Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation

Abstract

Cancer stem cells are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, for example, prominin 1 (PROM1, also called CD133). What remains unclear is whether cancer stem cells are the direct progeny of mutated stem cells or more mature cells that reacquire stem cell properties during tumour formation. Answering this question will require knowledge of whether normal stem cells are susceptible to cancer-causing mutations; however, this has proved difficult to test because the identity of most adult tissue stem cells is not known. Here, using an inducible Cre, nuclear LacZ reporter allele knocked into the Prom1 locus (Prom1(C-L)), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1(+/C-L) mice containing the Rosa26-YFP reporter allele showed that Prom1(+) cells are located at the base of crypts in the small intestine, co-express Lgr5 (ref. 2), generate the entire intestinal epithelium, and are therefore the small intestinal stem cell. Prom1 was reported recently to mark cancer stem cells of human intestinal tumours that arise frequently as a consequence of aberrant wingless (Wnt) signalling. Activation of endogenous Wnt signalling in Prom1(+/C-L) mice containing a Cre-dependent mutant allele of beta-catenin (Ctnnb1(lox(ex3))) resulted in a gross disruption of crypt architecture and a disproportionate expansion of Prom1(+) cells at the crypt base. Lineage tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1(+) cells in these mice, only 7% of tumour cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine that are susceptible to transformation into tumours retaining a fraction of mutant Prom1(+) tumour cells.

Figure 1. Generation of Prom1^C-L mice and reporter analysis

(a) Top to bottom: CreERT2-IRES-nuclear(n)LacZ-PGK-Neo was cloned into the ATG site of Prom1. pProm1 was subject to homologous recombination with wild type Prom1 to generate the Prom1^C-L targeted allele. PGK-Neo was excised by flip-recombination. p=Southern blot probe; A=ApaL1 sites; FRT=flip recombinase targets. Southern blot of mouse tissues heterozygous for the targeted allele (lane 1) and homozygous for the wild-type allele (lane 2). (b) Expression of nLacZ from the Prom1^C-L allele in embryonic tissues. NT, neural tube; R, rib; L, limb; RT, renal tubule. Scale bars=200μm. (c) Prom1 and nLacZ expression in adult mouse (3 month) tissues. β-galactosidase staining (left panel), Prom1 protein (middle panel, immunofluorescence) and markers (right panel) of neural stem and progenitor cells (Nestin), Clara cells (Clara cell specific protein [CCSP]), pancreatic ductal cells (Dolichos Biflorus Agglutinin [DBA]-fluorescein), proximal renal tubular endothelial cells (Lotus Tetragonolobus Lectin [LTL]-fluorescein), and photoreceptors (rhodopsin). Middle and right panels are adjacent sections of the boxed areas in left panels. V, ventricle; B, bronchiole; * vessel at bronchioalveolar junction; PD, pancreatic duct; RT, renal tubule; PR, photoreceptor. All scale bars=50μm.

Figure 2. Prom1 marks small intestinal crypt stem cells

Expression of (a) Prom1^C-L (β-galactosidase) and (b) Prom1 wild-type (Prom1 in situ hybridization) alleles is confined to the small intestinal crypt base in Prom1^+/C-L mice and overlaps with (c) Lgr5 expression (in situ hybridization). (d) Lgr5 (in situ hybridization, cyoplasmic dark stain) and Prom1 (green, nLacZ immunofluorescence) are co-expressed in the same cells (broken lines indicate cell boundaries). (e) nLacZ immunohistochemistry (brown nuclear immunostain) counterstained with Periodic Acidic Schiff (PAS [pink], to detect Paneth cells) and hematoxylin (blue) reveals the position of Prom1⁺ cells (numbers denote crypt cell positions) in Prom1^+/C-L mice. Scale bar=10μm. (f) Inset shows YFP⁺ cells in the crypt base of Prom1^+/C-L ; RosaYFP mice two days following tamoxifen-induction. Main panel, YFP-labeling of the crypts and epithelial surface of the villi, 60 days post tamoxifen-induction. Scale bar=50μm. (g) Two-photon laser-scanning microscopy (YFP, green) through 464μm of adult Prom1^+/C-L mouse small intestinal villus 10 days following tamoxifen-induction.

Figure 3. Tumorigenesis in the mouse small intestine is initiated in Prom1⁺ crypt stem cells

(a) View (394μm depth) into the base of a small intestinal crypt in a Prom1^+/C-L ; RosaYFP ; Ctnnb1^+/lox(ex3) mouse, two days following tamoxifen-induction. Mice had 8.4 YFP⁺ cells per crypt (range 2 to 16 >210 crypts counted). (b) Graph reporting the mean (±s.e.m., n=3) number of total and YFP⁺ cells isolated 2 days following tamoxifen induction, from Prom1^+/C-L ; RosaYFP ; Ctnnb1^+/+ (control) and Prom1^+/C-L ; RosaYFP ; Ctnnb1^+/lox(ex3) (Ctnnb1) small intestinal mucosae (***=P<0.0001, Wilcoxon test). Colonies formed following four weeks in stem cell culture medium by single cells isolated 2 days post tamoxifen induction from Prom1^+/C-L ; RosaYFP ; Ctnnb1^+/+ (c) and Prom1^+/C-L ; RosaYFP ; Ctnnb1^+/lox(ex3) (d) small intestinal mucosae, numbers=total colonies per small intestinal culture (±s.e.m., n=10, P<0.0001, Wilcoxon test). Scale bars=50μm.

Figure 4. Ctnnb1-mutant Prom1⁺ crypt stem cells produce diffuse small intestinal tumors in adult Prom1^+/C-L ; RosaYFP ; Ctnnb1^+/lox(ex3) mice

(a) Hematoxylin and eosin (H & E) staining of a dysplastic small intestinal crypt 10 days following tamoxifen-induction (left). Normal crypt in a no tamoxifen control mouse, right. (b) Ctnnb1 immunostaining of crypt cells 10 days following tamoxifen-induction. Scale bars=10μm. (c) Two-photon laser-scanning microscopy (578μm depth; YFP, green) of cells emerging from a small intestinal crypt, 10 days following tamoxifen-induction. (d) H & E stained section of tumorous small intestine 60 days following tamoxifen-induction. (e) YFP (green), nLacZ (red) co-immunofluorscence of neoplastic small intestine. Inset, arrows, nLacZ⁺ nuclei. nLacZ (f) and Ctnnb1 (g) immunostaining of adjacent sections of tumor. Note, Prom1⁺ (nLacZ⁺) cells enclosed by broken lines express nuclear Ctnnb1. (h) Ki67 (green) and nLacZ (red) co-immunofluorscence of tumor. Note the small fraction of proliferating (co-express Ki67⁺, arrow*) Prom1⁺ tumor cells (arrows). (d) to (h) scale bars=50μm.