RNA-seq analysis of Lgr6+ stem cells and identification of an Lgr6 isoform.

We studied Lgr6+ stem cells in experimental UV carcinogenesis in hairless mice. For further characterization through RNA-seq, these stem cells were isolated by FACS from transgenic hairless mice bearing an EGFP-Ires-CreERT2 reporter cassette inserted into exon 1 of the Lgr6 gene (purity confirmed by human ERT2 expression). Between Lgr6/EGFP+ and Lgr6/EGFP- basal cells (Tg/wt), 682 RNAs were differentially expressed, indicating stemness and expression of cancer-related pathways in Lgr6/EGFP+ cells. We discovered that suspected "Lgr6 null" mice (Tg/Tg) expressed RNA of an Lgr6 isoform (delta-Lgr6, lacking 74 N-terminal aa) which could be functional and explain the lack of a phenotype.

Lgr6+ stem cells and their progeny in mouse epidermis under regimens of exogenous skin carcinogenesis, and their absence in ensuing skin tumors.

Lgr6+ cells have been identified as a novel class of proliferating (Ki67+) stem cells in mouse epidermis. We investigated their response to UV exposure in Lgr6-EGFP-Ires-CreERT2/R26R-LacZ haired and hairless mice and whether they become initiating cells of UV- or chemically induced skin tumors. UV overexposure erased Lgr6+ cells (EGFP+) from the interfollicular epidermis (IFE), but - as after wounding - they apparently repopulated the IFE from the hair follicles. Under sub-sunburn chronic UV exposure, Lgr6+ cells and their progeny (LacZ+ after pulse of tamoxifen) diminished strongly in the IFE. Although the inter-tumoral IFE clearly showed Lgr6 progeny, none of the UV- or chemically induced tumors (n = 22 and 41, respectively) appeared to be clonal expansions of Lgr6+ stem cells; i.e. no Lgr6+ cells or progeny in the proliferating tumor bulk. In checking for promoter methylation we found it to occur stochastically for the EGFP-Cre cassette. Lgr6 mRNA measured by qPCR was found to be diminished in skin tumors (also in UV tumors from wt type mice). The ratio of Lgr6/Ki67 was significantly reduced, pointing at a loss of Lgr6+ cells from the proliferative pool. Our data show that Lgr6+ cells are not major tumor-initiating cells in skin carcinogenesis.

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

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.

Fractionation of a tumor-initiating UV dose introduces DNA damage-retaining cells in hairless mouse skin and renders subsequent TPA-promoted tumors non-regressing.

Sunburns and especially sub-sunburn chronic UV exposure are associated with increased risk of squamous cell carcinomas (SCCs). Here we focus on a possible difference in tumor initiation from a single severe-sunburn dose (on day 1, 21 hairless mice) and from an equal dose fractionated into very low sub-sunburn doses not causing any (growth-promoting) epidermal hyperplasia (40 days daily exposure, n=20). From day 47 all mice received 12-O-Tetradecanoylphorbol-13-acetate (TPA) applications (2x/wk) for 20 weeks to promote tumor development within the lifetime of the animals. After the sub-sunburn regimen sparse DNA damage-retaining basal cells (quiescent stem cells, QSCs) remained in the non-hyperplastic epidermis. These cells were forced to divide by TPA. After discontinuation of TPA tumors regressed and disappeared in the 'sunburn group' but persisted and grew in the 'sub-sunburn group' (0.06 vs 2.50 SCCs and precursors ≥4 mm/mouse after 280 days, p=0.03). As the tumors carried no mutations in p53, H/K/N-Ras and Notch1/2, these 'usual suspects' were not involved in the UV-driven tumor initiation. Although we could not selectively eliminate QSCs (unknown phenotype) to establish causality, our data suggest that forcing specifically DNA damage-retaining QSCs to divide--with high mutagenic risk--gives rise to persisting (mainly 'in situ') skin carcinomas.

Targeted delivery of TLR ligands to human and mouse dendritic cells strongly enhances adjuvanticity.

Effective vaccines consist of 2 components: immunodominant antigens and effective adjuvants. Whereas it has been demonstrated that targeted delivery of antigens to dendritic cells (DCs) improves vaccine efficacy, we report here that co-targeting of TLR ligands (TLRLs) to DCs strongly enhances adjuvanticity and immunity. We encapsulated ligands for intracellular TLRs within biodegradable nanoparticles coated with Abs recognizing DC-specific receptors. Targeted delivery of TLRLs to human DCs enhanced the maturation and production of immune stimulatory cytokines and the Ag-specific activation of naive CD8(+) T cells. In vivo studies demonstrated that nanoparticles carrying Ag induced cytotoxic T-lymphocyte responses at 100-fold lower adjuvant dose when TLRLs were co-encapsulated instead of administered in soluble form. Moreover, the efficacy of these targeted TLRLs reduced the serum cytokine storm and related toxicity that is associated with administration of soluble TLRLs. We conclude that the targeted delivery of adjuvants may improve the efficacy and safety of DC-based vaccines.