Abstract A01: Investigating clonal heterogeneity and transcriptional vulnerabilities in latent metastasis

Most cancer deaths are due to the poorly understood process of metastasis, which is seeded by disseminated tumor cells (DTCs). DTCs exhibit exceedingly variable latency, outgrowing immediately or lying dormant for months to years in a viable state of mass dormancy or replicative quiescence. The mechanisms enabling DTCs to survive and resist therapy are poorly understood. We have recently derived several models of latent metastasis that default to a slow-cycling state under stress, reserve the capacity for tumor-initiation, and evade immune surveillance. These DTCs resemble progenitor stem cell populations at the molecular level, actively inhibiting the pro-differentiation Wnt pathway and over-expressing Sox transcriptional factors. Tissue immunofluorescence demonstrates that latent DTCs typically manifest as solitary or clusters of infiltrating cancer cells without forming overt metastasis within several months after hematogenous dissemination in mice. This compelling observation begs the question, are these bursts truly monoclonal? Recent studies have shown that rapidly cycling DTCs occasionally migrate in groups and this improves their likelihood of establishing polyclonal metastases; however, we hypothesize that colonization in the context of delayed metastasis may in fact be monoclonal. To address this question, unique and heritable fluorescent markers were integrated into the host genome of isolated DTCs to interrogate the clonal dynamics of apparent proliferative bursts and to enable lineage tracing. A tricolor-marking strategy was employed, whereby latent derivatives were simultaneously transduced with three vectors encoding Cerulean, Venus and mCherry fluorescent proteins at equal multiplicities of infection. Transduced cells were stably marked by an extensive color palette, which was created by variations in vector copy number and insertion sites. An automated strategy for segmenting and analyzing tissue immunofluorescence was developed to measure the size and number of subclones present in indolent micro-metastases. Fluorescent lesions were represented by their angular polar hue distribution and the Shannon-Weaver diversity index (H) was used as an estimate of clonal complexity. Lineage tracing revealed latent DTCs give rise to largely monoclonal metastases, in stark contrast to their aggressive counterparts. The dynamic behavior of latent populations was analyzed according to variations in the morphology, topological growth patterns, proliferation rates and drug response of > 300 single-cell-derived subclones within latent and aggressive metastatic derivatives. Both intra- and inter-clonal heterogeneity was enriched in DTCs derived from latent populations. The most heterogeneous clones were also the most resistant to taxol. Our results support the hypothesis that DTCs undergo short bursts of proliferation followed by elimination, growth arrest and quiescence. Consequently, long-term latency may represent the sum of multiple distinct, but balanced, states. Citation Format: Ashley M. Laughney, Srinivas Malladi, Danilo Macalinao, Joan Massague. Investigating clonal heterogeneity and transcriptional vulnerabilities in latent metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A01.