Differences in cell shape, motility, and growth reflect chromosomal number variations that can be visualized with live-cell ChReporters.

Chromosome numbers often change dynamically in tumors and cultured cells, which complicates therapy as well as understanding genotype-mechanotype relationships. Here we use a live-cell "ChReporter" method to identify cells with a single chromosomal loss in efforts to better understand differences in cell shape, motility, and growth. We focus on a standard cancer line and first show clonal populations that retain the ChReporter exhibit large differences in cell and nuclear morphology as well as motility. Phenotype metrics follow simple rules, including migratory persistence scaling with speed, and cytoskeletal differences are evident from drug responses, imaging, and single-cell RNA sequencing. However, mechanotype-genotype relationships between fluorescent ChReporter-positive clones proved complex and motivated comparisons of clones that differ only in loss or retention of a Chromosome-5 ChReporter. When lost, fluorescence-null cells show low expression of Chromosome-5 genes, including a key tumor suppressor APC that regulates microtubules and proliferation. Colonies are compact, nuclei are rounded, and cells proliferate more, with drug results implicating APC, and patient survival data indicating an association in multiple tumor-types. Visual identification of genotype with ChReporters can thus help clarify mechanotype and mechano-evolution.

Confinement plus myosin-II suppression maximizes heritable loss of chromosomes, as revealed by live-cell ChReporters.

The mechanical environment of a cell can have many effects, but whether it impacts the DNA sequence of a cell has remained unexamined. To investigate this, we developed a live-cell method to measure changes in chromosome numbers. We edited constitutive genes with GFP or RFP tags on single alleles and discovered that cells that lose Chromosome reporters (ChReporters) become non-fluorescent. We applied our new tools to confined mitosis and to inhibition of the putative tumor suppressor myosin-II. We quantified compression of mitotic chromatin in vivo and demonstrated that similar compression in vitro resulted in cell death, but also rare and heritable ChReptorter loss. Myosin-II suppression rescued lethal multipolar divisions and maximized ChReporter loss during three-dimensional (3D) compression and two-dimensional (2D) lateral confinement, but not in standard 2D culture. ChReporter loss was associated with chromosome mis-segregation, rather than just the number of divisions, and loss in vitro and in mice was selected against in subsequent 2D cultures. Inhibition of the spindle assembly checkpoint (SAC) caused ChReporter loss in 2D culture, as expected, but not during 3D compression, suggesting a SAC perturbation. Thus, ChReporters enable diverse studies of viable genetic changes, and show that confinement and myosin-II affect DNA sequence and mechano-evolution.

Cooperative phagocytosis of solid tumours by macrophages triggers durable anti-tumour responses.

In solid tumours, the abundance of macrophages is typically associated with a poor prognosis. However, macrophage clusters in tumour-cell nests have been associated with survival in some tumour types. Here, by using tumour organoids comprising macrophages and cancer cells opsonized via a monoclonal antibody, we show that highly ordered clusters of macrophages cooperatively phagocytose cancer cells to suppress tumour growth. In mice with poorly immunogenic tumours, the systemic delivery of macrophages with signal-regulatory protein alpha (SIRPα) genetically knocked out or else with blockade of the CD47-SIRPα macrophage checkpoint was combined with the monoclonal antibody and subsequently triggered the production of endogenous tumour-opsonizing immunoglobulin G, substantially increased the survival of the animals and helped confer durable protection from tumour re-challenge and metastasis. Maximizing phagocytic potency by increasing macrophage numbers, by tumour-cell opsonization and by disrupting the phagocytic checkpoint CD47-SIRPα may lead to durable anti-tumour responses in solid cancers.

Nuclear mechanoprotection: From tissue atlases as blueprints to distinctive regulation of nuclear lamins.

Two meters of DNA in each of our cells must be protected against many types of damage. Mechanoprotection is increasingly understood to be conferred by the nuclear lamina of intermediate filament proteins, but very different patterns of expression and regulation between different cells and tissues remain a challenge to comprehend and translate into applications. We begin with a tutorial style presentation of "tissue blueprints" of lamin expression including single-cell RNA sequencing in major public datasets. Lamin-A, C profiles appear strikingly similar to those for the mechanosensitive factors Vinculin, Yap1, and Piezo1, whereas datasets for lamin-B1 align with and predict regulation by the cell cycle transcription factor, FOXM1, and further predict poor survival across multiple cancers. Various experiments support the distinction between the lamin types and add mechanistic insight into the mechano-regulation of lamin-A, C by both matrix elasticity and externally imposed tissue strain. Both A- and B-type lamins, nonetheless, protect the nucleus from rupture and damage. Ultimately, for mechanically active tissue constructs and organoids as well as cell therapies, lamin levels require particular attention as they help minimize nuclear damage and defects in a cell cycle.

Gaussian curvature dilutes the nuclear lamina, favoring nuclear rupture, especially at high strain rate.

Nuclear rupture has long been associated with deficits or defects in lamins, with recent results also indicating a role for actomyosin stress, but key physical determinants of rupture remain unclear. Here, lamin-B filaments stably interact with the nuclear membrane at sites of low Gaussian curvature yet dilute at high curvature to favor rupture, whereas lamin-A depletion requires high strain-rates. Live-cell imaging of lamin-B1 gene-edited cancer cells is complemented by fixed-cell imaging of rupture in: iPS-derived progeria patients cells, cells within beating chick embryo hearts, and cancer cells with multi-site rupture after migration through small pores. Data fit a model of stiff filaments that detach from a curved surface.Rupture is modestly suppressed by inhibiting myosin-II and by hypotonic stress, which slow the strain-rates. Lamin-A dilution and rupture probability indeed increase above a threshold rate of nuclear pulling. Curvature-sensing mechanisms of proteins at plasma membranes, including Piezo1, might thus apply at nuclear membranes.Summary statement: High nuclear curvature drives lamina dilution and nuclear envelope rupture even when myosin stress is inhibited. Stiff filaments generally dilute from sites of high Gaussian curvature, providing mathematical fits of experiments.

Scaling concepts in 'omics: Nuclear lamin-B scales with tumor growth and often predicts poor prognosis, unlike fibrosis.

Physicochemical principles such as stoichiometry and fractal assembly can give rise to characteristic scaling between components that potentially include coexpressed transcripts. For key structural factors within the nucleus and extracellular matrix, we discover specific gene-gene scaling exponents across many of the 32 tumor types in The Cancer Genome Atlas, and we demonstrate utility in predicting patient survival as well as scaling-informed machine learning (SIML). All tumors with adjacent tissue data show cancer-elevated proliferation genes, with some genes scaling with the nuclear filament LMNB1, including the transcription factor FOXM1 that we show directly regulates LMNB1 SIML shows that such regulated cancers cluster together with longer overall survival than dysregulated cancers, but high LMNB1 and FOXM1 in half of regulated cancers surprisingly predict poor survival, including for liver cancer. COL1A1 is also studied because it too increases in tumors, and a pan-cancer set of fibrosis genes shows substoichiometric scaling with COL1A1 but predicts patient outcome only for liver cancer-unexpectedly being prosurvival. Single-cell RNA-seq data show nontrivial scaling consistent with power laws from bulk RNA and protein analyses, and SIML segregates synthetic from contractile cancer fibroblasts. Our scaling approach thus yields fundamentals-based power laws relatable to survival, gene function, and experiments.

Nuclear failure, DNA damage, and cell cycle disruption after migration through small pores: a brief review.

In many contexts of development, regeneration, or disease such as cancer, a cell squeezes through a dense tissue or a basement membrane, constricting its nucleus. Here, we describe how the severity of nuclear deformation depends on a nucleus' mechanical properties that are mostly determined by the density of chromatin and by the nuclear lamina. We explain how constriction-induced nuclear deformation affects nuclear contents by causing (i) local density changes in chromatin and (ii) rupture of the nuclear lamina and envelope. Both processes mislocalize diffusible nuclear factors including key DNA repair and regulatory proteins. Importantly, these effects of constricted migration are accompanied by excess DNA damage, marked by phosphorylated histone γH2AX in fixed cells. Rupture has a number of downstream consequences that include a delayed cell cycle-consistent with a damage checkpoint-and modulation of differentiation, both of which are expected to affect migration-dependent processes ranging from wound healing to tumorigenic invasion.

Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest.

Whether cell forces or extracellular matrix (ECM) can impact genome integrity is largely unclear. Here, acute perturbations (∼1 h) to actomyosin stress or ECM elasticity cause rapid and reversible changes in lamin-A, DNA damage, and cell cycle. The findings are especially relevant to organs such as the heart because DNA damage permanently arrests cardiomyocyte proliferation shortly after birth and thereby eliminates regeneration after injury including heart attack. Embryonic hearts, cardiac-differentiated iPS cells (induced pluripotent stem cells), and various nonmuscle cell types all show that actomyosin-driven nuclear rupture causes cytoplasmic mis-localization of DNA repair factors and excess DNA damage. Binucleation and micronuclei increase as telomeres shorten, which all favor cell-cycle arrest. Deficiencies in lamin-A and repair factors exacerbate these effects, but lamin-A-associated defects are rescued by repair factor overexpression and also by contractility modulators in clinical trials. Contractile cells on stiff ECM normally exhibit low phosphorylation and slow degradation of lamin-A by matrix-metalloprotease-2 (MMP2), and inhibition of this lamin-A turnover and also actomyosin contractility are seen to minimize DNA damage. Lamin-A is thus stress stabilized to mechano-protect the genome.

Manipulating the mechanics of extracellular matrix to study effects on the nucleus and its structure.

Tissues such as brain, muscle, and bone differ greatly not only in their biological functions but also in their mechanical properties. Brain is far softer than muscle while bone is the stiffest tissue. Stiffness of extracellular microenvironments affects fundamental cell biological processes such as polarization and DNA replication, which affect nuclear size, shape, and levels of nuclear proteins such as the lamins that modulate gene expression. Reductionist approaches have helped dissect the effects of matrix mechanics away from confounding biochemical signals. Here, we summarize materials and methods for synthesizing and characterizing soft and stiff synthetic hydrogels widely used for mechanobiological studies. Such gels are also easily made to mimic the mechanical heterogeneity of fibrotic tissues. We further describe a nano-thin collagen fiber system, which enables control of anisotropy in addition to stiffness. With the different systems, we illustrate the effects of matrix mechanics on nuclear size, shape, and proteins including the lamins.