A Rare Subset of Primary Tumor Cells with Concomitant Hyperactivation of Extracellular Matrix Remodeling and dsRNA-IFN1 Signaling Metastasizes in Breast Cancer

Abstract

Metastatic breast cancer (BC) has a poor prognosis and is largely considered incurable. A better understanding of the molecular determinants of BC metastasis could facilitate development of improved prevention and treatment strategies. We used lentiviral barcoding coupled to single-cell RNA sequencing to trace clonal and transcriptional evolution during BC metastasis and showed that metastases derive from rare pro-metastatic clones that are under-represented in primary tumors. Both low clonal fitness and high metastatic potential were independent of clonal origin. Differential expression and classification analyses revealed that the pro-metastatic phenotype was acquired by rare cells characterized by the concomitant hyper-activation of extracellular matrix remodeling and dsRNA-interferon signaling pathways. Notably, genetic silencing of key genes in these pathways (KCNQ1OT1 or IFI6, respectively) significantly impaired migration in vitro and metastasis in vivo, with marginal effects on cell proliferation and tumor growth. Gene expression signatures derived from the identified pro-metastatic genes predict metastatic progression in BC patients, independently of known prognostic factors. This study elucidates previously unknown mechanisms of BC metastasis and provides prognostic predictors and therapeutic targets for metastasis prevention.

Indocyanine Green Imaging by Vital Tissue Slices Scanning Allows for the Isolation of Intact Liver Micro-Metastasis for Single-Cell Analysis

“…Clusters 8 and 10 showed upregulation of oxidative phosphorylation, while Cluster 5 was mainly glycolytic. Interestingly, our dataset displays a transcriptional heterogeneity which largely resembles what was reported in previous studies in BC (27). Therefore, ICG can be successfully exploited to guide manual coring of vital hepatic micrometastases which can be directly analyzed by scRNA-seq.…”

“…We found that each micro-metastasis harbors a subset of private upregulated genes, which can be involved in either inflammatory stress response, ribosomal translation, or interferon response. Of note, we previously demonstrated that the hyper-activation of interferon-related genes is fundamental for the successful accomplishment of the metastatic cascade (27). Our results suggest that the activation of these genes support the transition from micro-to macro-metastasis.…”