Cell-to-cell influence on growth in large populations.

Recent studies have revealed the importance of outlier cells in complex cellular systems. Quantifying heterogeneity in such systems may lead to a better understanding of organ engineering, microtumor growth, and disease models, as well as more precise drug design. We used the ability of quantitative phase imaging to perform long-term imaging of cell growth to estimate the "influence" of cellular clusters on their neighbors. We validated our approach by analyzing epithelial and fibroblast cultures imaged over the course of several days. Interestingly, we found that there is a significant number of cells characterized by a medium correlation between their growth rate and distance (modulus of the Pearson coefficient between 0.25-.5). Furthermore, we found a small percentage of cells exhibiting strong such correlations, which we label as "influencer" cellular clusters. Our approach might find important applications in studying dynamic phenomena, such as organogenesis and metastasis.

Small-scale displacement fluctuations of vesicles in fibroblasts.

The intracellular environment is a dynamic space filled with various organelles moving in all directions. Included in this diverse group of organelles are vesicles, which are involved in transport of molecular cargo throughout the cell. Vesicles move in either a directed or non-directed fashion, often depending on interactions with cytoskeletal proteins such as microtubules, actin filaments, and molecular motors. How these proteins affect the local fluctuations of vesicles in the cytoplasm is not clear since they have the potential to both facilitate and impede movement. Here we show that vesicle mobility is significantly affected by myosin-II, even though it is not a cargo transport motor. We find that myosin-II activity increases the effective diffusivity of vesicles and its inhibition facilitates longer states of non-directed motion. Our study suggests that altering myosin-II activity in the cytoplasm of cells can modulate the mobility of vesicles, providing a possible mechanism for cells to dynamically tune the cytoplasmic environment in space and time.

Perspective: The promise of multi-cellular engineered living systems.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of "multi-cellular engineered living systems," which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.

Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow.

UNLABELLED: Mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical and biochemical properties of the extracellular matrix. In this work we conduct a combinatorial study of matrix properties that influence adipogenesis and neurogenesis including: adhesion proteins, stiffness, and cell geometry, for mesenchymal stem cells derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs). We uncover distinct differences in integrin expression, the magnitude of traction stress, and lineage specification to adipocytes and neuron-like cells between cell sources. In the absence of media supplements, adipogenesis in AT-MSCs is not significantly influenced by matrix properties, while the converse is true in BM-MSCs. Both cell types show changes in the expression of neurogenesis markers as matrix cues are varied. When cultured on laminin conjugated microislands of the same adhesive area, BM-MSCs display elevated adipogenesis markers, while AT-MSCs display elevated neurogenesis markers; integrin analysis suggests neurogenesis in AT-MSCs is guided by adhesion through integrin αvß3. Overall, the properties of the extracellular matrix guides MSC adhesion and lineage specification to different degrees and outcomes, in spite of their similarities in general characteristics. This work will help guide the selection of MSCs and matrix components for applications where high fidelity of differentiation outcome is desired. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) are an attractive cell type for stem cell therapies; however, in order for these cells to be useful in medicine, we need to understand how they respond to the physical and chemical environments of tissue. Here, we explore how two promising sources of MSCs-those derived from bone marrow and from adipose tissue-respond to the compliance and composition of tissue using model extracellular matrices. Our results demonstrate a source-specific propensity to undergo adipogenesis and neurogenesis, and uncover a role for adhesion, and the degree of traction force exerted on the substrate in guiding these lineage outcomes.

Geometric guidance of integrin mediated traction stress during stem cell differentiation.

Cells sense and transduce the chemical and mechanical properties of their microenvironment through cell surface integrin receptors. Traction stress exerted by cells on the extracellular matrix mediates focal adhesion stabilization and regulation of the cytoskeleton for directing biological activity. Understanding how stem cells integrate biomaterials properties through focal adhesions during differentiation is important for the design of soft materials for regenerative medicine. In this paper we use micropatterned hydrogels containing fluorescent beads to explore force transmission through integrins from single mesenchymal stem cells (MSCs) during differentiation. When cultured on polyacrylamide gels, MSCs will express markers associated with osteogenesis and myogenesis in a stiffness dependent manner. The shape of single cells and the composition of tethered matrix protein both influence the magnitude of traction stress applied and the resultant differentiation outcome. We show how geometry guides the spatial positioning of focal adhesions to maximize interaction with the matrix, and uncover a relationship between αvß3, α5ß1 and mechanochemical regulation of osteogenesis.

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A novel cell traction force microscopy to study multi-cellular system.

Traction forces exerted by adherent cells on their microenvironment can mediate many critical cellular functions. Accurate quantification of these forces is essential for mechanistic understanding of mechanotransduction. However, most existing methods of quantifying cellular forces are limited to single cells in isolation, whereas most physiological processes are inherently multi-cellular in nature where cell-cell and cell-microenvironment interactions determine the emergent properties of cell clusters. In the present study, a robust finite-element-method-based cell traction force microscopy technique is developed to estimate the traction forces produced by multiple isolated cells as well as cell clusters on soft substrates. The method accounts for the finite thickness of the substrate. Hence, cell cluster size can be larger than substrate thickness. The method allows computing the traction field from the substrate displacements within the cells' and clusters' boundaries. The displacement data outside these boundaries are not necessary. The utility of the method is demonstrated by computing the traction generated by multiple monkey kidney fibroblasts (MKF) and human colon cancerous (HCT-8) cells in close proximity, as well as by large clusters. It is found that cells act as individual contractile groups within clusters for generating traction. There may be multiple of such groups in the cluster, or the entire cluster may behave a single group. Individual cells do not form dipoles, but serve as a conduit of force (transmission lines) over long distances in the cluster. The cell-cell force can be either tensile or compressive depending on the cell-microenvironment interactions.

A mechanically-induced colon cancer cell population shows increased metastatic potential.

BACKGROUND: Metastasis accounts for the majority of deaths from cancer. Although tumor microenvironment has been shown to have a significant impact on the initiation and/or promotion of metastasis, the mechanism remains elusive. We previously reported that HCT-8 colon cancer cells underwent a phenotypic transition from an adhesive epithelial type (E-cell) to a rounded dissociated type (R-cell) via soft substrate culture, which resembled the initiation of metastasis. The objective of current study was to investigate the molecular and metabolic mechanisms of the E-R transition. METHODS: Global gene expressions of HCT-8 E and R cells were measured by RNA Sequencing (RNA-seq); and the results were further confirmed by real-time PCR. Reactive oxygen species (ROS), anoikis resistance, enzyme activity of aldehyde dehydrogenase 3 family, member A1 (ALDH3A1), and in vitro invasion assay were tested on both E and R cells. The deformability of HCT-8 E and R cells was measured by atomic force microscopy (AFM). To study the in vivo invasiveness of two cell types, athymic nude mice were intra-splenically injected with HCT-8 E or R cells and sacrificed after 9 weeks. Incidences of tumor development and metastasis were histologically evaluated and analyzed with Fisher's exact test. RESULTS: Besides HCT-8, E-R transition on soft substrates was also seen in three other cancer cell lines (HCT116, SW480 colon and DU145 prostate cancer). The expression of some genes, such as ALDH3A1, TNS4, CLDN2, and AKR1B10, which are known to play important roles in cancer cell migration, invasion, proliferation and apoptosis, were increased in HCT-8 R cells. R cells also showed higher ALDH3A1 enzyme activity, higher ROS, higher anoikis resistance, and higher softness than E cells. More importantly, in vitro assay and in vivo animal models revealed that HCT-8 R cells were more invasive than E cells. CONCLUSIONS: Our comprehensive comparison of HCT-8 E and R cells revealed differences of molecular, phenotypical, and mechanical signatures between the two cell types. To our knowledge, this is the first study that explores the molecular mechanism of E-R transition, which may greatly increase our understanding of the mechanisms of cancer mechanical microenvironment and initiation of cancer metastasis.

Active transport of vesicles in neurons is modulated by mechanical tension.

Effective intracellular transport of proteins and organelles is critical in cells, and is especially important for ensuring proper neuron functionality. In neurons, most proteins are synthesized in the cell body and must be transported through thin structures over long distances where normal diffusion is insufficient. Neurons transport subcellular cargo along axons and neurites through a stochastic interplay of active and passive transport. Mechanical tension is critical in maintaining proper function in neurons, but its role in transport is not well understood. To this end, we investigate the active and passive transport of vesicles in Aplysia neurons while changing neurite tension via applied strain, and quantify the resulting dynamics. We found that tension in neurons modulates active transport of vesicles by increasing the probability of active motion, effective diffusivity, and induces a retrograde bias. We show that mechanical tension modulates active transport processes in neurons and that external forces can couple to internal (subcellular) forces and change the overall transport dynamics.

Measuring nonequilibrium vesicle dynamics in neurons under tension.

Vesicle transport in neurons is a highly complex nonequilibrium process. Their subcellular environment is undergoing constant fluctuations from thermal energy and molecular motors. Vesicle transport is an interplay between random motion (passive) and directed motion (active) driven by molecular motors along cytoskeletal filaments. It has been shown that growth, guidance, and vesicle dynamics of neurons is affected by mechanical tension. Here we present a method to analyze vesicle transport via a temporal Mean Square Displacement (tMSD) analysis while applying mechanical strain to neurons. The tMSD analysis allows characterization of active and passive vesicle motion as well as many other parameters including: power law scaling, velocity, direction, and flux. Our results suggest: (1) The tMSD analysis is able to capture vesicle motion alternating between passive and active states, and indicates that vesicle motion in Aplysia neurons is primarily passive (exhibiting active motion for ~8% of the time). (2) Under mechanical stretch (increased neurite tension), active transport of vesicles increases to ~13%, while vesicle velocity remains unchanged. (3) Upon unstretching (decreased tension), the level of active transport returns to normal but vesicle velocity decreases. These results suggest that vesicle transport in neurons is highly sensitive to mechanical stimulation. Our method allows precise characterization of vesicle dynamics in response to applied mechanical strain.

Attenuation of cell mechanosensitivity in colon cancer cells during in vitro metastasis.

Human colon carcinoma (HCT-8) cells show a stable transition from low to high metastatic state when cultured on appropriately soft substrates (21 kPa). Initially epithelial (E) in nature, the HCT-8 cells become rounded (R) after seven days of culture on soft substrate. R cells show a number of metastatic hallmarks [1]. Here, we use gradient stiffness substrates, a bio-MEMS force sensor, and Coulter counter assays to study mechanosensitivity and adhesion of E and R cells. We find that HCT-8 cells lose mechanosensitivity as they undergo E-to-R transition. HCT-8 R cells' stiffness, spread area, proliferation and migration become insensitive to substrate stiffness in contrast to their epithelial counterpart. They are softer, proliferative and migratory on all substrates. R cells show negligible cell-cell homotypic adhesion, as well as non-specific cell-substrate adhesion. Consequently they show the same spread area on all substrates in contrast to E cells. Taken together, these results indicate that R cells acquire autonomy and anchorage independence, and are thus potentially more invasive than E cells. To the best of our knowledge, this is the first report of quantitative data relating changes in cancer cell adhesion and stiffness during the expression of an in vitro metastasis-like phenotype.

Development of miniaturized walking biological machines.

The quest to 'forward-engineer' and fabricate biological machines remains a grand challenge. Towards this end, we have fabricated locomotive "bio-bots" from hydrogels and cardiomyocytes using a 3D printer. The multi-material bio-bot consisted of a 'biological bimorph' cantilever structure as the actuator to power the bio-bot, and a base structure to define the asymmetric shape for locomotion. The cantilever structure was seeded with a sheet of contractile cardiomyocytes. We evaluated the locomotive mechanisms of several designs of bio-bots by changing the cantilever thickness. The bio-bot that demonstrated the most efficient mechanism of locomotion maximized the use of contractile forces for overcoming friction of the supporting leg, while preventing backward movement of the actuating leg upon relaxation. The maximum recorded velocity of the bio-bot was ~236 µm s(-1), with an average displacement per power stroke of ~354 µm and average beating frequency of ~1.5 Hz.

Calcium signaling in live cells on elastic gels under mechanical vibration at subcellular levels.

A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels.

Top-down synthesis of versatile polyaspartamide linkers for single-step protein conjugation to materials.

Materials used in various biological applications are often modified with proteins to regulate biomolecular and cellular adhesion. Conventional strategies of protein conjugation accompany monovalent bifunctional protein linkers, which present several limitations in molecular synthesis and protein conjugation. Herein, we present a new strategy of preparing multivalent polyaspartamide linkers in a simple top-down manner, and also demonstrate that the resulting polymer linkers allow us to readily conjugate proteins to both organic and inorganic materials. The top-down synthesis of polyaspartamide linkers was performed by partially opening succinimidyl ring moieties of polysuccinimide (PSI) with the controlled number of nucleophiles reactive to photo-cross-linked hydrogel or gold-coated inorganic materials: (1) Poly(2-hydroxyethyl-co-2-methacryloxyethyl aspartamide) (PHMAA) presenting methacrylate was used to micropattern fibronectin or collagen on a hydrogel in order to regulate cell adhesion and growth area on a micrometer scale. (2) Poly(2-hydroxyethyl-co-2-mercaptoethyl aspartamide) (PHMCA) presenting thiol functional groups was used to link fibronectin to a gold-coated silicon microelectromechanical probe designed to measure cell traction force. Overall, these multivalent polyaspartamide protein linkers will greatly assist efforts to analyze and regulate the cellular adhesion to and phenotypic activities of a wide array of substrates and devices.

Mechanical force affects expression of an in vitro metastasis-like phenotype in HCT-8 cells.

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.

Stiffness of the substrate influences the phenotype of embryonic chicken cardiac myocytes.

We examined the effect of substrate stiffness on the beating rate, force of contraction, and cytoskeletal structure of embryonic chicken cardiac myocytes by culturing them on laminin-coated polyacrylamide (PA) substrates. Cells cultured on PA substrates with elasticity comparable to that of the native myocardium (18 kPa) exhibited the highest beating rate during the first few days of culture. The initial beating rate of individual cells on all the substrates varied significantly but began to converge within 5 days. We also examined the focal adhesions (FAs) and cytoskeletal structure on different substrates via confocal microscopy and found a higher percentage of FAs on tissue culture (TC) plastic dishes compared with the softer PA gels. Furthermore, highly aligned sarcomeric striations were clearly visible on 18 kPa, 50 kPa, and TC dish, whereas cells on 1 kPa only showed nonaligned diffused striations. The force of contraction on these substrates was measured using a micro-electromechanical system force sensor, which showed that the force of contraction for the cells on TC dishes (F = 71.30 ± 6.38 nN) was significantly larger than those cultured on the 18-kPa PA gel (F = 30.16 ± 3.83 nN). This is most likely due to the formation of higher percentage of FAs on the TC dishes compared with fewer FAs on the softer gels. Our cumulative findings can have a significant impact on the design of 3D cardiac tissue engineered scaffolds.

Myoblast morphology and organization on biochemically micro-patterned hydrogel coatings under cyclic mechanical strain.

Mechanical forces and geometric constraints play critical roles in determining cell functionality and tissue development. Novel experimental methods are essential to explore the underlying biological mechanisms of cell response. We present a versatile method to culture cells on adhesive micro-patterned substrates while applying long-term cyclic tensile strain (CTS). A polydimethysiloxane (PDMS) mold is coated with a cell repulsive NCO-sP(EO-stat-PO) hydrogel which in turn is covalently patterned by fibronectin using micro-contact printing. This results in two-dimensional, highly selective cell-adhesive micro-patterns. The substrates allow application of CTS to adherent cells for more than 4 days under cell culture conditions without unspecific adhesion. The applicability of our system is demonstrated by studying the adaptive response of C2C12 skeletal myoblasts seeded on fibronectin lines with different orientations relative to the strain direction. After application of CTS (amplitude of 7%, frequency of 0.5 Hz) we find that actin fiber organization is dominantly controlled by CTS. Nuclei shape is predominantly affected by the constraint of the adhesive lines, resulting in significant elongation. Morphologically, myotube formation was incomplete after 4 days of culture, but actin striations were observed exclusively on the 45 degrees line patterns subjected to CTS, the direction of maximum shear strain.