PremalignantNf1, Trp53-null Oligodendrocyte Precursor Cells Become Stalled in a Heterogeneous State of Replication Stress Before Gliomagenesis

Abstract

Cancer evolves from premalignant clones that accumulate mutations and adopt unusual cell states to achieve transformation. Tracking a cancer cell-of-origin through the cell-state alterations of premalignancy could provide clues for early-detection and cancer-prevention strategies. Previously we pinpointed the oligodendrocyte precursor cell (OPC) as a cell-of-origin for glioma. However, the early adaptations and cell-state changes of mutant OPCs during premalignancy are unknown. Using a genetically engineered mouse model (GEMM) of inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection and determined gene-expression changes in two ways: global changes in gene expression were measured by differential analysis of wild-type and mutant OPCs after bulk RNA sequencing; cell-to-cell state variations were identified by a fluctuation analysis, called stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. We chose two time points for the analysis. At 12 days after Nf1-Trp53 deletion, while bulk differences were mostly limited to increases in mitotic hallmarks and decreases in ribosome biosynthesis, stochastic profiling of mutant OPCs revealed a spectrum of stemprogenitor, proneural, and mesenchymal states as potential starting points for gliomagenesis.At 90 days after Nf1-Trp53 deletion, while bulk sequencing detected very few differentially expressed transcripts, stochastic profiling revealed multiple cell states that are absent from glial tumors, suggesting that they marked dead-ends for gliomagenesis. In parallel, we identified cells without dead-end markers but abundantly expressing key effectors of nonsense-mediated decay and homology-dependent DNA repair. This suggests that resolution of replication stress may pose a considerable bottleneck for glioma initiation in premalignant mutant OPCs. Statement of significance:In situ heterogeneity profiling of cell states in a mouse model of glioma uncovers regulatory confusion in a glioma cell-of-origin and defines a state of replication stress that precedes tumor initiation.

Pan-cancer Drivers are Recurrent Transcriptional Regulatory Heterogeneities in Early-stage Luminal Breast Cancer

“…This work combines 10cRNA-seq (16) and stochastic profiling (20) for disruption-free isolation of cancer cell-regulatory heterogeneities in a clinically practicable way. We targeted LCM isolation to breast-carcinoma cells here by using nuclear cytology or epithelial-targeted antibodies; the approach is also compatible with genetically encoded fluorophores (49,50). For 10cRNA-seq, artifactual cell stress (14) is avoided by LCM (49), and dominant cycling transcripts are mitigated by 10-cell averaging.…”

“…The approaches described and implemented here for late-onset, early-stage breast cancer are also a compromise, but one that triangulates differently by using cell pools to reduce handling and improve coverage. We see great potential in using stochastic profiling by 10cRNA-seq to deconstruct the very earliest stages of tumor initiation and premalignancy in engineered systems (50) and in precancerous in situ lesions of the breast where the need for treatment is actively debated (76). Figure 1.…”

“…4B and Supplementary Table ST1). RHEGs provide a conceptual framework for prioritizing cell-state regulatory heterogeneities identified in vivo (49,50).…”

Fragmentation of Small-Cell Lung Cancer Regulatory States in Heterotypic Microenvironments

“…Overdispersion values from the 2007 transcripts identified as candidate heterogeneities in either the KP1 spheres or in vivo conditions were recorded for 100 subsampling iterations. For the KP1 in vitro spheroids, 100 iterations of leave-one-out crossvalidation were performed as detailed in the accompanying contribution (37). Transcripts were retained if the 5th percentile of overdispersion in the C57BL/6 × 129S F 1 hybrid condition was greater than the 95th percentiles of the other two conditions.…”

Fragmentation of Small-cell Lung Cancer Regulatory States in Heterotypic Microenvironments

“…For the KP1 in vitro spheroids, 100 iterations of leave-one-out crossvalidation were performed as detailed in the accompanying contribution (47). Transcripts were retained if the 5th percentile of overdispersion in the C57/B6 x 129S F1 hybrid condition was greater than the 95th percentiles of the other two conditions.…”