Supplemental Figures 1-6, Supplemental Tables 1-2, Supplemental Materials & Methods, Supplemental References Figure S1. Increased tumour growth is not a result of decreased apoptosis. Figure S2. Pericytes increase OVCAR-5 cell proliferation in vitro. Figure S3. Pericytes promote metastasis in vivo. Figure S4. Pericytes promote growth and initiate metastasis in the non-metastatic OVCAR-8 cells in vivo. Figure S5. Pericytes recruit αSMAcells expressing BM-MSC markers to OVCAR-5 xenografts. Figure S6. CD34 and PDGFRβ expression do not correlate with early relapse in serous ovarian cancer patients. Table S1: List of probe sets for the 146 overlapping genes up-regulated in both the pericytes and ovarian stroma. Table S2A-D: Top GO cellular component, molecular function & biological processes terms; and KEGG pathways enriched in early versus late relapse patients. Supplemental Methods: Details of methods used for in silico analyses, GFP transduction of cells, immunostaining, invasion assays and morphometric analyses.

Pericytes have long been known to contribute indirectly to tumour growth by regulating angiogenesis. Thus, remodelling tumour blood vessels to maintain blood supply is critical for continued tumour growth. A role for pericytes in restricting leakage of tumour cells through blood vessels has also become evident given that adequate pericyte coverage of these blood vessels is critical for maintaining vascular permeability. Interestingly, the relocation of pericytes from blood vessels to the tumour microenvironment results in the emergence of different properties in these cells that actively promote tumour growth and metastasis-functions not associated with their well-studied role in vascular stability and permeability. These form the focus of this review.

Bioluminescence imaging (BLI) technology is an advanced method of carrying out molecular imaging on live laboratory animals in vivo. This powerful technique is widely-used in studying a variety of biological processes, and it has been an ideal tool in exploring tumor growth and metastatic spread in real-time. This technique ensures the optimal use of laboratory animal resources, particularly the ethical principle of reduction in animal use, given its non-invasive nature, ensuring that ongoing biological processes can be studied over time in the same animal, without the need to euthanize groups of mice at specific time points. In this protocol, the luciferase imaging technique was developed to study the effect of co-inoculating pericytes (contractile, αSMA+ mesenchymal stem cell-like cells, located abluminally in microvessels) on the growth and metastatic spread of ovarian cancers using an aggressive ovarian cancer cell line-OVCAR-5-as an example.

PURPOSECirculating tumor DNA (ctDNA) allows noninvasive disease monitoring across a range of malignancies. In metastatic melanoma, the extent to which ctDNA reflects changes in metabolic disease burden assessed by 18F-labeled fluorodeoxyglucose positron emission tomography (FDG-PET) is unknown. We assessed the role of ctDNA analysis in combination with FDG-PET to monitor tumor burden and genomic heterogeneity throughout treatment.PATIENTS AND METHODSWe performed a comprehensive analysis of serial ctDNA and FDG-PET in 52 patients who received systemic therapy for metastatic melanoma. Next-generation sequencing and digital polymerase chain reaction were used to analyze plasma samples from the cohort.RESULTSctDNA levels were monitored across patients with mutant BRAF, NRAS, and BRAF/NRAS wild type disease. Mutant BRAF and NRAS ctDNA levels correlated closely with changes in metabolic disease burden throughout treatment. TERT promoter mutant ctDNA levels also paralleled changes in tumor burden, which provide an alternative marker for disease monitoring. Of note, subcutaneous and cerebral disease sites were not well represented in plasma. Early changes in ctDNA and metabolic disease burden were important indicators of treatment response. Patients with an early decrease in ctDNA post-treatment had improved progression-free survival compared with patients in whom ctDNA levels remained unchanged or increased over time (hazard ratio, 2.6; P = .05). ctDNA analysis contributed key molecular information through the identification of putative resistance mechanisms to targeted therapy. A detailed comparison of the genomic architecture of plasma and multiregional tumor biopsy specimens at autopsy revealed the ability of ctDNA to comprehensively capture genomic heterogeneity across multiple disease sites.CONCLUSIONThe findings highlight the powerful role of ctDNA in metastatic melanoma as a complementary modality to functional imaging that allows real-time monitoring of both tumor burden and genomic changes throughout therapy.

Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richter's syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.

The diagnosis and monitoring of myelodysplastic syndromes (MDSs) are highly reliant on bone marrow morphology, which is associated with substantial interobserver variability. Although azacitidine is the mainstay of treatment in MDS, only half of all patients respond. Therefore, there is an urgent need for improved modalities for the diagnosis and monitoring of MDSs. The majority of MDS patients have either clonal somatic karyotypic abnormalities and/or gene mutations that aid in the diagnosis and can be used to monitor treatment response. Circulating cell-free DNA is primarily derived from hematopoietic cells, and we surmised that the malignant MDS genome would be a major contributor to cell-free DNA levels in MDS patients as a result of ineffective hematopoiesis. Through analysis of serial bone marrow and matched plasma samples (n = 75), we demonstrate that cell-free circulating tumor DNA (ctDNA) is directly comparable to bone marrow biopsy in representing the genomic heterogeneity of malignant clones in MDS. Remarkably, we demonstrate that serial monitoring of ctDNA allows concurrent tracking of both mutations and karyotypic abnormalities throughout therapy and is able to anticipate treatment failure. These data highlight the role of ctDNA as a minimally invasive molecular disease monitoring strategy in MDS.

Abstract Purpose: The aim of this study was to investigate the role of pericytes in regulating malignant ovarian cancer progression. Experimental Design: The pericyte mRNA signature was used to interrogate ovarian cancer patient datasets to determine its prognostic value for recurrence and mortality. Xenograft models of ovarian cancer were used to determine if co-injection with pericytes affected tumor growth rate and metastasis, whereas co-culture models were utilized to investigate the direct effect of pericytes on ovarian cancer cells. Pericyte markers were used to stain patient tissue samples to ascertain their use in prognosis. Results: Interrogation of two serous ovarian cancer patient datasets [the Australian Ovarian Cancer Study, n = 215; and the NCI TCGA (The Cancer Genome Atlas), n = 408] showed that a high pericyte score is highly predictive for poor patient prognosis. Co-injection of ovarian cancer (OVCAR-5 & -8) cells with pericytes in a xenograft model resulted in accelerated ovarian tumor growth, and aggressive metastases, without altering tumor vasculature. Pericyte co-culture in vitro promoted ovarian cancer cell proliferation and invasion. High αSMA protein levels in patient tissue microarrays were correlated with more aggressive disease and earlier recurrence. Conclusions: High pericyte score provides the best means to date of identifying patients with ovarian cancer at high risk of rapid relapse and mortality (mean progression-free survival time < 9 months). The stroma contains rare yet extremely potent locally resident mesenchymal stem cells—a subset of “cancer-associated fibroblasts” that promote aggressive tumor growth and metastatic dissemination, underlying the prognostic capacity of a high pericyte score to strongly predict earlier relapse and mortality. Clin Cancer Res; 22(7); 1813–24. ©2015 AACR.

9582 Background: While immunotherapy and MAPK-targeted therapies have improved patient outcome, the genomic heterogeneity of melanoma contributes to treatment resistance. Molecular approaches for monitoring tumor burden, treatment response and resistance has enormous potential. ctDNA may allow global representation of all disease sites, and serial ctDNA analysis can track responses and provide insights into the spatio-temporal profile of the disease. Methods: Fifty-two metastatic melanoma patients (pts) were serially monitored during sequential lines of therapy with FDG-PET scans, LDH and ctDNA. Serial tumor biopsies were collected. Five of these 52 pts consented to multiregional tumor sampling at autopsy. Allele specific digital PCR assays, customized targeted and whole exome sequencing was used to measure ctDNA levels and identify genomic alterations. Results: In BRAF and NRAS mutant patients, mutant ctDNA was detected in 72% of patients at baseline and encompassed between 0.4%-86% (median 8.2%) of total ctDNA levels. LDH and FDG-PET metabolic tumor volume correlated significantly with mutant BRAF and NRAS copies in plasma [(r=0.7818, p=0.0032) and (r=0.63, p=0.0001) respectively]. High ctDNA levels were associated with worse overall survival. ctDNA levels tracked with FDG-PET responses. Targeted sequencing of baseline ctDNA and tumor samples identified multiple mutations in clinically important genes such as MAP2K1, MAP2K2, PTEN, CDKN2A, RAC1, CTNNB1,BRAF and NRAS; high concordance was observed between ctDNA and tumor. ctDNA analysis also identified multiple mutations associated with primary and acquired resistance to MAPK-targeted therapy. Comprehensive exome sequencing of ctDNA in patients undergoing autopsy provided an accurate representation of spatial heterogeneity, when compared to multi-regional tumor sequencing. Conclusions: These findings highlight the potential clinical utility of ctDNA analysis to monitor tumor responses and disease progression in the management of metastatic melanoma patients.

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.

committed progenitor differentiating cell keratin keratinocyte stem cell label-retaining cell transient amplifying activating Wnt signal Wnt inhibitory factor-1 TO THE EDITOR Although it is possible to isolate keratinocyte stem cells (KSCs) of the human interfollicular epithelium by virtue of their composite cell surface phenotype (α6 integrin and CD71; Li et al., 1998Li A. Simmons P.J. Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.Proc Natl Acad Sci USA. 1998; 95: 3902-3907Crossref PubMed Scopus (424) Google Scholar; Schluter et al., 2011Schluter H. Paquet-Fifield S. Gangatirkar P. et al.Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis.Stem Cells. 2011; 29: 1256-1268Crossref PubMed Scopus (53) Google Scholar, it remains difficult to visualize them in situ within the skin. Although murine interfollicular KSCs can be visualized in situ by DNA-labeling protocols combined with long chase periods (termed DNA label-retaining cells or LRCs; Bickenbach, 1981Bickenbach J.R. Identification and behavior of label-retaining cells in oral mucosa and skin.J Dent Res. 1981; 60: 1611-1620Crossref PubMed Google Scholar, this approach is not possible in humans for ethical reasons. Our aim was to identify single markers for human interfollicular KSCs at the protein level, by mining microarray data previously generated by us to identify genes preferentially expressed by KSCs compared with their more committed progeny (Schluter et al., 2011Schluter H. Paquet-Fifield S. Gangatirkar P. et al.Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis.Stem Cells. 2011; 29: 1256-1268Crossref PubMed Scopus (53) Google Scholar. Interfollicular epidermal cells from neonatal human skin tissue can be resolved into quiescent KSCs, cycling committed progenitor (CP) or transient-amplifying (TA) cells, and early differentiating (D) cells on the basis of their cell surface phenotype, i.e., α6briCD71dim, α6briCD71bri, and α6dim, respectively (Li et al., 1998Li A. Simmons P.J. Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.Proc Natl Acad Sci USA. 1998; 95: 3902-3907Crossref PubMed Scopus (424) Google Scholar. Importantly, functional long-term tissue reconstitution assays confirmed the identity of α6briCD71dim cells as a bona fide stem cell population, given their potency in reconstituting human interfollicular epidermis for up to 10 weeks from as little as 100 cells (Schluter et al., 2011Schluter H. Paquet-Fifield S. Gangatirkar P. et al.Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis.Stem Cells. 2011; 29: 1256-1268Crossref PubMed Scopus (53) Google Scholar. Further, molecular characterization by microarray validated their quiescent nature (Schluter et al., 2011Schluter H. Paquet-Fifield S. Gangatirkar P. et al.Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis.Stem Cells. 2011; 29: 1256-1268Crossref PubMed Scopus (53) Google Scholar. Notably, KSCs overexpressed negative regulators of key signaling pathways including Wnt, insulin, sonic hedgehog, and transforming growth factor beta, shown in Figure 1a (taken from Schluter et al., 2011Schluter H. Paquet-Fifield S. Gangatirkar P. et al.Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis.Stem Cells. 2011; 29: 1256-1268Crossref PubMed Scopus (53) Google Scholar. Among the Wnt signaling pathway inhibitors expressed by KSCs were Sfrp-1, Dkk-3, and (Wnt inhibitory factor-1) WIF1 (Figure 1a). Notably, WIF1 was the single most overexpressed gene in KSCs (Schluter et al., 2011Schluter H. Paquet-Fifield S. Gangatirkar P. et al.Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis.Stem Cells. 2011; 29: 1256-1268Crossref PubMed Scopus (53) Google Scholar—its differential expression in KSCs was confirmed by reverse transcription–PCR (Figure 1b). These data suggested that WIF1 may be an excellent marker for interfollicular KSCs of human skin. We performed dual immunofluorescence staining in human neonatal and adult breast skin for WIF1, and keratin (K)14 or K15—markers of basal keratinocytes and α6 integrin—a basement membrane marker. Single basal keratinocytes coexpressing WIF1 and K14/K15 were found scattered throughout the basal layer in neonatal foreskin, but were more widely expressed in adult skin (Figure 1c–h; arrows). In addition, almost all dermal cells, an active site for Wnt signaling, were also positive for WIF1 (Figure 1c–h). WIF1 immunostaining on cytospins of α6briCD71dim/KSC, α6briCD71bri/TA or CP, and α6dim/D cells confirmed highest WIF1 protein expression in KSCs but not the cycling TA/CPs qualitatively (Figure 1k–m), and quantitatively (Figure 1n). Although the α6dim differentiating fraction has a higher level of WIF1 than TA/CP cells in cytospins, this population is somewhat heterogeneous, containing many suprabasal cells that are clearly WIF1 positive. Notably, cells positive for both K10 and WIF1 were consistently located in the suprabasal layers in both neonatal and adult skin in situ (Figure 1g and h). Further, dual staining for Ki67 and WIF1 in adult skin revealed that Ki67-positive cells do not express WIF1 (Figure 1c). Thus, WIF1 is highly expressed in non-cycling cells, indicating a role in negatively regulating keratinocyte proliferation. To further demonstrate that WIF1 is a bona fide interfollicular KSC marker, we sought to determine whether slow-cycling LRCs coexpressed WIF1 in human skin. LRCs can be visualized in human skin in situ in grafts on immunodeficient mice (Lyle et al., 1998Lyle S. Christofidou-Solomidou M. Liu Y. et al.The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.J Cell Sci. 1998; 111: 3179-3188PubMed Google Scholar, or alternately in long-term organotypic cultures of human keratinocytes, with an incidence of <1% after 6 weeks (Muffler et al., 2008Muffler S. Stark H.J. Amoros M. et al.A stable niche supports long-term maintenance of human epidermal stem cells in organotypic cultures.Stem Cells. 2008; 26: 2506-2515Crossref PubMed Scopus (61) Google Scholar. Using the latter approach validated by us previously, we stained histological sections of iododeoxyuridine-labeled long-term organotypic cultures for iododeoxyuridine and WIF1. All iododeoxyuridine-positive epidermal cells were also WIF1 positive (Figure 2f–k), whereas cycling Ki67-positive cells were not (Figure 2a and b), providing supportive evidence that WIF1 protein is an excellent marker for quiescent stem cells of the interfollicular epidermis. Notably, there was an absence of WIF1-positive cells in the hyperproliferative basal layer of organotypic cultures in the early 2-week post-labeling time points, as evidenced by the presence of many Ki67-positive cells (Figure 2a and c). WIF1-positive basal cells were detectable by 6 weeks (Figure 2b and d), a time point corresponding with the establishment of single LRCs (Muffler et al., 2008Muffler S. Stark H.J. Amoros M. et al.A stable niche supports long-term maintenance of human epidermal stem cells in organotypic cultures.Stem Cells. 2008; 26: 2506-2515Crossref PubMed Scopus (61) Google Scholar, although none of these were Ki67 positive. Notably, none of the WIF1-positive cells in the basal layer of organotypic cultures were K10 positive at all time points analyzed, and double-positive cells were only localized suprabasally (Figure 2c–e), confirming our observations in skin tissue. The functional significance of WIF1 overexpression in interfollicular KSCs is of great interest given the current evidence for an increase in Wnt signaling in wound healing, psoriasis, and basal cell carcinomas (Gudjonsson et al., 2010Gudjonsson J.E. Johnston A. Stoll S.W. et al.Evidence for altered Wnt signaling in psoriatic skin.J Invest Dermatol. 2010; 130: 1849-1859Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar; Arwert et al., 2012Arwert E.N. Hoste E. Watt F.M. Epithelial stem cells, wound healing and cancer.Nat Rev Cancer. 2012; 12: 170-180Crossref PubMed Scopus (322) Google Scholar. Indeed, activating mutations in Wnt pathway family members contribute to the progression of several major human cancers (Moon et al., 2004Moon R.T. Kohn A.D. De Ferrari G.V. et al.WNT and beta-catenin signalling: diseases and therapies.Nat Rev Genet. 2004; 5: 691-701Crossref PubMed Scopus (1585) Google Scholar. Surprisingly, Wnt/beta-catenin signaling is reportedly crucial for the development of epidermal appendages, but not the interfollicular epidermis (Huelsken et al., 2001Huelsken J. Vogel R. Erdmann B. et al.Beta-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin.Cell. 2001; 105: 533-545Abstract Full Text Full Text PDF PubMed Scopus (1107) Google Scholar; Nguyen et al., 2009Nguyen H. Merrill B.J. Polak L. et al.Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia.Nat Genet. 2009; 41: 1068-1075Crossref PubMed Scopus (165) Google Scholar. However, abundant expression of wnt 5a, one of several wnt ligands, has been reported in normal adult skin (Pourreyron et al., 2012Pourreyron C. Reilly L. Proby C. et al.Wnt5a is strongly expressed at the leading edge in non-melanoma skin cancer, forming active gradients, while canonical Wnt signalling is repressed.PLoS One. 2012; 7: e31827Crossref PubMed Scopus (53) Google Scholar. Immunostaining of both neonatal and adult skin also revealed abundant expression of the canonical wnt ligand activating Wnt signal (Wnt3A) throughout the epithelium and in some dermal cells (Figure 1i and j). Given the observation that WIF1 is expressed in quiescent LRCs of adult human skin and differentiating keratinocytes, we next addressed the possibility that it may negatively regulate keratinocyte proliferation in the presence of Wnt3A. Immunostaining cultured keratinocytes for WIF1 revealed that its expression was lost in proliferating cultured keratinocytes (data not shown), consistent with the mRNA data shown in Figure 1a and b (TA/CP fraction). Therefore, we looked at the effects of exogenous WIF1 on keratinocytes. We synchronized the cell cycle activity of neonatal human foreskin keratinocytes in culture using the double thymidine block method, arresting them in late G1 phase (Figure 2l). Subsequent treatment of arrested keratinocytes with 20ng of Wnt3A for 2hours at 37°C released them from the G1 arrest, i.e., a decrease in the number of cells in G1 of 26% and an increase in cells in % S+G2M of 19% (Figure 2m). Whereas treating cells with 3μg WIF1 alone did not maintain the cells in G1, simultaneous treatment with WIF1 and the Wnt ligand Wnt3A blocked the re-entry of cells into S-phase (16%, Figure 2n, compared with 25% with Wnt3A alone, Figure 2m). Concomitantly, the combined Wnt3A and WIF1 treatment resulted in the accumulation of cells in G1 (44 vs. 24% in Wnt3A control) and a decrease of cells in G2M (16 vs. 31% in Wnt3A control; Figure 2m and n). These data show that WIF1 negatively regulates cell cycle progression in human keratinocytes in the presence of Wnt ligands, indicating a potential role in keratinocyte quiescence and differentiation. It has been reported that WIF1 achieves G1 cell cycle arrest in cancer cells, by the transcriptional downregulation of cell cycle regulatory Wnt target genes such as c-myc and Skp2 (S-phase kinase associated protein 2) (Tang et al., 2009Tang Y. Simoneau A.R. Liao W.X. et al.WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells.Mol Cancer Ther. 2009; 8: 458-468Crossref PubMed Scopus (92) Google Scholar. c-myc is a repressor of p21/WAF1 (van de Wetering et al., 2002van de Wetering M. Sancho E. Verweij C. et al.The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar, whereas Skp2 regulates p27/Kip1 degradation (Gstaiger et al., 2001Gstaiger M. Jordan R. Lim M. et al.Skp2 is oncogenic and overexpressed in human cancers.Proc Natl Acad Sci USA. 2001; 98: 5043-5048Crossref PubMed Scopus (438) Google Scholar—thus, downregulation of c-myc and Skp2 results in accumulation of p21 and p27 (Tang et al., 2009Tang Y. Simoneau A.R. Liao W.X. et al.WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells.Mol Cancer Ther. 2009; 8: 458-468Crossref PubMed Scopus (92) Google Scholar—two major regulators of CDK2, the main cyclin-dependent kinase controlling G1- to S-phase progression (Pfeuty et al., 2008Pfeuty B. David-Pfeuty T. Kaneko K. Underlying principles of cell fate determination during G1 phase of the mammalian cell cycle.Cell Cycle. 2008; 7: 3246-3257Crossref PubMed Scopus (33) Google Scholar. Consistent with this, we observed that the downstream effectors of the Wnt pathway, i.e., c-myc and Skp2 were not expressed in KSCs by reverse transcription–PCR (Figure 1b). Further, probing cell lysates of WIF1-arrested keratinocytes in western blots revealed that Wnt3A/WIF1 treatment resulted in increased p21 protein levels (Figure 2o) demonstrable quantitatively (Figure 2p). Thus, WIF1 may achieve its cell cycle arrest in keratinocytes at least in part through derepression of p21 transcription. In conclusion, we report that WIF1 is, to our knowledge, previously unreported as a marker of interfollicular KSCs, and that it inhibits cell cycle progression in human keratinocytes even in the presence of activating Wnt signals (Wnt3A). Although canonical Wnt signaling appears to be dispensable during development in the interfollicular epidermis (Huelsken et al., 2001Huelsken J. Vogel R. Erdmann B. et al.Beta-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin.Cell. 2001; 105: 533-545Abstract Full Text Full Text PDF PubMed Scopus (1107) Google Scholar; Nguyen et al., 2009Nguyen H. Merrill B.J. Polak L. et al.Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia.Nat Genet. 2009; 41: 1068-1075Crossref PubMed Scopus (165) Google Scholar, our data suggest that inhibition of Wnt signaling may be required for keeping interfollicular stem cells quiescent and differentiating cells from proliferating during homeostasis. This work was supported by National Institutes of Health grant RO1 AR050013-01A2 to PK and, in part, by contract research 'Adulte Stammzellen II' of the Baden-Württemberg Stiftung to PB. We thank Dr Sarah Ellis for her skilled assistance with confocal microscopy.