Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution

Abstract

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO(-)) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.

Keywords: cancer stem cells; intestinal adenomas; intratumor heterogeneity; tumor growth; tumor life-history.

Fig. 1.

Multipotent stem cells reside within clonal CCO⁻ adenomatous crypts. (A) (i) H&E staining showing tubular adenoma C157 with low-grade dysplasia. (ii) CCO enzyme histochemistry identifying two patches of multiple, blue, CCO⁻ crypts. (iii and iv) Laser-capture microdissection of areas in ii outlined in red and (v) mtDNA sequencing of single cells from multiple blue crypts and within the same blue crypts versus adjacent brown, wild-type crypts demonstrated that all blue crypts shared a common, clonal point mutation in their mtDNA that was not present in adjacent brown crypts. (B) Immunofluorescence staining of serial sections from adenoma 160. Clonal CCO⁻ crypts contained cells positive for markers of neuroendocrine cells (chromogranin A) and secretory cells [mucin2 (MUC2) and mucin5AC (MUC5AC)], indicating these crypts contained a multipotential stem cell that had produced these distinct cell types. Detection of CCO expression was conducted on the same section as chromogranin A after visualization for chromogranin A expression. Detection of MUC2 and MUC5AC expression was conducted on the same section simultaneously. Negative controls were isotype-matched at the same concentration as the corresponding primary antibody. Asterisks indicate the crypt enlarged in high-power images. (Scale bars: ∼50 μm in low-power images and 25 μm in high-power images. (C) LGR5 mRNA detection in FFPE tissue from patients with familial adenomatous polyposis (left pair) and sporadic adenocarcinoma (right pair). Expression is detectable in bases of unaffected crypts, in patches of adenomatous epithelium, and, in this example, extensively through invasive adenocarcinoma. Bright-field and dark-field reflected light image pairs with Giemsa counterstain.

Fig. 2.

Epigenetic diversity in adenomatous crypts. (A) Topographical maps showing crypts sampled from adenoma 174 (wholly APC-mutant, KRAS-mutant subclone). A pairwise comparison of adenomatous crypts showed crypts had markedly different methylation patterns. Individual adenomatous crypts showed intracrypt epigenetic diversity. Boxes of dots represent methylation patterns; each row is a tag (molecule), each column a CpG site. An open circle denotes an unmethylated site; a filled circle denotes a methylated site. (B and C) Pairwise differences between each pair of crypts, sorted by adenoma. In some adenomas (e.g., 160 and 158), all crypts had similar methylation patterns, whereas in other adenomas (e.g., 162) some crypts had methylation patterns that were very different from those in the majority of crypts, illustrating the differences in the life-history of each adenoma. (B) ICD and (C) minimum distance between crypts. Colors indicate the dominant genotype within the adenoma; blue: APC–mutant KRAS–wild-type, red: APC/KRAS-mutant, green: APC/KRAS–wild-type).

Fig. 3.

Intercrypt diversity of the methylation patterns from intratumor clones compared with the diversity of the bulk of the adenoma. Methylation patterns of subclones tended to be significantly less diverse than the bulk of the tumor cell population. C, intraclone diversity (red); CN, diversity between clone and nonclone regions of the tumor (blue); W, diversity of whole tumor (green); ***P <.001, **P < 0.01, *P < 0.05 for Wilcoxon test between clone and nonclone; numbers at the top of the plot indicate the adenoma along with the mutation denoting the subclone.

Fig. 4.

Correlation between spatial and epigenetic distance in adenomas. (A) Spatial distance versus epigenetic distance for whole adenomas. No correlation between spatial distance and epigenetic distance was observed. Spatial distances between crypts were binned into intervals of five units, with a unit corresponding to the average crypt diameter. The average ICD of all pairs of crypts within the adenoma that fell within the spatial interval was computed. Each colored line represents a different adenoma. (B) Spatial distance versus cumulative epigenetic distance. The straight lines suggest that physically close crypt-pairs are no more similar in their methylation patterns than physically distant pairs of crypts. If physically close crypts were more epigenetically similar on average than distant pairs of crypts, the cumulative epigenetic distance would increase exponentially. The cumulative epigenetic distance was computed from the data in A to compensate for the sparse spatial sampling of adenomas.

Fig. 5.

Modeling epigenetic diversity during clonal expansions. The diversity of methylation patterns between a patch of five clonally derived crypts was simulated with a mathematical model. Two modeling assumptions were tested: (i) that the patch formed as burst (with very rapid division of the ancestral crypt) at the onset of tumor growth, and (ii) that the crypts within a patch continued to divide every f years. The epigenetic diversity of the crypts was greater in the initial burst model (cyan line) than in the continual growth model (orange line, shown for crypt divisions every f = 2 y), and this diversity increased as the clone aged. Dashed lines represent 95% quantiles of the simulated values. Boxplots represent the mean ICD of samples of repeated five crypts taken from the CCO⁻ clone in adenoma 157 (red box) or from the nonclonal area (blue box). The diversity of the subclone was significantly less than that of the nonclone, suggesting that the subclone shared a much more recent common ancestor than the bulk of the adenoma.

Fig. 6.

Diversity of the methylation pattern of individual adenomatous crypts compared with the adenoma bulk. (A) Intracrypt diversity of normal and adenomatous crypts as a function of age is consistent with a model of neutral stem cell competition within crypts. Points are intracrypt diversity measured in normal crypts (blue circles) or adenomatous crypts (red triangles); the x-axis (age) jitter is artificial to allow all data points to be seen. Solid black line shows mean intracrypt diversity as predicted by the mathematical model with parameters as described in Materials and Methods; dashed lines are 95% quantiles for the simulation results. (B) Intracrypt distances (A) versus ICDs (I) for each adenoma. The mean ICD was greater than the intracrypt distance for all adenomas. Statistical comparison was performed using a Wilcox test: **P < 0.001, *P < 0.05. Unmarked pairs did not show a statistically significant difference.

Fig. 7.

Cartoon of tumor evolution. (A) A stepwise model of tumor evolution in which sequential mutations trigger extensive clonal expansions of the new mutant clone within the tumor mass. (B) A model for the relative stasis scenario of colorectal adenoma evolution. Here, intratumor clones can form near the onset of neoplasia but do not sweep through the tumor and so appear as spatially localized clones with divergent intraclone methylation patterns. Rare subclones form later in tumor development, have relatively homogeneous methylation patterns, and also occupy only focal regions of the tumor. Colors denote distinct clones. Within a clone, methylation patterns diverge as the clone ages. MRCA, most recent common ancestor.