Mutational spectrum of Barrett's stem cells suggests paths to initiation of a precancerous lesion - PubMed (original) (raw)

doi: 10.1038/ncomms10380.

Xia Wang 2, Denis Bertrand 3, Florian Kern 3, Ting Zhang 3, Marcin Duleba 2, Supriya Srivastava 4, Chiea Chuen Khor 3, Yuanyu Hu 3, Lane H Wilson 5, Hagen Blaszyk 6, Daniil Rolshud 6, Ming Teh 4, Jianjun Liu 3, Brooke E Howitt 7, Matthew Vincent 8, Christopher P Crum 7, Niranjan Nagarajan 3, Khek Yu Ho 9, Frank McKeon 2 10 11, Wa Xian 7 11 12 13

Affiliations

Mutational spectrum of Barrett's stem cells suggests paths to initiation of a precancerous lesion

Yusuke Yamamoto et al. Nat Commun. 2016.

Abstract

The precancerous lesion known as Barrett's oesophagus can evolve to oesophageal adenocarcinoma in decades-long processes of regenerative growth. Here we report the isolation and propagation of distinct, patient-matched stem cells of Barrett's, gastric and oesophageal epithelia that yield divergent tumour types following in vitro transformation and xenografting. Genomic analyses reveal a broad mutational spectrum unique to Barrett's stem cells that likely reflects their risk for oncogenesis. Remarkably, 25% of cases show no cancer-related genomic changes, suggesting that Barrett's initiates without driver mutations. Most cases, however, sustain patterns of deletions almost identical to adenocarcinoma though tumour-associated gene amplifications were absent. Notably, those suspected of low-grade dysplasia have p53 mutations or undergo amplifications of proto-oncogenes and receptor tyrosine kinases, implicating these events in lethal transitions. Our findings suggest paths for the initiation and progression of Barrett's and define a discrete stem cell underlying its regenerative growth whose eradication could prevent oesophageal adenocarcinoma.

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Figures

Figure 1

Figure 1. Stem cells from the distal oesophagus of Barrett's patient.

(a) Left, Endoscopic image of distal oesophagus of a Barrett's patient and approximate locations of biopsies. Right, phase contrast images of typical colonies derived from biopsies. Inset diagram, schematic of cloning process to yield Barrett's or oesophageal stem cell pedigrees from mixed population from Barrett's biopsies. Scale bar, 150 μm. _n_=12 biological replicates. (b) Colony formation from a single stem cell over indicated days in culture. Scale bar, 50 μm. (c) Heatmap of differential gene expression of three independent pedigrees from each of oesophageal (EsoSC), Barrett's (BESC) and stomach (GSC) stem cells. (d) PCA of expression data of three pedigrees each of oesophageal, Barrett's and stomach stem cells. (e) Representative immunofluorescence labelling of colonies of EsoSC, BESC and GSC with antibodies to the indicated marker proteins (green or red) with nuclei counterstained with DAPI (blue). Scale bar, 50 μm. _n_=12 biological replicates. (f) Gene expression heatmap comparing those differentially expressed between immature GSC (_n_=3) and Barrett's stem cells (BESC; _n_=3) with those from endoscopic gastric biopsy and Barrett's biopsies (BE-biopsy) (GSE34619) (ref. 20).

Figure 2

Figure 2. Barrett's stem cell pedigree recapitulates intestinal metaplasia.

(a) Derivation of stem cell pedigree from pool of colonies (left) by selecting individual colony for replating and expansion (right). Scale bar, 200 μm. _n_=12 biological replicates. (b) Clonogenicity assay for BESC at passage 5 and passage 12 determined by plating 2,000 cells and counting colony formation following Rhodamine red staining. Histogram depicting colony counts. Error bars, s.d. _n_=3 biological replicates. (c) Left, schematic diagram of induced differentiation of stem cell pedigree in ALI culture. Right, histological section of differentiated p5 and p12 Barrett's stem cells with Alcian blue-positive goblet cells. Scale bar, 100 um. _n_=12 biological replicates. (d) Upper, Alcian blue staining of ALI culture differentiation of EsoSC, BESC and GSC, bottom, immunostainings of section of BESC-derived epithelium following ALI differentiation showed distribution of multiple types of cells positive for CHGA (endocrine cells), HD6 (Paneth cells) and TFF3 (goblet cells). Scale bar, 50 μm. _n_=12 biological replicates. (e) PCA of whole-genome expression data sets from EsoSC, BESC and GSC before and after ALI differentiation as indicated. (f) Expression heatmap of differentially expressed genes in ALI cultures as indicated.

Figure 3

Figure 3. Stem cells of the gastroesophageal junction.

(a) Comparison of gene expression microarray data from Barrett's and gastric stem cells by Log2 expression distribution and Venn diagrams. (b) Gene expression heatmap of patient-matched BESC and GSC pedigrees showing relative expression of the 131 most differentially expressed genes between BESC and GSC (>2.5-fold, P<0.0001). (c) Top, PCA of whole-genome expression microarray data of BESC, GSC and stem cells of human fetal intestine (ISC). Bottom, heatmap of differentially expressed genes between BESC, GSC and ISC. (d) Histological sections of ALI-differentiated ISC, BESC and GSC stained with (from top) H&E, E-cadherin (E-cad) and mucin 2 (Muc2) antibodies, mucin 5AC (Muc5AC) antibodies and Trefoil factor 2 (TFF2) antibodies. Scale bar, 50 μm. _n_=12 biological replicates.

Figure 4

Figure 4. Tumours derived from transformed BESC and EsoSC pedigrees.

(a) Schematic of stem cell transformation and xenografting in immunodeficient mice. (b) Dendritic relationship of gene expression profiles of EACs, ESCC and tumours developed in immunodeficient xenografted transformed Barrett's (BE tumour; _n_=7) and oesophageal (Eso tumour; _n_=3) stem cell pedigrees. (c) Correlation cluster analysis of genes expressed in BESC-derived tumours and those expressed in EAC. (d) Box plot of gene expression intensity from arrays for four genes marking tumours arising from xenografted transformed BESCs and EAC and squamous cell carcinomas. The top whisker shows maximum, the boxed zone shows 1st quartile, mediam and 3rd quartile, while the lower whisker shows minimum with outliers indicated (see Methods section). (e) Immunohistochemistry on sections of EAC, tumours derived from transformed BESC pedigrees (BE tumour), ESCC and tumours from transformed oesophageal stem cell pedigrees (Eso tumour). Scale bar, 7 mm.

Figure 5

Figure 5. SNV in Barrett's stem cell pedigrees.

(a) Histogram of somatic SNV allele frequency determined from exome sequencing of Barrett's stem cell pedigrees across the patient study group. (b) Distribution and median per cent overlap among SNVs determined from two independent BESC pedigrees (see Methods section). (c) Comparison of distribution of somatic mutation rates between BESC and GSC across the patient study group (see Methods section). (d) Summary of non-synonymous mutations (NS MUT), interstitial deletions (DEL) and interstitial amplifications (AMP) in Barrett's stem cells cloned from 12 cases without evidence of high-grade dysplasia. Histograms depict total number of events, whereas specific genes were listed beneath each histogram to highlight genes frequently altered in oesophageal and gastric adenocarcinoma. (e) List of average number of CNV events as amplifications (Amp) or deletions (Del) in Barrett's and gastric stem cells as well as EAC. (f) Graph depicting the distribution of BESC and GSC by mutation rate (SNV per Mb) and CNV (interstitial deletions).

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