Deterministic evolution and stringent selection during pre-neoplasia

Abstract

The earliest events during human tumor initiation are poorly characterized but may hold clues as to how to detect and prevent malignancy. Here we model this occult process by engineering TP53 deficiency in primary human gastric organoids and performing experimental evolution in multiple clonally derived cultures over two years, thereby defining causal relationships between this common initiating genetic lesion and resulting phenotypes. TP53 loss elicited progressive aneuploidy, including copy number alterations and complex structural variants that are common in gastric cancers and which follow preferred temporal orders. Longitudinal single cell sequencing of TP53 deficient gastric organoids similarly indicates progression towards malignant transcriptional programs. Moreover, lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programs repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a striking degree of phenotypic convergence in pre-malignant epithelial organoids, implying that the earliest stages of tumorigenesis may be predictable while illuminating evolutionary constraints and barriers to malignant transformation.

Engineered extracellular matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids

“…Genetically-barcoded PDAC organoids were generated as previously described 39 . For this work, genetically-barcoded PDAC organoids were expanded within Cultrex, HELP Low, or HELP High matrices for a total of three passages from the initial parent population established within Cultrex.…”

“…Barcode sequencing and analysis was performed similarly to previous work 39 . Briefly, the barcode region was amplified by PCR using 2X KAPA Hifi PCR Master Mix (Roche Sequencing Solutions KK2601) with a minimum of 200 ng of DNA input per sample.…”

“…Given that organoid models can retain cellular heterogeneity in vitro [35][36][37][38] and that our chemoresistant phenotype is preserved on the single cell-level, we hypothesized that stiffness-mediated chemoresistance could be due to the selection of inherently resistant subclones within our organoid population. To explore this hypothesis, we cultured genetically barcoded (via lentiviral transduction) 39 PDAC organoids within Cultrex, HELP Low, or HELP High matrices for three passages (Fig. 2g).…”

Frequent loss of metastatic ability in subclones of Apc, Kras, Tgfbr2, and Trp53 mutant intestinal tumor organoids

“…Consistently, organoid culture experiments confirmed the evolutional progression of TP53-deficient human gastric epithelial cells toward malignant programs with the accumulation of copy number alterations. 3 Moreover, we previously examined the mechanism of multistep tumorigenesis of colon cancer by using mouse models with multiple driver mutations, which demonstrated the mutation combination-specific malignant progression. [4][5][6] In particular, intestinal tumor-derived organoid cells carrying mutations in Apc, Kras, Tgfbr2, and Trp53 acquired metastatic ability by loss of wild-type Trp53 and achieved dominance in metastatic foci by positive selection.…”

Open questions in human lung organoid research

“…This could be due to sampling bias, selective clonal outgrowth, or tumour evolution driven by ongoing genetic instability. While the genetic landscape of organoids is relatively stable during long-term culture (Kim et al, 2019), there is evidence for ongoing genetic instability and clonal selection from single cell sequencing and barcoding studies (Bolhaqueiro et al, 2019;Karlsson et al, 2022;Kester et al, 2022). The establishment of organoids from separate tumour regions, possibly combined with the generation of clonal organoid lines at early passage, could prevent the loss of minor subclones (Fujii et al, 2016;Roerink et al, 2018).…”