In-vivo correlations of fluorescent or radioisotope glucose-analogs in imaging cancer metabolism.

OBJECTIVE: To investigate the impact of different tracer modifications on the imaging of cancer metabolism, focusing on the comparison of fluorescent glucose-analog tracers (2-NBDG and 2-DG-750) and the radiolabeled tracer 18F-FDG in both in-vitro and in-vivo settings. METHODS: We conducted an in-vitro comparative study using four cancer cell lines, each with unique glucose uptake characteristics. The study involved direct comparison of three tracers: 2-NBDG, 2-DG-750 and 18F-FDG, examining their internalization behaviors, metabolic functionality and localization effects in cancer metabolism imaging. RESULTS: The study revealed that each tracer exhibits distinct internalization behaviors correlated with imaging label size and type. 18F-FDG showed the highest uptake efficiency. Fluorescent molecules were found to accumulate in tumors primarily due to hydrophobic interactions and possible aggregation, indicating inefficiency in metabolism and suitability for imaging metabolic phenomena when compared to radiolabeled biomolecules. CONCLUSION: Our findings demonstrate that despite certain impracticalities, nuclear imaging, particularly using radiolabeled biomolecules like 18F-FDG, offers significant potential for accurately capturing biological phenomena. This is crucial for future advancements in both clinical and research settings. The study emphasizes the limitations of fluorescent molecules in imaging metabolic activities due to their inefficient metabolism and aggregation tendencies.

Cell Spheroid Formation on the Surface of Multi-Block Copolymers Composed of Poly(2-methoxyethyl acrylate) and Polyethylene Glycol.

3D structured cells have great drug screening potential because they mimic in vivo tissues better than 2D cultured cells. In this study, multi-block copolymers composed of poly(2-methoxyethyl acrylate) (PMEA) and polyethylene glycol (PEG) are developed as a new kind of biocompatible polymers. PEG imparts non-cell adhesion while PMEA acts as an anchoring segment to prepare the polymer coating surface. The multi-block copolymers show higher stability in water than PMEA. A specific micro-sized swelling structure composed of a PEG chain is observed in the multi-block copolymer film in water. A single NIH3T3-3-4 spheroid is formed in 3 h on the surface of the multi-block copolymers with 8.4 wt% PEG. However, at a PEG content of 0.7 wt%, spheroid formed after 4 days. The adenosine triphosphate (ATP) activity of cells and the internal necrotic state of the spheroid change depending on PEG loading in the multi-block copolymers. As the formation rate of cell spheroid on low-PEG-ratio multi-block copolymers is slow, internal necrosis of cell spheroid is less likely to occur. Consequently, the cell spheroid formation rate by changing the PEG chain content in multi-block copolymers is successfully controlled. These unique surfaces are suggested to be useful for 3D cell culture.

Cell Adhesion and Migration on Thickness Gradient Bilayer Polymer Brush Surfaces: Effects of Properties of Polymeric Materials of the Underlayer.

In the field of tissue engineering and biomaterials, controlling the surface properties and mechanical properties of scaffold materials is crucial and has attracted much attention. Here, two types of bilayer polymer brushes composed of a hydrophilic underlying layer and a cationic surface layer [made of poly(2-aminoethyl methacrylate)] with a thickness gradient were prepared by surface-initiated atom-transfer radical polymerization. To investigate the influence of the stiffness as a mechanical property of the polymer brush on cell behavior, the underlayer was prepared from either 2-methacryloyloxyethyl phosphorylcholine or oligo(ethylene glycol) methyl ether methacrylate, with the bilayers designated as gradient poly(2-methacryloyloxyethyl phosphorylcholine)-block-poly(2-aminoethyl methacrylate) [grad-pMbA] and gradient poly(oligo[ethylene glycol] methyl ether methacrylate)-block-poly(2-aminoethyl methacrylate) [grad-pEGbA], respectively. Characterization of these surfaces was performed by spectroscopic ellipsometry, X-ray reflectivity, and determination of the zeta potential, static contact angle, and force curve. These diblock copolymer brushes with a thickness gradient helped to distinguish the effects of the mechanical and surface properties of the brushes on cell behavior. The attachment and motility of L929 fibroblasts and epithelial MCF 10A cells on the fabricated brushes were then assessed. L929 cells had a round shape on the thin surface layer of grad-pMbA and spread well on thicker areas. In contrast, MCF 10A cells spread well in areas of any thickness of either grad-pMbA or grad-pEGbA. Single MCF 10A cells migrated randomly on grad-pMbA, whereas grouped cells started to climb up along the thickness gradient of grad-pMbA. In contrast, both single and grouped MCF 10A cells migrated randomly on grad-pEGbA. These thickness gradient diblock copolymer brushes are simple, reproducible, and reasonable platforms that can facilitate practical applications of biomaterials, for example, in tissue engineering and biomaterials.

Fluorescent polymeric nanoparticle for ratiometric temperature sensing allows real-time monitoring in influenza virus-infected cells.

Temperature is a key indicator of infection and disease, however, it is difficult to measure at a cellular level. Nanoparticles are applied to measure the cellular temperature, and enhancement of the stability and reliability of the signal and higher biocompatibility are demanded. We have developed fluorescent polymeric nanoparticles loaded with temperature-sensitive units (as rhodamine B) and internal reference units (as coumarin) for imaging and ratiometric sensing of the cellular temperature in the physiological range. The fluorescence signal of the nanoparticles was stable in the bio-environment and the ratiometric sensing strategy could overcome the concentration effect of nanoparticles. The nanoparticles were endocytosed by cells and partially presented in mitochondria. The fluorescence intensity ratio of rhodamine B and coumarin using nanoparticles showed good linear correlations in buffer solutions, cell suspensions, and imaging of living cells. Using the fluorescent polymeric nanoparticles, the change of temperature of cells during influenza virus infection could be individually monitored.

Unique Cancer Migratory Behaviors in Confined Spaces of Microgroove Topography with Acute Wall Angles.

In recent years, many types of micro-engineered platform have been fabricated to investigate the influences of surrounding microenvironments on cell migration. Previous researches demonstrated that microgroove-based topographies can influence cell motilities of normal and cancerous cells differently. In this study, the microgroove wall angle was altered from obtuse to acute angles and the resulting differences in the responses of normal and cancer cells were investigated to explore the geometrical characteristics that can efficiently distinguish normal and cancer cells. Interestingly, different trends in cell motilities of normal and cancer cells were observed as the wall angles were varied between 60-120°, and in particular, invasive cancer cells exhibited a unique, oscillatory migratory behavior. Results from the immunostaining of cell mechanotransduction components suggested that this difference stemmed from directional extensions and adhesion behaviors of each cell type. In addition, the specific behaviors of invasive cancer cells were found to be dependent on the myosin II activity, and modulating the activity could revert cancerous behaviors to normal ones. These novel findings on the interactions of acute angle walls and cancer cell migration provide a new perspective on cancer metastasis and additional strategies via microstructure geometries for the manipulations of cell behaviors in microscale biodevices.

[Negative regulation of gastric proton pump by desialylation suggested by fluorescent imaging with the sialic acid-specific nanoprobe].

Gastric proton pump (H+,K+-ATPase) which is responsible for H+ secretion of gastric acid (HCl) in gastric parietal cells is the major therapeutic target for treatment of acid-related diseases. H+,K+-ATPase consists of two subunits, a catalytic α-subunit (αHK) and a glycosylated ß-subunit (ßHK). N-glycosylation of ßHK is essential for trafficking and stability of αHK in apical membrane of gastric parietal cells. Terminal sialic acid residues on sugar chains have an important role in various cellular functions. Recently, we succeeded in visualizing the sialylation and desialylation dynamics of ßHK using a fluorescence bioimaging nanoprobe consisting of biocompatible polymers conjugated with lectins for detecting sialic acid. In H+,K+-ATPase-expressing cell lines, rat gastric mucosa, and primary culture of rat gastric parietal cells, fluorescence imaging of sialic acid with the nanoprobe showed that sialylation of ßHK is regulated by intragastric pH and that inhibition of gastric acid secretion induces desialylation of ßHK. In biochemical and pharmacological studies, we revealed that enzyme activity of αHK is negatively regulated by desialylation of ßHK. Our studies uncovered a novel negative-feedback mechanism of H+,K+-ATPase in which sialic acids of ßHK positively regulates H+,K+-ATPase activity, and acidic pH decreases the pump activity by cleaving sialic acids of ßHK. In this topic, we introduce the overview of our research using the bioimaging nanoprobe.

Influence of cell adhesive molecules attached onto PEG-lipid-modified fluid surfaces on cell adhesion.

The involvement of intercellular interactions in various biological events indicates the importance of studying cell-cell interactions using fluid model surfaces. Here, we propose a fluid surface composed of a self-assembled monolayer (SAM) and poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) derivatives, which can be an alternative to supported lipid membranes. The modification of SAM surfaces with PEG-lipids carrying functional peptides resulted in the formation of the fluid surfaces with different mobility, which was quantitatively determined by quartz crystal microbalance with dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP). Different types of fluid surfaces with calculated diffusion coefficients between 0.9 ± 0.25 and 0.16 ± 0.03 µm2/sec for PEG-lipids derivatives were fabricated, onto which arginylglycylaspartate (RGD) peptides were immobilized for cell adhesion, and compared to solid surfaces with the same surface density of RGD peptides. The fluid surfaces revealed that cell adhesions of epithelial cells (MCF-10 A) and human umbilical vein endothelial cells (HUVEC) could not be established on the surfaces with higher fluidity, while cells could adhere onto surfaces with lower fluidity, where the lateral diffusion of PEG-lipids was approximately 20 times lower, and solid surfaces. Interestingly, cells that adhered onto the surface with lower fluidity proliferated at a normal rate while maintaining their round morphology, which was a different shape from that observed on solid surfaces. Thus, the scaffold fluidity greatly influenced cell adhesion behaviors, demonstrating that it is an important parameter for designing novel biomimetic scaffolds for biomedical applications.

Rapid multiplex microfiber-based immunoassay for anti-MERS-CoV antibody detection.

On-site multiplex biosensors for innate immunity antibodies are ideal tools for monitoring health status of individuals against various diseases. This study introduces a novel antibody immunoassay testing platform incorporating microfiber-based arrays of antigens to capture specific antibodies. The fabrication and setup of the device revolved around electrospun polystyrene (ESPS) microfibers that act as three-dimensional membrane filters, capable of rapid and multifold analyte capture. In particular, the ESPS microfibers were patterned through localized oxygen plasma to create hydrophilic zones that facilitate fluid flows and immobilizations of antigens. The bulk of this robust antibody immunoassay platform could be installed into a compact syringe-driven cassette device, which could perform multiplex antibody immunoassay for antibodies specifically against Middle East respiratory syndrome coronavirus (MERS-CoV) with rapid preparation amounting to a total of 5 min, as well as high sensitivity and specificity for the MERS-CoV down to 200 µg/mL.

Fabrication and assessment of an electrospun polymeric microfiber-based platform under bulk flow conditions with rapid and efficient antigen capture.

Correction for 'Fabrication and assessment of an electrospun polymeric microfiber-based platform under bulk flow conditions with rapid and efficient antigen capture' by Carlton F. O. Hoy et al., Analyst, 2018, 143, 865-873.

Fabrication and assessment of an electrospun polymeric microfiber-based platform under bulk flow conditions with rapid and efficient antigen capture.

This study investigated the fabrication and proof of concept design demonstrating rapid and highly sensitive antigen capture utilizing electrospun polystyrene (PS) microfiber mat substrates paired with vacuum pump pressurization to induce bulk flow. In comparison with conventional flat PS surfaces used for immunoassay purposes, this system optimizes the increased surface area of the electrospun polystyrene (ESPS) fiber mat substrates and the accelerated propagation of the antigen through the detection platform by using a vacuum pump to enable efficient and rapid antigen capture. The novelty of this work was demonstrated through a parametric study detailing how a fiber substrate can capture antigen sensitively and at high speeds. In terms of sensitivity, the current system is comparable to the conventionally used flat PS substrates. Additionally, the amount of antigen captured on a flat PS substrate in 60 minutes was surpassed in under 5 seconds when utilizing the ESPS-vacuum system. Three-dimensional ESPS fiber mats were then noted as a comparison between Damkohler numbers and between flat PS and ESPS-vacuum systems. The bulk flow of the ESPS-vacuum system allows for a Damkohler number of 0.37 indicating a balance between the flow rate and the reaction rate as opposed to a PS flat platform of 5.80 × 104 which illustrates a diffusion rate limited system. Finally, the overall ESPS-vacuum system was tested for its immunoassay capability. A sandwich fluorescence-based immunoassay was performed on both PS flat-diffusion and ESPS-vacuum systems. The ESPS-vacuum system indicated a wider detection range capability from 5 to 1000 ng mL-1 in comparison with the PS flat-diffusion system at 5 to 100 ng mL-1.

Differences in Three-Dimensional Geometric Recognition by Non-Cancerous and Cancerous Epithelial Cells on Microgroove-Based Topography.

During metastasis, cancer cells are exposed to various three-dimensional microstructures within the body, but the relationship between cancer migration and three-dimensional geometry remain largely unclear. Here, such geometric effects on cancerous cells were investigated by characterizing the motility of various cancer cell types on microgroove-based topographies made of polydimethylsiloxane (PDMS), with particular emphasis on distinguishing cancerous and non-cancerous epithelial cells, as well as understanding the underlying mechanism behind such differences. The 90-degree walls enhanced motility for all cell lines, but the degrees of enhancements were less pronounced for the cancerous cells. Interestingly, while the non-cancerous epithelial cell types conformed to the three-dimensional geometrical cues and migrated along the walls, the cancerous cell types exhibited a unique behavior of climbing upright walls, and this was associated with the inability to form stable, polarized actin cytoskeleton along the walls of the microgrooves. Furthermore, when non-cancerous epithelial cell lines were altered to different levels of polarization capabilities and cancer malignancy or treated with inhibitory drugs, their three-dimensional geometry-dependent motility approached those of cancerous cell lines. Overall, the results suggest that cancerous cells may gradually lose geometrical recognition with increasing cancer malignancy, allowing them to roam freely ignoring three-dimensional geometrical cues during metastasis.

Analysis of the Changes in Expression Levels of Sialic Acid on Influenza-Virus-Infected Cells Using Lectin-Tagged Polymeric Nanoparticles.

Viral infections affect millions around the world, sometimes leading to severe consequences or even epidemics. Understanding the molecular dynamics during viral infections would provide crucial information for preventing or stopping the progress of infections. However, the current methods often involve the disruption of the infected cells or expensive and time-consuming procedures. In this study, fluorescent polymeric nanoparticles were fabricated and used as bioimaging nanoprobes that can monitor the progression of influenza viral infection through the changes in the expression levels of sialic acids expressed on the cell membrane. The nanoparticles were composed of a biocompatible monomer to prevent non-specific interactions, a hydrophobic monomer to form the core, a fluorescent monomer, and a protein-binding monomer to conjugate lectin, which binds sialic acids. It was shown that these lectin-tagged nanoparticles that specifically target sialic acids could track the changes in the expression levels of sialic acids caused by influenza viral infections in human lung epithelial cells. There was a sudden drop in the levels of sialic acid at the initial onset of virus infection (t = 0~1 h) and at approximately 4~5 h post-infection. The latter drop correlated with the production of viral proteins that was confirmed using traditional techniques. Thus, the accuracy, the rapidity and the efficacy of the nanoprobes were demonstrated. Such molecular bioimaging tools, which allow easy-handling and in situ monitoring, would be useful to directly observe and decipher the viral infection mechanisms.

Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy.

The conformation and dynamics of the unfolded state of ubiquitin doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated using single-molecule fluorescence spectroscopy. The line confocal fluorescence detection system combined with the rapid sample flow enabled the characterization of unfolded proteins at the improved structural and temporal resolutions compared to the conventional single-molecule methods. In the initial stage of the current investigation, however, the single-molecule Förster resonance energy transfer (sm-FRET) data of the labeled ubiquitin were flawed by artifacts caused by the adsorption of samples to the surfaces of the fused-silica flow chip and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl phosphorylcholine polymer to the flow chip surface was found to suppress the artifacts. The sm-FRET measurements based on the coated flow chip demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin at the native condition were narrowly distributed, which is comparable to the probability density function (PDF) expected from the shot noise, demonstrating the structural homogeneity of the native state. In contrast, the histogram of the sm-FRET efficiencies of the unfolded ubiquitin obtained at a time resolution of 100 µs was distributed significantly more broadly than the PDF expected from the shot noise, demonstrating the heterogeneity of the unfolded state conformation. The variety of the sm-FRET efficiencies of the unfolded state remained even after evaluating the moving average of traces with a window size of 1 ms, suggesting that conformational averaging of the heterogeneous conformations mostly occurs in the time domain slower than 1 ms. Local structural heterogeneity around the labeled fluorophores was inferred as the cause of the structural heterogeneity. The heterogeneity and slow dynamics revealed by the line confocal tracking of sm-FRET might be common properties of the unfolded proteins.

Non-Osmotic Hydrogels: A Rational Strategy for Safely Degradable Hydrogels.

Hydrogels are promising materials for biomedical applications, where timely degradation is often preferred. In the conventional design, however, the cleavage of polymer networks essentially causes considerable morphological changes (i.e., degradation-induced swelling), triggering various medical complications. Herein, we report a rational strategy to suppress the degradation-induced swelling based on the synthetic control of the polymer-solvent interaction parameter (χ) of constituent polymer networks. The resultant hydrogels with an optimal χ parameter (χ37 °C ≈0.53; non-osmostic hydrogels) displayed the capability to retain their original shape and degrade without generating significant swelling pressure under physiological conditions (Π37 °C <1 kPa). This concept of the safely degradable non-osmotic hydrogel is theoretically universal, and can be exploited for other types of synthetic hydrogels in various settings.

Simultaneous characterization of protein-material and cell-protein interactions using dynamic QCM-D analysis on SAM surfaces.

Understanding the interactions among materials, proteins and cells is critical for the development of novel biomaterials, and establishing a highly sensitive and quantitative method to standardize these interactions is desired. In this study, quartz crystal microbalance with dissipation (QCM-D) combined with microscopy was utilized to quantitatively monitor the entirety of the cell adhesion processes, starting from the protein adsorption, on various self-assembled monolayer (SAM) surfaces. Although the resulting cell adhesion morphologies were similar on most of the surfaces, the dynamic QCM-D signal patterns were unique on each surface, suggesting different forms of material-protein-cell interactions. The viscoelasticity and the density of the surface-adsorbed fibronectin (FN), as well as the relative exposure of the cell adhesive arginine-glycine-aspartic acid (RGD) motifs, were correlated to the different cell adhesion dynamics and mechanics. Some surfaces exhibited complicated behaviors alluding to the detachment/rearrangement of surface proteins or highly sparse but bioactive proteins that promote a slow adhesion process. This study underscores the potential use of the QCM-D signal pattern as a rule of thumb for delineating different protein-material and cell-protein interactions, and offers a rapid in vitro platform for the dynamic evaluation of protein and cell behaviors on novel biomaterials.

Positive regulation of the enzymatic activity of gastric H(+),K(+)-ATPase by sialylation of its ß-subunit.

The gastric proton pump (H(+),K(+)-ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated ß-subunit (ßHK). ßHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of ßHK. Sialylation of ßHK was found in LLC-PK1 cells stably expressing αHK and ßHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-ßHK antibody. The sialic acids of ßHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH5). Interestingly, the enzymatic activity of H(+),K(+)-ATPase was significantly decreased by cleavage of the sialic acids of ßHK. In contrast, ßHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H(+),K(+)-ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of ßHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H(+),K(+)-ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that ßHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of ßHK positively regulate the enzymatic activity of αHK.

Slope-Dependent Cell Motility Enhancements at the Walls of PEG-Hydrogel Microgroove Structures.

In recent years, research utilizing micro- and nanoscale geometries and structures on biomaterials to manipulate cellular behaviors, such as differentiation, proliferation, survival, and motility, have gained much popularity; however, how the surface microtopography of 3D objects, such as implantable devices, can affect these various cell behaviors still remains largely unknown. In this study, we discuss how the walls of microgroove topography can influence the morphology and the motility of unrestrained cells, in a different fashion from 2D line micropatterns. Here adhesive substrates made of tetra(polyethylene glycol) (tetra-PEG) hydrogels with microgroove structures or 2D line micropatterns were fabricated, and cell motility on these substrates was evaluated. Interestingly, despite being unconstrained, the cells exhibited drastically different migration behaviors at the edges of the 2D micropatterns and the walls of microgroove structures. In addition to acquiring a unilamellar morphology, the cells increased their motility by roughly 3-fold on the microgroove structures, compared with the 2D counterpart or the nonpatterned surface. Immunostaining revealed that this behavior was dependent on the alignment and the aggregation of the actin filaments, and by varying the slope of the microgroove walls, it was found that relatively upright walls are necessary for this cell morphology alterations. Further progress in this research will not only deepen our understanding of topography-assisted biological phenomena like cancer metastasis but also enable precise, topography-guided manipulation of cell motility for applications such as cancer diagnosis and cell sorting.

Effect of the distribution of adsorbed proteins on cellular adhesion behaviors using surfaces of nanoscale phase-reversed amphiphilic block copolymers.

In order to create suitable biocompatible materials for various tissue engineering applications, it is important to be able to understand protein adsorption and cell adhesion behaviors on the material's surfaces. It is known that the nanoscale distribution of adsorbed proteins affects cell adhesion behaviors. However, how nanoscale structures affect cell adhesion behaviors is still unclear. Therefore, in this study, we investigate the effect of the distribution of adsorbed proteins by the phase reversal of amphiphilic block copolymers composed of protein-non-adsorptive poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and protein-adsorptive poly(3-methacryloyloxy propyltris(trimethylsilyloxy) silane) (PMPTSSi) on cell adhesion behaviors. The nanodomain structures of phase-separated block copolymers were successfully confirmed using transmission electron microscopy and atomic force microscopy. Surfaces that had PMPC dot-like domains (23 ± 4 nm) and ones that had PMPTSSi dot-like domains (25 ± 6 nm) were made. From protein adsorption and L929 cell adhesion measurements, it was found that even on surfaces with equal quantities of protein adsorption, the number of cells on surfaces with PMPC dot-like domains was larger than those with PMPTSSi dot-like domains. This suggests that the simple phase-reversal of the distribution of adsorbed proteins can be used to affect cell adhesion behaviors for designing biomaterial surfaces for tissue engineering applications.

Lectin-tagged fluorescent polymeric nanoparticles for targeting of sialic acid on living cells.

In this study, we fabricated lectin-tagged fluorescent polymeric nanoparticles approximately 35 nm in diameter using biocompatible polymers conjugated with lectins for the purpose of detecting sialic acid on a living cell surface, which is one of the most important biomarkers for cancer diagnosis. Through cellular experiments, we successfully detected sialic acid overexpression on cancerous cells with high specificity. These fluorescent polymeric nanoparticles can be useful as a potential bioimaging probe for detecting diseased cells.

Modular design of micropattern geometry achieves combinatorial enhancements in cell motility.

Basic micropattern shapes, such as stripes and teardrops, affect individual facets of cell motility, such as migration speed and directional bias, respectively. Here, we test the idea that these individual effects on cell motility can be brought together to achieve multidimensional improvements in cell behavior through the modular reconstruction of the simpler "building block" micropatterns. While a modular design strategy is conceptually appealing, current evidence suggests that combining environmental cues, especially molecular cues, such as growth factors and matrix proteins, elicits a highly nonlinear, synergistic cell response. Here, we show that, unlike molecular cues, combining stripe and teardrop geometric cues into a hybrid, spear-shaped micropattern yields combinatorial benefits in cell speed, persistence, and directional bias. Furthermore, cell migration speed and persistence are enhanced in a predictable, additive manner on the modular spear-shaped design. Meanwhile, the spear micropattern also improved the directional bias of cell movement compared to the standard teardrop geometry, revealing that combining geometric features can also lead to unexpected synergistic effects in certain aspects of cell motility. Our findings demonstrate that the modular design of hybrid micropatterns from simpler building block shapes achieves combinatorial improvements in cell motility. These findings have implications for engineering biomaterials that effectively mix and match micropatterns to modulate and direct cell motility in applications, such as tissue engineering and lab-on-a-chip devices.