Fig. 1: Benign-to-invasive rewiring of the tumour-initiating CSC transcriptome fuels angiogenesis.
From: Ras drives malignancy through stem cell crosstalk with the microenvironment
a, The tumour model. Lentivirus containing a TGFβ mCherry reporter and transactivator rtTA3 was injected at a low titre in utero into the amniotic sacs of E9.5 TRE-HRASG12V mouse embryos to sparsely transduce individual skin progenitors. Postnatally, doxycycline activates rtTA3 and induces HRASG12V in these stem cells. Haematoxylin and eosin (H&E) staining reveals temporally distinct pathologies of benign and malignant SCCs. Tu, tumour; St, stroma. Scale bars, 300 µm. b, Quantification of a collapsed z-stack of 3D whole-mount immunofluorescence images and FACS-purified mCherry+ITGA6high basal progenitors reveals increased TGFβ signalling as tumours progress to invasive SCCs (Extended Data Fig. 1b). Bottom left: n = 7 (papilloma) and n = 10 (SCC); bottom right: n = 6 (papilloma) and n = 8 (SCC) tumours per stage. P < 0.0001 (left) and P = 0.0018 (right). Scale bars, 50 µm. c, UMAP representations and unsupervised k-nearest-neighbour-based clustering of single-cell transcriptomes performed on pooled FACS-isolated integrinlow (spiked, 159 total suprabasal) and integrinhigh (bulk, 1,346 total basal) cells from invasive SCC tumours. Clusters C2 and C3, basal progenitors; C1, suprabasal cells. Note that mCherry (TGFβ reporter, dotted box) is enriched in, but not exclusive to, C2 (35.8% of all basal cell progenitors). C2 is enriched for markers of SCC-CSCs with tumour-initiating and invasive properties. The UMAP plots show the relative expression levels (log2[TPM + 1]) of these genes across single cells. See also Extended Data Fig. 1g. d, Angiogenesis is the top GO biological process (BP) term of C2 CSC transcripts (UMAP displays clustering). P values were calculated using DAVID bioinformatic analysis. See also Extended Data Fig. 3. Dev., development; neg. reg., negative regulation; org., organization; prolif., proliferation; sig. trans., signal transduction. e, 3D collapsed whole-mount immunofluorescence images of the invasive fronts of tissue sections. Keratin 18 (K18) identifies CSCs; CD31 identifies vasculature. Scale bars, 150 µm. Quantifications are of keratin 18+ cell abundance, proximity to vessels and distances with vessels. n = 8 (top middle), n = 8 (bottom middle) and n = 8 (right) tumours per condition per stage. P < 0.0001 (top and bottom middle); and P0–25 = 0.0020, P25–50 = 0.0176, P50–75 = 0.1337, P75–100 = 0.1358. For b and e, statistical analysis was performed using unpaired two-tailed Student’s t-tests; NS, P ≥ 0.05; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are mean ± s.e.m. (b and e). See also Supplementary Tables 1–3. The diagram in a was created using BioRender.
