Deleterious Mechanical Deformation Selects Mechanoresilient Cancer Cells with Enhanced Proliferation and Chemoresistance.

Cancer cells in secondary tumors are found to form metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, the role of deleterious mechanical stresses in this change of metastatic potential is unclear. Here, by forcing cancer cells to flow through small capillary-sized constrictions, it is demonstrated that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. These results highlight a potential link between the microenvironmental physical stresses and the enhanced malignancy of metastasizing cancer cells which may be utilized as a therapeutic strategy in preventing the metastatic spread of cancer cells.

Microfluidic detection of human diseases: From liquid biopsy to COVID-19 diagnosis.

Microfluidic devices can be thought of as comprising interconnected miniaturized compartments performing multiple experimental tasks individually or in parallel in an integrated fashion. Due to its small size, portability, and low cost, attempts have been made to incorporate detection assays into microfluidic platforms for diseases such as cancer and infection. Some of these technologies have served as point-of-care and sample-to-answer devices. The methods for detecting biomarkers in different diseases usually share similar principles and can conveniently be adapted to cope with arising health challenges. The COVID-19 pandemic is one such challenge that is testing the performance of both our conventional and newly-developed disease diagnostic technologies. In this mini-review, we will first look at the progress made in the past few years in applying microfluidics for liquid biopsy and infectious disease detection. Following that, we will use the current pandemic as an example to discuss how such technological advancements can help in the current health challenge and better prepare us for future ones.

Multivariate analysis reveals activation-primed fibroblast geometric states in engineered 3D tumor microenvironments.

Fibroblasts are a heterogeneous group of cells comprising subpopulations that have been found to be activated in the stromal microenvironment that regulates tumor initiation and growth. The underlying mechanisms of such selective activation of fibroblasts are not understood. We propose that the intrinsic geometric heterogeneity of fibroblasts modulates the nuclear mechanotransduction of signals from the microenvironment, resulting in their selective activation. To test this, we developed an engineered 3D fibroblast tumor coculture system and used high resolution images to quantify multiple cell geometry sensitive nuclear morphological and chromatin organizational features. These features were then mapped to activation levels as measured by the nuclear abundance of transcription cofactor, megakaryoblastic leukemia, and protein levels of its target, αSMA. Importantly, our results indicate the presence of activation-"primed" cell geometries that present higher activation levels, which are further enhanced in the presence of stimuli from cancer cells. Further, we show that by enriching the population of activation-primed cell geometric states by either increasing matrix rigidity or micropatterning primed cell shapes, fibroblast activation levels can be increased. Collectively, our results reveal important cellular geometric states that select for fibroblast activation within the heterogenous tumor microenvironment.

Nuclear morphometrics and chromatin condensation patterns as disease biomarkers using a mobile microscope.

Current cancer diagnosis involves the use of nuclear morphology and chromatin condensation signatures for accurate advanced stage classification. While such diagnostic approaches rely on high resolution imaging of the cell nucleus using expensive microscopy systems, developing portable mobile microscopes to visualize nuclear and chromatin condensation patterns is desirable at clinical settings with limited infrastructure. In this study, we develop a portable fluorescent mobile microscope capable of acquiring high resolution images of the nucleus and chromatin. Using this we extracted nuclear morphometric and chromatin texture based features and were able to discriminate between normal and cancer cells with similar accuracy as wide-field fluorescence microscopy. We were also able to detect subtle changes in nuclear and chromatin features in cells subjected to compressive forces, cytoskeletal perturbations and cytokine stimulation, thereby highlighting the sensitivity of the portable microscope. Taken together, we present a versatile platform to exploit nuclear morphometrics and chromatin condensation features as physical biomarkers for point-of-care diagnostic solutions.

Regulation of nuclear architecture, mechanics, and nucleocytoplasmic shuttling of epigenetic factors by cell geometric constraints.

Cells sense mechanical signals from their microenvironment and transduce them to the nucleus to regulate gene expression programs. To elucidate the physical mechanisms involved in this regulation, we developed an active 3D chemomechanical model to describe the three-way feedback between the adhesions, the cytoskeleton, and the nucleus. The model shows local tensile stresses generated at the interface of the cell and the extracellular matrix regulate the properties of the nucleus, including nuclear morphology, levels of lamin A,C, and histone deacetylation, as these tensile stresses 1) are transmitted to the nucleus through cytoskeletal physical links and 2) trigger an actomyosin-dependent shuttling of epigenetic factors. We then show how cell geometric constraints affect the local tensile stresses and subsequently the three-way feedback and induce cytoskeleton-mediated alterations in the properties of the nucleus such as nuclear lamina softening, chromatin stiffening, nuclear lamina invaginations, increase in nuclear height, and shrinkage of nuclear volume. We predict a phase diagram that describes how the disruption of cytoskeletal components impacts the feedback and subsequently induce contractility-dependent alterations in the properties of the nucleus. Our simulations show that these changes in contractility levels can be also used as predictors of nucleocytoplasmic shuttling of transcription factors and the level of chromatin condensation. The predictions are experimentally validated by studying the properties of nuclei of fibroblasts on micropatterned substrates with different shapes and areas.

Actin Dynamics Couples Extracellular Signals to the Mobility and Molecular Stability of Telomeres.

Genome regulatory programs such as telomere functioning require extracellular signals to be transmitted from the microenvironment to the nucleus and chromatin. Although the cytoskeleton has been shown to directly transmit stresses, we show that the intrinsically dynamic nature of the actin cytoskeleton is important in relaying extracellular signals to telomeres. Interestingly, this mechanical pathway not only transmits physical stimuli but also chemical stimuli. The cytoskeletal network continuously reorganizes and applies dynamic forces on the nucleus and feeds into the regulation of telomere dynamics. We further found that distal telomeres are mechanically coupled in a length- and timescale-dependent manner and identified nesprin 2G as well as lamin A/C as being essential to regulate their translational dynamics. Finally, we demonstrated that such mechanotransduction events impinge on the binding dynamics of critical telomere binding proteins. Our results highlight an overarching physical pathway that regulates positional and molecular stability of telomeres.

Laterally confined growth of cells induces nuclear reprogramming in the absence of exogenous biochemical factors.

Cells in tissues undergo transdifferentiation programs when stimulated by specific mechanical and biochemical signals. While seminal studies have demonstrated that exogenous biochemical factors can reprogram somatic cells into pluripotent stem cells, the critical roles played by mechanical signals in such reprogramming process have not been well documented. In this paper, we show that laterally confined growth of fibroblasts on micropatterned substrates induces nuclear reprogramming with high efficiency in the absence of any exogenous reprogramming factors. We provide compelling evidence on the induction of stem cell-like properties using alkaline phosphatase assays and expression of pluripotent markers. Early onset of reprogramming was accompanied with enhanced nuclear dynamics and changes in chromosome intermingling degrees, potentially facilitating rewiring of the genome. Time-lapse analysis of promoter occupancy by immunoprecipitation of H3K9Ac chromatin fragments revealed that epithelial, proliferative, and reprogramming gene promoters were progressively acetylated, while mesenchymal promoters were deacetylated by 10 days. Consistently, RNA sequencing analysis showed a systematic progression from mesenchymal to stem cell transcriptome, highlighting pathways involving mechanisms underlying nuclear reprogramming. We then demonstrated that these mechanically reprogrammed cells could be maintained as stem cells and can be redifferentiated into multiple lineages with high efficiency. Importantly, we also demonstrate the induction of cancer stemness properties in MCF7 cells grown in such laterally confined conditions. Collectively, our results highlight an important generic property of somatic cells that, when grown in laterally confined conditions, acquire stemness. Such mechanical reprogramming of somatic cells demonstrated here has important implications in tissue regeneration and disease models.

Cell geometry dictates TNFα-induced genome response.

Cells in physiology integrate local soluble and mechanical signals to regulate genomic programs. Whereas the individual roles of these signals are well studied, the cellular responses to the combined chemical and physical signals are less explored. Here, we investigated the cross-talk between cellular geometry and TNFα signaling. We stabilized NIH 3T3 fibroblasts into rectangular anisotropic or circular isotropic geometries and stimulated them with TNFα and analyzed nuclear translocation of transcription regulators -NFκB (p65) and MKL and downstream gene-expression patterns. We found that TNFα induces geometry-dependent actin depolymerization, which enhances IκB degradation, p65 nuclear translocation, nuclear exit of MKL, and sequestration of p65 at the RNA-polymerase-II foci. Further, global transcription profile of cells under matrix-TNFα interplay reveals a geometry-dependent gene-expression pattern. At a functional level, we find cell geometry affects TNFα-induced cell proliferation. Our results provide compelling evidence that fibroblasts, depending on their geometries, elicit distinct cellular responses for the same cytokine.

Human embryonic stem cells derived keratinocyte as an in vitro research model for the study of immune response.

BACKGROUND: The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR. METHODS: The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity. RESULTS: The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar. CONCLUSION: Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency.