Abstract
The skin epidermis is constantly renewed throughout life1,2. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour initiation3. However, the ways in which oncogenic mutations affect the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumour development are unknown. Here, through multidisciplinary approaches that combine in vivo clonal analysis using intravital microscopy, single-cell analysis and functional analysis, we show how SmoM2—a constitutively active oncogenic mutant version of Smoothened (SMO) that induces the development of basal cell carcinoma—affects clonal competition and tumour initiation in real time. We found that expressing SmoM2 in the ear epidermis of mice induced clonal expansion together with tumour initiation and invasion. By contrast, expressing SmoM2 in the back-skin epidermis led to a clonal expansion that induced lateral cell competition without dermal invasion and tumour formation. Single-cell analysis showed that oncogene expression was associated with a cellular reprogramming of adult interfollicular cells into an embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparisons between the ear and the back skin revealed that the dermis has a very different composition in these two skin types, with increased stiffness and a denser collagen I network in the back skin. Decreasing the expression of collagen I in the back skin through treatment with collagenase, chronic UV exposure or natural ageing overcame the natural resistance of back-skin basal cells to undergoing EHFP reprogramming and tumour initiation after SmoM2 expression. Altogether, our study shows that the composition of the extracellular matrix regulates how susceptible different regions of the body are to tumour initiation and invasion.
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Data availability
All of the raw sequencing data have been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE228047. All of the proteomics data have been deposited to the ProteomeXchange with the dataset identifier PXD041644 and https://doi.org/10.6019/PXD041644. Source data are provided with this paper.
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Acknowledgements
We thank the ULB animal facility and genomics core facility (F. Libert and A. Lefort); DIAPath–CMMI, which is supported by the European Regional Development Fund and the Walloon Region; E. Zindy for help and expertise in immunohistochemistry analyses; K. Midner for help with sample preparation for electron microscopy; V. Greco, P. Friedl and P. Rompolas for help and advice on the intravital imaging; and C. Dubois for her help with FACS sorting. N.B. is supported by the FRIA. P.V. is supported by the TELEVIE. The Department of Pathology at Erasme Hospital acknowledges support from ‘Fonds Yvonne Boël’ and ‘Fonds Erasme pour la recherche médicale’ (Convention d’Excellence de Gaiffier d’Emeville 2015–2021). S.A.W. was supported by the Max Planck Society and the Sigrid Juselius Foundation; Y.A.M. is the recipient of Human Frontier Science Program fellowship LT000861/2018; and C.B. is supported by the WEL Research Institute, FNRS, TELEVIE, Fond Erasme, Fondation Contre le Cancer, ULB Foundation, Fondation Julie et Francoise Drion, EOS FNRS/FWO and European Research Council.
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N.B. and C.B. designed the experiments and performed data analysis. N.B. performed most of the biological experiments. P.V. and N.B. initiated the project. R.S. performed experiments and analyses of proliferation, cell density, symmetric renewal, differentiation and apoptosis. Y.S. and A.S. performed bioinformatic analysis. D.Z., Y.A.M. and S.A.W. performed stiffness and electron microscopy experiments. I.S. and E.M. performed vital coloration and microscopic analysis. J.A. and A.C. performed immunostaining for collagen I. E.T., D.V.H. and F.I. performed proteomic analysis. N.B., R.S., P.V., A.-L.D. and M.L. performed genotyping, immunostaining and follow-ups of the mice. N.B. and C.B. wrote the manuscript. All authors read and approved the final manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Intravital microscopy set-up to follow the same clone over time in living mice.
a, Set-up of intravital microscope. b,c, Making tattoos in the mouse ear (b) and back skin (c). d, Method combining tattoo and the stable pattern of hair follicles to establish spatial coordinate to monitor and track the same clones in the skin by intravital microscopy over time. For the ear, we used a 30g needle to make three punctiform tattoos. The tattoos are visible at 10× magnification. At 10× magnification, we can select an easily identifiable area using the hair follicles (yellow dashed square). At 40× magnification, we can image an area at the single-cell resolution. This area will be revisited over time to monitor cell dynamics. e, The same strategy is used to follow the same clones over time in the back skin, with the difference that tattooing (white dashed line) is performed using a tattoo machine. For more details, see Methods. White dashed line: delimitation of the tattoo. Hair follicles are numbered.
Extended Data Fig. 2 Clonal dynamics of normal epidermis and mode of cell division of SmoM2 cells in the ear and in the back skin.
a,b, Microscopy analysis of β4 integrin (β4) (a) and Laminin-332 (Lam5) (b) in SmoM2 clones in the ear and the back skin 6 weeks following tamoxifen administration. Note the small disruption of the basal lamina (BL) at the leading edge of SmoM2 clones invading the dermis (yellow arrows). c, Quantification of the fluorescence intensity of the immunostainings of Lam5 in control and K14CreER/Rosa-SmoM2-YFP mice 6 weeks after SmoM2 expression. Measurements were performed along the BL in control and at the leading edge of invading BCC using ImageJ. For the control, 20 areas Lam5 were analysed (n = 3). For SmoM2, 56 BCC for Lam5 (n = 3). Two-tailed z-test. d, Quantification of the fluorescence intensity of the immunostainings of β4 in control and K14CreER/Rosa-SmoM2-YFP mice 6 weeks after SmoM2 expression. Measurements were performed along the BL in control and at the leading edge of invading BCC using ImageJ. For the control, 21 areas for β4 were analysed (n = 3). For SmoM2, 41 BCC for β4 (n = 3). Two-tailed z-test. e, Transmission electron microscopical images of ear and back skin ultrathin sections from control and K14CreER/Rosa-SmoM2-YFP mice 6 weeks after SmoM2 expression. Representative images show the remodelling of the BL (increased (blue arrow) or reduced (yellow arrow)) observed at the leading edge of BCC in the ear epidermis whereas the BL remains intact in the back skin after SmoM2 expression. EC: epidermal cell. BL: basal lamina. Scale bar: 200 nm. f, Quantification of the total number of cells per clone in WT ear epidermis at 2, 4, and 6 weeks after clonal marking following tamoxifen injection by intravital microscopy (n = 4). Kruskal–Wallis test. g, Intravital imaging of the same clones in the outer part of the ear skin at 2, 4, and 6 weeks after tamoxifen administration to K14CreER/Ichr-Ctrl-mosaic mice. h, Whole-mount images of epidermis of K14CreER/Ichr-Ctrl-mosaic mice from the inner and outer part of the ear, the back and the tail at 45 days after tamoxifen administration. 40x Magnification. i, Quantification of the number of cells per clones at 6 weeks on whole mount of epidermis of K14CreER/Ichr-Ctrl-mosaic. The number of basal cells found by intravital microscopy was similar to the one found by whole-mount immunostaining. (n = 4 mice). Kruskal–Wallis test. j,k, BrdU pulse-chase analysis to analyse the fate of two dividing daughter cells (doublets). Whole-mount images of SmoM2 clones in the ear (j) and the back skin (k) showing the location of BrdU doublets: two BrdU+ basal cells, one basal and one suprabasal BrdU+ cells or two suprabasal BrdU+ cells. l, Percentage of cell-fate outcome in WT and SmoM2 cells in the ear and in the back skin. WT ear cells (61 BrdU+ doublets from 3 mice), ear 6 weeks SmoM2 clones (110 BrdU+ doublets from 4 mice), ear 12 weeks SmoM2- clones (85 BrdU+ doublets from 3 mice), WT back skin cells (113 BrdU+ doublets from 3 mice), 6 weeks SmoM2 clones (137 BrdU+ doublets from 4 mice) and 12 weeks SmoM2 clones (115 BrdU+ doublets from 3 mice). These data show that in WT ear and back skin, most division present asymmetric cell-fate outcome, whereas most SmoM2 cells in WT ear and back skin divide symmetrically. a,b,g,h, Scale bars, 20 µm. f,i, In parentheses, the number of clones. c,d,f,i, Mean ± s.e.m.
Extended Data Fig. 3 Expression of actin, phospho-myosin and active YAP in SmoM2 clones in the ear and in the back-skin epidermis.
a,b, Immunostainings for actin (phalloidin) (a) and phospho-myosin II (P-Myosin II) (b) in control and K14CreER/Rosa-SmoM2-YFP mice at 6 weeks after SmoM2 expression, revealing an increase in the level of F-actin (a) and phospho-myosin II (b) in SmoM2 cells in the ear. In addition, some WT cells at the border of the ear clones were also positive for phospho-myosin II. No change of phalloidin or phospho-myosin II was found in the back skin. Red: Phalloidin or phospho-myosin II; Blue: Hoechst. Scale bars, 20 µm. c, Immunostainings for active Yap (aYap) in control and K14CreER/Rosa-SmoM2-YFP mice at 6 weeks after SmoM2 activation, revealing an increase expression of nuclear YAP at the leading edge of SmoM2-expressing clones and in the WT cells at the border of the clone in the ear. Only rare nuclear YAP expressing cells were found in the SmoM2-expressing cells of the back skin. Red: aYAP (d) Green: SmoM2; Blue: Hoechst. Scale bars, 20 µm. d,e, Immunostainings on whole mount for actin (phalloidin) (d) and active Yap (aYap) (e) in K14CreER/Rosa-SmoM2-YFP/Rosa-mT/mG 3 weeks after SmoM2 activation, showing a higher level of actin expression and aYap in SmoM2 cells in the ear. No change of actin or aYap was found in the back skin. Blue: Hoechst. Scale bars, 20 µm.
Extended Data Fig. 4 Cell competition, proliferation and apoptosis surrounding SmoM2 clones in the back skin and in the ear.
a, Immunostainings of EdU in the ear and the back skin of K14CreER/Rosa-SmoM2-YFP 6 weeks after SmoM2 expression. b, Quantification EdU-positive cells in SmoM2 clones in K14CreER/Rosa-SmoM2-YFP at 3 (n = 3), 6 (n = 4 for the ear and n = 5 for the back skin), and 12 weeks (n = 3) after SmoM2 activation showing an increase of proliferation in SmoM2 clones compared to the control (n = 3). Mean ± s.e.m. t-test. c, Intravital microscopy analysis of the cell density in WT cells and SmoM2 clones in the ear and the back skin of K14CreER/ Rosa-mT/mG and K14CreER/Rosa-SmoM2-YFP/Rosa-mT/mG before and at 6 weeks after SmoM2 expression. d,e, Quantification of aspect ratio of the SmoM2 cells at the border (d) and at the centre (e) of the clone 3 and 6 weeks after SmoM2 expression. These data show that the SmoM2 cells adopt an elongated shape in the ear whereas in the back skin, SmoM2 cells are more deformed in the centre of the clones than at the periphery. In blue, the average. n = 3 mice for each conditions. Mean ± s.e.m. Two-tailed z-test. f, Time-lapse intravital microscopy analysis of a SmoM2-clone in the back skin of K14CreER/Rosa-SmoM2-YFP/Rosa-mT/mG mouse 10 weeks after tamoxifen administration at different time points for 56 h showing the clonal expansion of SmoM2 cells and out competition of WT cells surrounding the SmoM2-clone. g, Immunostaining of cleaved Caspase 3 (CC3) in ear and the back skin of K14CreER/Rosa-SmoM2-YFP at 6 weeks after oncogenic expression. Scale bar, 20 µm. h, Quantification of CC3+ cells in WT cells and SmoM2 –clones at 3, 6, and 12 weeks after SmoM2 activation showing the increase of apoptosis in SmoM2 clones. Mean ± s.e.m. t-test. a,c,f g, Scale bars, 20 µm. b,d,e,h, In parentheses, the number of cells.
Extended Data Fig. 5 Cell competition, proliferation and apoptosis surrounding SmoM2 clones in the back skin and in the ear by whole-mount imaging.
a–c, Immunostaining on whole-mount back skin and ear epidermis for cleaved caspase 3 (CC3) (a), keratin 10 (Krt10) (b) and EdU (c) in K14CreER/Rosa-SmoM2-YFP/Rosa-mT/mG 6 weeks after SmoM2 activation. These data show WT apoptotic cells and basal cells expressing K10 at the edge of the SmoM2-clone in the back skin and not in the ear. In the ear and the back skin, Edu-positive cells are more present at the periphery of the clone. Scale bars, 20 μm.
Extended Data Fig. 6 Single-cell RNA-seq analysis of WT cells and SmoM2 epidermal cells, and FACS sorting strategy.
a,b. Fluorescence activated cell sorting (FACS) plots showing the gating strategy used to FACS isolate the proportion of SmoM2-YFP in control mice (a) and K14CreER/Rosa-SmoM2-YFP mice (b) 6 weeks after oncogenic expression. c, Table showing the genes used to annotate the different clusters. d, UMAP graphic of the clustering analysis for single-cell RNA-seq of the WT ear and back-skin epidermis after data integration using Seurat.
Extended Data Fig. 7 Gene ontology of EHFP-associated genes.
a, Gene ontology analysis of the genes upregulated in the ear 6 W EHFP cluster. Statistics are based on the permutation method. b–g, UMAP plot of the ear and back samples coloured by the level of normalized gene expression values for genes regulating ECM organization or basal lamina (b), migration (c), cell proliferation (d), the WNT pathway (e), cell–cell adhesion (f) or the Hedgehog pathway (g).
Extended Data Fig. 8 EHFP reprogramming and block of normal IFE differentiation in SmoM2-expressing cells in the ear but not in the back-skin epidermis.
a,b, Immunostaining of ear skin (a) and back skin (b) for K15, LEF1 and SmoM2–YFP in control and in K14CreER/Rosa-SmoM2 mice at 6 and 12 weeks after tamoxifen induction. SmoM2 cells in the ear skin are reprogrammed into EHFP-like cells. c,d, Immunostaining of ear skin (c) and back skin (d) for K1, K10 and SmoM2–YFP in control and in K14CreER/Rosa-SmoM2-YFP mice at 6 and 12 weeks after tamoxifen induction. SmoM2 cells in the back skin differentiate into K1/K10-positive cells. These immunostaining analyses also confirmed the expression of normal differentiation marker K1 in the suprabasal cells of the SmoM2-expressing cells in the back skin and the repression of the differentiation in SmoM2-expressing clones in the ear epidermis. IFE, interfollicular epidermis; HF, hair follicle. a–d, Scale bars, 20 μm.
Extended Data Fig. 9 Orientation of collagen-fibre bundles in the ear and in the back skin.
a–f, Angular distribution of collagen-fibre bundles measured by OrientationJ software from SHG images of the dermis of the WT ear (a,b) and the back skin before (c,d) and after (e,f) collagenase administration. HBS colour-coded map (a,c,e) and histogram of local angles (b,d,f) showing the orientation of collagen-fibre bundles. The orientation of the collagen bundles was relatively similar in the two locations.
Extended Data Fig. 10 ECM composition and remodelling in the ear and in the back skin after SmoM2 expression.
a,b, Quantitative proteomic analysis of the dermis after SmoM2 expression in the ear (a) and the back skin (b). Volcano plot indicating the differentially expressed proteins. Proteins upregulated in SmoM2 dermis (red dot) or in WT dermis (blue dot). A total of 742 proteins were significantly downregulated, and 7 proteins were significantly upregulated in SmoM2 back-skin dermis samples. Proteomic analysis shows that only very few proteins were more upregulated in the dermis of back skin after SmoM2 expression. Twenty-seven proteins were significantly upregulated in WT ear dermis and 40 proteins were significantly upregulated in SmoM2 ear dermis, which contains a higher number of proteins expressed by inflammatory cells after oncogenic expression in the ear. Statistical significance for differential regulation is set at FDR < 0.05 and fold change of 2 (|log2FC| ≥ 1). c, Gene ontology analysis of the proteins upregulated in the SmoM2 ear at 6 weeks after oncogenic expression. Statistics are based on the permutation method. d, H&E staining in control and K14CreER/Rosa-SmoM2-YFP mice of the ear and back skin at 1.5 and 3 months after SmoM2 activation. e, Quantification of CD45+ cells in the dermis of the ear and the back skin in WT and SmoM2 mice at 6 weeks after SmoM2 expression showing the increase of the inflammation in the dermis of the ear after SmoM2 expression but not in the back skin. For each condition, at least 10 areas (40×) per mice (n = 3) were analysed. In parentheses, number of CD45+ cells. Mean ± s.e.m. Paired t-test, with two-sample unequal variance (heteroscedastic). f, Immunostaining for CD45 (pan-immune cells), Vimentin (fibroblasts and inflammatory cells), CD68 (macrophages), CD4 (T helper lymphocytes), CD8 (T cytotoxic lymphocytes) and SmoM2 in control and K14CreER/Rosa-SmoM2-YFP mice at 3, 6, and 12 weeks after SmoM2 expression, revealing a strong recruitment of immune cells in particular macrophages (CD45+ CD68+) and fibroblasts (Vimentin+) in the SmoM2-expressing cells that invade the ear dermis. No inflammatory cell infiltration is observed in the dermis of the back skin. Scale bars, 20 µm.
Extended Data Fig. 11 The level of collagen I expression correlates with the competence for BCC initiation in the tail skin.
a, Collagen I immunohistochemistry of the scale and interscale regions of the tail. The level of collagen I is higher in the scale compared to the interscale of the tail. Scale bar, 50 µm. b, H&E stainings in the tail skin of K14CreER/Rosa-SmoM2-YFP mice at 6 weeks after SmoM2 activation, showing that BCC arises from the interscale epidermis whereas the tail scale epidermis (as the back skin) is resistant to SmoM2-induced tumorigenesis. c, Immunostaining of LHX2 in the tail of K14CreER/Rosa-SmoM2-YFP mice 6 weeks after SmoM2 activation. Scale bars, 20 µm. d, Quantification of tumour invasion measured by the vertical distance between WT cells and SmoM2 cells in the tail dermis at 6 weeks after SmoM2 activation. SmoM2 clones in the interscale but not in the scale of the tail skin invade the dermis. A total of 366 clones in the scale region and 318 clones in the interscale region of the tail have been quantified from 3 mice. Mean ± s.e.m. Kruskal–Wallis test.
Extended Data Fig. 12 Inflammation in the back skin does not promote the invasion of SmoM2-expressing cells.
a, Immunostaining of back skin after intradermal injection of PBS (left) or collagenase (right) in control mouse (K14CreER/Rosa-mT/mG), showing the absence of dermal invasion of normal epidermis. b,c. Immunostaining for β4 and Lam5 (b) and electron microscopy imaging (c) of the back skin of WT mice (control) and after collagenase injection showing that the BL remains intact after collagenase injection. Scale bars, 20 µm. EC, epidermal cell; BL, basal lamina. Scale bars for EM images: 500 nm (left) and 200 nm (right). d–h, H&E and immunostaining for CD45 (immune cells) and vimentin (immune cells and fibroblasts) of back skin in control mice (d), one day after collagenase injection (e), of back skin of K14CreER/Rosa-SmoM2-YFP mice at 6 weeks after oncogene expression following bleomycin administration for 1 month (f), of back skin of K14CreER/Rosa-SmoM2-YFP mice at 6 weeks after oncogene expression following daily application of imiquimod for 1 month (g), of back skin in K14CreER/Rosa-SmoM2-YFP 1.5-year-old mice at 6 weeks after oncogene expression (h). Scale bars, 20 µm. i, Quantification of CD45+ cells in the dermis of the back skin in WT and SmoM2 mice in different inflammatory conditions (collagenase, imiquimod, bleomycin) and in old mice. For the control SmoM2, 472 CD45+ cells for WT (n = 3). For the old mice, 569 CD45+ cells from 41 area (n = 2). For imiquimod, 2,801 CD45+ cells from 30 areas (n = 2). For collagenase, 2,225 CD45+ cells from 30 areas (n = 2). For bleomycin, 3,094 CD45+ cells from 45 areas (n = 3). These data show that inflammation induced by imiquimod and bleomycin does not promote invasion of SmoM2-expressing cells. Mean ± s.e.m. Paired t-test, with two-sample unequal variance.
Extended Data Fig. 13 Gene ontology of SmoM2 cells of the back skin.
a, Gene ontology analysis of the genes upregulated in the all of the SmoM2 cells of the back skin 6 weeks after oncogenic expression versus the WT cells of the back skin. Statistics are based on the permutation method.
Extended Data Fig. 14 Model summarizing how ECM composition dictates the mode of cell competition and competence for tumour initiation.
a, Oncogenic Hedgehog activation by the SmoM2 oncogene induces BCC in the ear epidermis. SmoM2 cells adopt a placode-like shape, lose their ability to differentiate and are reprogrammed into an EHFP-like fate. The SmoM2 clones compress the WT cells at the edge. SmoM2 clones invade the dermis until they form a branch-like structure and become invasive BCCs. b, Oncogene-expressing cells in the back-skin epidermis undergo clonal expansion in a lateral manner without leading to tumour invasion. SmoM2 cells are very efficient at outcompeting the WT cells but lack the ability to become invasive. The high level of collagen I in the dermis of the back skin restricts BCC development and imposes a lateral clonal expansion of oncogene-expressing cells. c, Decreasing the abundance of collagen by collagenase injection, during natural ageing and after chronic UVA exposure overcomes the natural resistance to BCC development in the back skin.
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Bansaccal, N., Vieugue, P., Sarate, R. et al. The extracellular matrix dictates regional competence for tumour initiation. Nature 623, 828–835 (2023). https://doi.org/10.1038/s41586-023-06740-y
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DOI: https://doi.org/10.1038/s41586-023-06740-y
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