Identification of EMT signaling cross-talk and gene regulatory networks by single-cell RNA sequencing.

The epithelial-to-mesenchymal transition (EMT) plays a critical role during normal development and in cancer progression. EMT is induced by various signaling pathways, including TGF-ß, BMP, Wnt-ß-catenin, NOTCH, Shh, and receptor tyrosine kinases. In this study, we performed single-cell RNA sequencing on MCF10A cells undergoing EMT by TGF-ß1 stimulation. Our comprehensive analysis revealed that cells progress through EMT at different paces. Using pseudotime clustering reconstruction of gene-expression profiles during EMT, we found sequential and parallel activation of EMT signaling pathways. We also observed various transitional cellular states during EMT. We identified regulatory signaling nodes that drive EMT with the expression of important microRNAs and transcription factors. Using a random circuit perturbation methodology, we demonstrate that the NOTCH signaling pathway acts as a key driver of TGF-ß-induced EMT. Furthermore, we demonstrate that the gene signatures of pseudotime clusters corresponding to the intermediate hybrid EMT state are associated with poor patient outcome. Overall, this study provides insight into context-specific drivers of cancer progression and highlights the complexities of the EMT process.

EMTome: a resource for pan-cancer analysis of epithelial-mesenchymal transition genes and signatures.

BACKGROUND: The epithelial-mesenchymal transition (EMT) enables dissociation of tumour cells from the primary tumour mass, invasion through the extracellular matrix, intravasation into blood vessels and colonisation of distant organs. Cells that revert to the epithelial state via the mesenchymal-epithelial transition cause metastases, the primary cause of death in cancer patients. EMT also empowers cancer cells with stem-cell properties and induces resistance to chemotherapeutic drugs. Understanding the driving factors of EMT is critical for the development of effective therapeutic interventions. METHODS: This manuscript describes the generation of a database containing EMT gene signatures derived from cell lines, patient-derived xenografts and patient studies across cancer types and multiomics data and the creation of a web-based portal to provide a comprehensive analysis resource. RESULTS: EMTome incorporates (i) EMT gene signatures; (ii) EMT-related genes with multiomics features across different cancer types; (iii) interactomes of EMT-related genes (miRNAs, transcription factors, and proteins); (iv) immune profiles identified from The Cancer Genome Atlas (TCGA) cohorts by exploring transcriptomics, epigenomics, and proteomics, and drug sensitivity and (iv) clinical outcomes of cancer cohorts linked to EMT gene signatures. CONCLUSION: The web-based EMTome portal is a resource for primary and metastatic tumour research publicly available at www.emtome.org .

Enrichment of Cancer Stem Cells in a Tumorsphere Assay.

Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. CSCs possess tumor initiation potential as well as the ability to resist toxic compounds and chemotherapeutic agents through the upregulation of drug efflux transporters, DNA repair pathways, and survival cascades. Accumulating evidence suggests that CSCs are responsible for tumor relapse and resistance to chemotherapeutic agents and that targeting CSCs is critical to inhibition of cancer progression. Therefore, isolation and characterization of CSCs is important in studying tumor initiation and progression. In this chapter, we provide a detailed method for the identification and isolation of CSCs.

Limiting Dilution Tumor Initiation Assay: An In Vivo Approach for the Study of Cancer Stem Cells.

Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. Calculating the frequency of tumor-initiating cells is important in the assessment of the number of CSCs present in a cell population. In this chapter, we present a protocol developed for quantification of CSCs from breast cancer tumors that can be adapted to CSCs from other types of tumors.

In Vitro Quantification of Cancer Stem Cells Using a Mammosphere Formation Assay.

Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. In this chapter, we provide a detailed method for the quantification of CSCs in vitro through mammosphere formation.

Breast cancer dormancy: need for clinically relevant models to address current gaps in knowledge.

Breast cancer is the most commonly diagnosed cancer in the USA. Although advances in treatment over the past several decades have significantly improved the outlook for this disease, most women who are diagnosed with estrogen receptor positive disease remain at risk of metastatic relapse for the remainder of their life. The cellular source of late relapse in these patients is thought to be disseminated tumor cells that reactivate after a long period of dormancy. The biology of these dormant cells and their natural history over a patient's lifetime is largely unclear. We posit that research on tumor dormancy has been significantly limited by the lack of clinically relevant models. This review will discuss existing dormancy models, gaps in biological understanding, and propose criteria for future models to enhance their clinical relevance.