Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts

Abstract

N.Y.).One Sentence Summary: Detailed single-cell phylogenies capture the frequency, tissue routes, and seeding patterns of metastasis in vivo. Abstract:25 2 Consequential events in cancer progression are typically rare and occur in the unobserved past. Detailed cell phylogenies can capture the history and chronology of such transient events -including metastasis. Here, we applied our Cas9-based lineage tracer to study metastatic progression in a lung cancer xenograft mouse model, revealing the underlying rates, routes, and patterns of metastasis. We report deeply resolved phylogenies for tens of thousands of metastatically disseminated cancer cells. We observe surprisingly diverse metastatic phenotypes, 5 ranging from metastasis-incompetent to highly aggressive populations, and these differences are associated with characteristic changes in transcriptional state, including differential expression of metastasis-related genes like IFI27 and ID3. We further show that metastases transit via tissue routes that are diverse, complex, and multidirectional, and identify examples of reseeding, seeding cascades, and parallel seeding topologies. More broadly, we demonstrate the power of next-generation lineage tracers to record cancer evolution at high resolution 10 and vast scale. Main Text:Cancer progression is governed by evolutionary principles (reviewed in (1)), which leave clear phylogenetic signatures upon every step of this process (2, 3), from early acquisition of oncogenic mutations (i.e., the relationships between normal and malignantly transformed cells (4)), to metastatic colonization of distant 15 tissues (i.e., the relationship between the primary tumor and metastases (5)), and finally adaptation to therapeutic challenges (i.e., the relationship between sensitive and resistant clones (6)). Metastasis is a particularly important step in cancer progression to study because it is chiefly responsible for disease relapse and mortality (7). Yet because metastatic events are intrinsically rare, transient, and stochastic (8,9), they are challenging to monitor in real time. Analogous to the cell fate maps that have played an essential role in deepening our understanding of 20 organismal development and cell type differentiation (10, 11), accurately reconstructed phylogenetic trees of tumors and metastases can reveal key features of this process, such as the clonality, timing, frequency, origins, and destinations of metastatic seeding (12).Lineage tracing techniques allow one to map the genealogy of related cells, providing a critical tool for exploring the phylogenetic principles of biological processes like cancer progression and metastasis. Classical

Single-cell lineage and transcriptome reconstruction of metastatic cancer reveals selection of aggressive hybrid EMT states

“…Such methods can be readily coupled to single-cell RNA sequencing (scRNA-seq) to explicitly relate cell lineage histories with transcriptional states [6][7][8] . Until recently 9,10 , GESTALT and related methods have primarily been applied to early development, e.g. by injection of CRISPR/Cas9 reagents into zygotes and subsequent profiling of edited barcodes and single cell transcriptomes from the resulting multicellular organism.…”

Single-cell ATAC and RNA sequencing reveal pre-existing and persistent subpopulations of cells associated with relapse of prostate cancer

“…Karthaus and colleagues recently found that luminal prostate cells that persist after ADT in a mouse model can contribute to tissue regeneration of the normal prostate epithelium by assuming stem-like transcriptional properties (Karthaus et al, 2020). Similar phenotypic features important for routing and rates of metastasis formation in lung cancer models have been found to be driven by differences in gene expression (Quinn et al, 2021). Similarly, here we find that during exposure to AR targeting agents, a small proportion of persistent cells remain transcriptionally unperturbed by the treatment, consistent with our recent findings of subclones with high metastatic potential interlayered within the primary untreated lesions (Woodcock et al, 2020).…”

“…Using PC specimen tumor DNA, we recently showed the presence of subclones within the primary tumors that preserve the ability to expand and metastasize years after treatment and are found interlayered within different lesions of multifocal tumors (Woodcock et al, 2020). Similarly, a recent work studying lung cancer metastases found that metastatic capacity arises from pre-existing and heritable differences in gene expression (Quinn et al, 2021). Therefore, we hypothesized that the persistent cluster 11, Stem-Like, initial cluster 10, and PROSGenesis signatures could be able to capture signals from such types of pre-existing subclones with metastatic potential in primary untreated tumors.…”

Progression of Metastasis through Lymphatic System

“…The authors concluded that axillary LN metastases contribute minimally to distant seeding in breast cancer patients [ 146 , 147 ]. Further, using a Cas9-based method that allows the long-term single-cell lineage tracing of cancer cells in an orthotopic lung cancer model in mice, some distant metastasis was traced back to an origin in mediastinal lymph nodes [ 148 ]. Collectively, these data show that in some—but not all—cancer models and patients, LNMs can serve as a source of distant metastasis.…”