Acoustic Force Elastography Microscopy.

OBJECTIVE: Hydrogel scaffolds have attracted attention to develop cellular therapy and tissue engineering platforms for regenerative medicine applications. Among factors, local mechanical properties of scaffolds drive the functionalities of cell niche. Dynamic mechanical analysis (DMA), the standard method to characterize mechanical properties of hydrogels, restricts development in tissue engineering because the measurement provides a single elasticity value for the sample, requires direct contact, and represents a destructive evaluation preventing longitudinal studies on the same sample. We propose a novel technique, acoustic force elastography microscopy (AFEM), to evaluate elastic properties of tissue engineering scaffolds. RESULTS: AFEM can resolve localized and two-dimensional (2D) elastic properties of both transparent and opaque materials with advantages of being non-contact and non-destructive. Gelatin hydrogels, neat synthetic oligo[poly(ethylene glycol)fumarate] (OPF) scaffolds, OPF hydroxyapatite nanocomposite scaffolds and ex vivo biological tissue were examined with AFEM to evaluate the elastic modulus. These measurements of Young's modulus range from approximately 2 kPa to over 100 kPa were evaluated and are in good agreement with finite element simulations, surface wave measurements, and DMA tests. CONCLUSION: The AFEM can resolve localized and 2D elastic properties of hydrogels, scaffolds and thin biological tissues. These materials can either be transparent or non-transparent and their evaluation can be done in a non-contact and non-destructive manner, thereby facilitating longitudinal evaluation. SIGNIFICANCE: AFEM is a promising technique to quantify elastic properties of scaffolds for tissue engineering and will be applied to provide new insights for exploring elastic changes of cell-laden scaffolds for tissue engineering and material science.

In vitro and in vivo effects of AVA4746, a novel competitive antagonist of the ligand binding of VLA-4, in B-cell acute lymphoblastic leukemia.

Treatment of resistant or recurrent acute lymphoblastic leukemia (ALL) remains a challenge. It was previously demonstrated that the adhesion molecule integrin α4, referred to hereafter as α4, mediates the cell adhesion-mediated drug resistance (CAM-DR) of B-cell ALL by binding to vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stroma. In addition, it was previously observed that the blockade of α4 with natalizumab or inhibition using the small molecule antagonist TBC3486 sensitized relapsed ALL cells to chemotherapy. However, α4-targeted therapy is not clinically available for the treatment of leukemia to date. In the present study, the use of a novel non-peptidic small molecule integrin α4 antagonist, AVA4746, as a potential new approach to combat drug-resistant B-ALL was explored. An in vitro co-culture = model of primary B-ALL cells and an in vivo xenograft model of patient-derived B-ALL cells were utilized for evaluation of AVA4746. VLA-4 conformation activation, cell adhesion/de-adhesion, endothelial tube formation, in vivo leukemia cell mobilization and survival assays were performed. AVA4746 exhibited high affinity for binding to B-ALL cells, where it also efficiently blocked ligand-binding to VCAM-1. In addition, AVA4746 caused the functional de-adhesion of primary B-ALL cells from VCAM-1. Inhibition of α4 using AVA4746 also prevented angiogenesis in vitro and when applied in combination with chemotherapy consisting of Vincristine, Dexamethasone and L-asparaginase, it prolonged the survival of ~33% of the mice in an in vivo xenograft model of B-ALL. These data implicate the potential of targeting the α4-VCAM-1 interaction using AVA4746 for the treatment of drug-resistant B-lineage ALL.

Characterizing blood clots using acoustic radiation force optical coherence elastography and ultrasound shear wave elastography.

Thromboembolism in a cerebral blood vessel is associated with high morbidity and mortality. Mechanical thrombectomy (MT) is one of the emergenc proceduresperformed to remove emboli. However, the interventional approaches such as aspiration catheters or stent retriever are empirically selected. An inappropriate selection of surgical devices can influence the success rate during embolectomy, which can lead to an increase in brain damage. There has been growing interest in the study of clot composition and using a priori knowledge of clot composition to provide guidance for an appropriate treatment strategy for interventional physicians. Developing imaging tools which can allow interventionalists to understand clot composition could affect management and device strategy. In this study, we investigated how clots of different compositions can be characterized by using acoustic radiation force optical coherence elastography (ARF-OCE) and compared with ultrasound shear wave elastography (SWE). Five different clots compositions using human blood were fabricated into cylindrical forms from fibrin-rich (21% red blood cells, RBCs) to RBC-rich (95% RBCs). Using the ARF-OCE and SWE, we characterized the wave velocities measured in the time-domain. In addition, the semi-analytical finite element model was used to explore the relationship between the phase velocities with various frequency ranges and diameters of the clots. The study demonstrated that the wave group velocities generally decrease as RBC content increases in ARF-OCE and SWE. The correlation of the group velocities from the OCE and SWE methods represented a good agreement as RBC composition is larger than 39%. Using the phase velocity dispersion analysis applied to ARF-OCE data, we estimated the shear wave velocities decoupling the effects of the geometry and material properties of the clots. The study demonstrated that the composition of the clots can be characterized by elastographic methods using ARF-OCE and SWE, and OCE demonstrated better ability to discriminate between clots of different RBC compositions, compared to the ultrasound-based approach, especially in clots with low RBC compositions.

Characterizing the Motility of Chemotherapeutics-Treated Acute Lymphoblastic Leukemia Cells by Time-Lapse Imaging.

Drug resistance is an obstacle in the therapy of acute lymphoblastic leukemia (ALL). Whether the physical properties such as the motility of the cells contribute to the survival of ALL cells after drug treatment has recently been of increasing interest, as they could potentially allow the metastasis of solid tumor cells and the migration of leukemia cells. We hypothesized that chemotherapeutic treatment may alter these physical cellular properties. To investigate the motility of chemotherapeutics-treated B-cell ALL (B-ALL) cells, patient-derived B-ALL cells were treated with chemotherapy for 7 days and left for 12 h without chemotherapeutic treatment. Two parameters of motility were studied, velocity and migration distance, using a time-lapse imaging system. The study revealed that compared to non-chemotherapeutically treated B-ALL cells, B-ALL cells that survived chemotherapy treatment after 7 days showed reduced motility. We had previously shown that Tysabri and P5G10, antibodies against the adhesion molecules integrins α4 and α6, respectively, may overcome drug resistance mediated through leukemia cell adhesion to bone marrow stromal cells. Therefore, we tested the effect of integrin α4 or α6 blockade on the motility of chemotherapeutics-treated ALL cells. Only integrin α4 blockade decreased the motility and velocity of two chemotherapeutics-treated ALL cell lines. Interestingly, integrin α6 blockade did not affect the velocity of chemoresistant ALL cells. This study explores the physical properties of the movements of chemoresistant B-ALL cells and highlights a potential link to integrins. Further studies to investigate the underlying mechanism are warranted.

Investigation of cell mechanics using single-beam acoustic tweezers as a versatile tool for the diagnosis and treatment of highly invasive breast cancer cell lines: an in vitro study.

Advancements in diagnostic systems for metastatic cancer over the last few decades have played a significant role in providing patients with effective treatment by evaluating the characteristics of cancer cells. Despite the progress made in cancer prognosis, we still rely on the visual analysis of tissues or cells from histopathologists, where the subjectivity of traditional manual interpretation persists. This paper presents the development of a dual diagnosis and treatment tool using an in vitro acoustic tweezers platform with a 50 MHz ultrasonic transducer for label-free trapping and bursting of human breast cancer cells. For cancer cell detection and classification, the mechanical properties of a single cancer cell were quantified by single-beam acoustic tweezers (SBAT), a noncontact assessment tool using a focused acoustic beam. Cell-mimicking phantoms and agarose hydrogel spheres (AHSs) served to standardize the biomechanical characteristics of the cells. Based on the analytical comparison of deformability levels between the cells and the AHSs, the mechanical properties of the cells could be indirectly measured by interpolating the Young's moduli of the AHSs. As a result, the calculated Young's moduli, i.e., 1.527 kPa for MDA-MB-231 (highly invasive breast cancer cells), 2.650 kPa for MCF-7 (weakly invasive breast cancer cells), and 2.772 kPa for SKBR-3 (weakly invasive breast cancer cells), indicate that highly invasive cancer cells exhibited a lower Young's moduli than weakly invasive cells, which indicates a higher deformability of highly invasive cancer cells, leading to a higher metastasis rate. Single-cell treatment may also be carried out by bursting a highly invasive cell with high-intensity, focused ultrasound.

Integrin Antibody Decreases Deformability of Patient-Derived Pre-B Acute Lymphocytic Leukemia Cells as Measured by High-Frequency Acoustic Tweezers.

OBJECTIVES: This article reports a study of cell mechanics in patient-derived (primary) B-cell acute lymphocytic leukemia (ALL) cells treated with antibodies against integrins. Leukemia cell adhesion to stromal cells mediates chemotherapeutic drug resistance, also known as cell adhesion-mediated chemotherapeutic drug resistance. We have previously shown that antibodies against integrin α4 and α6 adhesion molecules can de-adhere ALL cells from stromal cells or counter-receptors. Because drug-resistant cells are more deformable, as evaluated by single-beam acoustic tweezers, we hypothesized that changes in cell mechanics might contribute to the de-adhesive effect of integrin-targeting antibodies. METHODS: In this study, the deformability of primary pre-B ALL cells was evaluated by single-beam acoustic tweezers after treatments with the de-adhering antibody Tysabri or P5G10 against integrin α4 and α6 adhesion molecules. RESULTS: We demonstrated that primary ALL cells treated with P5G10 expressed decreased deformability compared with immunoglobulin G1 -treated control cells (P < .05). Tysabri did not show an effect on deformability (P > .05). CONCLUSIONS: These results suggest that decreased deformability is associated with an integrin α6 blockade. Further assessments of the functional roles of deformability and integrin blockades in B-ALL cell drug resistance and deformability, respectively, are necessary.

Acoustic radiation force optical coherence elastography for evaluating mechanical properties of soft condensed matters and its biological applications.

Evaluating mechanical properties of biological soft tissues and viscous mucus is challenging because of complicated dynamic behaviors. Soft condensed matter models have been successfully used to explain a number of dynamical behaviors. Here, we reported that optical coherence elastography (OCE) is capable of quantifying mechanical properties of soft condensed matters, micellar fluids. A 7.5 MHz focused transducer was utilized to generate acoustic radiation force exerted on the surface of soft condensed matters in order to produce Rayleigh waves. The waves were recorded by optical coherence tomography (OCT). The Kelvin-Voigt model was adopted to evaluate shear modulus and loss modulus of soft condensed matters. The results reported that various concentrations of micellar fluids can provide reasonable ranges of elasticity from 65.71 to 428.78 Pa and viscosity from 0.035 to 0.283 Pa·s, which are close to ranges for actual biological samples, like mucus. OCE might be a promising tool to differentiate pathologic mucus samples from healthy cases as advanced applications in the future.

Characterizing Deformability of Drug Resistant Patient-Derived Acute Lymphoblastic Leukemia (ALL) Cells Using Acoustic Tweezers.

The role of cell mechanics in cancer cells is a novel research area that has resulted in the identification of new mechanisms of therapy resistance. Single beam acoustic (SBA) tweezers are a promising technology for the quantification of the mechanical phenotype of cells. Our previous study showed that SBA tweezers can be used to quantify the deformability of adherent breast cancer cell lines. The physical properties of patient-derived (primary) pre-B acute lymphoblastic leukemia (ALL) cells involved in chemotherapeutic resistance have not been widely investigated. Here, we demonstrate the feasibility of analyzing primary pre-B ALL cells from four cases using SBA tweezers. ALL cells showed increased deformability with increasing acoustic pressure of the SBA tweezers. Moreover, ALL cells that are resistant to chemotherapeutic drugs were more deformable than were untreated ALL cells. We demonstrated that SBA tweezers can quantify the deformability of nonadherent leukemia cells and discriminate this mechanical phenotype in chemotherapy-resistant leukemia cells in a contact- and label-free manner.

Effects of CD49d-targeted antisense-oligonucleotide on α4 integrin expression and function of acute lymphoblastic leukemia cells: Results of in vitro and in vivo studies.

We recently demonstrated the effectiveness of blocking CD49d with anti-functional antibodies or small molecule inhibitors as a rational targeted approach to the treatment of acute leukemia in combination with chemotherapy. Antisense oligonucleotide promises to be no less specific than antibodies and inhibitors, but more interesting for pharmacokinetics and pharmacodynamics. We addressed this using the published CD49d antisense drug ATL1102. In vitro, we incubated/nucleofected the ALL cell line Kasumi-2 with ATL1102. In vivo, immunodeficient hosts were engrafted with primary ALL cells and treated with ATL1102. Changes in expression of CD49d mRNA and CD49d protein, and of cooperating gene products, including ß1 integrin and CXCR4, as well as survival in the mouse experiments were quantified. We observed dose-dependent down-regulation of CD49d mRNA and protein levels and its partner integrin ß1 cell surface protein level and, up-regulation of CXCR4 surface expression. The suppression was more pronounced after nucleofection than after incubation, where down-regulation was significant only at the higher doses. In vivo effects of ATL1102 were not sufficient to translate into "clinical" benefit in the leukemia model. In summary, antisense oligonucleotides are successful tools for specifically modulating gene expression but sufficient delivery to down-regulate CD49d in vivo may be difficult to achieve.