Measurement of Cancer Cell Growth Heterogeneity through Lentiviral Barcoding Identifies Clonal Dominance as a Characteristic of In Vivo Tumor Engraftment

Abstract

Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term “clonal dominance”. We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780cis) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.

Lentiviral Fluorescent Genetic Barcoding for Multiplex Fate Tracking of Leukemic Cells

“…The advantage of in vitro multiplexing became apparent, when single color codes occasionally outcompeted the other five populations (Figure 1F), despite stable maintenance of all color codes when monitored in isolation. Since DNA barcoding of cell lines and mesenchymal stem cells previously detected clonal restriction, color coding may represent a simple method to investigate heterogeneous growth properties in vitro, albeit requiring integration site analyses for the assessment of clonal complexities 44, 45, 46…”

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm

“…One thousand cells/ shRNA were exposed to ADC for 96 hours and remaining cells were expanded for an additional 96 hours in the absence of ADC. Cells were harvested and barcodes were PCR-amplified and sequenced using an Illumina sequencer (Cellecta Inc) as previously described (17). The number of reads of each shRNA barcode was normalized to reads per 40 million total reads (the typical read depth in each experiment) to correct for variations in read depth in each experiment.…”

Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution

“…Expansion of minor subclones has been suggested in previous xenotransplantation studies using malignant epithelial 10,[21][22][23] or hematopoietic 24,25 cells, without formal resolution of the clonal genotypes or pattern of subsequent clonal dynamics. In contrast with preliminary studies of xenoengraftment, we find correlated dynamics of clones defined by SNVs or copy number aberrations as clonal marks.…”