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.

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15-20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro R , copGFP, or H-2K k as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.

No abstract

Genes modulating proliferation and survival in oncogenic cells have been identified using pooled lentiviral-based libraries expressing many thousands of shRNAs. Viability assays with human isogenic mammary epithelial cells (HMEC) and blood cancer cell lines transduced with these shRNA libraries targeting thousands of genes identified hundreds of essential genes for each panel of cells. Subsequent validation using single shRNA-expressing constructs showed that about 80% of the shRNAs identified in each complex library screen did in fact lead to cell death when transduced in cells. Analysis of the identified essential genes for known biological interactions revealed non-random clusters of interacting proteins that provide some insight into signaling pathways and protein networks specific to these cancers. Also, we have recently adapted the approach to combinatorially screen shRNA sequences targeting hundreds of genes to discover additive and synergistic combinations that generate a synthetic-lethal phenotype. Analysis of the lethal combinations indicates redundant, complementary, and compensatory responses in cancer cells. This presentation will provide an overview of the screening platform, our approach to shRNA library design, and results from the screens for cancer cell-specific lethal and synthetic-lethal genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C159.

We have developed an open-source RNAi genetic screening technology, funded in part by NIH grants and freely available to the scientific community. This platform can be used for functional discovery of drug targets associated with viability and regulation of signaling pathways in cancer cells. Here we present a novel, free public resource for genome-wide RNAi screens: DECIPHER pooled lentiviral shRNA libraries. Results are shown from two RNAi screens with DECIPHER shRNA libraries: one “drop-out” screen to identify genes essential for viability in a panel of leukemic cells, and a second “rescue” screen to identify genes required for FAS induced apoptosis. Both screens found a combination of known and novel signaling pathway and regulatory genes whose functions were confirmed to be required to produce the biological responses. From the viability screens in the panel of leukemia cell lines, subsequent validation using single shRNA-expressing constructs showed that about 80% of the shRNAs identified in each complex library screen did in fact lead to cell death when transduced in cells. Analysis of the identified essential genes for known biological interactions revealed non-random clusters of interacting proteins that provide a useful strategy for prioritization of potential targets. Analysis of the lethal combinations indicates redundant, complementary, and compensatory responses in cancer cells. In the case of the FAS-induced apoptosis, in vitro screening data also enabled us to select targets that protected mice from FAS-induced hepatic failure. These results demonstrate that complex pooled shRNA libraries provide a highly efficient, flexible, and cost-effective alternative to array-based RNAi screening methods for identifying genes regulating biological responses and possible new therapeutic targets. Citation Format: Andrei Komarov, Mikhail Makhanov, Elena Komarova, Lilya Novototzkaja, Michael Yeluashvili, Dmitry Suchkov, Kyle Bonneau, Donato Tedesco, Costa G. Frangou, Paul Diehl, Debbie Deng, Karim Hyder, Pavel Komarov, Andrei Gudkov, Alex Chenchik. Open-source RNAi genetic screen platform: discovery of drug targets in cancer cell models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3160. doi:10.1158/1538-7445.AM2013-3160