Mitotic progression under confinement drives heritable loss of chromosomes

Introduction: Unlike most liquid tumors, nearly all solid tumors exhibit chromosome losses and gains caused by chromosomal instability (CIN) during various phases of the cell cycle. Chromosome losses are also considered early events in oncogenesis, leading to decreased expression of key tumor suppressor genes. CIN-related factors and chromosome number changes, or aneuploidy, contribute to tumor heterogeneity and associate with poor prognosis. Compression of mitotic cells increases chromosome mis-segregation and increases cell death (Lancaster et al., 2013). However, evidence is thus far lacking for micronuclei that harbor mis-segregated chromosomes and for any heritable changes in chromosome numbers. Strong compression of interphase cells causes nuclear rupture with chromatin blebs and DNA damage, which if not repaired properly, can contribute to CIN (Nader et al., 2021). We recently developed a live cell imaging method to track heritable loss of chromosomes (Hayes et al., 2023), but whether heritable changes during cell compression reflect interphase processes or mitotic processes is again unclear. We hypothesized that confinement drives chromosome loss in mitosis more so than in interphase, and we compressed live cells to heights as small as 2um in combination with various molecular perturbations to test this hypothesis.

Materials and

Methods: For our chromosome loss studies, we use our chromosome reporter (ChReporter) system, in which we edit a GFP/RFP tag into constitutive gene found on chromosomes 5, 9, 12, and 19 of solid-tumor derived A549 lung adenocarcinoma or U2OS osteosarcoma lines. Thus, the loss of the GFP/RFP signal (quantified by flow cytometry) allows us to track chromosome loss in living cells. To study mechanically induced chromosome loss, we physically confined A549 cells using microfabricated PDMS micropillars with heights ranging from 19um to 2um. We increase interphase nuclear envelope rupture by both deeper confinement and knockdown of nuclear envelope protein, LMNA. To look at mitotic effects, we use a CDK4/6 inhibitor to prevent mitosis or an MPS1 inhibitor to promote mitotic errors.

Results, Conclusions, and Discussions: Following confinement and a 48h rest, we see a ~2-fold increase in ChReporter loss relative to the 2D control for confining heights ≤10um (Fig.1). The upper range restricts mitotic rounding but has no significant effect on interphase nuclei. Remarkably, we see no significant differences in overall reporter loss within this range of confining heights even though nuclear rupture increases several fold over this range of confinement and is evident in the majority of cells under deep (2um) compression. Knockdown of the nuclear envelope protein Lamin-A,C also increases nuclear rupture, but again showed no difference in chromosome loss. These results begin to suggest that nuclear envelope rupture of interphase cells – which leads to some extrusion of chromatin into cytoplasm and DNA damage – does not contribute to heritable chromosome loss.
To examine the effect of mitotic perturbations on chromosome loss, we combined compression with drugs affecting cell cycle. Arrest of G1 phase with CDK4/6 inhibition shows chromosome loss is decreased relative to 2D control cells and independent of confinement. The experiments included both deep (2um) and intermediate confinement (6um) which once again confirmed the lack of effect of nuclear rupture on chromosome loss. The result is consistent with a requirement for cell cycle progression for the chromosome loss caused by compression. Mitotic errors can be increased in 2D culture by disrupting the spindle assembly checkpoint through MPS1 inhibition, which accelerates exit from mitosis and increases chromosome mis-segregation events leading to formation of micronuclei. Such inhibition shows an increase in chromosome loss in 2D equivalent to loss from 2-10um confinement. Moreover, combination of MPS1i with confinement does not further increase chromosome loss, which suggests that chromosome loss induced by either perturbation shares a common pathway related to spindle-chromatin CIN. Novel evidence of confinement-induced increases in the formation of micronuclei is also clear, but their complex kinetics is a focus of ongoing studies.
In conclusion, the results here suggest that compression during mitosis physically contributes to induction of the frequently observed changes in chromosome numbers. The findings add new perspective to the fact that solid tumors typically grow within confining tissue microenvironments.

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