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. 2017 Jul 3;214(7):2073-2088.
doi: 10.1084/jem.20162017. Epub 2017 Jun 1.

Genetic Subclone Architecture of Tumor Clone-Initiating Cells in Colorectal Cancer

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Free PMC article

Genetic Subclone Architecture of Tumor Clone-Initiating Cells in Colorectal Cancer

Klara M Giessler et al. J Exp Med. .
Free PMC article

Abstract

A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.

Figures

Figure 1.
Figure 1.
Genetic makeup of primary CRCs, derived xenografts, and spheroids. (A) High-coverage WGS of primary CRC tissue (TU; n = 3 patients), derived serial xenografts (n = 6; 2 serial mice/patient), and parallel spheroids (n = 6; 2 serial passages/patient). (B) Number of SNVs in three primary tumors. (C) Distribution of SNVs in different genomic regions. (D) CNAs in patient tumors. (Top left) copy numbers with baseline ploidy (black), gains (green), and losses (red). (Bottom left) Raw BAF. (Right) Segments harboring noninteger copy numbers. X axis, genomic location; y axis, copy numbers; light-blue lines, allele-specific copy numbers; dark-blue lines, total copy number; Chrom, chromosome; Mbp, mega base pair. (E) Concordance and discordance of SNVs in serial patient-derived samples. X-axis, number of SNVs. (F–H) Copy number profiles of patient-derived xenografts and spheroid cultures from P1 (F), P2 (G), and P3 (H) as shown in D. (A–H) All experiments were performed independently for three CRC patients.
Figure 2.
Figure 2.
Patient tumors, spheroids, and xenografts harbor distinct SNV- and CNV-based growth clones. (A) Each panel shows a pairwise comparison of CFs of the SNVs. Each dot represents one SNV. SNVs with similar kinetics over time were assigned to the same growth clone, colored accordingly, and either assigned to the main SNV-based growth clone (main; gray color), which is present in all cells, or to SNV-based growth clones, which are present in only a fraction of cells (q1-qx; colors as indicated). X and y axes, CF. (B) Representative chromosomal segments harboring subclonal copy numbers. Each small vertical panel shows the allele-specific copy number (light-blue line) and TCN (dark-blue line) for the chromosomal region indicated in the gray box above for one sample. Chrom, chromosome; Mbp, mega base pair. (C) Representative mutational profiles for growth clones of P1-X1. Each panel shows the relative signature exposures of one SNV-based growth clone. AC, Alexandrov COSMIC (nomenclature referring to http://cancer.sanger.ac.uk/cosmic/signatures (Alexandrov et al., 2013); Chrom, chromosome. See also Figs. S1 and S3 and Table S1. (A–C) Experiments were performed independently with tumor material from three CRC patients.
Figure 3.
Figure 3.
Genomic subclone kinetics in serial in vivo and in vitro passaging. (A) Ancestral trees of identified genomic subclones in patient tumors, early spheroids, and serial xenografts. (B) Relative contribution of genomic subclones from A in each sample. (C) Relative contribution of genomic subclones in patient tumors and serial spheroids. (D) Ancestral trees for genomic subclones shown in (C). Colors, defined genomic subclones; light gray, unidentified genomic subclones; gray circles, common ancestors; dashed lines, phylogeny constructed applying maximum parsimony. See also Fig. S2. (A–D) Experiments were performed independently with tumor material from three CRC patients.
Figure 4.
Figure 4.
Functional heterogeneity of TcIC early and late in serial transplantation. (A) Tumor cells of early and late xenograft tumors, used for assessing genetic heterogeneity by WGS, were genetically barcoded and serially transplanted for three mouse generations. The contribution of individually marked cell clones was assessed by LAM-PCR and high-throughput sequencing. Relative contribution of individually marked cell clones to tumor formation in serial transplantation from P1 (B), P2 (C), and P3 (D). Each row displays one unique IS; each column displays one xenograft in serial transplantation. Dotted lines, 2° n/a. Arrows indicate serial transplantation steps. (E) Total number of ISs detected in serial transplants derived from X1 or X2 and corresponding mean GFP expression (green). (F) Relative contribution of functional tumor-initiating cell classes to early and late TcIC compartments in individual xenografts, and (G) on average, from three patients. Error bars represent the SEM. (A–G) All experiments were performed independently with tumor material from three CRC patients.

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