Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma

Journal name:
Nature Cell Biology
Volume:
16,
Pages:
469–478
Year published:
DOI:
doi:10.1038/ncb2956
Received
Accepted
Published online
Corrected online

Abstract

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.

At a glance

Figures

  1. Histopathology of murine nitrosamine-induced bladder carcinoma mimics progression of human urothelial CIS to invasive carcinoma.
    Figure 1: Histopathology of murine nitrosamine-induced bladder carcinoma mimics progression of human urothelial CIS to invasive carcinoma.

    (a,b) Histopathological analysis (haematoxylin and eosin (H&E) staining) of early (a) and late (b) stages of N-butyl-N-4-hydroxybutyl nitrosamine (BBN)-induced bladder carcinogenesis (see also Supplementary Table 1). The lower panels show magnified views of the outlined regions in the upper panels. Note the appearance, beginning at 3 months of BBN exposure, of histologic abnormalities, including nuclear atypia, crowding, and architectural disarray histologically identical to human CIS, and leading to invasive carcinoma by 6 months of BBN exposure. L, bladder lumen. Scale bars, 50 μm. Repeated experimental results are shown in Supplementary Table 1.

  2. Shh expression marks basal stem cells that give rise to CIS and invasive bladder carcinoma.
    Figure 2: Shh expression marks basal stem cells that give rise to CIS and invasive bladder carcinoma.

    (a,b) Schematic diagrams (top left) show the experimental strategies for marking Shh-expressing cells and their progeny before BBN induction of CIS (a) or invasive carcinoma (b). ShhCreER; R26mTmG mice injected with tamoxifen (TM) on 3 consecutive days were treated with BBN for 4 months to induce CIS lesions (a) or 6 months to induce invasive carcinoma (b), followed by histological analysis (H&E) or analysis of mG/mT expression. All tumours and CIS lesions are marked by expression of mG, indicating that invasive carcinoma and CIS originate from Shh-expressing basal cells, not intermediate or luminal cells. L, bladder lumen; mG, membrane-targeted enhanced green fluorescent protein (EGFP); mT, membrane-targeted tdTomato. Repeated experimental results are shown in Supplementary Fig. 3. Scale bars, 50 μm.

  3. Ablation of Shh-expressing basal stem cells confers resistance to nitrosamine-induced formation of invasive bladder carcinoma.
    Figure 3: Ablation of Shh-expressing basal stem cells confers resistance to nitrosamine-induced formation of invasive bladder carcinoma.

    (a) TM was injected into ShhCreER; R26DTA mice on 5 consecutive days. Five days after the last TM injection, bladders were analysed by immunostaining. Sections from the bladders of control vehicle-injected or TM-injected mice (left and right panels, respectively) were stained for CK5 (green). Note that TM treatment effectively ablates basal epithelial cells in the ShhCreER; R26DTA mouse bladder. (b) ShhCreER; R26DTA mice were injected with TM on 5 consecutive days to ablate Shh-expressing basal cells and were maintained for 8 months without BBN exposure. Histology of the bladder from a mouse 8 months after TM injection shows the failure of maintenance of normal bladder epithelium, resulting from loss of stem cells. The right panel shows an enlarged view of the outlined region in the left panel. (c,d) TM was injected into ShhCreER; R26DTA mice on 5 consecutive days to ablate Shh-expressing basal cells, and mice were exposed to BBN for 6 (c) or 4 (d) months. Bladder tissues were analysed by H&E staining (left panels) and immunostaining for CK5 (green, right panels). The upper panels show bladders from vehicle controls (no TM). L, bladder lumen. Repeated experimental results for c are shown in Supplementary Fig. 4. Scale bars, 50 μm.

  4. Ablation of Shh-expressing basal stem cells reduces CIS during nitrosamine-induced carcinogenesis while preserving urothelial architecture.
    Figure 4: Ablation of Shh-expressing basal stem cells reduces CIS during nitrosamine-induced carcinogenesis while preserving urothelial architecture.

    (a) Histopathological analysis (H&E) of TM-injected ShhCreER; R26DTA mice after 1–4 months of BBN exposure. Repeated experimental results are shown in Supplementary Tables 2 and 3. (b) Histopathological evaluation of CIS in bladders from ShhCreER; R26DTA mice (Supplementary Tables 2 and 3). Scores from 0 to 3 are based on the extent of CIS within urothelium (0, none; 1, <10%; 2, 10–30%; 3, >30%). No invasive carcinoma and little or no CIS was induced by BBN exposure in animals with ablated stem cells. n = 4 (1 month), n = 4 (2 months), n = 8 (3 months), n = 8 (4 months) for vehicle-treated ShhCreER; R26DTA mice; n = 4 (1 month), n = 4 (2 months), n = 8 (3 months), n = 9 (4 months) for TM-treated ShhCreER; R26DTA mice. Data are presented as mean ± s.e.m. (c,d) ShhCreER; R26DTA mice were injected with TM on 5 consecutive days to ablate Shh-expressing basal cells and exposed to BBN for 3 (c) and 4 (d) months. Sections from the bladders of control vehicle-injected or TM-injected mice (top and bottom panels, respectively) were stained for the luminal and basal markers, CK18 and CK5 (green and red, respectively). Note that luminal cells are well preserved at intermediate stages of BBN exposure in stem-cell-ablated animals. L, bladder lumen. Scale bars, 50 μm.

  5. Shh-positive and-negative cells in the CIS lesion contribute to invasive carcinoma, but tumour-propagating cells derive exclusively from Shh-positive cells.
    Figure 5: Shh-positive and-negative cells in the CIS lesion contribute to invasive carcinoma, but tumour-propagating cells derive exclusively from Shh-positive cells.

    (a) Experimental scheme (top left panel) to label Shh-expressing cells in the CIS lesion. Following 4 months of BBN exposure to induce CIS, ShhCreER;R26mTmG mice were injected with TM on 3 consecutive days to label Shh-expressing cells before euthanization. Bladder tissues were analysed by H&E staining (top right panel) or by immunostaining for mG and CK5 (green and red, respectively). The lower panels show magnified views of the regions highlighted by white squares in the panels immediately above. (b) Experimental scheme for marking Shh-positive and-negative cells in CIS lesions, and to track them into carcinoma formation. (c) ShhCreER; R26mTmG mice exposed to BBN for 4 months and injected with TM on 3 consecutive days were subsequently exposed to BBN for 2 more months. Invasive carcinomas from these animals were analysed by H&E staining (left panel) or for expression of mG or mT, which respectively marks cells that expressed (green) or did not express (red) Shh at the time of TM injection. (d) Experimental scheme to determine the tumour-propagating ability of carcinoma cells. mG/EpCAM-positive and mT/EpCAM-positive cells from invasive carcinomas generated as described in b were isolated by FACS and transplanted intramurally into the dome of the bladder. (e) Orthotopic transplantation with serial dilutions of mG/EpCAM-positive and mT/EpCAM-positive cells from a single tumour. Results of transplantations from three different tumours, summarized below, show that only Shh-expressing cells in the CIS lesion will subsequently become tumour-propagating cells in transplantation experiments. L, bladder lumen. Repeated experimental results for a and e are shown in Supplementary Fig. 5a, d, respectively. Scale bars, 50 μm.

  6. Shh expression is lost in invasive carcinoma of murine bladder.
    Figure 6: Shh expression is lost in invasive carcinoma of murine bladder.

    (a) Experimental scheme for marking of Shh-expressing cells in invasive carcinoma induced by 6 months of BBN exposure. (b) ShhCreER; R26mTmG mice exposed to BBN for 6 months were injected with TM on 3 consecutive days to label Shh-expressing cells before euthanization, and the bladder carcinoma was analysed by H&E staining (left panel) or by immunostaining for mG and CK5 (green and red, respectively, in middle and right panels), with DAPI staining to visualize nuclei. Note the lack of mG expression in the carcinoma, indicating the absence of Shh expression, whereas CK5 expression is seen in all cancer cells. (c) Laser capture microdissection (LCM) of normal basal cell layer (upper panels) in untreated bladder and of carcinoma cells within tumour (lower panels). Nine tumour areas were assessed (3 different tumour areas from 3 distinct tumours) and representative images are shown here (area no. 1 from tumour no. 1). Other areas from 3 distinct tumours (area nos 2–3 in tumour no. 1; area nos 1–3 for tumour no. 2; area nos 1–3 from tumour no. 3) are shown in Supplementary Fig. 7a. (d) Shh mRNA is detected in cells microdissected from normal basal cells but absent from 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, 50 μm.

  7. Four-colour marking reveals monoclonal and oligoclonal urothelial colonization and carcinoma formation on nitrosamine exposure.
    Figure 7: Four-colour marking reveals monoclonal and oligoclonal urothelial colonization and carcinoma formation on nitrosamine exposure.

    (a) Schematic diagram of the Rainbow allele. By virtue of three different lox sites (lox2272, loxN and loxP), individual cells express either EGFP (before Cre-mediated recombination) or mCerulean, mOrange or mCherry (after Cre-mediated recombination). (b) Experimental scheme to investigate clonal relationships during bladder carcinogenesis. A low dose of TM injected into ActinCreER; R26Rainbow mice stochastically labelled bladder cells with one of four fluorescence colours, and these animals then were exposed to BBN until they developed CIS (c) or invasive carcinoma (d, e). (c) Bladders from TM-injected ActinCreER; R26Rainbow mice exposed to BBN for 4 months (CIS no. 1) or 5 months (CIS no. 2) and analysed by H&E staining (left panels) or for four-colour fluorescence. The right panels show magnified views of the regions highlighted by white rectangles in the middle panels. CIS lesions covering the entire urothelium arise from a single (no. 1, monoclonal) or two cells (no. 2, oligoclonal). A small carcinoma arising within the blue-marked CIS no. 2 is outlined by a dotted line. (d,e) Bladders from TM-injected ActinCreER; R26Rainbow mice exposed to BBN for 6 months were analysed by H&E staining (top left panels) or for four-colour fluorescence (top right panels). The bottom panels show magnified views of the regions highlighted by white rectangles in the top right panels. Carcinomas within a single bladder arise from one (d, monoclonal) or several (e, oligoclonal) cells, respectively. In e, tumours are outlined by solid lines with asterisks and nearby CIS regions are outlined by dotted lines. L, bladder lumen. Scale bars, 50 μm.

  8. Model for progression of nitrosamine-induced bladder carcinogenesis.
    Figure 8: Model for progression of nitrosamine-induced bladder carcinogenesis.

    BBN-induced invasive carcinoma of the mouse bladder arises from basal stem cells that express Shh and CK5. Normal basal cells accumulate mutations at early stages of carcinogenesis and initiate clonal expansions to form intermediate CIS lesions, as indicated by green, yellow and red colours. During this process, one or two clones become dominant and expand to repopulate the entire urothelium, generating mono/oligo-clonal CIS lesions. CIS basal cells in one of these lesions then lose expression of Shh on establishment of invasive carcinoma.

  9. Mouse model of bladder cancer.
    Supplementary Fig. 1: Mouse model of bladder cancer.

    (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.

  10. Lack of urothelial proliferation at early stages of BBN exposure.
    Supplementary Fig. 2: Lack of urothelial proliferation at early stages of BBN exposure.

    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.

  11. Marked Shh-expressing basal stem cells give rise to CIS and invasive carcinoma.
    Supplementary Fig. 3: Marked Shh-expressing basal stem cells give rise to CIS and invasive carcinoma.

    (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.

  12. Ablation of Shh-expressing basal cells renders bladder resistant to nitrosamine-induced formation of invasive carcinoma.
    Supplementary Fig. 4: Ablation of Shh-expressing basal cells renders bladder resistant to nitrosamine-induced formation of invasive carcinoma.

    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.

  13. Tumour-propagating cells derive exclusively from Shh-positive cells in the CIS lesion.
    Supplementary Fig. 5: Tumour-propagating cells derive exclusively from Shh-positive cells in the CIS lesion.

    (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.

  14. Shh-negative cells in CIS lesion do not contribute to tumour-propagating cells in invasive carcinoma.
    Supplementary Fig. 6: Shh-negative cells in CIS lesion do not contribute to tumour-propagating cells in invasive carcinoma.

    (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.

  15. Loss of Shh expression in invasive carcinoma.
    Supplementary Fig. 7: Loss of Shh expression in invasive carcinoma.

    (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.

  16. Stochastic four colour fluorescence marking of normal bladder and intestinal cells.
    Supplementary Fig. 8: Stochastic four colour fluorescence marking of normal bladder and intestinal cells.

    (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.

Change history

Corrected online 24 April 2014
In the version of this article originally published, the bottom panel of Fig. 4b should have read: 'ShhCreER/WT; R26DTA/WT (tamoxifen)'. In Fig. 7a the three shaded triangles on the left should have been in the sequence: dark, intermediate, light. These errors have now been corrected in the online versions of the Article.

References

  1. Reya, T., Morrison, S. J., Clarke, M. F. & Weissman, I. L. Stem cells, cancer, and cancer stem cells. Nature 414, 105111 (2001).
  2. Taipale, J. & Beachy, P. A. The Hedgehog and Wnt signalling pathways in cancer. Nature 411, 349354 (2001).
  3. Bonnet, D. & Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature Med. 3, 730737 (1997).
  4. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J. & Clarke, M. F. Prospective identification of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. USA 100, 39833988 (2003).
  5. Blanpain, C. Tracing the cellular origin of cancer. Nat. Cell Biol. 15, 126134 (2013).
  6. Barker, N. et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608611 (2009).
  7. Schepers, A. G. et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337, 730735 (2012).
  8. Flesken-Nikitin, A. et al. Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche. Nature 495, 241245 (2013).
  9. Chen, J. et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488, 522526 (2012).
  10. Driessens, G., Beck, B., Caauwe, A., Simons, B. D. & Blanpain, C. Defining the mode of tumour growth by clonal analysis. Nature 488, 527530 (2012).
  11. Molyneux, G. et al. BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. Cell Stem Cell 7, 403417 (2010).
  12. Lim, E. et al. Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat. Med. 15, 907913 (2009).
  13. Lawson, D. A. et al. Basal epithelial stem cells are efficient targets for prostate cancer initiation. Proc. Natl Acad. Sci. USA 107, 26102615 (2010).
  14. Mulholland, D. J. et al. Lin-Sca-1+CD49fhigh stem/progenitors are tumor-initiating cells in the Pten-null prostate cancer model. Cancer Res. 69, 85558562 (2009).
  15. Goldstein, A. S., Huang, J., Guo, C., Garraway, I. P. & Witte, O. N. Identification of a cell of origin for human prostate cancer. Science 329, 568571 (2010).
  16. Wang, X. et al. A luminal epithelial stem cell that is a cell of origin for prostate cancer. Nature 461, 495500 (2009).
  17. Wang, Z. A. et al. Lineage analysis of basal epithelial cells reveals their unexpected plasticity and supports a cell-of-origin model for prostate cancer heterogeneity. Nat. Cell Biol. 15, 274283 (2013).
  18. Miyamoto, T., Weissman, I. L. & Akashi, K. AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc. Natl Acad. Sci. USA 97, 75217526 (2000).
  19. Shin, K. et al. Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder. Nature 472, 110114 (2011).
  20. Nagao, M., Suzuki, E., Yasuo, K., Yahagi, T. & Seino, Y. Mutagenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine, a bladder carcinogen, and related compounds. Cancer Res. 37, 399407 (1977).
  21. Bryan, G. T. The pathogenesis of experimental bladder cancer. Cancer Res. 37, 28132816 (1977).
  22. Hecht, S. S. Cigarette smoking: cancer risks, carcinogens, and mechanisms. Langenbecks Arch. Surg. 391, 603613 (2006).
  23. Zeegers, M. P., Tan, F. E., Dorant, E. & van Den Brandt, P. A. The impact of characteristics of cigarette smoking on urinary tract cancer risk: a meta-analysis of epidemiologic studies. Cancer 89, 630639 (2000).
  24. Bryan, G. T. Pathogenesis of human urinary bladder cancer. Environ. Health Perspect. 49, 201207 (1983).
  25. Spruck, C. H., 3rd et al. Two molecular pathways to transitional cell carcinoma of the bladder. Cancer Res. 54, 784788 (1994).
  26. Chan, K. S. et al. Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells. Proc. Natl Acad. Sci. USA 106, 1401614021 (2009).
  27. He, X. et al. Differentiation of a highly tumorigenic basal cell compartment in urothelial carcinoma. Stem Cells 27, 14871495 (2009).
  28. Yamaizumi, M., Mekada, E., Uchida, T. & Okada, Y. One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15, 245250 (1978).
  29. Maxwell, F., Maxwell, I. H. & Glode, L. M. Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain. Mol. Cell. Biol. 7, 15761579 (1987).
  30. Wu, S., Wu, Y. & Capecchi, M. R. Motoneurons and oligodendrocytes are sequentially generated from neural stem cells but do not appear to share common lineage-restricted progenitors in vivo. Development 133, 581590 (2006).
  31. Jost, S. P. Cell cycle of normal bladder urothelium in developing and adult mice. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 57, 2736 (1989).
  32. Thievessen, I., Wolter, M., Prior, A., Seifert, H. H. & Schulz, W. A. Hedgehog signaling in normal urothelial cells and in urothelial carcinoma cell lines. J. Cell. Physiol. 203, 372377 (2005).
  33. Kimura, F. et al. Decrease of DNA methyltransferase 1 expression relative to cell proliferation in transitional cell carcinoma. Int. J. Cancer [J. International du Cancer] 104, 568578 (2003).
  34. Sidransky, D. et al. Clonal origin bladder cancer. N. Engl. J. Med. 326, 737740 (1992).
  35. Simon, R. et al. Cytogenetic analysis of multifocal bladder cancer supports a monoclonal origin and intraepithelial spread of tumor cells. Cancer Res. 61, 355362 (2001).
  36. Fadl-Elmula, I. et al. Cytogenetic monoclonality in multifocal uroepithelial carcinomas: evidence of intraluminal tumour seeding. British J. Cancer 81, 612 (1999).
  37. Yamamoto, S., Tatematsu, M., Yamamoto, M., Fukami, H. & Fukushima, S. Clonal analysis of urothelial carcinomas in C3H/HeN <- - >BALB/c chimeric mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine. Carcinogenesis 19, 855860 (1998).
  38. Hafner, C. et al. Evidence for oligoclonality and tumor spread by intraluminal seeding in multifocal urothelial carcinomas of the upper and lower urinary tract. Oncogene 20, 49104915 (2001).
  39. Trkova, M. et al. Analysis of genetic events in 17p13 and 9p21 regions supports predominant monoclonal origin of multifocal and recurrent bladder cancer. Cancer Lett. 242, 6876 (2006).
  40. Duggan, B. J. et al. Oligoclonality in bladder cancer: the implication for molecular therapies. J. Urol. 171, 419425 (2004).
  41. Dahse, R., Gartner, D., Werner, W., Schubert, J. & Junker, K. P53 mutations as an identification marker for the clonal origin of bladder tumors and its recurrences. Oncol. Rep. 10, 20332037 (2003).
  42. Paiss, T. et al. Some tumors of the bladder are polyclonal in origin. J. Urol. 167, 718723 (2002).
  43. Vriesema, J. L., Aben, K. K., Witjes, J. A., Kiemeney, L. A. & Schalken, J. A. Superficial and metachronous invasive bladder carcinomas are clonally related. Int. J. Cancer 93, 699702 (2001).
  44. Rinkevich, Y., Lindau, P., Ueno, H., Longaker, M. T. & Weissman, I. L. Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip. Nature 476, 409413 (2011).
  45. Hisha, H. et al. Establishment of a novel lingual organoid culture system: generation of organoids having mature keratinized epithelium from adult epithelial stem cells. Sci. Rep. 3, 3224 (2013).
  46. Yanai, H., Tanaka, T. & Ueno, H. Multicolor lineage tracing methods and intestinal tumors. J. Gastroenterol. 48, 423433 (2013).
  47. Montironi, R., Mazzucchelli, R., Lopez-Beltran, A., Cheng, L. & Scarpelli, M. Mechanisms of disease: high-grade prostatic intraepithelial neoplasia and other proposed preneoplastic lesions in the prostate. Nat. Clin. Practice Urol. 4, 321332 (2007).
  48. Ayala, A. G. & Ro, J. Y. Prostatic intraepithelial neoplasia: recent advances. Arch. Pathol. Lab Med. 131, 12571266 (2007).
  49. Scarlett, C. J., Salisbury, E. L., Biankin, A. V. & Kench, J. Precursor lesions in pancreatic cancer: morphological and molecular pathology. Pathology 43, 183200 (2011).
  50. Maitra, A., Fukushima, N., Takaori, K. & Hruban, R. H. Precursors to invasive pancreatic cancer. Adv. Anat. Pathol. 12, 8191 (2005).
  51. Allred, D. C. Ductal carcinoma in situ: terminology, classification, and natural history. J. Natl Cancer Inst. Monogr. 2010, 134138 (2010).
  52. Fearon, E. R. & Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 61, 759767 (1990).
  53. Hoglund, M. Bladder cancer, a two phased disease? Semin. Cancer Biol. 17, 225232 (2007).
  54. Cheng, L. et al. Precise microdissection of human bladder carcinomas reveals divergent tumor subclones in the same tumor. Cancer 94, 104110 (2002).
  55. Slaughter, D. P., Southwick, H. W. & Smejkal, W. Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 6, 963968 (1953).
  56. Forsti, A., Louhelainen, J., Soderberg, M., Wijkstrom, H. & Hemminki, K. Loss of heterozygosity in tumour-adjacent normal tissue of breast and bladder cancer. Eur. J. Cancer 37, 13721380 (2001).
  57. Prevo, L. J., Sanchez, C. A., Galipeau, P. C. & Reid, B. J. p53-mutant clones and field effects in Barrett’s esophagus. Cancer Res. 59, 47844787 (1999).

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Author information

Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

    • Kunyoo Shin,
    • Sally Kawano &
    • Philip A. Beachy
  2. Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Agnes Lim &
    • Philip A. Beachy
  3. Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Justin I. Odegaard
  4. Stanford Immunology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Jared D. Honeycutt
  5. Department of Urology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Michael H. Hsieh
  6. Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA

    • Philip A. Beachy
  7. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA

    • Philip A. Beachy

Contributions

K.S. and P.A.B. conceived ideas and experimental design. K.S. and A.L. performed the experiments. J.I.O. performed the histopathological analysis, J.D.H. aided in orthotopic injection, S.K. performed the genotyping of experimental mice, and M.H.H. helped analyse data. K.S. and P.A.B. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

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