Cellular and Molecular Bioengineering

Elastic and viscoelastic properties of extracellular matrices (ECM) are known to regulate cellular behavior and mechanosensation differently, with implications for morphogenesis, wound healing, and pathophysiology. Most in vitro cellular processes, including cell migration, are studied on linear-elastic substrates to mimic extracellular matrices. However, most tissues are viscoelastic and display a loss modulus (G) that may be 10-20% of their storage modulus (G) under biophysically relevant conditions. Recent research has shown that cells can distinguish between elastic and viscoelastic ECM, leading to alterations in their cellular morphology, migration rates, and contractility. Here, we present a protocol for creating PAH-based model ECMs that enables the fabrication of viscoelastic substrates with storage moduli similar to those of their elastic counterparts. To explore how G influences epithelial cell mechanobiology, we fabricated tunable viscoelastic model ECMs with G of 3 kPa, 8 kPa, and 12 kPa, and for each, independently tuned G values to approximately 300 Pa, 500 Pa, and 700 Pa, respectively. We found that A549 cells cultured on stiff elastic model ECMs migrated [~]30% slower and formed larger focal adhesions compared to their viscoelastic counterparts. Conversely, A549 cells on intermediate viscoelastic model ECMs exhibited a [~]54% reduction in migration speed, with no significant difference in focal adhesion size relative to their elastic counterparts. These findings highlight the complex interplay between substrate (ECM) elastic and viscoelastic properties in regulating epithelial cell mechanobiology and emphasize the importance of time-dependent matrix mechanics in governing epithelial responses.

Cardiac fibrosis is a pathological process in which the myocardium stiffens due to the overproduction of extracellular matrix (ECM) proteins. Cardiac fibroblasts activate to myofibroblasts in response to the inflammatory cytokine transforming growth factor beta1 (TGF-{beta}1) to promote fibrotic scarring. Biological sex also influences cardiac fibrosis progression and patient outcomes, where males exhibit increased fibrotic scarring after acute inflammation relative to females. At the cellular level, sex differences in TGF-{beta}1-mediated cardiac myofibroblast activation processes have not been clearly defined. We hypothesized that TGF-{beta}1 would cause sex-specific cardiac myofibroblast activation levels and alter the secretion of bioactive molecules to modulate sex differences in cardiac fibrosis. Primary left ventricle cardiac fibroblasts were isolated from male and female C57BL/6J mice and cultured on hydrogel biomaterials mimicking native myocardial ECM stiffness and treated with TGF-{beta}1 and/or the TGF-{beta}1 receptor inhibitor SD208. Male myofibroblasts exhibited increased -SMA stress fiber formation, increased SMAD2/3 localization, and greater resistance to SD208 inhibition compared to female myofibroblasts on hydrogels at various time points tested. Sex differences in relative secreted cytokine abundance were also determined, with male CFs secreting increased vascular endothelial growth factor (VEGF) and female CFs producing increased periostin and fibroblast growth factor 21 in response to TGF-{beta}1. Our findings establish that TGF-{beta}1 mediates sex differences in cardiac myofibroblast activation on hydrogels and secreted factors that may modulate the myocardial microenvironment. Our work underscores the importance of using hydrogels as cell culture platforms to recapitulate sex-specific cardiac fibrosis phenotypes as a steppingstone towards identifying sex-dependent therapeutic interventions for cardiac fibrosis.

PurposeFibrosis is the pathological remodeling of the extracellular matrix (ECM) that is largely orchestrated by activated fibroblasts. The mechanical properties of the ECM change drastically during fibrosis, and fibroblasts become increasingly activated by mechanical environments that mimic the properties of fibrotic tissues. While the effects of increased elastic modulus (stiffness) on fibroblast activation have been well-studied, the impact of changes in viscoelasticity are less clear. Here, we sought to determine how fibroblast activation is altered by changes in viscoelasticity in a three-dimensional, fibrillar microenvironment. MethodsWe employed 3D alginate collagen I hydrogels with independently tunable stiffness and stress relaxation rates. Dermal fibroblasts were encapsulated in hydrogels with four distinct mechanical profiles (soft: 3 kPa or stiff: 10 kPa, fast stress relaxing: {tau}1/2 {approx} 160 s or slow stress relaxing: {tau}1/2 {approx} 1600 s). We assessed fibroblast activation by changes in cell morphology, expression of key activation markers, and evidence of ECM remodeling. ResultsFibrillar alginate collagen networks enhanced fibroblast spreading, -smooth muscle actin stress fiber formation, and fibroblast activation protein- expression in matrices that were slow relaxing or stiff. The presence of the fibrillar network further enhanced fibroblast activation, independent of the changes driven by matrix viscoelasticity. ECM remodeling was also promoted by slow relaxing matrices, with increased fibronectin deposition and more remodeling of the local collagen fiber network. ConclusionsOur results demonstrate that fibroblast activation is highly responsive to matrix stress relaxation rate, and that models incorporating fibrillar, viscoelastic networks can provide new insights into the role of ECM mechanics driving fibroblast activation.

BackgroundActivated platelets release polyphosphate (polyP), a linear polymer of inorganic phosphate residues, from dense granules. Experiments performed under no-flow conditions show that polyP alters the kinetics of tissue factor (TF) pathway reactions, accelerating FXI activation by thrombin and FV activation by FXa and thrombin, and may impact inhibition by tissue factor pathway inhibitor (TFPI). How polyP influences this pathway in conjunction with platelet deposition under flow remains understudied. ObjectivesTo investigate how polyP-mediated acceleration of FV and FXI activation modulates thrombin generation under flow in TF-initiated coagulation. MethodsWe extended a previously validated mathematical model of platelet deposition and coagulation under flow to examine polyP-mediated effects following a small vascular injury during intravascular clotting. Simulations varied the surface density of TF exposed, wall shear rate, and plasma TFPI concentration. ResultsPolyP shifts the threshold TF density for a thrombin burst to lower TF densities. For TF densities above this threshold, polyP shortens the lag time to thrombin generation in a TF- and shear-rate-dependent manner. Although no explicit effect of polyP on TFPI function was included in the model, thrombin generation was much less sensitive to TFPI concentration with polyP, in a TF-dependent manner. Relative contributions of accelerations of FV and FXI activations depend on incompletely known enhancements by polyP. ConclusionsThe experimentally observed influence of polyP on TFPI function depends on TF density and may arise indirectly from accelerated FV and FXI activation, with the dominant effect arising through accelerated thrombin-mediated conversion of FV to FVa.

The extracellular matrix (ECM) plays a pivotal role in lymphatic vasculature physiology, yet the specific contribution of individual ECM components to lymphatic endothelial permeability remains poorly understood, limiting the development of physiologically relevant in vitro models for lymphatic disease research and therapeutic development. Here, we used an in vitro transwell platform to systematically investigate how four clinically relevant ECM proteins, collagen I, fibronectin, fibrin, and laminin, regulate human lymphatic endothelial cell (LEC) barrier function and junctional integrity. Fibrin and collagen I substrates enhanced barrier integrity, demonstrating 80% and 67% increases in transendothelial electrical resistance (TEER), respectively, compared to uncoated controls. FITC-dextran transport assays confirmed these findings, with fibrin and collagen I reducing permeability by 20% and 10%, respectively. Immunofluorescence analysis revealed elevated ZO-1 expression on fibrin, fibronectin, and laminin matrices, while VE-cadherin levels remained unchanged across conditions. Quantitative junctional analysis demonstrated that fibrin increased ZO-1 junction continuity by [~]35%, while collagen I and fibronectin enhanced continuity by [~]22%, with all ECM coatings reducing discontinuous junctions by 60-80%. Mechanistically, RhoA expression was reduced in LECs cultured on fibrin, suggesting decreased stress fiber formation contributes to enhanced barrier function, though overall actin cytoskeletal anisotropy remained unchanged. These findings demonstrate that ECM composition modulates LEC junctional organization and barrier integrity, with fibrin and collagen I exerting the most pronounced barrier-enhancing effects. This engineered platform provides a foundation for developing next-generation in vitro models of lymphatic vasculature that more accurately recapitulate physiological conditions, with applications in lymphedema research, cancer metastasis studies, and immune cell trafficking investigations.

Cell-penetrating peptides (CPPs) can deliver biomacromolecular cargos into cells, potentially enabling a new mode of intracellular drug delivery. However, a major problem with CPP-mediated delivery is entrapment of CPPs within endosomes and covalent linkages ensure CPPs and cargos share a common fate. We previously developed a CPP-adaptor system based on reversible, calcium-dependent cargo binding that produces cargo release from adaptors as complexes dissociate following internalization and Ca2+ efflux from early endosomes. Having employed CPP-adaptors with an array of protein cargos of differing charges, it became apparent that positively charged cargos often appeared to dominate internalization and that association with the adaptor had little effect. To systematically address the effects of cargo charge and CPP function, we tested the ability of several adaptors to increase internalization of a set of adaptor binding GFP cargos having net charges of +9, +15, +20, +25 and +36. Intrinsic internalization of these cargos reproduced reported patterns showing that positive charge increases internalization. However, labeling these cargos with a chemical fluorophore revealed that GFP fluorescence grossly underestimated total internalization. Internalization was charge and concentration dependent with more positive cargos showing apparent saturation of internalization at 100-400 nM, well below the concentrations at which covalently linked CPP-cargos are dosed. We tested the ability of 5 adaptors to internalize these cargos. Our prototype adaptor, TAT-CaM, was completely ineffective with the +9 cargo, but internalized moderately charged cargos extremely efficiently at concentrations far below the {micro}M range. A derivative adaptor, TAT-LAH4-CaM, was highly effective with all cargos and produced similar maximal internalization at 100-400 nM. However, two adaptors specifically designed with increased positive charge inhibited internalization of the most positive cargos. One of these, GFP-CaM, based on the supercharged GFP with net charge of +36, did increase internalization of the least positive cargos, demonstrating an adaptor with high affinity for the cell surface can increase internalization of a neutral cargo at very low concentration. The common maximal level of intrinsic GFP cargo internalization correlated with surface loading of these cargos, suggesting a limit to the beneficial effects of increased plasma membrane association. However, TAT-CaM further increased internalization via an apparently distinct mechanism. In this limited study of the interaction of cargo charge and adaptor efficacy, we found diverse behaviors that hint at the power and flexibility possible with adaptor/cargo internalization.

Methods to visualize and quantify the molecular responses of cells to local forces exerted at adhesions are crucial to elucidate how physical forces control cellular behavior. Of the many proteins involved in focal adhesions, vinculin plays a key role in mediating force-sensitive processes. Here, we combined optical tweezers and Forster resonance energy transfer (FRET) microscopy to measure the intensity and FRET efficiency of the vinculin tension sensor, VinTS, in response to a force. Fibroblasts expressing VinTS formed adhesions on fibronectin-coated, 3m-diameter, polystyrene beads. As the beads were displaced by the cell, we applied an optical trap to counteract this movement and increase the traction force required by the cell to maintain the bead displacement. The optical trap stiffness varied from zero (no laser) up to 0.26 pN/nm. In this range, the median bead displacement after 5 min was ~200nm in all trapping conditions inducing counteracting forces in the 10-100pN range. To maintain this displacement, vinculin recruitment increased (up to 35% in relative intensity at high stiffness) while tension increased but more moderately (1-2% decrease in absolute FRET efficiency). For higher trap stiffness, the main response was an increase in vinculin recruitment, while the tension did not increase significantly. The increase in vinculin intensity was correlated with the decrease in FRET efficiency at 0.26 pN/nm but not at lower stiffness. Thus, the presence of the high stiffness optical trap over 5 min appears to induce a positive correlation between vinculin recruitment and vinculin tension. In a few instances, vinculin puncta migrated a few microns away from the bead exceeding the bead movement speed while experiencing an increase in both vinculin intensity and tension. Taken together, the results suggest that combining an optical trap with vinculin tension measurements uncovers novel vinculin dynamics in the presence of a force.

Mechanical homeostasis indicates the remarkable ability displayed by cells in tissues to maintain their mechanical properties near a stable homeostatic set-point. Experimental investigations and theoretical studies indicate that mechanical stress represents a key homeostatic target that stromal cells, such as fibroblasts, seek to maintain by tuning the intracellular structure and by remodeling the extracellular matrix. Much of what is known about mechanical homeostasis of tissues under tension, or tensional homeostasis, is based on experiments on tissue equivalents, that is fibroblast-populated collagen gels. However, existing platforms used to investigate tensional homeostasis cannot infer mechanical stress dynamically. Here we developed an integrated biomechanical bioreactor combining force sensing with confocal microscopy to dissect the mechanobiological mechanisms of tensional homeostasis. We used our novel platform to test the hypothesis that fibroblasts maintain a constant state of stress across varying collagen concentrations. Contrary to this assumption, synchronized force and imaging measurements revealed that stress is not constant but rather elevated at low collagen concentrations, where fibroblast contraction drives earlier collagen fiber alignment and greater tissue compaction. Conversely, force generation and -SMA expression increase with increasing collagen concentration, accompanied by modest transcriptional changes. However, at the highest collagen concentration, this homeostatic balance is disrupted, with lower force generation and -SMA expression, as gene expression shifts toward VEGFC-mediated autocrine survival signaling. These findings demonstrate that tensional homeostasis emerges from a dynamic balance between cellular contractility and extracellular matrix densification rather than stress maintenance, and reveal that excessive matrix density disrupts this balance by triggering a pro-survival response.

Extracellular vesicles (EVs) are lipid spheres released from cells. Research utilizing EVs has met several hurdles owing to the small size of the majority of EVs and other nanoparticles (<150 nm) and the lack of detection technologies capable of providing high-throughput single particle measurements at this scale. The use of high-throughput single particle measurements is critical for the assessment of EV heterogeneity and abundance which are features often used to assess the development of isolation protocols or particle characterization. The Coulter principle, known in the field as resistive pulse sensing (RPS), has been used for several decades to size and count cells. More recently, this technology has evolved to accommodate nanoparticle analysis. In the last decade a platform utilizing microfluidic resistive pulse sensing (MRPS) has been demonstrated for nanoparticles, offering ergonomic characterization of nanoparticles along with utilizing open format data. To date, assessment of MRPS accuracy and reporting standards have not been assessed. With the aim of increasing data accuracy, ergonomics, and reporting transparency, we developed a microfluidic resistive pulse sensing post-acquisition analysis software (RPSPASS) application for automated cohort calibration, population gating, statistical output, QC plot generation, alternative data file outputs, and standardized reporting templates.

BackgroundDespite improved cancer outcomes with kinase inhibitors (KIs), their cardiotoxicity remains a significant clinical challenge. Current approaches to predict and prevent KI-induced cardiac adverse events (CAEs) are limited by an incomplete understanding of underlying mechanisms, including the contribution of off-target kinase engagement. ObjectivesTo establish links between kinase inhibition profiles and cardiotoxic phenotypes using empirical proteomic data, and to leverage these profiles in machine learning (ML) models capable of predicting KI cardiotoxicity. MethodsWe curated a database connecting kinome-wide target binding profiles of FDA-approved KIs (n=44) with documented incidence rates of six distinct CAEs. Binding profiles were derived from unbiased chemoproteomics and used to assess associations between KI selectivity, specific kinase targets, and CAEs. Profiles were further used to develop ML models to predict CAE risk, with SHAP-based model interpretation applied to identify cardiotoxicity-associated kinases. ResultsKI promiscuity was not a significant predictor of cardiotoxicity across all six CAEs. Frequency analysis revealed that kinases including RET, PDGFRB, and DDR1 are recurrently inhibited across CAE-linked compounds, with nearly all identified as off-targets not annotated by the FDA. Network and pathway enrichment analyses supported a systems-level model in which cardiotoxicity arises from coordinated disruption of cardiac-relevant signaling networks. ML models achieved 66-84% cross-validated accuracy (ROC-AUC 0.75-0.8) across CAE endpoints, with SHAP analysis identifying PDGFRB, EGFR, and MEK1/2 among the most predictive kinases. ConclusionsProteomic kinome profiling combined with machine learning provides a mechanistically grounded framework for predicting KI cardiotoxicity and supports off-target-aware drug design to minimize cardiovascular risk.

Most cellular therapies, like CAR T cells, remain ineffective in solid tumors. This is primarily due to a complex tumor microenvironment (TME), which creates biochemically hostile and often immunosuppressive conditions that limit efficacy of immunotherapies. Besides, cellular therapy efficacy is still often established in traditional 2D cultures that fail to simulate relevant aspects of solid tumor biology. Recent advances in three-dimensional (3D) and organ-on-chip culture systems have provided more physiologically relevant models for immunotherapy testing. These microphysiological systems (MPS) not only offer a 3D environment that alters tumor cell sensitivity to therapy but also enable inclusion of TME components and assessment of processes such as extravasation and infiltration, key steps in CAR T cell activity in vivo. This study focuses on applying an advanced culture technique and further building on the use of a scalable on-chip platform, the OrganoPlate, to grow EpCAM-positive and EpCAM-negative tumor cells in co-culture with an endothelial vessel to study EpCAM-targeting CAR T cell migration and killing kinetics. The CAR T cells specifically targeted and killed EpCAM-positive HT-29 tumor cells while EpCAM-negative A375 tumor cells were not affected. In addition, target cell killing was dependent on the ratio between CAR T and tumor cells (E:T ratio) and was enhanced by addition of IL-2. Inflammatory cytokines like INF-{gamma}, TNF and IL-6 increased overtime in cultures containing CAR T cells. Morphometric analyses of the endothelial compartment showed E:T ratio dependent disruption of endothelial vessels. Additionally, this system was able to distinguish EpCAM ScFv-CD28-CD3z and EpCAM ScFv-TM-4-1BB-CD3z CAR T cells killing abilities and was used for studying the effect of immune checkpoint inhibitors and Temozolomide, a DNA targeting drug, on CAR T cell performance. Altogether, this work adds to the available advanced culture techniques for immunotherapy developers by describing a model that is modular, scalable, and suitable for phenotypic and functional characterization of CAR T cells.

Microfluidic devices with surface-bound biomolecular patterns enable localised detection arrays, enzymatic catalysis, and gene expression. Photolithography is a contactless patterning method with high spatial control. However, while patterning open surfaces by photolithography is well-established, patterning enclosed microfluidic channels remains technically challenging. Such capability would enable in situ surface modification and precise pattern alignment to channel geometries. Here, we present a photolithographic method using commercially available reagents to pattern sealed microfluidic devices. We first coat surfaces with (3-Aminopropyl)triethoxysilane (APTES) to bond microfluidic chips and provide surface amine groups onto which photocleavable polyethylene glycol (PC PEG) compounds are bound. UV exposure using standard photolithography equipment selectively deprotects the amine groups, which can subsequently bind amine-reactive cargos. We demonstrate this methods versatility by patterning both glass and poly(dimethylsiloxane) (PDMS) surfaces with diverse cargoes: DNA, proteins and gold nanoparticles. We also compare covalent versus noncovalent DNA patterning. Covalently bound DNA patterns were denser and could be used for sequence-specific target DNA capture. However, noncovalently bound DNA yielded higher cell-free gene expression from surface-bound GFP templates.

CRISPR-Cas-based diagnostics utilize the Cas enzymes trans-cleavage activity to generate signal and have become popular platforms for sensitive nucleic acid detection. Recently, autocatalytic systems have been demonstrated to improve the time to response and sensitivity in some cases. However, mechanistic description of these assays is limited and optimization relies on simple trial-and-error. In this work, we present the first comprehensive kinetic model that integrates all major biochemical processes involved in these assays, including cleavage reactions, nucleic acid equilibrium kinetics, inhibition of trans-cleavage by single-stranded DNA, and degradation of single-stranded reaction components. We discuss the biochemical foundations and implementation of the ordinary differential equation model, which is built for adaptation to different reaction schemes. We use the full model to investigate the role of nucleic acid stability in assay performance for a typical nucleic acid design and show that our model demonstrates inhibition effects consistent with experimental data. We describe the reaction behavior, derive a simplified analytical model and compare its performance to the full analytical model. Finally, we demonstrate tools developed for rapid in silico optimization to guide the rational design of future target-amplification-free CRISPR-Cas-based autocatalysis assays.

BackgroundMET overexpression is associated with poor prognosis in many solid tumors due to its central role in tumor survival, invasion, metastasis, and chemoresistance. While targeting MET with antibody-drug conjugates has shown promising results, engineered cellular immunotherapeutic approaches have not been extensively explored. Compared to conventional single-chain variable fragments (scFv), naturally occurring single-domain antibodies consisting of variable heavy chains only (VHH or nanobodies) are smaller, retain high specificity, and exhibit remarkable biochemical stability. In this study, we tested the efficacy of MET-targeting VHH-CAR-T (chimeric antigen receptor T cells). MethodsWe generated a panel of VHH-CAR-Ts using mRNA electroporation. VHH-CAR-T cells were evaluated in functional assays including cell binding avidity, cytokine production profiles, hydrogel microwell-based cellular kinetics, and in vitro cytotoxicity. We also assessed the therapeutic efficacy of VHH-CAR-T in an in vivo mouse model of metastatic triple negative breast cancer (TNBC). ResultsAmong the tested VHH, we identified those with intermediate avidity as most effective for in vitro tumor killing. VHH-CAR-Ts with CD28 costimulatory domains demonstrated augmented cytotoxicity with favorable selectivity, requiring a minimum antigen density threshold for activation. Mechanistically, VHH-CAR-Ts demonstrated low tonic signaling, high avidity, potent cytokine production, and rapid tumor killing kinetics. When administered in an mRNA format, VHH-CAR-Ts exhibited potent and prolonged control of tumor growth in an in vivo metastatic model of TNBC. ConclusionTaken together, these results demonstrate that VHH-CAR-Ts exhibit robust MET specificity and potent therapeutic efficacy both in vitro and in vivo. Thus, VHH-CAR-T cell therapy represents a promising immunotherapeutic strategy for targeting MET-overexpressing solid tumors. What is already known on this topicMET signaling is an important contributor to the aggressiveness of many solid tumors, and targeting MET by antibody-drug conjugates has shown efficacy and safety. Targeting MET by CAR-T cells has been under study, though with limited potency. What this study addsThis study is the first to demonstrate effectiveness of anti-MET VHH-CAR-T cells. Compared with other antigen binding domains, VHH-incorporated CAR-T cells show low tonic signaling, a favorable cytokine profile, and potent tumor killing. How this study might affect research, practice or policyWith the multiple advantages of VHHs including small size, stability, and low potential for tonic signaling, VHH-CAR-T cells represent a promising approach for CAR-T design against solid tumors.

Chemotaxis plays a critical role in the metastatic progression of breast cancer. The chemokine CXCL12 is well recognized as an essential component of chemotactic migration in triple-negative breast cancer (TNBC) cells in vivo. The purpose of this study is to determine how the highly metastatic TNBC cell line, MDA-MB-231, migrates in response to well-defined CXCL12 gradients in vitro. Traditional 2D transwell migration assays were optimized to gauge the MDA-MB-231 cells responsiveness to various CXCL12 concentrations. The optimum chemoattractant concentrations were applied to a commercially available 3D chemotaxis assay as stable linearly diffused gradients. Cells were embedded in type 1 bovine collagen at two different collagen concentrations, and individual unlabeled cells were monitored for 24 hours using brightfield microscopy. Time-lapse videos were used to track cell movement and shape. Quantitative data analysis was performed using an automated tracking software to measure chemotactic parameters based on cell morphology. MDA-MB-231 cells were responsive to CXCL12 concentrations greater than 200 ng/mL in 2D and 3D systems. In 3D systems, significant directed migration was observed in denser collagen matrices. It was observed that in 3D matrices a range of cell morphologies was present. Therefore, chemotaxis was evaluated as a function of cell shape revealing some differences between sub cellular populations. Our findings show the cells shape influences the chemotactic sensing towards CXCL12 gradients.

Anticancer drug resistance is challenging to overcome because it can arise through both intrinsic and acquired mechanisms, each driven by distinct cellular machinery. In particular, there is a sharp need for therapies that target hormone-insensitive prostate tumors due to the growing incidence of castration-resistant prostate cancer. Optimizing the pathways that regulate apoptosis in prostate cancer offers a promising strategy to induce apoptosis and inhibit tumor progression, since these mechanisms do not depend on hormonal signaling. Here, we identified strategies to enhance apoptosis in prostate cancer cells. We used several computational tools (including sensitivity analysis, particle swarm optimization, and ImageJ) to design an ordinary differential equation model of caspase-mediated prostate cancer apoptosis signaling. We apply the model to identify key modalities that increase the propensity toward apoptosis across three separate pro-apoptotic drugs (Tocopheryloxybutyrate, Narciclasine, and Celecoxib). Overall, we demonstrate that apoptosis dynamics can be accurately captured in response to each of the three drugs and identify which features of the model represent viable targets for overcoming intrinsic drug resistance.

Cell mechanics can serve as an important biomarker for cell state and phenotype, such as metastatic ability. While some molecular mechanisms underlying cell mechanical properties have been investigated through targeted analyses, a genome-wide study of human genes and gene networks that modulate cell biophysical properties has not been attempted. In this work, we combined a microfluidic stiffness-based sorting device with a genome-scale CRISPR knockout (GeCKO) screen in order to investigate the effect of individual gene knockouts on cell stiffening and cell softening across the entire protein-coding genome. We processed approximately 150 million Cas9-expressing ovarian cancer cells that had been transduced with a library of 76,000 single guide RNAs (sgRNAs) against the 19,000 protein-coding genes in the genome. The cells were sorted into 5 mechanical subsets. We identified 7 gene knockouts that were significantly depleted in the softer subsets and over 700 gene knockouts that were significantly enriched in the stiffer subsets. Of these significant genes of interest, we selected 3 genes that were highly expressed in our ovarian cancer cell line with greater than 100-fold enrichment in the stiff outlet and resulted in significant changes in ovarian cancer patient survival. These genes, PIK3R4, CCDC88A, and GSK3B, when knocked out result in a significant and predicted increase in cell stiffness. This study is the first to explore the relation between human gene expression and cell mechanics at the genome-scale to generate datasets at the intersection between cell genotype, mechanotype, and phenotype for metastatic cancer cells. The method could also be applied to study the effect of genes on other biophysical cell processes as well as for identifying pathways for the control of cellular mechanics across many cell types.

Extracellular matrix (ECM) proteins assemble to form a heterogeneous connective scaffold that supports cells. Physical interactions between cells and the matrix regulate cellular behaviors and influence subsequent tissue construction. However, there is a lack of fundamental understanding regarding the contributions of individual native ECM proteins to the matrix. This gap arises from the need for nanoscopic characterization, which operates on a much smaller length scale than typical assessments in cell and tissue cultures, as well as in tissue reconstruction and clinical implantation. This study aims to systematically investigate how individual ECM proteins affect lipid membranes structurally and mechanically, and how these influences regulate cell migration. Results from Langmuir isotherm analysis, X-ray reflectivity measurements, and cell scratch assays demonstrate that strong collagen adsorption on the membrane surface disrupts lipid packing. However, its rigid network provides a sturdy scaffold for cell adhesion, thereby enhancing cell attachment and promoting cell migration. In contrast, elastin has a minimal structural or mechanical impact on the membrane during both adsorption and compression, but it benefits cells by facilitating migration and reducing the risk of infection. Fibronectin, on the other hand, exhibits complex mechanical responses to compression, characterized by significant structural rearrangements that occur during adsorption. This strong interaction with the membrane can result in excessively high adhesion forces, ultimately limiting cell motility. These findings lay the foundation for the design of artificial scaffolds that can manipulate cellular responses, a critical step toward advancing regenerative medicine and tissue engineering. SignificanceFabricating extracellular matrix (ECM) scaffolds from cells offers advantages over traditional approaches, such as decellularized tissues, which face donor limitations, and artificial scaffolds, which may hinder cellular communication. However, the slow harvesting process of cell-derived ECM has limited its clinical applications. This research is part of a larger mission to engineer ECM prescaffolds on lipid carriers tailored to cell requirements, enhancing ECM production and regulating cell behavior. The first step involves systematically analyzing the structural and mechanical effects of ECM on lipid membranes and how these effects regulate cellular behavior. This work confirms distinct characteristics of ECM proteins, advancing fundamental understanding of cell-matrix interactions and paving the way for scaffold engineering.

Cardiovascular disease (CVD) is the leading cause of death in the United States and worldwide. While most of these deaths are the result of chronic heart diseases, some CVDs are induced artificially. Doxorubicin (DOX) is a chemotherapeutic that is commonly used to treat breast cancer which is one of the most common types of cancer in the United States. While DOX is an effective anti-cancer agent, over 10% of treated women show signs of acute cardiotoxicity immediately following treatment, and approximately 2% develop severe cardiotoxicity up to 10 years after the end of treatment. Despite this prevalence, the mechanism by which the onset of this cardiotoxicity occurs over time is not well understood. Here, we show that treatment of cardiac cells with DOX changes the cardiac function and the resulting paracrine signaling profile. Subsequent exposure of healthy cells to these altered paracrine agents can recapitulate the effects of direct DOX exposure in 2D and 3D in vitro models. We suggest that this is the result of an altered paracrine miRNA profile and other paracrine factors that propagate the initial disruption caused by direct DOX exposure. Plasma EV miRNA profiling of blinded patient samples revealed distinct clustering by DOX-cardiotoxicity risk, with high-risk patients exhibiting miRNA signatures similar to those from DOX-treated tissue-engineered models. Pathway analysis of the most distinguishing miRNAs linked them to cardiac homeostasis and cardiotoxicity-related mechanisms, supporting the potential of plasma EV miRNAs as noninvasive biomarkers for early risk stratification and personalized cardioprotective interventions in oncological care, and the targeting of key clusters of miRNAs to enhance both understanding of and intervention strategies for preventing the onset of DOX cardiotoxicity.

Anthracycline induced cardiotoxicity is a significant problem for oncologists and cancer patients. The leading cause of non-cancer death in cancer patients and survivors is heart failure, which is frequently attributed to the exposure to chemotherapeutics like anthracyclines. The most notorious of these chemotherapies is doxorubicin, which causes cardiac contractile dysfunction that in some cases is irreversible. In this study, we report the development of NanoDMX, a phosphatidylserine-containing liposomal formulation of DMX5804, a small molecule inhibitor of MAP4K4, and demonstrate that its administration prevents doxorubicin-induced left ventricular dysfunction in mice. Additionally, we demonstrate that DMX-5804 protects cardiomyocytes in vitro through a combination of mechanisms outside of the expected route of suppressing the JNK pathway. Overall, we demonstrate that the use of NanoDMX, a novel liposomal system using both DMX-5804 and phosphatidylserine, can prevent the damage induced by doxorubicin over the course of a single high dose in vivo model.