prime candidates to transform into skin tumor-initiating cells. This was investigated by subjecting Lgr5-EGFP-Ires-CreERT2/R26R-LacZ mice (haired and hairless) to chemical and UV carcinogenic regimens. In the course of these regimens Lgr5+ cells (EGFP+) remained exclusively located in HFs, and in deep-seated cysts of hairless skin. In haired mice, progeny of Lgr5+ stem cells (LacZ+ after a pulse of tamoxifen) appeared in the interfollicular epidermis upon UV-induced sunburn and in TPA-induced hyperplasia. In hairless mice the progeny remained located in deep-seated cysts and in HF remnants. Progeny in hairless skin was only detected interfollicularly at a late stage, in between outgrowing tumors. Lgr5+ stem cells were absent in the ultimate tumor masses, and no tumor appeared to be a (clonal) expansion of Lgr5+ cells (52 tumors with tamoxifen at the start of carcinogenesis, 42 tumors with tamoxifen late during tumor outgrowth). In contrast to CD34/K15+ quiescent bulge stem cells, actively proliferating Lgr5+ stem cells do therefore not appear to be tumor drivers in experimental skin carcinogenesis.

Keywords: Lgr5; UV; lineage tracing; skin carcinogenesis; stem cells.

Figure 1. Lgr5+ stem cells remain in their homeostatic location, but Lgr5 progeny repopulated the epidermal basal layer after UV overexposure in haired mice

Paraffin sections of control mice and mice that received an UV overexposure were stained with an anti-EGFP antibody A-E; whole mounts F-J were stained for LacZ expression (representative pictures are shown). A+F show Lgr5+ stem cells and their progeny in control mice without any UV exposure (homeostasis). B-E show the location of Lgr5+ stem cells at different time points after UV overexposure; no differences were observed compared to the control mice (see arrows). G-J show the Lgr5 progeny at different time points after overexposure. One week after overexposure progeny clearly migrated out of the hair follicle into the epidermal basal layer (I), this was not observed in control mice. Hair follicle orifice (in H+I) contoured; and arrow in H points at rim staining. scale bar = 100μm (in A-G and J), scale bar = 50μm (H+I).

Figure 2. Lgr5 progeny migrates out of the hair follicle into the IFE after hyperplasia induced by TPA in haired mice

Paraffin sections of haired mice were stained with an anti-EGFP antibody A-C and frozen sections D-F were stained for LacZ expression (representative pictures are shown). B and C show the location of EGFP-expressing Lgr5+ stem cells after hyperplasia induced by UV (B) or chemically by TPA (C), the location is the same as in untreated control mice (A). After UV induced hyperplasia (E) the Lgr5 progeny was found in the same location as in de control mice (D). However, after TPA induced hyperplasia Lgr5 progeny migrated out of the hair follicle into the epidermal basal layer (F). scale bar = 100μm (in A-C and F), scale bar = 50μm (D+E).

Figure 3. Progeny of Lgr5+ stem cells is (largely) absent in skin tumors; in hairless mice progeny of Lgr5+ stem cells migrated into the IFE only after prolonged treatment (over 6 months) with carcinogenic stimuli

Whole mount skin samples of haired mice after chemocarcinogenesis A and of hairless mice after UV-carcinogenesis D were stained for LacZ and showed interfollicular Lgr5 stem cell progeny. Hair follicle orifices are contoured (dotted lines). Tumors were induced by chemocarcinogenesis B+C in haired mice or by UV exposure E+F in hairless mice, respectively. LacZ lineage tracing was induced at the start of the experiment B+E or when tumors were formed (C+F), and tumor sections were stained for LacZ expression. Sparse inclusions of Lgr5 progeny in terminally differentiated parts were only observed in haired mice after chemocarcinogenesis when tracing was induced at the start of the experiment (B, some positivity seen near and in keratin pearls in 7/29 tumors). Scale bar in D = 50 μm, all other scale bars represent 100 μm.

Figure 4. CD34 and Sox2 expression in skin tumors

Sections of tumors were stained with anti-CD34 A+C or anti-Sox2 B+D. Both chemically induced tumors in haired mice (A+B) (CD34+ 15 of 21 tumors and Sox2+ 7 of 8 tumors) as well as UV-induced tumors in hairless mice (C+D) showed CD34 (6 of 14 tumors) and Sox2 expression (10 of 11 tumors). The CD34 expression in hairless mice was located in the differentiated compartments and more granular (C) compared to the haired mice (A). Scale bar = 100μm.