Abstract 5334: Novel lentiviral barcoding strategy for lineage tracing of cancer stem cells

Abstract

Abstract The cancer stem cell theory posits the existence of a subset of tumorigenic cells with self-renewal potential and the ability to wholly reconstitute original tumors by lineage restriction. The corollary follows that these tumor-initiating cells (TICs) may be defined in part by their progeny, non-TICs that can contribute to tumor bulk, but lose their self-renewal potential. Although the expression of surface markers have been employed to identify highly enriched populations of TICs, no lineage-tracing experiments have been performed to date to trace the fate of TICs. To characterize TIC growth kinetics in mammary tumor cells transplanted into syngeneic recipient mice containing intact immune system, we developed a lentiviral barcoding system as a means of genetic lineage tracing of cell subpopulations sorted by surface marker expression. Combining antibody-based flow sort analysis with lentiviral-borne 60mer genetic barcodes, we have examined both clonality and propagation kinetics of barcodes in the tumor outgrowth. These studies are based on the hypothesis that TICs represent a limited subpopulation in breast cancers that give rise to tumor heterogeneity. By uniquely barcoding cells sorted by candidate surface marker expression (e.g. CD29 and CD24), then re-pooling barcoded cell fractions for transplantation into recipient mice, the abundance of each barcode in the reconstituted tumor reflects the frequency of TIC progeny from individual sorted cell fractions. In theory, only barcodes integrated into TICs should propagate throughout the heterogeneous tumor cell population. Thus, lineage tracing of each barcode is possible when the transplant-derived tumors are again FACS-analyzed using the same antibodies of the original sort. Alternatively, if there is extensive plasticity in tumors, non-TIC may acquire self-renewal properties and give rise to a new population of TICs with altered genetic and epigenetic properties. Here we present preliminary results in the previously characterized mouse p53 null stochastic model of basal-like breast cancer. Using qPCR, we demonstrate that barcodes integrate stably and are present throughout the reconstituted tumor cell population. We further determined that CD29hi/CD24hi subfraction consistently out-compete other subpopulations while giving rise to self and progeny populations not of the CD29hi/CD24hi immuno-profile. Our results indicate that CD29hi/CD24hi subfraction retains the two cardinal features of self-renewal and ability to give rise to progeny through lineage restriction ascribed to TICs. Our novel lentiviral barcoding strategy not only directly tests the CSC hypothesis but also provides future framework for defining specific biomarkers and evaluating therapeutics directed towards cancer stem cells. This lineage tracing methodology should be applicable not only to genetically engineered mouse models but also to primary human breast cancer xenografts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5334. doi:1538-7445.AM2012-5334