Cell confinement memory initiates heritable loss of chromosomes and a persistently dysregulated division program

Heritable genetic changes continually arise in cancer, especially in solid tumors where cells sometimes appear compressed. Rare heritable losses of chromosomes in live cells are quantified here with chromosome reporters (ChReporters) that show similar losses after mild or strong compression. Mild compression (to <40% of interphase height) perturbs mitotic spindles and prolongs pro/metaphase but ruptures few nuclei compared to strong compression. Unexpectedly, chromosome mis-segregation into micronuclei emerges only after release from mild confinement, but arrest and death predominate. All such effects are phenocopied by Nocodazole washout that induces a 'memory' of prolonged mitosis. The results differ from transient inhibition of the spindle assembly checkpoint and cell cycle entry with a CDK4/6-inhibitor – both of which quickly generate micronuclei. Single-cell-RNA-sequencing confirms chromosome loss days after confinement and reveals persistent upregulation of chromosome segregation. The results suggest a mitotic memory of confinement, addressing a significant gap in mechanobiology and cancer evolution. Overall design: Our focus on the very rare cells that are ChReporter-negative (~1-per-500 cells). Therefore we sorted live cells for ChReporter loss after the 48h recovery from confinement or 2D controls, and then we used single-cell RNA-seq (scRNAseq) to quantify transcript changes. RNA libraries were constructed using the Chromium Single Cell Gene Expression kit (v3.1, single index, Catalog no. PN-1000128; PN-1000127; PN-1000213) from 10X Genomics per the manufacturer's instructions. The libraries were submitted to the University of Pennsylvania's Next Generation Sequencing Core for sequencing using NovaSeq 6000 (100 cycles) from Illumina. Raw base call (BCL) files were analyzed using CellRanger (version 5.0.1) to generate FASTQ files and the 'count' command was used to generate raw count matrices aligned to GRCh38 provided by 10X genomics.