Abstract 3957: Characterization of functional heterogeneity and <i>in vivo</i> dynamics of clonal cell populations derived from the triple-negative breast cancer cell line MDA-MB-468
Authors: Hendrik J. Kuiken, Sabin Dhakal, Laura M. Selfors, Jett P. Crowdis, Hyo-eun C. Bhang, Frank Stegmeier, Gordon B. Mills, Joan S. Brugge
Journal: Cancer Research (2017)

Abstract Tumors evolve through progressive accumulation of (epi)genetic alterations, favoring expansion of the fittest cell populations as a result of stimuli and selective pressures in the microenvironment. Intratumor heterogeneity and the interplay of tumor cells with the microenvironment present an order of complexity that can have profound effects on tumor progression and drug sensitivity. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of functional heterogeneity within a single tumor, and the dynamics and spatial organization of distinct cell populations over time. The overall objective of this study is to characterize the functional heterogeneity of breast tumor cells and evaluate the dynamics of clonal populations within mixtures during tumor progression. We have generated and characterized 31 clonal cell populations (SCP) from the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable phenotypic heterogeneity with respect to morphology, proliferation rate, survival in suspension, colony formation in soft agar, and tumorigenicity. We have also performed whole-exome sequencing for 7 SCPs, and RNA-seq and RPPA analysis for all SCPs and the parental cell line. These analyses revealed SCP-unique single nucleotide variants, and differential expression of numerous genes. To study the dynamics of SCP mixtures during the development of xenograft tumors, we have transduced 22 SCPs with unique DNA barcodes derived from the ClonTracer DNA barcode library. The 22 barcoded SCPs were mixed in equal proportions and subsequently injected into the mammary fat pad of NOD/SCID mice. Tumors and lungs were collected at different time points (3 weeks, 2 months and 4 months) to determine the relative changes in clonal representation during tumor progression by next generation sequencing. We identified highly reproducible patterns of clonal expansion, with SCP01 and SCP03 being temporarily enriched for in tumor samples collected at 3 weeks and 2 months respectively, and SCP32 becoming progressively enriched for in tumors over time. In addition, we found recurrent enrichment for SCP01 in most lung samples. Moreover, in contrast to the tumor samples, we found a slight enrichment for SCP13, but not SCP32, during in vitro propagation of the SCP mixture. Together these results suggest the existence of distinct competitive advantages of individual clonal populations within certain spatial and temporal windows. In addition, the discordance in SCP dynamics between the in vivo and in vitro arms suggests that enrichment for SCP01, SCP03 and SCP32 is the result of stimuli or selective pressures that are specific to the tumor microenvironment. Guided by the RNA-seq and RPPA analyses, we are currently testing several hypotheses that may explain the observed enrichment patterns. Citation Format: Hendrik J. Kuiken, Sabin Dhakal, Laura M. Selfors, Jett P. Crowdis, Hyo-eun C. Bhang, Frank Stegmeier, Gordon B. Mills, Joan S. Brugge. Characterization of functional heterogeneity and in vivo dynamics of clonal cell populations derived from the triple-negative breast cancer cell line MDA-MB-468 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3957. doi:10.1158/1538-7445.AM2017-3957