Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics

Esophageal adenocarcinoma (EAC) incidence is increasing while 5-year survival rates remain less than 15%. A lack of experimental models has hampered progress. We have generated clinically annotated EAC organoid cultures that recapitulate the morphology, genomic, and transcriptomic landscape of the primary tumor including point mutations, copy number alterations, and mutational signatures. Karyotyping of organoid cultures has confirmed polyclonality reflecting the clonal architecture of the primary tumor. Furthermore, subclones underwent clonal selection associated with driver gene status. Medium throughput drug sensitivity testing demonstrates the potential of targeting receptor tyrosine kinases and downstream mediators. EAC organoid cultures provide a pre-clinical tool for studies of clonal evolution and precision therapeutics.


DistanceToAlignmentEndMedian
The median shortest distance of the variant position within the read to either aligned end is less than 10

DistanceToAlignmentEndMAD
The median absolute deviation of the shortest distance of the variant position within the read to either aligned end is less than 3

LowMapQual
The proportion of reads at the variant position with low mapping quality (less than 1) is greater than 10%

MapQualDiffMedian
The difference in the median mapping quality of variant reads (in the tumour) and reference reads (in the normal) is greater than 5 VariantMapQualMedian The median mapping quality of variant reads is less than 40 VariantBaseQualMedian The median base quality at the variant position of variant reads is less than 30 VariantAlleleCount The number of variant-supporting reads in the tumour is less than 4 VariantAlleleCountControl The number of variant-supporting reads in the normal is greater than 1

StrandBias
The strand bias for variant reads covering the variant position, i.e. the fraction of reads in either direction, is less than 0.02, unless the strand bias for all reads is also less than 0.02.

Repeat
The length of repetitive sequence adjacent to the variant position, where repeats can be 1-, 2-, 3-, or 4mers, is 12 or more

SNVCluster50
The largest number of variant positions within any 50 base pair window surrounding, but excluding, the variant position is greater than 2; variant positions are those in which the number of alternate allele is supported by at least 2 reads and at least 5% of all reads covering that position.

SNVCluster100
The largest number of variant positions within any 100 base pair window surrounding, but excluding, the variant position is greater than 4; variant positions are those in which the number of alternate allele is supported by at least 2 reads and at least 5% of all reads covering that position.

Multiplex-FISH karyotyping
The organoid cultures were incubated for 3 hours with 0.1 µg/ml Karyomax Colcemid (Gibco) before being harvested and dissociated using TrypLE (Gibco). Cells were incubated with buffered hypotonic solution (0.4% KCl in 10 MM HEPES) for 8-12 minutes at 37 oC. The cells were then fixed and washed in a 6:1 (v/v) methanol:glacial fixatives and stored at -20 o C until use.
For multiplex-fluorescence in situ hybridization (M-FISH), chromosome-specific DNA libraries were generated from 5,000 copies of flow-sorted chromosomes, using GenomePlex Whole Genome Amplification (WGA2) kit (Sigma-Aldrich). Human 24-color painting probe was made following the pooling strategy (Geigl et al., 2006). Five human chromosome pools were labelled with ATTO 425-, ATTO 488-, CY3-, CY5-, and Texas Red-dUTPs (Jena Bioscience), respectively, using WGA 3 re-amplification kit (Sigma-Aldrich) and home-made dNTP mixtures optimised for the incorporation of the aforementioned labelled dUTPs by Taq polymerase. The labelled products were pooled and sonicated to achieve a size range of 200-1,000 bp, optimal for chromosome painting. The sonicated DNA sample was precipitated with ethanol together with human Cot-1 DNA (Invitrogen) and resuspended in a hybridization buffer (50% formamide, 2 × SSC, 10% dextran sulfate, 0.5 M phosphate buffer, 1 × Denhardt's solution [pH 7.4]). Metaphase preparations were dropped onto precleaned microscopic slides, followed by fixation in acetone (Sigma-Aldrich) for 10 min and dehydration through an ethanol series (70%, 90%, and 100%). Metaphase spreads on slides were denatured by immersion in an alkaline denaturation solution (0.5 M NaOH, 1.0 M NaCl) for 7-8 minutes, followed by rinsing in 1M Tris-HCl (pH 7.4) solution for 3 min, 1 × PBS for 3 min, and dehydration through a 70%, 90%, and 100% ethanol series. The M-FISH probe was denatured at 65°C for 10 min before being applied onto the denatured slides. The hybridization area was sealed with a 22 × 22-mm coverslip and rubber cement. Hybridization was carried out in a 37°C incubator for 2 nights. The post-hybridization washes included a 5-min stringent wash in 0.5 × SSC at 75°C, followed by a 5-min rinse in 2 × SSC containing 0.05% Tween20 (VWR) and a 2-min rinse in 1 × PBS, both at room temperature. Finally, slides were mounted with SlowFade Gold mounting solution containing 4′6-diamidino-2-phenylindole (Invitrogen). Images were visualized on a Zeiss AxioImager D1 fluorescent microscope equipped with narrow band-pass filters for DAPI, DEAC, FITC, CY3, TEXAS RED, and CY5 fluorescence and an ORCA-EA CCD camera (Hamamatsu). M-FISH digital images were captured using the SmartCapture software (Digital Scientific UK) and processed using the SmartType Karyotyper software (Digital Scientific UK). Approximately 20 metaphase chromosomes from each organoid culture were fully karyotyped based on M-FISH classification.

Clonality analysis
Segmental copy number information was derived for each sample using the Battenberg algorithm as previously described 1 . Briefly, the algorithm phases heterozygous SNPs with use of the 1000 genomes genotypes as a reference panel. The resulting haplotypes are corrected for occasional errors in phasing in regions with low linkage disequilibrium. After segmentation of the resulting b-allele frequency (BAF) values, t-tests are performed on the BAFs of each copy number segment to identify whether they correspond to the value resulting from a fully clonal copy number change. If not, the copy number segment is represented as a mixture of 2 different copy number states, with the fraction of cells bearing each copy number state estimated from the average BAF of the heterozygous SNPs in that segment.