Abstract
Metastasis-initiating cells dynamically adapt to the distinct microenvironments of different organs, but these early adaptations are poorly understood due to the limited sensitivity of in situ transcriptomics. We developed fluorouracil-labeled RNA sequencing (Flura-seq) for in situ analysis with high sensitivity. Flura-seq utilizes cytosine deaminase (CD) to convert fluorocytosine to fluorouracil, metabolically labeling nascent RNA in rare cell populations in situ for purification and sequencing. Flura-seq revealed hundreds of unique, dynamic organ-specific gene signatures depending on the microenvironment in mouse xenograft breast cancer micrometastases. Specifically, the mitochondrial electron transport Complex I, oxidative stress and counteracting antioxidant programs were induced in pulmonary micrometastases, compared to mammary tumors or brain micrometastases. We confirmed lung metastasis-specific increase in oxidative stress and upregulation of antioxidants in clinical samples, thus validating Flura-seq's utility in identifying clinically actionable microenvironmental adaptations in early metastasis. The sensitivity, robustness and economy of Flura-seq are broadly applicable beyond cancer research.
Highlights
Metastasis is a multi-step process that begins with migration of cancer cells from the primary tumor into the circulation to reach lymph nodes and the parenchyma of distant organs (Massagueand Obenauf, 2016; Lambert et al, 2017)
The progression from micro- to macrometastasis is thought to entail a dynamic interaction between disseminated cancer cells and the host microenvironment, which determines an organ-specific pattern of metastatic relapse characteristic of each type of cancer (Obenauf and Massague, 2015; Celia-Terrassa and Kang, 2018)
Flura-seq identifies both a dynamically induced organ-specific stress program activated by metastasis-initiating cancer cells in the pulmonary microenvironment, as well as an adaptive transcriptional program that ensures cancer cell survival, which could be targeted to therapeutic advantage
Summary
Metastasis is a multi-step process that begins with migration of cancer cells from the primary tumor into the circulation to reach lymph nodes and the parenchyma of distant organs (Massagueand Obenauf, 2016; Lambert et al, 2017). Cancer cells in the early stages of metastatic colonization may dynamically alter their gene expression profiles in response to specific stresses experienced in distant organs as they adapt to the host tissue microenvironment and form long-lasting metastatic seeds These early disseminated cells represent a crucial transition state and may be vulnerable to therapy since they can sometimes be eliminated using adjuvant therapy after surgical resection of primary tumors, unlike established macrometastases. We describe the development of a CD-based method for in situ transcriptomic profiling of rare cell populations with high sensitivity (less than 0.01% of an organ), and the application of this method to the analysis of organ-specific micrometastatic adaptation Using this approach, we define microenvironment-dependent transcriptional programs in micrometastatic pulmonary and brain metastases from breast cancer, identify oxidative stress as a lung-specific liability of disseminated cancer cells, and demonstrate that NRF2 activation and upregulation of distinct antioxidant genes are adaptive responses to this stress in lung micrometastases. Flura-seq identifies both a dynamically induced organ-specific stress program activated by metastasis-initiating cancer cells in the pulmonary microenvironment, as well as an adaptive transcriptional program that ensures cancer cell survival, which could be targeted to therapeutic advantage
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