Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Abstract

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14 + cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs.uring metastasis, cancer cells escape the primary tumor, travel through the circulation, and colonize distant organs. Conventional models of cancer progression propose that each metastasis arises from the clonal outgrowth of a single tumor cell and this conceptual framework is a foundation for models, such as epithelial-mesenchymal transition (EMT) and migratory cancer stem cells (1).Challenging the generality of the single-cell/single-metastasis model are long-standing clinical observations that tumor cell clusters (also termed "tumor clumps") are also observed across the stages of metastasis. Tumor cell clusters are detected in the bloodstream of cancer patients (2), clusters can efficiently seed metastases (3), and though rare, circulating tumor cell (CTC) clusters have prognostic significance (4, 5). Furthermore, metastases are composed of multiple genetically distinct tumor cell clones, in mouse models of breast, pancreas, and small cell carcinoma (5-7), and in human metastatic prostate cancer patients (8). Taken together, these observations provide accumulating evidence that tumor cell clusters contribute to metastasis. However, they leave unresolved two important questions: how do tumor cell clusters emerge from the primary tumor, and which molecular features identify cell clusters that metastasize?An important clinical observation is that cancer cells invade the surrounding stroma as cohesive clusters in the majority of ...

Modeling and analysis of collective cell migration in an in vivo three-dimensional environment

“…43). Collective migration may be the rule rather than the exception in metastasis, despite the physical challenges posed by squeezing groups of cells into blood and lymphatic vessels (44). Additional examples of collective migrations include blood vessel sprouting, epithelial closures, and innumerable embryonic morphogenetic movements.…”

Cancer evolution and heterogeneity

“…Considering the fate of cancer cells, a malignant cell will meet and surpass various pressures during cancer progression. According to a mouse model, recurrence of a primary tumor demands multiple propensities for each clone, such as invasion, local dissemination, vascular embolus, circulating tumor cells, and micrometastasis 42. Therefore, ITH might provide advantages for metastasis.…”

“…LOH is an immune escape mechanism that is subject to strong microenvironment later in tumor evolution. Subclonal LOH was observed on chromosome 6, which harbored HLA haplotypes, and the number of putative neoantigens presented to T cells was clearly impaired 42. Therefore, cells with a high number of somatic mutations might not present neoantigens and evade the immune response.…”

Circulating tumor cells and their clinical significance

“…Presumably, this gives cancer cells an advantage in efficient seeding, given that they have been shown to have higher metastatic potential than single CTCs [13]. Indeed, polyclonal metastases have been documented in prostate cancer, and in mouse models of breast cancer [13,14]. Although CTC clusters retain cell-cell contacts, studies have shown that they are not fully epithelial, but rather, display a hybrid epithelial--mesenchymal phenotype [13,15].…”