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Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas

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Science  10 Aug 2012:
Vol. 337, Issue 6095, pp. 730-735
DOI: 10.1126/science.1224676

Cancer Stem Cells in Color

One of the liveliest debates in contemporary cancer research centers on whether cancer stem cells (CSCs) exist and, if so, how these cells are defined phenotypically. CSCs are hypothesized to be a small population of cells within a tumor that are endowed with the unique capacity to drive tumor growth—a scenario that in principle would offer important therapeutic opportunities. By studying mice expressing multicolor reporter genes, Schepers et al. (p. 730, published online 1 August) were able to visualize and monitor the fate of a candidate stem cell for intestinal adenomas, an early stage of cancer. This “lineage tracing” analysis suggests that tumor cells expressing the intestinal crypt stem cell marker Lgr5 (leucine-rich repeat containing G protein–coupled receptor 5) are the cells that fuel the growth of intestinal adenomas.

Abstract

The concept that tumors are maintained by dedicated stem cells, the so-called cancer stem cell hypothesis, has attracted great interest but remains controversial. Studying mouse models, we provide direct, functional evidence for the presence of stem cell activity within primary intestinal adenomas, a precursor to intestinal cancer. By “lineage retracing” using the multicolor Cre-reporter R26R-Confetti, we demonstrate that the crypt stem cell marker Lgr5 (leucine-rich repeat–containing heterotrimeric guanine nucleotide–binding protein–coupled receptor 5) also marks a subpopulation of adenoma cells that fuel the growth of established intestinal adenomas. These Lgr5+ cells, which represent about 5 to 10% of the cells in the adenomas, generate additional Lgr5+ cells as well as all other adenoma cell types. The Lgr5+ cells are intermingled with Paneth cells near the adenoma base, a pattern reminiscent of the architecture of the normal crypt niche.

Intestinal tumorigenesis is thought to result from sequentially acquired mutations in specific genes, driving progression from premalignant precursor lesions called adenomas to invasive malignancies (1). Adenomas are formed by mutational activation of the Wnt signaling pathway, most notably by loss of the APC (adenomatous polyposis coli) tumor suppressor gene (2). By using the knock-in allele Lgr5EGFP-Ires-CreERT2, we have shown that the cell surface receptor Lgr5 (leucine-rich repeat–containing heterotrimeric guanine nucleotide–binding protein–coupled receptor 5) marks normal tissue stem cells in stomach, small intestine, colon, and hair follicles (3). This allele allows visualization of Lgr5+ stem cells by GFP (green fluorescent protein). Moreover, the coexpressed Cre fusion protein can be activated at will in these stem cells by tamoxifen injection and will then recombine two adjacent LoxP sites When a so-called Cre reporter allele is crossed into the Lgr5 knock-in mouse strain, tamoxifen injection can result in a visible change (e.g., a color) in an individual Lgr5+ cell that is inherited by all of its daughter cells. This strategy to document the behavior of stem cells is called lineage tracing. Although deletion of Apc in other cells of the intestinal epithelium does not lead to adenoma formation, we have shown that Apc-mutant Lgr5+ stem cells form progressively growing adenomas (4). Comparable adenomas were obtained by using another Cre driver (i.e., CD133CreERT2) to induce Apc gene deletion in these same stem cells (5). We noted that macroscopic adenomas in these mice contained low numbers of Lgr5+ cells, suggesting the preservation of a stem cell hierarchy within the tumors (4).

To investigate the existence of such a cellular hierarchy in adenomas, we exploited the multicolor Cre reporter termed R26R-Confetti (6). When R26R-Confetti is crossed into the Lgr5 knock-in mouse strain, tamoxifen injection will allow single Lgr5+ stem cells to randomly adopt one of the four fluorescent colors encoded in the R26R-Confetti allele. Of importance for the current study, two of the four colors remain in the R26R-Confetti allele after tamoxifen injection (blue with red or green with yellow). One of these is active, the other silent (Fig. 1A). In theory, a second tamoxifen injection can induce “flipping” from the active color to the silent color (here termed retracing). To test this, we globally activated R26R-Confetti in the intestinal epithelium by using the β-naphtoflavone–inducible Ah-Cre mouse strain (7). As hypothesized, the already rearranged R26R-Confetti reporter could be flipped to the remaining color by a second tamoxifen injection (fig. S1). Spontaneous color conversions were never observed.

Fig. 1

Large adenomas consist of individually colored segments with stem cells located adjacent to Paneth cells. (A) Possible outcomes of the R26R-Confetti locus after the first Cre recombination (tracing) and second Cre recombination (retracing). (B) Confocal imaging of an adenoma in a Lgr5EGFP-Ires-CreERT2/Apcfl/fl/R26R-Confetti mouse after 4 weeks of tracing. The adenoma consists of multiple segments (white dashed lines), which are marked uniformly by a single color (or are unmarked). RFP (red fluorescent protein) and YFP (yellow fluorescent protein) tracings are shown in red and yellow, respectively. Lgr5+ stem cells (green) are located toward the base of adenoma segments. Colors are overlaid on a differential interference contrast (DIC) image in gray. (C) Distribution of stem cells marked by Lgr5-GFP and Paneth cells, marked by lysozyme (purple) at the base of wild-type (WT) crypts (left). (Right) Lgr5+ stem cells (green) are located adjacent to Paneth cells (purple) toward the base of an adenoma segment (indicated by the red dashed line). (D) Schematic representation of the intermingled stem cells and Paneth cells in wild-type crypts (left) and adenomas (right). Scale bars indicate 50 μm.

To study the behavior of Lgr5+ cells within Apc-mutant adenomas, we then crossed Lgr5EGFP-Ires-CreERT2/Apcfl/fl mice with the R26R-Confetti strain. Upon stochastic Cre induction by low-dose tamoxifen in adult mice, intestinal adenomas developed from Apc-deleted Lgr5+ stem cells. After 4 weeks, we observed large adenomas (fig. S2, A to C) that either uniformly expressed a single Confetti color or consisted of anatomically separate segments that were each marked uniformly in a single Confetti color (Fig. 1B). The latter result indicates that such large adenomas were derived from several independent Apc-mutant stem cells, most likely located in adjacent crypts. Indeed, such segmental oligo-clonality of adenomas has been described (8, 9). Of note, recombination of the floxed Apc allele was more efficient than the activation of R26R-Confetti in the same stem cell, because not all adenomas or adenoma segments expressed a Confetti color.

In normal small intestinal crypts, stem cells are always adjacent to Paneth cells. These Paneth cells serve as niche cells to the stem cells by producing signaling molecules such as Wnt, epidermal growth factor (EGF), and Notch ligands (10). The Apc-mutant adenomas contained Paneth cells (Fig. 1C) but typically lacked mature goblet cells and enterocytes (fig. S2, D and E). Lgr5+ adenoma cells were often located adjacent to adenoma Paneth cells, suggesting the existence of an adenoma stem cell niche (Fig. 1C). Indeed, these clusters of Lgr5+ cells and Paneth cells tended to be located toward the base of the wedge-shaped adenoma segments, a situation reminiscent of normal crypt architecture (Fig. 1D). We noted a similar arrangement in colon adenomas, in which areas of Lgr5+ cells coincided with the location of the colon-counterpart of the Paneth cell, the deep crypt secretory cell, marked by Reg4 (10, 11) (fig. S3). To mimic the next step in the tumorigenic process, we additionally crossed in an allele of K-ras that can be oncogenically activated by Cre (K-rasLSL-G12D) (12). We observed a dramatic acceleration of the growth of these Apc/Kras mutant tumors compared with Apc-mutant adenomas. The mice became moribund within 7 to 10 days, yet the cryptlike architecture was preserved in the Apc/Kras double-mutant tumors (fig. S4).

Next, we set out to determine the transcriptional profile of Lgr5-GFPhi cells in adenomas to allow comparison to normal crypt Lgr5-GFPhi stem cells. We sorted Lgr5-GFPhi and Lgr5-GFPlo cells from 30-day-old adenomas and performed comparative gene expression analyses of the two populations. Gene set enrichment analyses demonstrated that the Lgr5-GFPhi stem cell signature determined for normal crypts (13) was strongly enriched in the Lgr5-GFPhi adenoma cells (Fig. 2). This observation suggested that the Lgr5-GFPhi adenoma cells retain stem cell properties. To substantiate this notion, we compared the clonogenic potential of Lgr5-GFPhi and Lgr5-GFPlo adenoma cells in a defined culture system developed for normal tissue Lgr5-GFPhi stem cells (14, 15). In this assay, normal Lgr5-GFPhi cells grow out into crypt-villus organoids, whereas Lgr5-GFPlo rarely grow out, consistent with the idea that Lgr5-GFPhi cells constitute multipotent stem cells. Analysis of the clonogenic potential of Lgr5-GFPhi and Lgr5-GFPlo adenoma cells in this culture system recapitulated the observations made in normal tissue: The colony-forming efficiency of Lgr5-GFPhi adenoma cells was about 20 times greater than that of Lgr5-GFPlo adenoma cells (Fig. 2, D and E).

Fig. 2

Gene expression profile and clonogenic potential of adenoma stem cells. (A) Heat map of log2 ratios of three independent microarray experiments performed in dye swap comparing adenoma stem cells (Lgr5-GFPhi) to immediate daughter cells (Lgr5-GFPlo) within the adenoma. All array probes with consistent expression throughout the six arrays are shown (n = 27313). Known intestinal stem cell markers (e.g., Olfm4, Ascl2) are highly enriched in adenoma stem cells, whereas their daughter cells express markers for the four cell lineages in the gut: goblet cells (Gob5, Tff3), enteroendocrine cells (Chgb), enterocytes (Fabp2, Afp), and Paneth cells (Lyz1/2, Dfa21). (B) Gene set enrichment analysis (GSEA). Genes are ranked according to their differential expression between Lgr5-GFPlo cells and Lgr5-GFPhi cells. Black bars below the graph depict the position of 291 genes significantly enriched in normal Lgr5+ intestinal stem cells [GEO data set GSE23672 (13)]. A highly significant enrichment of this gene set was detected toward the genes highly expressed in Lgr5-GFPhi adenoma cells. (C) GSEA analysis on the same data set as in (B), now analyzed for the top 500 genes significantly enriched in daughter cells of normal Lgr5+ stem cells. A highly significant enrichment of this gene set was detected toward the genes with higher expression in Lgr5-GFPlo adenoma cells. ES, enrichment score. (D) Fluorescence-activated cell sorting profile of Lgr5-EGFP-Ires-CreERT2/Apcfl/fl/R26R-Confetti mice 7 days after induction. Cells positive for RFP are adenoma cells. Gates I and II represent the Lgr5-GFPlo and Lgr5-GFPhi adenoma cells, respectively. Gates III and IV represent WT Lgr5-GFPlo and Lgr5-GFPhi cells. (E) Colony-forming efficiency of sorted adenoma cells. The outgrowth efficiency of Lgr5-GFPhi adenoma cells is about 20 times higher than that of Lgr5-GFPlo adenoma cells.

To trace the fate of Lgr5+ adenoma cells in vivo, we injected 10-week-old Lgr5EGFP-Ires-CreERT2/Apcfl/fl/R26R-Confetti mice with tamoxifen. After allowing Confetti-marked adenomas to develop for 24 days, we induced lineage retracing by a second tamoxifen injection. Adenomas were analyzed by confocal microscopy at different time points after the second Cre pulse. We observed rare retracings within large adenomas (~six color retracings per 100 adenoma segments), which invariably involved switching to the silent color in that particular adenoma. As an example, rare blue cells were observed within red adenomas early after lineage tracing (Fig. 3). Because we observed retracing events in only ~6% of Confetti-marked adenomas and these presented typically as a single cell at one day after the second tamoxifen injection (Fig. 3, D and E), we considered these to represent clonal retracing events. In other words, these cells were derived from a single adenoma stem cell. As expected, the blue cells were mostly located near the base of the wedge-shaped adenoma segments, the location of the Lgr5+ cells. No spontaneous color conversion was observed in control mice that were not given a second Cre pulse, in a total of 107 adenomas analyzed 27 days after one pulse of tamoxifen.

Fig. 3

Retracing of Lgr5+ adenoma cells. (A) Apc-deficient adenomas were allowed to develop for 24 days in Lgr5-EGFP-Ires-CreERT2/Apcfl/fl/R26R-Confetti mice before retracing of Lgr5+ adenoma cells was induced. Two days after retracing, CFP [cyan fluorescent protein (blue)]–positive cells (arrows) were observed at the bottom of a red adenoma segment. Lgr5-EGFP is shown in green. (B) Six days after retracing, blue ribbons of cells were present within red adenoma segments. (C) Twenty-four days after retracing, blue cells comprised a large part of the adenoma. (D and E) Quantification of the total amounts of retraced cells (D) and the amounts of Lgr5-GFPhi+ retraced cells (E) over time. (F) Calculated volume of GFPhi cells as a percentage of the total volume of the adenoma segment. Error bars represent SEM. A linear trend line is given, R2 = 0.41. (G) Calculated volume of the retraced cells as a percentage of the total volume of the adenoma segment. Error bars represent SEM. A second-order polynomial trend line is given, R2 = 0.86.

Six days after retracing, the number of blue cells within red adenomas had increased. Moreover, the growing blue clones presented as ribbonlike structures emanating from the base of the segments and projecting toward to the intestinal lumen (Fig. 3B), thus resembling, in a crude and somewhat chaotic way, the more-organized tracing patterns observed in healthy intestinal tissue (3). From day 9 to day 24 after retracing, the blue cells expanded to compose up to 30% of the volume of the adenomas (Fig. 3, C and G). A quantification of clone size over time is given in Fig. 3D. The number of Lgr5-GFPhi stem cells within the retraced population also increased over time, indicating that Lgr5+ adenoma stem cells clonally expanded within established adenomas (Fig. 3E). The total number of Lgr5-GFPhi cells comprised a minority of the adenoma cells. We found that around 5 to 10% of the adenoma cells were Lgr5-GFPhi (Fig. 3F). This number is similar to that of normal crypts, which consist of 150 to 200 cells and harbor about 15 stem cells each, thus reinforcing the notion that adenomas retain characteristics of the crypt stem cell niche.

To address whether Lgr5-GFPhi adenoma cells are able to generate all cell types of the adenoma, we studied the phenotype of individual cells within the clonal offspring of an Lgr5+ adenoma cell. As an example, Fig. 4 depicts a red adenoma after 35 days of tracing, with a subsequent retracing for 9 days, in which an Lgr5+ adenoma cell gave rise to a blue ribbonlike clone (see also fig. S5). Multiple Lgr5-GFP+ adenoma cells were observed within the clonal ribbon (arrowheads), demonstrating the production of additional Lgr5+ adenoma cells within the ribbon. In addition, at least two blue Paneth cells (arrows) and multiple blue Lgr5 cells, resembling transit-amplifying cells (asterisks), could be observed after 9 days of retracing within the clonal ribbonlike structure. These results indicate that Lgr5+ adenoma cells give rise to the other cell types in the adenoma. Combined with the observation that large parts of an adenoma can emerge from a single Lgr5+ adenoma cell, these observations qualify these cells as the multipotent stem cells of the adenoma.

Fig. 4

Lgr5+ adenoma cells expand and give rise to adenoma Paneth cells. A red adenoma segment retraced for 9 days after 35 days of adenoma development in a Lgr5-EGFP-Ires-CreERT2/Apcfl/fl/R26R-Confetti mouse. The left image is an overlay of four channels; DIC is shown in gray, the original segment in red, Lgr5+ cells in green, and the retraced cells in blue. Within the red segment, a blue clone appears, originating at the Lgr5-EGFP+ base of the cryptlike structure. Multiple Lgr5-GFP+ adenoma cells occur within the clonal ribbon (arrowhead), demonstrating the expansion of Lgr5+ adenoma cells. In addition, at least two blue Paneth cells (arrows) and multiple blue Lgr5 cells, resembling transit-amplifying cells (asterisks), are present. Scale bars, 50 μm.

The original studies that propose the existence of cancer stem cells in human colorectal cancer have their basis in transplantation of sorted cell populations into immunodeficient mice (1618). The manipulations required for sorting, combined with the xenotransplantation setting, impose inherent limitations to the interpretation of the outcome of this assay (19). In an alternative approach, single-cell sorting of cultured primary colorectal cancers has been used to demonstrate that about 1 in 20 cells has the capacity in vitro of multilineage differentiation, considered an attribute of stem cells (20). In a follow-up study, this culture-based assay was combined with the classic xenograft approach to demonstrate that colon cancer stem cells display high Wnt pathway activity (21). The stem cell marker used in the current study, Lgr5, is encoded by a Wnt target gene and itself constitutes a Wnt receptor component (22).

Batlle and colleagues used markers enriched in normal colon Lgr5+ stem cells to visualize a stemlike cell population in human colon cancers, residing at the base of structures that resemble normal crypts (21). Indeed, the presence of Lgr5+ stem cells and of all differentiated lineages within colon cancers was confirmed by single-cell polymerase chain reaction (23). These latter two studies imply that our current functional observations on Lgr5+ stem cells in mouse adenomas extend to human colon carcinomas. Although adenomas represent only the first stage of intestinal tumorigenesis, the composition of these tumors, as revealed in this study, is fully compatible with the original notion of Pierce and Speers (24) that “carcinomas are caricatures of tissue renewal, in that they are composed of a mixture of malignant stem cells, which have a marked capacity for proliferation and a limited capacity for differentiation under normal homeostatic conditions, and of the differentiated, possibly benign, progeny of these malignant cells.”

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1224676/DC1

Materials and Methods

Figs. S1 to S5

References (25, 26)

References and Notes

  1. Acknowledgments: We thank C. Kroon-Veenboer for help with mouse experiments. A.G.S and H.J.S. were funded by KWF (the Dutch Cancer Society); D.E.S. by CBG (the Centre for Biomedical Genetics); and M.vdB., J.vE., and M.vdW. by TI Pharma. Array data sets are deposited at Gene Expression Omnibus (accession no. GSE37926).
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