Stiffness-tunable substrate fabrication by DMDbased optical projection lithography for cancer cell invasion studies.

OBJECTIVE: Cancer cell invasion is a critical cause of fatality in cancer patients. Physiologically relevant tumor models play a key role in revealing the mechanisms underlying the invasive behavior of cancer cells. However, most existing models only consider interactions between cells and extracellular matrix (ECM) components while neglecting the role of matrix stiffness in tumor invasion. Here, we propose an effective approach that can construct stiffness-tunable substrates using digital mirror device (DMD)-based optical projection lithography to explore the invasion behavior of cancer cells. The printability, mechanical properties, and cell viability of three-dimensional (3D) models can be tuned by the concentration of prepolymer and the exposure time. The invasion trajectories of gastric cancer cells in tumor models of different stiffness were automatically detected and tracked in real-time using a deep learning algorithm. The results show that tumor models of different mechanical stiffness can yield distinct regulatory effects. Moreover, owing to the biophysical characteristics of the 3D in vitro model, different cellular substructures of cancer cells were induced. The proposed tunable substrate construction method can be used to build various microstructures to achieve simulation of cancer invasion and antitumor screening, which has great potential in promoting personalized therapy.

Nanomechanical Analysis of Living Small Extracellular Vesicles to Identify Gastric Cancer Cell Malignancy Based on a Biomimetic Peritoneum.

Gastric cancer is one of the most prevalent digestive malignancies. The lack of effective in vitro peritoneal models has hindered the exploration of the potential mechanisms behind gastric cancer's peritoneal metastasis. An accumulating body of research indicates that small extracellular vesicles (sEVs) play an indispensable role in peritoneal metastasis of gastric cancer cells. In this study, a biomimetic peritoneum was constructed. The biomimetic model is similar to real peritoneum in internal microstructure, composition, and primary function, and it enables the recurrence of peritoneal metastasis process in vitro. Based on this model, the association between the mechanical properties of sEVs and the invasiveness of gastric cancer was identified. By performing nanomechanical analysis on sEVs, we found that the Young's modulus of sEVs can be utilized to differentiate between malignant clinical samples (ascites) and nonmalignant clinical samples (peritoneal lavage). Furthermore, patients' ascites-derived sEVs were verified to stimulate the mesothelial-to-mesenchymal transition, thereby promoting peritoneal metastasis. In summary, nanomechanical analysis of living sEVs could be utilized for the noninvasive diagnosis of malignant degree and peritoneal metastasis of gastric cancer. This finding is expected to contribute future treatments.

Novel Operation Mechanism and Multifunctional Applications of Bubble Microrobots.

Microrobots have emerged as powerful tools for manipulating particles, cells, and assembling biological tissue structures at the microscale. However, achieving precise and flexible operation of arbitrary-shaped microstructures in 3D space remains a challenge. In this study, three novel operation methods based on bubble microrobots are proposed to enable delicate and multifunctional manipulation of various microstructures. These methods include 3D turnover, fixed-point rotation, and 3D ejection. By harnessing the combined principles of the effect of the heat flow field and surface tension of an optothermally generated bubble, the bubble microrobot can perform tasks such as flipping an SIA humanoid structure, rotating a bird-like structure, and launching a hollow rocket-like structure. The proposed multi-mode operation of bubble microrobots enables diverse attitude adjustments of microstructures with different sizes and shapes in both 2D and 3D spaces. As a demonstration, a biological microenvironment of brain glioblastoma is constructed by the bubble microrobot. The simplicity, versatility, and flexibility of this proposed method hold great promise for applications in micromanipulation, assembly, and tissue engineering.

Dual-Responsive Nanorobot-Based Marsupial Robotic System for Intracranial Cross-Scale Targeting Drug Delivery.

Nanorobots capable of active movement are an exciting technology for targeted therapeutic intervention. However, the extensive motion range and hindrance of the blood-brain barrier impeded their clinical translation in glioblastoma therapy. Here, a marsupial robotic system constructed by integrating chemical/magnetic hybrid nanorobots (child robots) with a miniature magnetic continuum robot (mother robot) for intracranial cross-scale targeting drug delivery is reported. For primary targeting on macroscale, the continuum robot enters the cranial cavity through a minimally invasive channel (e.g., Ommaya device) in the skull and transports the nanorobots to pathogenic regions. Upon circumventing the blood-brain barrier, the released nanorobots perform secondary targeting on microscale to further enhance the spatial resolution of drug delivery. In vitro experiments against primary glioblastoma cells derived from different patients are conducted for personalized treatment guidance. The operation feasibility within organisms is shown in ex vivo swine brain experiments. The biosafety of the treatment system is suggested in in vivo experiments. Owing to the hierarchical targeting method, the targeting rate, targeting accuracy, and treatment efficacy have improved greatly. The marsupial robotic system offers a novel intracranial local therapeutic strategy and constitutes a key milestone in the development of glioblastoma treatment platforms.

Nanomechanical Signatures of Extracellular Vesicles from Hematologic Cancer Patients Unraveled by Atomic Force Microscopy for Liquid Biopsy.

Cells release extracellular vesicles (EVs) as the carriers for intercellular communications to regulate life activities. Particularly, it is increasingly apparent that mechanical forces play an essential role in biological systems. The nanomechanical properties of EVs and their dynamics in cancer development are still not fully understood. Herein, with the use of atomic force microscopy (AFM), the nanomechanical signatures of EVs from the liquid biopsies of hematologic cancer patients were unraveled. Single native EVs were probed by AFM under aqueous conditions. The elastic and viscous properties of EVs were measured and visualized to correlate EV mechanics with EV geometry. Experimental results remarkably reveal the significant differences in EV mechanics among multiple myeloma patients, lymphoma patients, and healthy volunteers. The study unveils the unique nanomechanical signatures of EVs in hematologic cancers, which will benefit the studies of liquid biopsies for cancer diagnosis and prognosis with translational significance.

Dynamic Characterization of Single Cells Based on Temporal Cellular Mechanical Properties.

The mechanical properties of cells play important roles in regulating the physiological activities of cells and reflect the state of macro-organisms. Although many approaches are available for investigating the mechanical properties of cells, the fluidity of cytoplasm across cell boundaries makes characterizing the dynamics of mechanical properties of single cells exceedingly difficult. In this study, we present a single cell characterization method by modelling the dynamics of cellular mechanical properties measured with an atomic force microscope (AFM). The mechanical dynamics of a single cell system was described by a linear model with a mechanical stimulus as virtual input and mechanical property parameters as outputs. The dynamic mechanical properties of a single cell were characterized by the system matrix of the single cell system. The method was used to classify different types of cells, and the experimental results show that the proposed method outperformed conventional methods by achieving an average classification accuracy of over 90%. The developed method can be used to classify different cancer types according to the mechanical properties of tumour cells, which is of great significance for clinically assisted pathological diagnosis.

A diatom-based biohybrid microrobot with a high drug-loading capacity and pH-sensitive drug release for target therapy.

Targeted delivery is a promising mean for various biomedical applications, and various micro/nano robots have been created for drug delivery. Mesoporous silica has been shown to be successful as a drug delivery carrier in numerous studies. However, mesoporous silica preparation usually requires expensive and toxic chemicals, which limits its biomedical applications. Diatoms, as the naturally porous silica structure, are promising substitutes for the artificial mesoporous silica preparation. However, the current studies utilizing intact diatom frustules as drug delivery packets lack flexible and controllable locomotion. Herein, we propose a biohybrid magnetic microrobot based on Thalassiosira weissflogii frustules (TWFs) as a cargo packet for targeted drug delivery using a simple preparation method. Biohybrid microrobots are fabricated in large quantities by attaching magnetic nanoparticles (Fe3O4) to the surface of diatoms via electrostatic adsorption. Biohybrid microrobots are agile and controllable under the influence of external magnetic fields. They could be precisely controlled to follow specific trajectories or to move as swarms. The cooperation of the two motion modes of the biohybrid microrobots increased microrobots' environmental adaptability. Microrobots have a high drug-loading capacity and pH-sensitive drug release. In vitro cancer cell experiments further demonstrated the controllability of diatom microrobots for targeted drug delivery. The biohybrid microrobots reported in this paper convert natural diatoms into cargo packets for biomedical applications, which possess active and controllable properties and show huge potential for targeted anticancer therapy. STATEMENT OF SIGNIFICANCE: In this study, diatoms with good biocompatibility were used to prepare biohybrid magnetic microrobots. Compared with the current diatom-based systems for drug delivery, the microrobots prepared in this study for targeted drug delivery have more flexible motion characteristics and exhibit certain swarming behaviors. Under the same magnetic field strength, by changing the magnetic field frequency, the movement state of the diatoms can be changed to pass through the narrow channel, so that it has better environmental adaptability.

Bubble-based microrobots enable digital assembly of heterogeneous microtissue modules.

The specific spatial distribution of tissue generates a heterogeneous micromechanical environment that provides ideal conditions for diverse functions such as regeneration and angiogenesis. However, to manufacture microscale multicellular heterogeneous tissue modulesin vitroand then assemble them into specific functional units is still a challenging task. In this study, a novel method for the digital assembly of heterogeneous microtissue modules is proposed. This technique utilizes the flexibility of digital micromirror device-based optical projection lithography and the manipulability of bubble-based microrobots in a liquid environment. The results indicate that multicellular microstructures can be fabricated by increasing the inlets of the microfluidic chip. Upon altering the exposure time, the Young's modulus of the entire module and different regions of each module can be fine-tuned to mimic normal tissue. The surface morphology, mechanical properties, and internal structure of the constructed bionic peritoneum were similar to those of the real peritoneum. Overall, this work demonstrates the potential of this system to produce and control the posture of modules and simulate peritoneal metastasis using reconfigurable manipulation.

Atomic Force Microscopy for Tumor Research at Cell and Molecule Levels.

Tumors have posed a serious threat to human life and health. Researchers can determine whether or not cells are cancerous, whether the cancer cells are invasive or metastatic, and what the effects of drugs are on cancer cells by the physical properties such as hardness, adhesion, and Young's modulus. The atomic force microscope (AFM) has emerged as a key important tool for biomechanics research on tumor cells due to its ability to image and collect force spectroscopy information of biological samples with nano-level spatial resolution and under near-physiological conditions. This article reviews the existing results of the study of cancer cells with AFM. The main foci are the operating principle of AFM and research advances in mechanical property measurement, ultra-microtopography, and molecular recognition of tumor cells, which allows us to outline what we do know it in a systematic way and to summarize and to discuss future directions.

In leukemia, knock-down of the death inducer-obliterator gene would inhibit the proliferation of endothelial cells by inhibiting the expression of CDK6 and CCND1.

BACKGROUND: Endothelial cells (ECs) are a critical component of the hematopoietic niche, and the cross-talk between ECs and leukemia was reported recently. This study aimed to determine the genes involved in the proliferation inhibition of endothelial cells in leukemia. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured alone or co-cultured with K562 cell lines. GeneChip assays were performed to identify the differentially expressed genes. The Celigo, MTT assay, and flow cytometric analysis were used to determine the effect of RNAi DIDO on cell growth and apoptosis. The differently expressed genes were verified by qRT-PCR (quantitative real-time PCR) and western-blot. RESULTS: In K562-HUVEC co-cultured cell lines, 323 down-regulated probes were identified and the extracellular signal-regulated kinase 5 (ERK5) signaling pathway was significantly inhibited. Among the down-regulated genes, the death inducer-obliterator gene (DIDO) is a part of the centrosome protein and may be involved in cell mitosis. As shown in the public data, leukemia patients with lower expression of DIDO showed a better overall survival (OS). The HUVEC cells were infected with shDIDO lentivirus, and reduced expression, inhibited proliferation, and increased apoptosis was observed in shDIDO cells. In addition, the expression of Cyclin-Dependent Kinase 6 (CDK6) and Cyclin D1 (CCND1) genes was inhibited in shDIDO cells. Finally, the public ChIP-seq data were used to analyze the regulators that bind with DIDO, and the H3K4me3 and PolII (RNA polymerase II) signals were found near the Exon1 and exon2 sites of DIDO. CONCLUSION: The knock-down of DIDO will inhibit the proliferation of endothelial cells in the leukemia environment. The expression of DIDO may be regulated by H3K4me3 and the inhibition of DIDO may lead to the down-regulation of CDK6 and CCND1. However, how DIDO interacts with CDK6 and CCND1 requires further study.

Density Regulation and Localization of Cell Clusters by Self-Assembled Femtosecond-Laser-Fabricated Micropillar Arrays.

Tumor cell clusters of varying sizes and densities have different metastatic potentials. Three-dimensional (3D) patterned structures with rational topographical and mechanical properties are capable of guiding the 3D clustering of tumor cells. In this study, single femtosecond laser pulses were used to fabricate individual high-aspect-ratio micropillars via two-photon polymerization (TPP). By combining this approach with capillary-force self-assembly, complex 3D microstructure patterns were constructed with a high efficiency. The microstructures were able to regulate the formation of cell clusters at different cell seeding densities and direct self-guided 3D assembly of cell clusters of various sizes and densities. Localization of cell clusters was achieved using grid-indexed samples to address individual cell clusters, which holds great promise for in situ cell cluster culture and monitoring and for applications such as RNA sequencing of cell clusters.

Concentration optimization of combinatorial drugs using Markov chain-based models.

BACKGROUND: Combinatorial drug therapy for complex diseases, such as HSV infection and cancers, has a more significant efficacy than single-drug treatment. However, one key challenge is how to effectively and efficiently determine the optimal concentrations of combinatorial drugs because the number of drug combinations increases exponentially with the types of drugs. RESULTS: In this study, a searching method based on Markov chain is presented to optimize the combinatorial drug concentrations. In this method, the searching process of the optimal drug concentrations is converted into a Markov chain process with state variables representing all possible combinations of discretized drug concentrations. The transition probability matrix is updated by comparing the drug responses of the adjacent states in the network of the Markov chain and the drug concentration optimization is turned to seek the state with maximum value in the stationary distribution vector. Its performance is compared with five stochastic optimization algorithms as benchmark methods by simulation and biological experiments. Both simulation results and experimental data demonstrate that the Markov chain-based approach is more reliable and efficient in seeking global optimum than the benchmark algorithms. Furthermore, the Markov chain-based approach allows parallel implementation of all drug testing experiments, and largely reduces the times in the biological experiments. CONCLUSION: This article provides a versatile method for combinatorial drug screening, which is of great significance for clinical drug combination therapy.

Enzymatic/Magnetic Hybrid Micromotors for Synergistic Anticancer Therapy.

Micro/nanomotors (MNMs), which propel by transforming various forms of energy into kinetic energy, have emerged as promising therapeutic nanosystems in biomedical applications. However, most MNMs used for anticancer treatment are only powered by one engine or provide a single therapeutic strategy. Although double-engined micromotors for synergistic anticancer therapy can achieve more flexible movement and efficient treatment efficacy, their design remains challenging. In this study, we used a facile preparation method to develop enzymatic/magnetic micromotors for synergetic cancer treatment via chemotherapy and starvation therapy (ST), and the size of micromotors can be easily regulated during the synthetic process. The enzymatic reaction of glucose oxidase, which served as the chemical engine, led to self-propulsion using glucose as a fuel and ST via a reduction in the energy available to cancer cells. Moreover, the incorporation of Fe3O4 nanoparticles as a magnetic engine enhanced the kinetic power and provided control over the direction of movement. Inherent pH-responsive drug release behavior was observed owing to the acidic decomposition of drug carriers in the intracellular microenvironment of cancer cells. This system displayed enhanced anticancer efficacy owing to the synergetic therapeutic strategies and increased cellular uptake in a targeted area because of the improved motion behavior provided by the double engines. Therefore, the demonstrated micromotors are promising candidates for anticancer biomedical microsystems.

Hierarchical Micro-/Nanotopography for Tuning Structures and Mechanics of Cells Probed by Atomic Force Microscopy.

Extracellular matrix plays an important role in regulating the behaviors of cells, and utilizing matrix physics to control cell fate has been a promising way for cell and tissue engineering. However, the nanoscale situations taking place during the topography-regulated cell-matrix interactions are still not fully understood to the best of our knowledge. The invention of atomic force microscopy (AFM) provides a powerful tool to characterize the structures and properties of living biological systems under aqueous conditions with unprecedented spatial resolution. In this work, with the use of AFM, structural and mechanical dynamics of individual cells grown on micro-/nanotopographical surface were revealed. First, the microgroove patterned silicon substrates were fabricated by photolithography. Next, nanogranular topography was formed on microgroove substrates by cell culture medium protein deposition, which was visualized by in situ AFM imaging. The micro-/nanotopographical substrates were then used to grow two types of cells (3T3 cell or MCF-7 cell). AFM morphological imaging and mechanical measurements were applied to characterize the changes of cells grown on the micro-/nanotopographical substrates. The experimental results showed the significant alterations in cellular structures and cellular mechanics caused by micro-/nanotopography. The study provides a novel way based on AFM to unveil the native nanostructures and mechanical properties of cell-matrix interfaces with high spatial resolution in liquids, which will have potential impacts on the studies of topography-tuned cell behaviors.

Characterization of microbiota in acute leukemia patients following successful remission induction chemotherapy without antimicrobial prophylaxis.

PURPOSE: In the present study, we characterized the microbiomes of acute leukemia (AL) patients who achieved complete remission following remission induction chemotherapy (RIC) as outpatients, but who did not receive antimicrobials to treat or prevent febrile neutropenia. METHODS: Saliva and stool samples from 9 patients with acute myeloid leukemia, 11 patients with acute lymphoblastic leukemia, and 5 healthy controls were subjected to 16S ribosomal RNA sequencing at baseline and at 3 months following RIC. Only patients who achieved remission at 3 months post-treatment were included. We excluded anyone who used antimicrobials within 2 months of enrollment or at any time during the study period. RESULTS: At baseline, the relative abundances of species of Prevotella maculosa (P=0.001), Megasphaera micronuciformis (P=0.014), Roseburia inulinivorans (P=0.021), and Bacteroides uniformis (P=0.004) in saliva and Prevotella copri (P=0.002) in the stools of controls were significantly higher than in AL patients. Following RIC, the relative abundances of Eubacterium sp. oral clone DO008 (P=0.012), Leptotrichia sp. oral clone IK040 (P=0.002), Oribacterium sp. oral taxon 108 (P=0.029), Megasphaera micronuciformis (P=0.016), TM7 phylum sp. oral clone DR034 (P<0.001), Roseburia inulinivorans (P=0.034), Actinomyces odontolyticus (P=0.014), Leptotrichia buccalis (P=0.005), and Prevotella melaninogenica (P=0.046) in saliva and Lactobacillus fermentum (P=0.046), Coprococcus catus (P=0.050), butyrate-producing bacterium SS3/4 (P=0.013), and Bacteroides coprocola (P=0.027) in the stools of AL patients were significantly greater than in controls. CONCLUSION: Following RIC, several taxa are changed in stool and salvia samples of AL patients. Our results warrant future large-scale multicenter studies to examine whether the microbiota might have an effect on clinical outcomes of AL patients.

Recent Advances in Three-Dimensional Multicellular Spheroid Culture and Future Development.

Three-dimensional multicellular spheroids (MCSs) have received extensive attention in the field of biomedicine due to their ability to simulate the structure and function of tissues in vivo more accurately than traditional in vitro two-dimensional models and to simulate cell-cell and cell extracellular matrix (ECM) interactions. It has become an important in vitro three-dimensional model for tumor research, high-throughput drug screening, tissue engineering, and basic biology research. In the review, we first summarize methods for MCSs generation and their respective advantages and disadvantages and highlight the advances of hydrogel and microfluidic systems in the generation of spheroids. Then, we look at the application of MCSs in cancer research and other aspects. Finally, we discuss the development direction and prospects of MCSs.

Atomic force microscopy for revealing micro/nanoscale mechanics in tumor metastasis: from single cells to microenvironmental cues.

Mechanics are intrinsic properties which appears throughout the formation, development, and aging processes of biological systems. Mechanics have been shown to play important roles in regulating the development and metastasis of tumors, and understanding tumor mechanics has emerged as a promising way to reveal the underlying mechanisms guiding tumor behaviors. In particular, tumors are highly complex diseases associated with multifaceted factors, including alterations in cancerous cells, tissues, and organs as well as microenvironmental cues, indicating that investigating tumor mechanics on multiple levels is significantly helpful for comprehensively understanding the effects of mechanics on tumor progression. Recently, diverse techniques have been developed for probing the mechanics of tumors, among which atomic force microscopy (AFM) has appeared as an excellent platform enabling simultaneously characterizing the structures and mechanical properties of living biological systems ranging from individual molecules and cells to tissue samples with unprecedented spatiotemporal resolution, offering novel possibilities for understanding tumor physics and contributing much to the studies of cancer. In this review, we survey the recent progress that has been achieved with the use of AFM for revealing micro/nanoscale mechanics in tumor development and metastasis. Challenges and future progress are also discussed.

Detection and isolation of free cancer cells from ascites and peritoneal lavages using optically induced electrokinetics (OEK).

Detection of free gastric cancer cells in peritoneal lavages and ascites plays a vital role in gastric cancer. However, due to the low content of cancer cells in patients' peritoneal lavages, traditional detection methods lack sensitivity and cannot satisfy clinical demand. In this study, we used an optically induced electrokinetics (OEK) microfluidic method for label-free separation and characterization of patient gastric cancer cells. This method showed high effectiveness and sensitivity. We successfully separated cancer cells from a simulated peritoneal lavage mixture of gastric cancer cell lines and peritoneal lavage cells in a ratio of 1:1000. We further separated gastric cancer cells from six patients' ascites with purity up to 71%. In addition, we measured the cell membrane capacitances, which may be used as a biomarker for gastric cancer cells. Thus, our method can be used to effectively and rapidly detect peritoneal metastasis and to acquire cellular electrical information.

Biomimetic construction of peritoneum to imitate peritoneal metastasis using digital micromirror device-based optical projection lithography.

Currently, the mechanisms underlying the peritoneal metastasis of gastric cancer cells and the function of mesothelial cells during this process are unclear, primarily due to the absence of an effective in vitro peritoneal model. In this study, we constructed a biomimetic peritoneal model using a digital micromirror device-based optical projection lithography system. This model enabled the simulation of a damaged peritoneum, which allowed for a comparison of the characteristics of an undamaged peritoneum, such as porosity, mechanical properties, and surface morphology, with those of a damaged peritoneum. Biological inertness and removability of the polyethylene glycol dimethacrylate hydrogel were exploited to fabricate an arrayed heterogeneous interface that imitated a damaged human peritoneum. The porous structure of the peritoneum was achieved by adjusting the ratio of collagen I to gelatin methacryloyl; this structure of the peritoneum might contribute to its shock absorption property. Atomic force microscopy characterization showed that the outermost layers of the model peritoneum and real peritoneum were similar in surface morphology and mechanical properties. Furthermore, we reproduced the process of peritoneal metastasis in vitro. The numbers of gastric cancer cells that adhered to the heterogeneous interface were different, and mesothelial cells played an essential role in peritoneal metastasis. Our findings indicate that this model can be utilized in preclinical drug screening and personalized therapy.