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Lgr6 is a stem cell marker in mouse skin squamous cell carcinoma

Abstract

The G-protein-coupled receptors LGR4, LGR5 and LGR6 are Wnt signaling mediators, but their functions in squamous cell carcinoma (SCC) are unclear. Using lineage tracing in Lgr5-EGFP-CreERT2/Rosa26-Tomato and Lgr6-EGFP-CreERT2/Rosa26-Tomato reporter mice, we demonstrate that Lgr6, but not Lgr5, acts as an epithelial stem cell marker in SCCs in vivo. We identify, by single-molecule in situ hybridization and cell sorting, rare cells positive for Lgr6 expression in immortalized keratinocytes and show that their frequency increases in advanced SCCs. Lgr6 expression is enriched in cells with stem cell characteristics, and Lgr6 downregulation in vivo causes increased epidermal proliferation with expanded lineage tracing from epidermal stem cells positive for Lgr6 expression. Surprisingly, mice with germline knockout of Lgr6 are predisposed to SCC development, through a mechanism that includes compensatory upregulation of Lgr5. These data provide a model for human patients with germline loss-of-function mutations in Wnt pathway genes, including RSPO1 or LGR4, who show increased susceptibility to squamous tumor development.

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Figure 1: Lgr6 expression increases with squamous tumor progression and Lgr6-GFP+ cells, not Lgr5-GFP+ cells, are localized within tumor epithelium.
Figure 2: Clonal expansion derived from Lgr6+ cells is observed in benign squamous tumors.
Figure 3: Clonal expansion derived from Lgr6+ cells in primary SCCs.
Figure 4: Lgr5 is expressed in rare single BCC cells but not in SCC cells, and Lgr6, and not Lgr5, is an SCC CSC marker.
Figure 5: Lgr6 is enriched in epidermal cells with stem cell properties and has growth-inhibitory effects in epidermal cell lines.
Figure 6: Lgr6 depletion increases proliferation in the epidermis and leads to expanded Lgr6-derived lineage tracing into the epidermis.

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Acknowledgements

We thank D. Wu, P. T. Menchavez and P. Vuong for assistance with handling of mice and RNA samples and D.A. Quigley for help with analysis of microarray data. This work was supported by NCI grants CA084244, CA141455, CA176287 and R01CA184510 and by California Institute for Regenerative Medicine (CIRM) grant TG2-01153 to P.Y.H. Additionally, A.B. acknowledges support from the Barbara Bass Bakar Chair of Cancer Genetics, and P.Y.H. acknowledges support from the Agency for Science, Technology and Research (A*STAR). We are grateful to F. de Sauvage (Genentech) for provision of the Lgr6−/− mouse strain. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of CIRM or any other agency of the state of California.

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Contributions

P.Y.H., E.K. and A.B. designed experiments. J. Seibler and V.B. generated and provided ES cells for creation of mouse strains. P.Y.H., E.K., J. Sjolund, A.K., R.D., H.C.K., A.J., M.P., A.S. and C.E.W. performed experiments. P.Y.H., E.K. and A.B. analyzed data. K.H. and M.M. carried out statistical analyses. P.Y.H., E.K. and A.B. wrote the manuscript, with contributions from the other authors.

Corresponding author

Correspondence to Allan Balmain.

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Competing interests

D.J.E., M.P. and A.S. are employees of Mission Bio, Inc. A.B. is a member of the Scientific Advisory Board for Mission Bio, Inc.

Integrated supplementary information

Supplementary Figure 1 Representative localization of Lgr5-GFP and Lgr6-GFP expression within primary squamous carcinoma tissue.

Localized expression of Lgr5-GFP and Lgr6-GFP was investigated in primary squamous carcinomas (at 25 weeks after initial TPA treatment; n = 3 biological replicates each) by immunostaining against GFP (green) or keratin 14 (red) to identify cell populations specifically expressing stem cell and basal cell markers. (a,b) Representative sections from squamous tumors demonstrating that epithelial Lgr5-GFP expression is absent in SCC tissue. (c,d) H&E staining of serial sections of the immunostaining depicted in a and b. (e,f) Representative sections from squamous tumors revealing abundant Lgr6-GFP+ cells (green) localized in epithelial squamous carcinoma tissue. (g,h) H&E staining of the serial sections of immunostaining depicted in e and f. (i) Magnified region of Lgr6-GFP+ squamous carcinoma cells (green) that display reduced K14 expression (red). The white arrow indicates Lgr6-GFP+ (green) cells within the tumor tissue. The yellow box denotes the region of interest displayed as single-color channels in the panel to the right with nuclear DAPI staining (blue). The experiment was carried out using three independent biological replicates of Lgr5-GFP and Lgr6-GFP primary SCC tumors; representative images are provided. Scale bar, 50 μm.

Supplementary Figure 2 Lgr5+ cells are capable of giving rise to spindle carcinoma, but Lgr5 expression is not maintained in the resulting tumors.

Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice developed papillomas when challenged with a back wound stimulus, and some of the papillomas eventually progressed to malignancy. (a) H&E-stained section of unwounded adult back skin from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice showing SG hyperplasia. (b) H&E-stained section of a typical papilloma harvested from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. The inset is a high-power view showing the epithelial morphology of the component tumor cells. (c) H&E-stained section of a malignant tumor harvested from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. The inset is a high-power view showing the spindle morphology of the component tumor cells. (d) Representative section showing anti-GFP immunofluorescence of normal adult skin from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. (e) Representative section showing anti-GFP immunofluorescence of a spindle carcinoma from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. (f,g) Three separate tumor cell lines were independently derived from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ spindle carcinomas. (f) TaqMan analysis showing Lgr4, Lgr5 and Lgr6 expression levels in the tumor cell lines and control normal back skin samples. Two different exon-spanning Lgr5 TaqMan probes that target different regions of the Lgr5 transcript were used. Data are presented as mean ± s.e. ***P < 0.001. n = 3. (g) Image analysis showing native GFP fluorescence in cells grown on chamber slides. Clockwise from top right: Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ tumor line 1, tumor line 2, tumor line 3 and positive-control spindle carcinoma cell line (D3) infected with the GFP-expressing F1HtUTG lentiviral vector89. Dotted lines demarcate the basement membrane and the outline of the hair follicles. Asterisks denote the autofluorescent hair shaft. White arrowheads point to specific GFP (Lgr5) expression in the outer root sheath of the hair follicles. Scale bar, 50 μm.

Supplementary Figure 3 LGR reporter–Tomato lineage-tracing treatment regime and sample collection.

(a,b) Lineage tracing using Tomato expression driven from Lgr5 (a) and Lgr6 (b) was performed in chemically induced skin tumors from Lgr6-EGFP-CreERT2 and Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 8 weeks of tumor promotion using TPA. Mice were then treated with tamoxifen (TAM), and papillomas were harvested at 2 d, 3 weeks and 6 weeks thereafter, to measure the extent of lineage tracing from either Lgr5-GFP- or Lgr6-GFP-positive stem cells.

Supplementary Figure 4 Dermal Tomato+ cells derived from Lgr5-GFP- and Lgr6-GFP-positive cells are observed in benign squamous tumors.

Lineage tracing of cells derived from Lgr5- and Lgr6-expressing cells was performed in chemically induced skin tumors from Lgr6-EGFP-CreERT2 and Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 8 weeks of tumor promotion using TPA. Mice were then treated with tamoxifen (TAM), and papillomas were harvested at 3 weeks and 6 weeks thereafter, to measure the extent of lineage tracing from either Lgr5-GFP- or Lgr6-GFP-positive stem cells. (ad) At 3 and 6 weeks after tamoxifen treatment, Tomato+ cells derived from Lgr5-expressing cells (a,c) and Lgr6-expressing cells (b,d) were both observed in the dermal component of papilloma tissue. The yellow dotted boxes demarcate the magnified regions of dermal Tomato+ cells in the insets. The white dotted line indicates epithelial–dermal border. The white arrow indicates dermal Tomato+ cells. DAPI staining (blue) was performed to localize nuclear staining in sections. The experiment was preformed once using three independent tumors; representative images are provided. Scale bar, 50 μm.

Supplementary Figure 5 Clonal expansion of cells derived from Lgr5-expressing cells is not observed in primary SCCs in vivo.

Lineage tracing of Lgr5-expressing cells was performed in the same chemically induced primary (1°) SCC from Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 20 weeks of tumor promotion using TPA. Once the SCC was readily visible, the mouse was treated with tamoxifen (TAM), and 2 d later initial labeling of Tomato+ cells derived from Lgr5-expressing cells was examined in the tumor tissue. (ad) At 2 d after tamoxifen treatment, only rare Tomato+ cells derived from Lgr5-expressing cells (red; b) were readily observed in SCC epithelium, suggesting that Lgr5 is not a CSC marker in primary SCCs in vivo. The yellow dotted boxes demarcate the magnified regions in the insets. The white line indicates the epithelial–dermal border. DAPI (blue) staining was used to visualize cell nuclei (b,d). (a,c) Serial H&E sections of the images depicted in b and d to provide tumor histology. One SCC was used in this study to specifically trace progeny derived from Lgr5-expressing cells in the same tumor in vivo over time. Scale bar, 50 μm.

Supplementary Figure 6 Lgr6-GFP-positive cells are found in carcinoma tissue but do not coexpress SOX2.

(a) Lgr6-GFP+ and Lgr5-GFP+ cells were quantified in primary carcinoma tissue sections (n = 15, 5 FOVs analyzed from 3 independent carcinoma samples). (b) Immunofluorescence for nuclear SOX2 (red) in papilloma tissue. (c) Nuclear SOX2 (red) expression in Lgr6-GFP+ (green) primary carcinoma tissue by immunofluorescence. DAPI (blue) counterstaining was preformed to detect cell nuclei. FOV, field of view; SCC, squamous cell carcinoma. Scale bar, 50 μm.

Supplementary Figure 7 Lgr5 is not expressed in immortalized keratinocyte and SCC cell lines.

Microarray expression analysis was performed on a panel of immortalized keratinocyte (NK and C5N), papilloma (P6, Mscp5, MscP1) and carcinoma cell lines. The cell lines used have been described previously (refs. 57, 90, 91 and references therein). All of the cell lines apart from NK, C5 and Hras-null cells have mutations in the Hras gene. The NK and C5 cells are wild type at Hras and Kras, and the Hras-null cell line has a mutation in Kras. Log2-transformed expression ≤4 corresponds to the background expression level.

Supplementary Figure 8 Lgr5 is not an SCC CSC marker.

(ae) Lgr4 and Lgr6 multiplex in situ hybridization was performed in a selection of skin cell lines—the immortalized keratinocyte cell line NK (a), the SCC cell line A5 (b) and the spindle carcinoma cell line CarB (c), and PACS analysis was performed for in vivo Lgr4, Lgr5 and Lgr6 expression in primary squamous carcinoma epithelial tissue (d,e). The Lgr4 (purple, a,b; red, c) expression patterns are shown in the left panels, while Lgr6 (red) expression patterns are shown in the right panels. DAPI staining (blue) localizes cell nuclei. The yellow dotted boxes identify the magnified area in image insets. (d) PACS-based expression profile for Lgr4, Lgr5 and Lgr6 in cells derived from primary squamous carcinoma tissue. (e) In vivo PACS-based analysis of Lgr4-, Lgr5- and Lgr6-positive cell populations derived from primary squamous carcinoma tissue. LGR coexpression profiles are shown for each individual LGR-positive cell population in vivo. The experiments were repeated twice, and representative images provided. Scale bar, 50 μm.

Supplementary Figure 9 Non-leakiness of the Lgr6-EGFP-IRES-CreERT2 promoter.

Representative section showing back skin harvested from non-4OHT-treated Lgr6-EGFP-IRES-CreERT2+/–/R26RLacZ+/– mice that was processed for X-gal staining. Scale bar, 50 μm.

Supplementary Figure 10 Wnt signaling in SCCs from Lgr6-knockout mice.

Original western blot images used to display β-catenin (phospho- b-catenin, green box; total β catenin, red box) and β-actin (blue box) protein levels in Figure 6k.

Supplementary Figure 11 Proposed model for the role of Lgr6 in tumor fate decisions.

Lgr6+ stem cells in the skin are capable of giving rise to progeny that differentiate down either the epidermal or hair follicle lineage. Within these stem cells, Lgr6 positively regulates Wnt signaling and serves to restrain commitment down the epidermal lineage.

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Supplementary Table 1

Gene Ontology (GO) enrichment results for LGR family members in human HNSCC expression data. (XLSX 9 kb)

Supplementary Table 2

Correlation r values between genes in the canonical Wnt signaling pathway (GO:0060070) and LGR family members. (XLSX 9 kb)

Supplementary Table 3

Primers for qPCR-based expression analysis. (XLSX 8 kb)

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Huang, P., Kandyba, E., Jabouille, A. et al. Lgr6 is a stem cell marker in mouse skin squamous cell carcinoma. Nat Genet 49, 1624–1632 (2017). https://doi.org/10.1038/ng.3957

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