Lineage Recording Reveals the Phylodynamics, Plasticity and Paths of Tumor Evolution

Abstract

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth, expansion to neighboring and distal tissues, and therapeutic resistance. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma which enabled us to track tumor evolution from single transformed cells to metastatic tumors at unprecedented resolution. We found that loss of the initial, stable alveolar-type2-like state was accompanied by transient increase in plasticity. This was followed by adoption of distinct fitness associated transcriptional programs which enable rapid expansion and ultimately clonal sweep of rare, stable subclones capable of metastasizing to distant sites. Finally, we showed that tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates tumor progression by creating novel evolutionary paths. Overall, our study elucidates the hierarchical nature of tumor evolution, and more broadly enables the in-depth study of tumor progression.

Interactive, integrated analysis of single-cell transcriptomic and phylogenetic data with PhyloVision

“…With this analysis, a user can identify gene signatures that are significantly associated with the tree structure, suggesting evolutionary patterns of interest. Our PhyloVision pipeline also includes modules to analyze user-provided meta-data, (e.g., the tissue of origin, extent of somatic mutations, or cell-level fitnesses inferred with external models; Neher et al, 2014 ), quantify cell-level plasticities with respect to categorical data ( Yang et al, 2021 ), and interactively visualize these cell-level data ( STAR Methods ). Together, these “signature-level” analyses enable users to identify cell-level properties whose variation is consistent with the tree structure and to highlight phenotypes that represent subclonal, heritable phenotypes.…”

Theoretical Guarantees for Phylogeny Inference from Single-Cell Lineage Tracing

“…Although having more characters is preferable, we recognize that currently there are practical limits on the number of recording sites that can be incorporated into CRISPR-Cas9 systems. Current methods to incorporate recording sites into the genome (such as lentiviral transduction [3,10,12] or transposition [5,6,9,11,14]) are limited by the low uptake of these sites into the progenitor cell, only offering on the scale of tens of recording sites [10,6,5,12,3,9,8,7]. One alternate technology of particular interest is the baseeditor, which uses a modified Cas9 complex to induce direct base-pair substitutions [39].…”

“…We discuss here current and potential strategies for engineering the discussed parameters: the editing rate, the collision probability, and the missing data probability. There is a large body of literature showing that the editing activity of Cas9 (as in lineage tracing experiments) can be tuned with relative precision using mismatches between the guide RNA and recording site [9,18,10,12,14]. In regards to the collision probability, experimenters are currently unable to dictate the collision rate in state outcomes due to the random indel outcomes of Cas9 editing.…”

“…These indel mutations are subsequently inherited by future descendants, and the accumulation of these mutations is used to infer the clonal relationships between the observed cells, stratifying them into clades of increasing resolution. Thus far, studies have paired these technologies with single-cell transcriptomic profiling [13] to study questions in development (e.g., inferring lineage relationships between cellular compartments) [7,8,6,5,9,11] and cancer progression (e.g., inferring rates and routes of metastases) [12,14].…”

FoxA1 and FoxA2 regulate growth and cellular identity in NKX2-1-positive lung adenocarcinoma

“…We have shown previously that FoxA1/2 are critical activators of gastric differentiation in NKX2-1-negative LUAD 15,49 . Recent single cell analyses of the KP LUAD GEMM, including in vivo lineage recording of cancer progression, defined multiple transcriptional programs associated with distinct cellular identities that emerge as tumors progress 11,12,50 . Early low grade tumor cells most closely resemble normal alveolar type 2 (AT2) cells, as evidenced by high levels of Nkx2-1 and its canonical AT2 targets.…”