6b, bottom panel). and minor subclones in metastasis. Tumors are mixtures of cells with distinct characteristics1. High intratumor diversity increases the likelihood of disease progression2, as different subclones respond differently to microenvironmental cues. Treatment of heterogeneous tumors favors selection of resistant subclones, leading to therapeutic failure. Heterogeneous tumors also display phenotypes different from those of individual clones; thus, intratumor heterogeneity has a significant impact on tumor progression and therapeutic resistance. Metastatic disease is responsible for most cancer-associated mortality; therefore, understanding drivers of metastatic progression is key for improving clinical outcomes. Cancer genome sequencing studies identified limited genetic differences between primary and metastatic tumors and exhibited extensive subclonal heterogeneity in Cathepsin Inhibitor 1 both primary and distant lesions3,4. However, the mechanism(s) by which polyclonal primary tumors produce polyclonal metastases remains elusive. Moreover, several recent studies implicated microenvironmental changes as key mediators of metastatic dissemination and outgrowth5,6, highlighting the role of non-cell-autonomous factors in tumor evolution. Clonal cooperation drives polyclonal metastasis We have been investigating the effect of subclonal interactions on tumor phenotypes using a human breast cancer cell line (MDA-MB-468)-derived xenograft model of intratumor heterogeneity. We previously established that a minor subclone can drive tumor growth through non-cell-autonomous interactions, supporting long-term subclonal heterogeneity7. Briefly, we tested 18 subclones, each expressing a secreted protein implicated in metastasis and angiogenesis, and found that polyclonal tumors with all 18 subclones grew the fastest, while in monoclonal tumors only IL11 and CCL5 were able to drive tumor growth. We also decided that a mixture of two subclones expressing IL11 (interleukin 11) and FIGF (FOS-induced growth factor, also known as VEGFD) was largely able to reproduce this phenotype. Omitting IL11+ cells from polyclonal tumors decreased tumor growth, suggesting that IL11 and FIGF may cooperate. In addition, both polyclonal tumors and tumors comprised of only IL11 and FIGF subclones were highly metastatic, but the underlying mechanism remained undefined. To dissect the molecular basis of this metastasis-driving subclonal cooperation, we first investigated the clonality of metastases of primary MDA-MB-468 tumors comprising IL11+ and FIGF+ driver subclones, as well as neutral subclones. Monoclonal or polyclonal mixtures of Cathepsin Inhibitor 1 green fluorescent protein (GFP) and luciferase-expressing parental cells, red fluorescent protein (RFP) and V5-tagged IL11+ cells, and RFP+ FIGF+ cells were implanted into the mammary fat pads of immunodeficient NOG mice. We monitored primary tumor growth by weekly caliper measurements and macrometastatic lesions by weekly bioluminescence imaging. Polyclonal tumors initiated from 5% IL11+ and 5% FIGF+ RFP+ cells with 90% GFP+ parental cells grew faster and were more metastatic than monoclonal and parental tumors Cathepsin Inhibitor 1 (Fig. 1a-c, Supplementary Table 1). Immunohistochemistry-based quantification of human cytokeratin+ (CK+) cells in the lungs revealed an increased number of metastatic lesions in mice with FIGF+ primary tumors (Fig. 1d,e) despite small primary tumors. However, most of these were micrometastases, detectable as single cells only by Rabbit Polyclonal to TBC1D3 immunohistochemistry, while the lungs of mice with polyclonal primary tumors were filled with macrometastases emitting high bioluminescence-signal (Fig. 1b). The increased metastases by polyclonal tumors were not simply due to their faster growth, as this trend was still observed when primary tumors were surgically removed upon reaching 1 cm in diameter (Supplementary Fig. 1a, b). Furthermore, IL11+ monoclonal tumors grew faster than parental and neutral clones but were not as metastatic as polyclonal tumors. Cathepsin Inhibitor 1 Thus, macrometastatic outgrowth is usually a phenotype unique to polyclonal tumors. Immunofluorescence exhibited that metastases of polyclonal tumors were also mostly polyclonal, composed of V5-tag+ IL11 and FIGF driver and GFP+ parental subclones, the latter of which did not produce significant macrometastases on their own except for rare GFP+ monoclonal metastases (Fig. 1f,g). These results show that polyclonal tumors behave differently than monoclonal tumors and that IL11+ and FIGF+ subclones driving tumor growth and dissemination, respectively, cooperate to generate frequent large polyclonal metastases. This cooperation is likely to Cathepsin Inhibitor 1 be indirect, as close cell contact between driver and neutral subclones was not commonly observed and clonal distribution within metastatic lesions was random (Fig. 1g,h). Open in.