Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumour progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma in situ. Shh-expressing basal cells within this precursor lesion become tumour-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumour cell phenotype can diverge significantly from that of the cancer cell of origin.
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- Supplementary Figure 1: Mouse model of bladder cancer. (252 KB)
(a) Schematic diagram describing mouse model of BBN-induced bladder cancer. (b) Histopathological analysis (H&E) of invasive carcinoma after 6 months of BBN exposure from three different animals. Scale bars represent 50 μm.
- Supplementary Figure 2: Lack of urothelial proliferation at early stages of BBN exposure. (187 KB)
Serial sections made from bladder exposed to BBN for 5 weeks were stained with Ki67 (left panel, green) and CK5 (right panel, green). Sections were co-stained with laminin (red) and DAPI (blue). Scale bars represent 50 μm.
- Supplementary Figure 3: Marked Shh-expressing basal stem cells give rise to CIS and invasive carcinoma. (784 KB)
(a) ShhCreER; R26mTmG mice (four different animals, bladder #1–4) injected with TM on three consecutive days were exposed to BBN for 4 months to induce development of CIS and bladder tissues analysed (H&E, left panel; mG/mT expression, right panel). (b) ShhCreER; R26mTmG mice (five different animals, bladder #1–5) injected with TM on three consecutive days were exposed to BBN for 6 months and bladder tumours analysed (H&E, left panels; mG/mT expression, right panels). L, bladder lumen. Representative images are shown in Fig. 2. Scale bars represent 50 μm.
- Supplementary Figure 4: Ablation of Shh-expressing basal cells renders bladder resistant to nitrosamine-induced formation of invasive carcinoma. (1,806 KB)
TM was injected into ShhCreER; R26DTA mice (twelve different animals; bladder #1–6, with TM; bladder #7–12, without TM) on five consecutive days to ablate Shh-expressing basal cells, and these mice subsequently were exposed to BBN for 6 months (right panels; without TM) and 8 months (left panels; with TM). Bladder tissues were analysed by H&E staining. L, bladder lumen. Representative images are shown in Fig. 3c. Scale bars represent 50 μm.
- Supplementary Figure 5: Tumour-propagating cells derive exclusively from Shh-positive cells in the CIS lesion. (457 KB)
(a) ShhCreER; R26mTmG mice (four different animals: Bladder #1–4) were exposed to BBN for 4 months to induce development of CIS. TM was injected on three consecutive days to label Shh-expressing CIS cells prior to sacrifice and analysis of bladder tissues by H&E staining (left panel) or by immunostaining for mG and CK5 (green and red, respectively, in three right panels). L, bladder lumen. (b) mG/EpCAM-positive and mT/EpCAM-positive populations from six different invasive carcinomas generated as described in Fig. 5b were separated using FACS. (c) BBN-induced bladder tumour cells were injected intramurally into the dome of the bladder. (d) Orthotopic transplantation with serial dilutions of mG/EpCAM-positive and mT/EpCAM-positive cells from invasive carcinomas #2 and #3 (invasive carcinoma #1 is shown in Fig. 5e). Representative images for (a) and (d) are shown in Fig. 5a, e, respectively. Scale bars represent 50 μm.
- Supplementary Figure 6: Shh-negative cells in CIS lesion do not contribute to tumour-propagating cells in invasive carcinoma. (214 KB)
(a) ShhCreER;R26mTmG mice were exposed to BBN for 4 months to induce CIS lesions, then injected with TM on three consecutive days, which heritably labels Shh-positive cells with EGFP whereas cells that do not express Shh remain labeled with tdTomato. Mice were subsequently treated with BBN for two more months, and EGFP and tdTomato positive cancer cells in invasive carcinoma from the resulting animals were separated by FACS. EGFP and tdTomato positive cells were then transplanted subcutaneously into immunocompromised mice (NOD/SCID/IL2Rgnull). (b) Allografts from transplantation of tdTomato- and EGFP-positive cells are shown in left and right panels, respectively. (c) Experimental strategy to investigate the tumorigenic capacity of mixed cancer cells originating from Shh-positive or-negative CIS cells. ShhCreER; R26mTmG mice were treated with BBN for 4 months to induce CIS, then injected with TM on three consecutive days to mark Shh-positive and-negative cells with EGFP and tdTomato, respectively. Mice were subsequently treated with BBN for two more months, and EGFP- and tdTomato-positive cancer cells from the resulting animals were separated by FACS. Equal numbers of EGFP- and tdTomato-positive cells were then mixed and subcutaneously transplanted into immunocompromised mice (NOD/SCID/IL2Rgnull). (d) Allograft tumour from the experiment described in (c) was analysed by H&E staining (left panel) and immunostaining for EGFP and tdTomato (green and red, respectively in middle and right panels). Note only EGFP, not tdTomato, is expressed in the tumour allograft. Scale bar represents 50 μm.
- Supplementary Figure 7: Loss of Shh expression in invasive carcinoma. (266 KB)
(a) Laser capture microdissection of three different tumour areas from three distinct bladder tumours. Nine tumour areas were assessed; 3 different tumour areas from 3 distinct tumours. Representative images (area #1 from tumour #1) are shown in Fig. 6c. (b) Analysis of Shh mRNA expression by qRT-PCR in microdissected basal urothelium and carcinoma cells. ND, not detected. Data are presented as mean ± s.e.m. from 3 technical replicates; 9 tumour areas were assessed (3 different tumour areas from 3 distinct tumours). Scale bars represent 50 μm.
- Supplementary Figure 8: Stochastic four colour fluorescence marking of normal bladder and intestinal cells. (535 KB)
(a) TM was injected into ActinCreER; R26Rainbow mouse to label all cells in the bladder with one of four fluorescence colours prior to BBN exposure. Right panels show magnified views of the regions highlighted by white boxes in the left panel. (b) Mouse intestine 4 months after TM injection into ActinCreER; R26Rainbow mouse. Note clonal expansions of intestinal stem cells from crypts into the villi, as expected, thus validating four-colour marking with the Rainbow mouse. L, lumen. Scale bars represent 50 μm.
- Supplementary Information (2,230 KB)