Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 ...in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4
and SCF
couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4
, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability.
Abstract During metastatic dissemination, circulating tumour cells (CTCs) enter capillary beds, where they experience mechanical constriction forces. The transient and persistent effects of these ...forces on CTCs behaviour remain poorly understood. Here, we developed a high-throughput microfluidic platform mimicking human capillaries to investigate the impact of mechanical constriction forces on malignant and normal breast cell lines. We observed that capillary constrictions induced nuclear envelope rupture in both cancer and normal cells, leading to transient changes in nuclear and cytoplasmic area. Constriction forces transiently activated cGAS/STING and pathways involved in inflammation (NF-κB, STAT and IRF3), especially in the non-malignant cell line. Furthermore, the non-malignant cell line experienced transcriptional changes, particularly downregulation of epithelial markers, while the metastatic cell lines showed minimal alterations. These findings suggest that mechanical constriction forces within capillaries may promote differential effects in malignant and normal cell lines.
Although classical genetic and biochemical approaches have identified hundreds of proteins that function in the dynamic remodeling of cell shape in response to upstream signals, there is currently ...little systems-level understanding of the organization and composition of signaling networks that regulate cell morphology. We have developed quantitative morphological profiling methods to systematically investigate the role of individual genes in the regulation of cell morphology in a fast, robust, and cost-efficient manner. We analyzed a compendium of quantitative morphological signatures and described the existence of local signaling networks that act to regulate cell protrusion, adhesion, and tension.
Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and ...investigated mechanisms of sensitivity.
A panel of cell lines representative of TNBC was tested for
and
sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging.
The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both
(
< 0.001 LAR vs. basal-like) and
in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in
-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes.
Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC.
.
Human cells that suffer mild DNA damage can enter a reversible state of growth arrest known as quiescence. This decision to temporarily exit the cell cycle is essential to prevent the propagation of ...mutations, and most cancer cells harbor defects in the underlying control system. Here we present a mechanistic mathematical model to study the proliferation–quiescence decision in nontransformed human cells. We show that two bistable switches, the restriction point (RP) and the G1/S transition, mediate this decision by integrating DNA damage and mitogen signals. In particular, our data suggest that the cyclin-dependent kinase inhibitor p21 (Cip1/Waf1), which is expressed in response to DNA damage, promotes quiescence by blocking positive feedback loops that facilitate G1 progression downstream of serum stimulation. Intriguingly, cells exploit bistability in the RP to convert graded p21 and mitogen signals into an all-or-nothing cell-cycle response. The same mechanism creates a window of opportunity where G1 cells that have passed the RP can revert to quiescence if exposed to DNA damage. We present experimental evidence that cells gradually lose this ability to revert to quiescence as they progress through G1 and that the onset of rapid p21 degradation at the G1/S transition prevents this response altogether, insulating S phase from mild, endogenous DNA damage. Thus, two bistable switches conspire in the early cell cycle to provide both sensitivity and robustness to external stimuli.
Abstract
Loss-of-function (LOF) methods such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing provide unparalleled power for studying the biological function of ...genes of interest. However, a major concern is non-specific targeting, which involves depletion of transcripts other than those intended. Little work has been performed to characterize the off-target effects of these common LOF methods at the whole-transcriptome level. Here, we experimentally compared the non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible off-target effects in gene expression, with CRISPRi also exhibiting strong clonal effects. As an illustrative example, we evaluated the performance of each method for determining the role of an uncharacterized long noncoding RNA (lncRNA). Several LOF methods successfully depleted the candidate lncRNA but yielded different sets of differentially expressed genes as well as a different cellular phenotype upon depletion. Similar discrepancies between methods were observed with a protein-coding gene (Ch-TOG/CKAP5) and another lncRNA (MALAT1). We suggest that the differences between methods arise due to method-specific off-target effects and provide guidelines for mitigating such effects in functional studies. Our recommendations provide a framework with which off-target effects can be managed to improve functional characterization of genes of interest.
We present a new folded dual-view oblique plane microscopy (OPM) technique termed dOPM that enables two orthogonal views of the sample to be obtained by translating a pair of tilted mirrors in ...refocussing space. Using a water immersion 40× 1.15 NA primary objective, deconvolved image volumes of 200 nm beads were measured to have full width at half maxima (FWHM) of 0.35 ± 0.04 µm and 0.39 ± 0.02 µm laterally and 0.81 ± 0.07 µm axially. The measured z-sectioning value was 1.33 ± 0.45 µm using light-sheet FWHM in the frames of the two views of 4.99 ± 0.58 µm and 4.89 ± 0.63 µm. To qualitatively demonstrate that the system can reduce shadow artefacts while providing a more isotropic resolution, a multi-cellular spheroid approximately 100 µm in diameter was imaged.
Small interfering RNA (siRNA) screening approaches used with quantitative single-cell analysis can uncover the roles of genes in cell morphogenesis. Here, we present a high-throughput automated ...phenotypic screening technique to quantify a single cell shape in cancer cells cultured on top of soft 3D hydrogels. We describe reverse transfection of cells with siRNAs and seeding of these cells on top of collagen, followed by image analysis to quantify morphology of a single cell and population levels in low-elasticity matrices.
For complete details on the use and execution of this protocol, please refer to Bousgouni et al. (2022).1
Display omitted
•High-throughput imaging of cells cultured on top of soft 3D collagen hydrogel•Screening in a 96-well format for genes that regulate shape determination•Automated quantitative single-cell phenotyping•Cell shape determination on top of soft collagen
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Small interfering RNA (siRNA) screening approaches used with quantitative single-cell analysis can uncover the roles of genes in cell morphogenesis. Here, we present a high-throughput automated phenotypic screening technique to quantify a single cell shape in cancer cells cultured on top of soft 3D hydrogels. We describe reverse transfection of cells with siRNAs and seeding of these cells on top of collagen, followed by image analysis to quantify morphology of a single cell and population levels in low-elasticity matrices.
Although a great deal is known about the signaling events that promote nuclear translocation of NF‐κB, how cellular biophysics and the microenvironment might regulate the dynamics of this pathway is ...poorly understood. In this study, we used high‐content image analysis and Bayesian network modeling to ask whether cell shape and context features influence NF‐κB activation using the inherent variability present in unperturbed populations of breast tumor and non‐tumor cell lines. Cell–cell contact, cell and nuclear area, and protrusiveness all contributed to variability in NF‐κB localization in the absence and presence of TNFα. Higher levels of nuclear NF‐κB were associated with mesenchymal‐like versus epithelial‐like morphologies, and RhoA‐ROCK‐myosin II signaling was critical for mediating shape‐based differences in NF‐κB localization and oscillations. Thus, mechanical factors such as cell shape and the microenvironment can influence NF‐κB signaling and may in part explain how different phenotypic outcomes can arise from the same chemical cues.
Synopsis
High‐content image analysis of hundreds of thousands of single cells in combination with statistical modeling reveals relationships between cell shape and NF‐κB localization in normal and tumor breast epithelial cells.
Cell shape and microenvironment influence NF‐κB nuclear translocation in normal and tumor breast epithelial cells.
Bayesian network models infer statistical dependencies between NF‐κB and YAP nuclear localization and cell shape features based on inherent cell‐to‐cell variability within populations.
RhoA‐ROCK‐myosin II activity suppresses NF‐κB nuclear translocation and cycling via effects on cell tension.
High content image analysis of hundreds of thousands of single cells in combination with statistical modeling reveals relationships between cell shape and NF‐κB localization in normal and tumor breast epithelial cells.
The intra-tumor diversity of cancer cells is under intense investigation; however, little is known about the heterogeneity of the tumor microenvironment that is key to cancer progression and ...evolution. We aimed to assess the degree of microenvironmental heterogeneity in breast cancer and correlate this with genomic and clinical parameters.
We developed a quantitative measure of microenvironmental heterogeneity along three spatial dimensions (3-D) in solid tumors, termed the tumor ecosystem diversity index (EDI), using fully automated histology image analysis coupled with statistical measures commonly used in ecology. This measure was compared with disease-specific survival, key mutations, genome-wide copy number, and expression profiling data in a retrospective study of 510 breast cancer patients as a test set and 516 breast cancer patients as an independent validation set. In high-grade (grade 3) breast cancers, we uncovered a striking link between high microenvironmental heterogeneity measured by EDI and a poor prognosis that cannot be explained by tumor size, genomics, or any other data types. However, this association was not observed in low-grade (grade 1 and 2) breast cancers. The prognostic value of EDI was superior to known prognostic factors and was enhanced with the addition of TP53 mutation status (multivariate analysis test set, p = 9 × 10-4, hazard ratio = 1.47, 95% CI 1.17-1.84; validation set, p = 0.0011, hazard ratio = 1.78, 95% CI 1.26-2.52). Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. Limitations of this study include the number of cell types included in the model, that EDI has prognostic value only in grade 3 tumors, and that our spatial heterogeneity measure was dependent on spatial scale and tumor size.
To our knowledge, this is the first study to couple unbiased measures of microenvironmental heterogeneity with genomic alterations to predict breast cancer clinical outcome. We propose a clinically relevant role of microenvironmental heterogeneity for advanced breast tumors, and highlight that ecological statistics can be translated into medical advances for identifying a new type of biomarker and, furthermore, for understanding the synergistic interplay of microenvironmental heterogeneity with genomic alterations in cancer cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
