Computational Modeling to Determine the Effect of Phenotypic Heterogeneity in Tumors on the Collective Tumor-Immune Interactions.

Tumor immunotherapy aims to maintain or enhance the killing capability of CD8+ T cells to clear tumor cells. The tumor-immune interactions affect the function of CD8+ T cells. However, the effect of phenotype heterogeneity of a tumor mass on the collective tumor-immune interactions is insufficiently investigated. We developed the cellular-level computational model based on the principle of cellular Potts model to solve the case mentioned above. We considered how asymmetric division and glucose distribution jointly regulated the transient changes in the proportion of proliferating/quiescent tumor cells in a solid tumor mass. The evolution of a tumor mass in contact with T cells was explored and validated by comparing it with previous studies. Our modeling exhibited that proliferating/quiescent tumor cells, exhibiting distinct anti-apoptotic and suppressive behaviors, redistributed within the domain accompanied by the evolution of a tumor mass. Collectively, a tumor mass prone to a quiescent state weakened the collective suppressive functions of a tumor mass on cytotoxic T cells and triggered a decline of apoptosis of tumor cells. Although quiescent tumor cells did not sufficiently do their inhibitory functions, the possibility of long-term survival was improved due to their interior location within a mass. Overall, the proposed model provides a useful framework to investigate collective-targeted strategies for improving the efficiency of immunotherapy.

The modeling study of the effect of morphological behaviors of extracellular matrix fibers on the dynamic interaction between tumor cells and antitumor immune response.

Low response rate limits the effective application of immunotherapy, in which the interactions between tumor cells and immune cells play a significant role. The strength of regulation could be mediated by extracellular matrix (ECM) fibers, which is still insufficiently investigated. In the study, the cellular potts model was utilized to explore the role of morphological properties of ECM in tumor-immune interactions. It was observed that high-density random ECM fibers delayed the interaction between tumor cells and T cells. Moreover, the tumor-immune interactions were ECM morphology-specific. Radial ECM fibers exhibited weaker inhibitory role in the process of contact between tumor cells and T cells. This study provided the useful mechanism of tumor-immune interactions from the viewpoint of morphological effect of ECM fibers, facilitating improving the efficiency of immunotherapy.

The Measurement and Analysis of Impedance Response of HeLa Cells to Distinct Chemotherapy Drugs.

Electric cell-substrate impedance sensing exhibits a real-time and label-free feature to monitor the response of cells stimulated by various biochemical and mechanical signals. Alterations in the currents passing through the cell-electrode system characterize the impedance variations of cells. The impedance responses of HeLa cells under distinct chemotherapy drugs combine the effects of cell proliferation and cell-substrate adhesion. Optimal interdigitated electrodes were selected to explore the impedance responses of HeLa cells. Measurements of impedance of cells in response to three widely used chemotherapy drugs in clinical practice, namely cisplatin, doxorubicin, 5-fluorouracil, were performed. The results demonstrated that distinct impedance responses of HeLa cells to drugs were exhibited and a decrease in measured impedance was observed after drug treatment, accompanied by alterations in the distribution and intensity of the adhesion-related protein vinculin and the rate of cell proliferation. The link between the impedance profiles of HeLa cells and their biological functions was developed based on the circuit model. This study demonstrated the weights of cell proliferation and adhesion of HeLa cells under the treatments of DDP, DOX, and 5-FU, resulted in distinct impedance responses of cells, providing an impedance-based evaluation methodology for cervical cancer treatment.

Effect of cell-nanostructured substrate interactions on the capture efficiency of HeLa cells.

Circulating tumor cells (CTCs) are regarded as an effective biomarker for cancer detection, diagnosis and prognosis monitoring. CTCs capture based on nanostructured substrates is a powerful technique. Some specific adhesion molecule antibody coated on the surface of nanostructured substrates, such as EpCAM, is commonly used to enhance the CTCs capture efficiency. Substrate nanotopographies regulate the interaction between the substrates and captured cells, further influencing cell capture efficiency. However, the relationship between cell capture efficiency and cell-substrate interaction remains poorly understood. Here, we explored the relationship between cell capture efficiency and cell-substrate interaction based on two sets of nanostructures with different nanotopographies without antibody conjugation. Given the urgent demand for improving the capture efficiency of EpCAM-negative cells, we used HeLa (EpCAM-negative) cells as the main targets. We demonstrated that HeLa cells could be more effectively captured by two nanostructural substrates, especially by double-layer composite nanoforests. Therefore, the morphological and migrating interaction between HeLa cells and distinct substrates was associated with cell capture efficiency. Our findings demonstrated the potential mechanism for optimizing the nanotopography for higher capture efficiency, and provide a potential foundation for cancer detection, diagnosis and treatment.

Inhibiting Cell Viability and Motility by Layer-by-Layer Assembly and Biomineralization.

Herein, we proposed a drug-free strategy named cell surface shellization to inhibit the motility of SKOV-3 and HeLa cells. We alternately deposited two- or three-layer cationic polyelectrolyte (PE) and anionic PE films on the surface of SKOV-3 and HeLa cells. Then, a mineral shell (calcium carbonate, CaCO3) was formed on the surface of polymer shells via electrostatic force and biomineralization. The CCK-8 assay results and live/dead staining showed that the surface shells strongly aggravated the cytotoxicity. The monolayer scratch wound migration assay results and immunofluorescence staining results showed that the shells, especially the mineral shells, could efficiently inhibit the migration of SKOV-3 and HeLa cells without any anticancer drugs. The immunofluorescence results of the three small G proteins of the cells showed that the immunofluorescence intensity in SKOV-3 did not change. Preliminary results from our laboratory showed an increase in MMP-9 secreted by cancer cells after coating with films or mineral shells. It suggests that mechanisms that inhibit cell migration are related to the MMP signaling pathway. All the results indicated that shellization (films or nanomineral shells) but not limited to calcification can be used as one of the tools to change the function of cells.

Preparation and characterization of porous hydroxyapatite/ß-cyclodextrin-based polyurethane composite scaffolds for bone tissue engineering.

In this study, novel porous scaffolds containing hydroxyapatite and ß-cyclodextrin-based polyurethane were first successfully fabricated by polymerizing ß-cyclodextrin with hexamethylene diisocyanate and hydroxyapatite in situ for bone tissue engineering. The physicochemical and mechanical properties as well as cytocompatibility of porous scaffolds were investigated. The results showed that polyurethane reinforced with hydroxyapatite composites had cancellous bone-like porous structure. The mechanical strength of the scaffolds increased with increasing the hydroxyapatite content in scaffolds. Synthesized scaffolds (PU1, PUHA1, PU2, and PUHA2) presented compressive strength values of 0.87 ± 0.24 MPa, 1.81 ± 0.10 MPa, 6.16 ± 0.89 MPa, and 12.95 ± 2.05 MPa, respectively. The pore size and porosity of these scaffolds were suitable for bone regeneration. Cytocompatibility of composite scaffolds was proven via favorable interactions with MC3T3-E1 cells. The addition of hydroxyapatite into CD-based polyurethane scaffolds improved cell attachment, well-spread morphology, and higher proliferation. The hydroxyapatite-polyurethane scaffolds have the potential to be applied in bone repair and regeneration.

Effect of hydroxyapatite fillers on the mechanical properties and osteogenesis capacity of bio-based polyurethane composite scaffolds.

A newly designed hydroxyapatite-polyurethane (HA-PU) composite scaffold was prepared by polymerizing glyceride of castor oil (GCO) with isophorone diisocyanate (IPDI) and HA as fillers. The aim of this study was to determine the effect of HA fillers on the mechanical properties and osteogenesis capacity of the composite scaffolds. The physical and biological properties of the scaffold were evaluated by SEM observation, mechanical testing, cell culture and animal experiments. The results showed that HA fillers enhanced the mechanical properties of PU composite scaffolds such as compressive strength and elastic modulus. The mechanical properties of the scaffolds were seen to increase with increase in HA loading. The compressive strength of composite scaffold with 0 wt%, 20 wt%, 40 wt% of HA was 0.6 ±â€¯0.1 MPa, 2.1 ±â€¯0.1 MPa, and 4.6 ±â€¯0.3 MPa, respectively. In vitro biodegradation studies of scaffolds were carried out. The results showed that all of the scaffolds were susceptible to cholesterol esterase (CE) -catalyzed degradation. HA-PU composite scaffolds exhibited a high affinity to osteoblastic cells and were good template for cell growth and proliferation. When implanted in bone defects of rats, PU scaffolds incorporated HA were biocompatible with the tissue host and had no immune rejection. Moreover, the higher the loading of HA in the composite scaffold, the better chances of osteogenesis. It confirmed that the prepared HA-PU composite scaffolds can be promising candidate for bone repair and bone tissue engineering.

Reinforced chitosan membranes by microspheres for guided bone regeneration.

In order to improve the osteogenic activity and mechanical strength of the guided bone regeneration (GBR) membrane for repairing bone defect, nano-hydroxyapatite/chitosan (nHA/CS) composite microspheres were prepared through in situ biomimetic method, then composite microspheres were incorporated into CS membrane. The morphologies and mechanical properties of the composite membranes were investigated through scanning electronic microscopy (SEM) and universal mechanical testing machine. The results show that the in situ biomimetic nHA/CS microspheres were embedded in CS membrane and were integrated tightly with CS matrix. The mechanical properties of GBR membranes containing in situ nHA/CS microspheres is significantly higher than that of membranes containing pure CS microspheres and blending nHA/CS microspheres. Its elongation rate at break reaches 5.61 ±â€¯0.95%. The elastic modulus and strength of the GBR membranes can reach 766.27 ±â€¯20.68 and 43.32 ±â€¯0.95 MPa, respectively. Further, The work-of-fracture of the membranes with in situ microspheres approaches 2.71 ±â€¯0.25 J/m2, which is about 3 times of the pure CS membrane. The cell culture results display that the GBR membranes containing in situ biomimetic nHA/CS microspheres exhibit good cytocompatibility.

Characterization of biomechanical properties of cells through dielectrophoresis-based cell stretching and actin cytoskeleton modeling.

BACKGROUND: Cytoskeleton is a highly dynamic network that helps to maintain the rigidity of a cell, and the mechanical properties of a cell are closely related to many cellular functions. This paper presents a new method to probe and characterize cell mechanical properties through dielectrophoresis (DEP)-based cell stretching manipulation and actin cytoskeleton modeling. METHODS: Leukemia NB4 cells were used as cell line, and changes in their biological properties were examined after chemotherapy treatment with doxorubicin (DOX). DEP-integrated microfluidic chip was utilized as a low-cost and efficient tool to study the deformability of cells. DEP forces used in cell stretching were first evaluated through computer simulation, and the results were compared with modeling equations and with the results of optical stretching (OT) experiments. Structural parameters were then extracted by fitting the experimental data into the actin cytoskeleton model, and the underlying mechanical properties of the cells were subsequently characterized. RESULTS: The DEP forces generated under different voltage inputs were calculated and the results from different approaches demonstrate good approximations to the force estimation. Both DEP and OT stretching experiments confirmed that DOX-treated NB4 cells were stiffer than the untreated cells. The structural parameters extracted from the model and the confocal images indicated significant change in actin network after DOX treatment. CONCLUSION: The proposed DEP method combined with actin cytoskeleton modeling is a simple engineering tool to characterize the mechanical properties of cells.

Reorganization of cytoskeleton and transient activation of Ca2+ channels in mesenchymal stem cells cultured on silicon nanowire arrays.

Tissue engineering combines biological cells and synthetic materials containing chemical signaling molecules to form scaffolds for tissue regeneration. Mesenchymal stem cells (MSCs) provide an attractive source for tissue engineering due to their versatility of multipotent differentiation. Recently, it has been recognized that both chemical and mechanical stimulations are essential mediators of adhesion and differentiation of MSCs. While significant progress has been made on the understanding of chemical regulatory factors within the extracellular matrix, the effects of mechanical stimulation exerted by nanomaterials on MSCs and the underlying mechanisms are less well-known. The present study showed that the adhesion, proliferation, and differentiation of MSCs cultured on vertically aligned silicon nanowire (SiNW) arrays were significantly different from those on flat silicon wafer and control substrates. The interactions between MSCs and the SiNW arrays caused the stem cells to preferentially differentiate toward osteocytes and chondrocytes but not adipocytes in the absence of supplementary growth factors. Our study demonstrated that Ca(2+) ion channels were transiently activated in MSCs upon mechanical stimulation, which eventually led to activation of Ras/Raf/MEK/ERK signaling cascades to regulate adhesion, proliferation, and differentiation of MSCs. The stretch-mediated transient Ca(2+) ion channel activation and cytoskeleton reorganization during stem cell-nanowire interaction may be early events of lineage-specific potentiation of MSCs in determining the fates of mesenchymal stem cells cultured on microenvironments with specific mechanical properties.

Influence of semiflexible structural features of actin cytoskeleton on cell stiffness based on actin microstructural modeling.

A new actin cytoskeleton microstructural model based on the semiflexible polymer nature of the actin filament is proposed. The relationship between the stretching force and the mechanical properties of cells was examined. Experiments on deforming hematopoietic cells with distinct primitiveness from normal and leukemic sources were conducted via optical tweezer manipulation at single-cell level. The modeling results were demonstrated to be in good agreement with the experimental data. We characterized how the structural properties of the actin cytoskeleton, such as prestress, density of cross-links, and actin concentration, affect the mechanical behavior of cells based on the proposed model. Increasing prestress, actin concentration, and density of cross-links reduced cell deformation, and the cell also exhibited strain stiffening behavior with an increase in the stretching force. Compared with existing models, the proposed model exhibits a distinct feature in probing the influence of semiflexible polymer nature of the actin filament on cell mechanical behavior.