Hybrid AI models allow label-free identification and classification of pancreatic tumor repopulating cell population.

Human pancreatic cancer cell lines harbor a small population of tumor repopulating cells (TRCs). Soft 3D fibrin gel allows efficient selection and growth of these tumorigenic TRCs. However, rapid and high-throughput identification and classification of pancreatic TRCs remain technically challenging. Here, we developed deep learning (DL) models paired with machine learning (ML) models to readily identify and classify 3D fibrin gel-selected TRCs into sub-types. Using four different human pancreatic cell lines, namely, MIA PaCa-2, PANC-1, CFPAC-1, and HPAF-II, we classified 3 main sub-types to be present within the TRC population. Our best model was an Inception-v3 convolutional neural network (CNN) used as a feature extractor paired with a Support Vector Machine (SVM) classifier with radial basis function (rbf) kernel which obtained a test accuracy of 90%. In addition, we compared this hybrid method of supervised classification with other methods of supervised classifications and showed that our working model outperforms others. With the help of unsupervised machine learning algorithms, we also validated that the pancreatic TRC subpopulation can be clustered into 3 sub-types. Collectively, our robust model can detect and readily classify tumorigenic TRC subpopulation label-free in a high-throughput fashion which can be very beneficial in clinical settings.

Single-Cell RNA Sequencing with Spatial Transcriptomics of Cancer Tissues.

Single-cell RNA sequencing (RNA-seq) techniques can perform analysis of transcriptome at the single-cell level and possess an unprecedented potential for exploring signatures involved in tumor development and progression. These techniques can perform sequence analysis of transcripts with a better resolution that could increase understanding of the cellular diversity found in the tumor microenvironment and how the cells interact with each other in complex heterogeneous cancerous tissues. Identifying the changes occurring in the genome and transcriptome in the spatial context is considered to increase knowledge of molecular factors fueling cancers. It may help develop better monitoring strategies and innovative approaches for cancer treatment. Recently, there has been a growing trend in the integration of RNA-seq techniques with contemporary omics technologies to study the tumor microenvironment. There has been a realization that this area of research has a huge scope of application in translational research. This review article presents an overview of various types of single-cell RNA-seq techniques used currently for analysis of cancer tissues, their pros and cons in bulk profiling of transcriptome, and recent advances in the techniques in exploring heterogeneity of various types of cancer tissues. Furthermore, we have highlighted the integration of single-cell RNA-seq techniques with other omics technologies for analysis of transcriptome in their spatial context, which is considered to revolutionize the understanding of tumor microenvironment.

A quartz crystal microbalance based study reveals living cell loading rate via αvß3 integrins.

Cellular interactions with the microenvironment are mediated by ligand-receptor bonds. Such ligand-receptor bond dynamics is known to be heavily dependent on the loading rate. However, the physiologically-relevant loading rate of living cells remains unknown. Here, using a quartz crystal microbalance (QCM), we developed a bulk-force sensing platform to semi-quantitatively detect the rate of cellular force application during early stages of cell adhesion and spreading. Atop an Au-coated quartz crystal, covalently linked self-assembled monolayers (SAM) were used to immobilize cyclic-RGDfK peptides that can interact with the αvß3 integrins on cells. The QCM detects the changes in resonant frequency of the vibrating crystal due to the cellular activity/probing (force application) on the QCM surface. The corresponding changes in mass on the surface, proportional to the rate of force application, arise from the cellular interactions with the functionalized surface were calculated. The loading rate of living cells was found to be ∼80-115 pN/s. Collectively, our results revealed a fundamental feature of cell adhesion and spreading providing valuable information regarding cellular interactions with the extracellular matrix.

Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells.

Recently, a robust mechanical method has been established to isolate a small subpopulation of highly tumorigenic tumor repopulating cells (TRCs) from parental melanoma cells. In order to characterize the molecular and mechanical properties of TRCs, we utilized the tension gauge tether (TGT) single-molecule platform and investigated force requirements during early cell spreading events. TRCs required the peak single molecular tension of around 40 pN through integrins for initial adhesion like the parental control cells, but unlike the control cells, they did not spread and formed very few mature focal adhesions (FAs). Single molecule resolution RNA quantification of three Rho GTPases showed that downregulation of Cdc42, but not Rac1, is responsible for the unusual biophysical features of TRCs and that a threshold level of Cdc42 transcripts per unit cell area is required to initiate cell spreading. Cdc42 overexpression rescued TRC spreading through FA formation and restored the sensitivity to tension cues such that TRCs, like parental control cells, increase cell spreading with increasing single-molecular tension cues. Our single molecule studies identified an unusual biophysical feature of suppressed spreading of TRCs that may enable us to distinguish TRC population from a pool of heterogeneous tumor cell population.

Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo.

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation.

Integrin Molecular Tension within Motile Focal Adhesions.

Forces transmitted by integrins regulate many important cellular functions. Previously, we developed tension gauge tether (TGT) as a molecular force sensor and determined the threshold tension across a single integrin-ligand bond, termed integrin tension, required for initial cell adhesion. Here, we used fluorescently labeled TGTs to study the magnitude and spatial distribution of integrin tension on the cell-substratum interface. We observed two distinct levels of integrin tension. A >54 pN molecular tension is transmitted by clustered integrins in motile focal adhesions (FAs) and such force is generated by actomyosin, whereas the previously reported ∼40 pN integrin tension is transmitted by integrins before FA formation and is independent of actomyosin. We then studied FA motility using a TGT-coated surface as a fluorescent canvas, which records the history of integrin force activity. Our data suggest that the region of the strongest integrin force overlaps with the center of a motile FA within 0.2 µm resolution. We also found that FAs move in pairs and that the asymmetry in the motility of an FA pair is dependent on the initial FA locations on the cell-substratum interface.

Deep Learning Powered Identification of Differentiated Early Mesoderm Cells from Pluripotent Stem Cells.

Pluripotent stem cells can be differentiated into all three germ-layers including ecto-, endo-, and mesoderm in vitro. However, the early identification and rapid characterization of each germ-layer in response to chemical and physical induction of differentiation is limited. This is a long-standing issue for rapid and high-throughput screening to determine lineage specification efficiency. Here, we present deep learning (DL) methodologies for predicting and classifying early mesoderm cells differentiated from embryoid bodies (EBs) based on cellular and nuclear morphologies. Using a transgenic murine embryonic stem cell (mESC) line, namely OGTR1, we validated the upregulation of mesodermal genes (Brachyury (T): DsRed) in cells derived from EBs for the deep learning model training. Cells were classified into mesodermal and non-mesodermal (representing endo- and ectoderm) classes using a convolutional neural network (CNN) model called InceptionV3 which achieved a very high classification accuracy of 97% for phase images and 90% for nuclei images. In addition, we also performed image segmentation using an Attention U-Net CNN and obtained a mean intersection over union of 61% and 69% for phase-contrast and nuclear images, respectively. This work highlights the potential of integrating cell culture, imaging technologies, and deep learning methodologies in identifying lineage specification, thus contributing to the advancements in regenerative medicine. Collectively, our trained deep learning models can predict the mesoderm cells with high accuracy based on cellular and nuclear morphologies.

pH-Responsive Degradable Electro-Spun Nanofibers Crosslinked via Boronic Ester Chemistry for Smart Wound Dressings.

Recent advances in the treatment of chronic wounds have focused on the development of effective strategies for cutting-edge wound dressings based on nanostructured materials, particularly biocompatible poly(vinyl alcohol) (PVA)-based electro-spun (e-spun) nanofibers. However, PVA nanofibers need to be chemically crosslinked to ensure their dimensional stability in aqueous environment and their capability to encapsulate bioactive molecules. Herein, a robust approach for the fabrication of pH-degradable e-spun PVA nanofibers crosslinked with dynamic boronic ester (BE) linkages through a coupling reaction of PVA hydroxyl groups with the boronic acid groups of a phenyl diboronic acid crosslinker is reported. This comprehensive analysis reveals the importance of the mole ratio of boronic acid to hydroxyl group for the fabrication of well-defined BE-crosslinked fibrous mats with not only dimensional stability but also the ability to retain uniform fibrous form in aqueous solutions. These nanofibers degrade in both acidic and basic conditions that mimic wound environments, leading to controlled/enhanced release of encapsulated antimicrobial drug molecules. More importantly, drug-loaded BE-crosslinked fibers show excellent antimicrobial activities against both Gram-positive and Gram-negative bacteria, suggesting that this approach of exploring dynamic BE chemistry is amenable to the development of smart wound dressings with controlled/enhanced drug release.

Fitness Costs of Antibiotic Resistance Impede the Evolution of Resistance to Other Antibiotics.

Antibiotic resistance is a major threat to global health, claiming the lives of millions every year. With a nearly dry antibiotic development pipeline, novel strategies are urgently needed to combat resistant pathogens. One emerging strategy is the use of sequential antibiotic therapy, postulated to reduce the rate at which antibiotic resistance evolves. Here, we use the soft agar gradient evolution (SAGE) system to carry out high-throughput in vitro bacterial evolution against antibiotic pressure. We find that evolution of resistance to the antibiotic chloramphenicol (CHL) severely affects bacterial fitness, slowing the rate at which resistance to the antibiotics nitrofurantoin and streptomycin emerges. In vitro acquisition of compensatory mutations in the CHL-resistant cells markedly improves fitness and nitrofurantoin adaptation rates but fails to restore rates to wild-type levels against streptomycin. Genome sequencing reveals distinct evolutionary paths to resistance in fitness-impaired populations, suggesting resistance trade-offs in favor of mitigation of fitness costs. We show that the speed of bacterial fronts in SAGE plates is a reliable indicator of adaptation rates and evolutionary trajectories to resistance. Identification of antibiotics whose mutational resistance mechanisms confer stable impairments may help clinicians prescribe sequential antibiotic therapies that are less prone to resistance evolution.

Reduced Cell-ECM Interactions in the EpiSC Colony Center Cause Heterogeneous Differentiation.

Mechanoregulation of cell-extracellular matrix (ECM) interactions are crucial for dictating pluripotent stem cell differentiation. However, not all pluripotent cells respond homogeneously which results in heterogeneous cell populations. When cells, such as mouse epiblast stem cells (EpiSCs), are cultured in clusters, the heterogeneity effect during differentiation is even more pronounced. While past studies implicated variations in signaling pathways to be the root cause of heterogeneity, the biophysical aspects of differentiation have not been thoroughly considered. Here, we demonstrate that the heterogeneity of EpiSC differentiation arises from differences in the colony size and varying degrees of interactions between cells within the colonies and the ECM. Confocal imaging demonstrates that cells in the colony periphery established good contact with the surface while the cells in the colony center were separated by an average of 1-2 µm from the surface. Traction force measurements of the cells within the EpiSC colonies show that peripheral cells generate large tractions while the colony center cells do not. A finite element modeling of EpiSC colonies shows that tractions generated by the cells at the colony periphery lift off the colony center preventing the colony center from undergoing differentiation. Together, our results demonstrate a biophysical regulation of heterogeneous EpiSC colony differentiation.

Spatiotemporal organization, regulation, and functions of tractions during neutrophil chemotaxis.

Despite recent advances in our understanding of biochemical regulation of neutrophil chemotaxis, little is known about how mechanical factors control neutrophils' persistent polarity and rapid motility. Here, using a human neutrophil-like cell line and human primary neutrophils, we describe a dynamic spatiotemporal pattern of tractions during chemotaxis. Tractions are located at both the leading and the trailing edge of neutrophils, where they oscillate with a defined periodicity. Interestingly, traction oscillations at the leading and the trailing edge are out of phase with the tractions at the front leading those at the back, suggesting a temporal mechanism that coordinates leading edge and trailing edge activities. The magnitude and periodicity of tractions depend on the activity of nonmuscle myosin IIA. Specifically, traction development at the leading edge requires myosin light chain kinase-mediated myosin II contractility and is necessary for α5ß1-integrin activation and leading edge adhesion. Localized myosin II activation induced by spatially activated small GTPase Rho, and its downstream kinase p160-ROCK, as previously reported, leads to contraction of actin-myosin II complexes at the trailing edge, causing it to de-adhere. Our data identify a key biomechanical mechanism for persistent cell polarity and motility.

Forces in stem cells and cancer stem cells.

Endogenous and exogenous forces are critical in physiology and pathology of the human body. Increasing evidence suggests that these forces, mechanics, and force-associated signaling are essential in regulating functions of living cells. Here we review advances in understanding the impact of forces and mechanics on functions and fate of embryonic stem cells, adult stem cells, and cancer stem cells and the pathways of mechanotransduction in cells. Stem-cells based models are useful in understanding how forces influence physiology, pathology, and embryonic development, which is incompletely understood, especially for mammals. We highlight increasing efforts and emerging favorable clinical outcomes in mechanomedicine, application of mechanobiology to medicine. Major progresses in mechanobiology, the pillar of mechanomedicine and mechanohealth (application of mechanobiology to health), are pivotal in understanding the life of force and making substantial advances in medicine and health.

Driven Radical Motion Enhances Cryptochrome Magnetoreception: Toward Live Quantum Sensing.

The mechanism underlying magnetoreception has long eluded explanation. A popular hypothesis attributes this sense to the quantum coherent spin dynamics and spin-selective recombination reactions of radical pairs in the protein cryptochrome. However, concerns about the validity of the hypothesis have been raised because unavoidable inter-radical interactions, such as the strong electron-electron dipolar coupling, appear to suppress its sensitivity. We demonstrate that sensitivity can be restored by driving the spin system through a modulation of the inter-radical distance. It is shown that this dynamical process markedly enhances geomagnetic field sensitivity in strongly coupled radical pairs via Landau-Zener-Stückelberg-Majorana transitions between singlet and triplet states. These findings suggest that a "live" harmonically driven magnetoreceptor can be more sensitive than its "dead" static counterpart.

Diisonitrile Lipopeptides Mediate Resistance to Copper Starvation in Pathogenic Mycobacteria.

Bacterial pathogens and their hosts engage in intense competition for critical nutrients during infection, including metals such as iron, copper, and zinc. Some metals are limited by the host, and some are deployed by the host as antimicrobials. To counter metal limitation, pathogens deploy high-affinity metal acquisition systems, best exemplified by siderophores to acquire iron. Although pathogen strategies to resist the toxic effects of high Cu have been elucidated, the role of Cu starvation and the existence of Cu acquisition systems are less well characterized. In this study, we examined the role of diisonitrile chalkophores of pathogenic mycobacteria, synthesized by the enzymes encoded by the virulence-associated nrp gene cluster, in metal acquisition. nrp gene cluster expression is strongly induced by starvation or chelation of Cu but not starvation of Zn or excess Cu. Mycobacterium tuberculosis and Mycobacterium marinum strains lacking the nrp-encoded nonribosomal peptide sythetase, the fadD10 adenylate-forming enzyme, or the uncharacterized upstream gene ppe1 are all sensitized to Cu, but not Zn, starvation. This low Cu sensitivity is rescued by genetic complementation or by provision of a synthetic diisonitrile chalkophore. These data demonstrate that diisonitrile lipopeptides in mycobacteria are chalkophores that facilitate survival under Cu-limiting conditions and suggest that Cu starvation is a relevant stress for M. tuberculosis in the host. IMPORTANCE Bacterial pathogens and their hosts engage in intense competition for nutrients, including metals. Mycobacterium tuberculosis, the cause of tuberculosis, lives within host macrophages and is subject to diverse stresses, including metal excess and metal limitation. In this study, we demonstrated that the nrp gene cluster, required for M. tuberculosis virulence and which directs synthesis of diisonitrile lipopeptides, mediates copper acquisition. Copper, but not zinc, deprivation strongly induces diisonitrile biosynthesis, and M. tuberculosis strains lacking the nrp gene, or the associated genes fadD10 or ppe1, are all sensitized to copper chelation or copper deprivation. These results establish a copper binding, or chalkophore, system in M. tuberculosis and indicate that resistance to copper restriction plays an important role in the ability of this global pathogen to cause infection.

Additive Manufacturing of Viscoelastic Polyacrylamide Substrates for Mechanosensing Studies.

Polymerized polyacrylamide (PAA) substrates are linearly elastic hydrogels that are widely used in mechanosensing studies due to their biocompatibility, wide range of functionalization capability, and tunable mechanical properties. However, such cellular response on purely elastic substrates, which do not mimic the viscoelastic living tissues, may not be physiologically relevant. Because the cellular response on 2D viscoelastic PAA substrates remains largely unknown, we used stereolithography (SLA)-based additive manufacturing technique to create viscoelastic PAA substrates with tunable mechanical properties that allow us to identify physiologically relevant cellular behaviors. Three PAA substrates of different complex moduli were fabricated by SLA. By embedding fluorescent markers during the additive manufacturing of the substrates, we show a homogeneous and uniform composition throughout, which conventional manufacturing techniques cannot produce. Rheological investigation of the additively manufactured PAA substrates shows a viscoelastic behavior with a 5-10% loss moduli compared to their elastic moduli, mimicking the living tissues. To understand the cell mechanosensing on the dissipative PAA substrates, single live cells were seeded on PAA substrates to establish the basic relationships between cell traction, cytoskeletal prestress, and cell spreading. With the increasing substrate moduli, we observed a concomitant increase in cellular traction and prestress, but not cell spreading, suggesting that cell spreading can be decoupled from traction and intracellular prestress in physiologically relevant environments. Together, additively manufactured PAA substrates fill the void of lacking real tissue like viscoelastic materials that can be used in a variety of mechanosensing studies with superior reproducibility.

Klebsiella pneumoniae pathogenicity in silk moth larvae infection model.

The emergence of antibiotic resistant bacteria is a major health concern worldwide in recent years. The objective of this study is to establish the larvae of the silk moth (commonly known as silkworm), Bombyx mori as an infection model to study antibacterial effect of antibiotics against Klebsiella pneumoniae. In this study, the pathogenicity of a K. pneumoniae strain isolated from food to silkworm larvae was examined. Within 72 h of bacterial injection, all silkworm larvae were killed in a dose-dependent manner with their body color turning into black due to increased melanization. Bacterial numbers in the larval hemolymph (blood) significantly increased after 9 h of infection with a decrease in viable circulatory hemocytes in hemolymph. When presented with bacteria laden leaves, larvae did not eat but injection of bacteria directly into the midgut killed larvae within 12 h with a higher load required in comparison to that required for the killing by hemolymph injection. Administration of four different antibiotics into larval hemolymph showed therapeutic effect at different doses with varying efficacies against hemolymph-injected K. pneumoniae. These results indicate that the silkworm larvae can be used as an infection model not only to study the pathogenicity of K. pneumoniae but also to perform rapid screening for the identification of antibiotics effective against multidrug-resistant strains of K. pneumoniae.

Self-organized podosomes are dynamic mechanosensors.

Podosomes are self-organized, dynamic, actin-containing structures that adhere to the extracellular matrix via integrins [1-5]. Yet, it is not clear what regulates podosome dynamics and whether podosomes can function as direct mechanosensors, like focal adhesions [6-9]. We show here that myosin-II proteins form circular structures outside and at the podosome actin ring to regulate podosome dynamics. Inhibiting myosin-II-dependent tension dissipated podosome actin rings before dissipating the myosin-ring structure. As podosome rings changed size or shape, tractions underneath the podosomes were exerted onto the substrate and were abolished when myosin-light-chain activity was inhibited. The magnitudes of tractions were comparable to those generated underneath focal adhesions, and they increased with substrate stiffness. The dynamics of podosomes and of focal adhesions were different. Torsional tractions underneath the podosome rings were generated with rotations of podosome rings in a nonmotile, nonrotating cell, suggesting a unique feature of these circular structures. Stresses applied via integrins at the apical surface directly displaced podosomes near the basal surface. Stress-induced podosome displacements increased nonlinearly with applied stresses. Our results suggest that podosomes are dynamic mechanosensors in which interactions of myosin tension and actin dynamics are crucial for regulating these self-organized structures in living cells.

Force via integrins but not E-cadherin decreases Oct3/4 expression in embryonic stem cells.

Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. Recently we have demonstrated that a local force applied via Arg-Gly-Asp (RGD) peptides coated magnetic beads to mouse embryonic stem (ES) cells increases cell spreading and cell stiffness and decreases Oct3/4 (Pou5f1) gene expression. However, it is not clear whether the effects of the applied stress on these functions of ES cells can be extended to natural extracellular matrix proteins or cell-cell adhesion molecules. Here we show that a local cyclic shear force applied via fibronectin or laminin to integrin receptors increased cell spreading and stiffness, downregulated Oct3/4 gene expression, and decreased cell proliferation rate. In contrast, the same cyclic force applied via cell-cell adhesion molecule E-cadherin (Cdh1) had no effects on cell spreading, Oct3/4 gene expression, and the self-renewal of mouse ES cells, but induced significant cell stiffening. Our findings demonstrate that biological responses of ES cells to force applied via integrins are different from those to force via E-cadherin, suggesting that mechanical forces might play different roles in different force transduction pathways to shape early embryogenesis.

Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells.

Growing evidence suggests that physical microenvironments and mechanical stresses, in addition to soluble factors, help direct mesenchymal-stem-cell fate. However, biological responses to a local force in embryonic stem cells remain elusive. Here we show that a local cyclic stress through focal adhesions induced spreading in mouse embryonic stem cells but not in mouse embryonic stem-cell-differentiated cells, which were ten times stiffer. This response was dictated by the cell material property (cell softness), suggesting that a threshold cell deformation is the key setpoint for triggering spreading responses. Traction quantification and pharmacological or shRNA intervention revealed that myosin II contractility, F-actin, Src or cdc42 were essential in the spreading response. The applied stress led to oct3/4 gene downregulation in mES cells. Our findings demonstrate that cell softness dictates cellular sensitivity to force, suggesting that local small forces might have far more important roles in early development of soft embryos than previously appreciated.

Rapid signal transduction in living cells is a unique feature of mechanotransduction.

It is widely postulated that mechanotransduction is initiated at the local force-membrane interface by inducing local conformational changes of proteins, similar to soluble ligand-induced signal transduction. However, all published reports are limited in time scale to address this fundamental issue. Using a FRET-based cytosolic Src reporter in a living cell, we quantified changes of Src activities as a local stress via activated integrins was applied. The stress induced rapid (<0.3 s) activation of Src at remote cytoplasmic sites, which depends on the cytoskeletal prestress. In contrast, there was no Src activation within 12 s of soluble epidermal growth factor (EGF) stimulation. A 1.8-Pa stress over a focal adhesion activated Src to the same extent as 0.4 ng/ml EGF at long times (minutes), and the energy levels for mechanical stimulation and chemical stimulation were comparable. The effect of both stress and EGF was less than additive. Nanometer-scale cytoskeletal deformation analyses revealed that the strong activation sites of Src by stress colocalized with large deformation sites of microtubules, suggesting that microtubules are essential structures for transmitting stresses to activate cytoplasmic proteins. These results demonstrate that rapid signal transduction via the prestressed cytoskeleton is a unique feature of mechanotransduction.