Differential Contributions of Pre- and Post-EMT Tumor Cells in Breast Cancer Metastasis

Abstract

◥ Metastases are responsible for the majority of breast cancerassociated deaths. The contribution of epithelial-to-mesenchymal transition (EMT) in the establishment of metastases is still controversial. To obtain in vivo evidence of EMT in metastasis, we established an EMT lineage tracing (Tri-PyMT) model, in which tumor cells undergoing EMT would irreversibly switch their fluorescent marker from RFP þ to GFP þ due to mesenchymal-specific Cre expression. Surprisingly, we found that lung metastases were predominantly derived from the epithelial compartment of breast tumors. However, concerns were raised on the fidelity and sensitivity of RFP-to-GFP switch of this model in reporting EMT of metastatic tumor cells. Here, we evaluated Tri-PyMT cells at the single-cell level using single-cell RNAsequencing and found that the Tri-PyMT cells exhibited a spectrum of EMT phenotypes, with EMT-related genes concomitantly expressed with the activation of GFP. The fluorescent color switch in these cells precisely marked an unequivocal change in EMT status, defining the pre-EMT and post-EMT compartments within the tumor. Consistently, the pre-EMT cells played dominant roles in metastasis, while the post-EMT cells were supportive in promoting tumor invasion and angiogenesis. Importantly, the post-EMT (GFP þ) cells in the Tri-PyMT model were not permanently committed to the mesenchymal phenotype; they were still capable of reverting to the epithelial phenotype and giving rise to secondary tumors, suggesting their persistent EMT plasticity. Our study addressed major concerns with the Tri-PyMT EMT lineage tracing model, which provides us with a powerful tool to investigate the dynamic EMT process in tumor biology. Significance: These findings confirm the fidelity and sensitivity of the EMT lineage tracing (Tri-PyMT) model and highlight the differential contributions of pre-and post-EMT tumor cells in breast cancer metastasis. See related commentary by Bunz, p.

Comparative Study of Transcriptomics-Based Scoring Metrics for the Epithelial-Hybrid-Mesenchymal Spectrum

“…Third, our current efforts focus on microarray data because the gene signatures utilized by all three methods were identified on this platform. Although the MLR method has been implemented on RNA-seq datasets by regressing the values obtained from microarray and RNA-seq analysis on a case-bycase basis (Kilinc et al, 2019;Lourenco et al, 2020), varying sensitivity of microarray and RNA-seq methods needs to be incorporated for future efforts in assessing these EMT scoring methods systematically.…”

New Insights Into the Role of Phenotypic Plasticity and EMT in Driving Cancer Progression

“…The number of mesenchymal cells observed in primary cancers in many xenograft studies had been observed to be less than 10%. Although the specific dissemination process of these cells is not yet well documented (Bhatia et al, 2019;Lourenco et al, 2020), enrichment of EMT in circulating tumor cells has supported a role for EMT in the initial steps of metastasis. Various studies have highlighted the role of key EMT TFs, such as Slug and Zeb1, in promoting metastasis of breast and colorectal cancer to liver and lung, respectively (Spaderna et al, 2008;Guo et al, 2012).…”

“…Orthotopic implantation strategies, such as inoculation into the mammary fat pad, has also improved the recapitulation of the breast cancer in mice (Proia et al, 2011). In conjunction with intravital imaging and fluorophore chemistry, various Cre-Lox lineage tracing approaches have been employed in cell lines, and in injected mouse and zebrafish models, to delineate EMP status of the cells at primary and metastatic sites, and also of encaptured CTCs (Lourenco et al, 2020). These reporter tags are valuable in identification of CTCs and in scenarios of low numbers of cells seeding at secondary niches during metastasis (Zheng et al, 2015;Sikandar et al, 2017).…”

The Role of p53-Mediated Signaling in the Therapeutic Response of Colorectal Cancer to 9F, a Spermine-Modified Naphthalene Diimide Derivative

“…A correlation has been identified between p53 and the death receptor [29,30], which suggests a significant role of p53 in death receptor-mediated cell death. Metastasis is the main cause of cancer death, and the epithelial-to-mesenchymal transition (EMT) is a crucial process for metastasis [31,32]. Recently, emerging evidence has demonstrated that p53 acts as a negative regulator of EMT via by mediating the expression of EMT markers [33,34] such as Snail, Twist, Slug and Vimentin [35][36][37][38].…”