Acute myeloid leukemia (AML) is the most common and lethal leukemia in adults. AML consists of many genetic subtypes, which limits broad applicability of targeted therapy. We discovered that the hematopoiesis-restricted tetraspanin CD37 is expressed on the majority of primary AML blasts and thus may represent a common therapeutic target for AML regardless of subtype. We demonstrate that the internalization properties of CD37 are distinct in AML blasts when compared with normal blood cells, and that CD37 rapidly accumulates inside AML blasts via dynamin-dependent endocytosis. Our work revealed that the clinically relevant anti-CD37 antibody-drug conjugate (ADC) Debio 1562 (alpha CD37DM1) is highly cytotoxic to AML blasts, but not normal hematopoietic stem cells. We found that alpha CD37-DM1 improved clinical outcomes and overall survival in multiple in vivo models of AML. Together, these data demonstrate that targeting CD37 with an ADC such as alpha CD37DM1 is a feasible and promising therapeutic option for the treatment of AML.

Mechanical loading of the intervertebral disc induces biophysical cues at the cellular level that regulate cellular biology and tissue maintenance [1–3]. Of these cues, fluid shear, hydrostatic pressure, and osmotic swelling are key to tissue function, and as such are of importance to measure. The goal of this study was to develop and characterize the response of DNA origami biosensors to these biophysical cues. Tunable DNA origami sensors were created with 2 barrels connected by 6 radially oriented fluctuating connectors.

Comprehending cellular interactions within the extracellular matrix (ECM) is crucial for unraveling the intricacies of cell fate and functions, with far-reaching implications for diseases like metabolic disorders and cancer. These interactions are influenced by the physical and biochemical properties of the surrounding ECM, which constantly remodels through crosstalk between its constituents, maintaining microenvironmental homeostasis. Consequently, investigating intercellular interactions within a 3D tissue model and between ECM components becomes pivotal for elucidating the underlying mechanisms driving disease progression.

Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. Here we characterized and decoupled the effects of the GAG molecules chondroitin sulfate (CS) dermatan sulfate (DS) and hyaluronic acid (HA) on the stiffness (indentation modulus), transport (hydraulic permeability), and matrix microarchitecture (pore size and fiber radius) properties of collagen-based hydrogels. We complement these biophysical measurements of collagen hydrogels with turbidity assays to profile collagen aggregate formation. Here we show that CS, DS, and HA differentially regulate the biophysical properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs play significant roles in defining key physical properties of the ECM, this work shows new ways in which stiffness measurements, microscopy, microfluidics, and turbidity kinetics can be used complementary to reveal details of collagen self-assembly and structure.

Biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces, but they are not optimized for studies in constrained environments as they require attachment to micron-scale probes such as beads or cantilevers. Nanomechanical devices are a promising alternative, but this requires versatile designs that can be tuned to respond to a wide range of forces. We investigate the properties of a nanoscale force sensitive DNA origami device which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, has a binary (open or closed) response to an applied force by undergoing a reversible structural transition. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). The DNA oligonucleotide design parameters also strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show the opening force is tunable in real time by adding a single DNA oligonucleotide. These results establish the potential of the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

in our authorship and readership.In the past year, we had authors from across the globe (Figure 3), with China and the United States leading with the highest share of all published articles.We are also thrilled to see that Advanced NanoBiomed Research has readers all over the world, as shown in the country distribution of article downloads (Figure 4).One of the most exciting developments in Advanced NanoBiomed Research in the past year was the acceptance in the Emerging Sources Citation Index (ESCI) database of Web of Science, which is a testament to its quality and impact.

ABSTRACT DNA origami is a rapidly emerging nanotechnology that enables researchers to create nanostructures with unprecedented geometric precision that have tremendous potential to advance a variety of fields, including molecular sensing, robotics, and nanomedicine. Hence, many students could benefit from exposure to basic knowledge of DNA origami nanotechnology. However, due to the complexity of design, cost of materials, and cost of equipment, experiments with DNA origami have been limited mainly to research institutions in graduate-level laboratories with significant prior expertise and well-equipped laboratories. This work focuses on overcoming critical barriers to translating DNA origami methods to educational laboratory settings. In particular, we present a streamlined protocol for fabrication and analysis of DNA origami nanostructures that can be carried out within a 2-h laboratory course using low-cost equipment, much of which is readily available in educational laboratories and science classrooms. We focus this educational experiment module on a DNA origami nanorod structure that was previously developed for drug delivery applications. In addition to fabricating nanostructures, we demonstrate a protocol for students to analyze structures via gel electrophoresis using classroom-ready gel equipment. These results establish a basis to expose students to DNA origami nanotechnology and can enable or reinforce valuable learning milestones in fields such as biomaterials, biological engineering, and nanomedicine. Furthermore, introducing students to DNA nanotechnology and related fields can also have the potential to increase interest and future involvement by young students.

Sprouting angiogenesis is orchestrated by an intricate balance of biochemical and mechanical cues in the local tissue microenvironment. Interstitial flow has been established as a potent regulator of angiogenesis. Similarly, extracellular matrix (ECM) physical properties, such as stiffness and microarchitecture, have also emerged as important mediators of angiogenesis. However, the interplay between interstitial flow and ECM physical properties in the initiation and control of angiogenesis is poorly understood. Using a three-dimensional (3D) microfluidic tissue analogue of angiogenic sprouting with defined interstitial flow superimposed over ECM with well-characterized physical properties, we found that the addition of hyaluronan (HA) to collagen-based matrices significantly enhances sprouting induced by interstitial flow compared to responses in collagen-only hydrogels. We confirmed that both the stiffness and matrix pore size of collagen-only hydrogels were increased by the addition of HA. Interestingly, interstitial flow-potentiated sprouting responses in collagen/HA matrices were not affected when functionally blocking the HA receptor CD44. In contrast, enzymatic depletion of HA in collagen/HA matrices with hyaluronidase (HAdase) resulted in decreased stiffness, pore size, and interstitial flow-mediated sprouting to the levels observed in collagen-only matrices. Taken together, these results suggest that HA enhances interstitial flow-mediated angiogenic sprouting through its alterations to collagen ECM stiffness and pore size.

DNA origami (DO) nanotechnology has tremendous promise in developing nanodevices for complex functions including mechanically aided drug delivery, molecular sensing, force sensing, and probing single molecule dynamics. Complex and dynamic 3-dimensional DO nanodevices can perform prescribed functions through controlled actuation making their use precise and reproducible. In this study, we focused on the NanoDyn, a previously reported force sensor. This device consists of two origami bundles linked by six parallel 116 base single strand connections that can exist in either an open or closed configuration using a single DNA oligonucleotide closing strand.

This chapter provides an overview of the common procedures used in making functional DNA origami devices. These procedures include the design, assembly, purification, and characterization of the DNA origami structures, with a focus on dynamic devices.

DNA origami (DO) nanotechnology has strong potential for applications including molecular sensing, drug delivery, and nanorobotics that rely on nanoscale structural precision and the ability to tune mechanical and dynamic properties. Given these emerging applications, there is a need to broaden access to and training on DO concepts, which would also provide an avenue to demonstrate engineering concepts such as kinematic motion and mechanical deformation as applied to nanotechnology and molecular systems. However, broader use in educational settings is hindered by the excessive cost and time of fabrication and analysis. Compliant, or deformable, DO is especially difficult to design and characterize in a cost-effective manner, because analysis often relies on advanced imaging methods to quantify structure conformations. Building on recent work establishing classroom-ready methods for DO fabrication and analysis, we developed an experiment module for classroom implementation focused on a DO compliant hinge joint. The module consists of folding three distinct joint conformations that can be evaluated via gel electrophoresis using portable and cost-effective equipment within ~120 min. To highlight the mechanical design, we present two beam-based models for describing the deformation that controls the joint angle. We envision that this module can broaden access to and interest in the mechanical design of DO.

In most solid tumors, malignant cells coexist with non-cancerous host tissue comprised of a variety of extracellular matrix components and cell types, notably fibroblasts, immune cells, and endothelial cells. It is becoming increasingly evident that the non-cancerous host tissue, often referred to as the tumor stroma or the tumor microenvironment, wields tremendous influence in the proliferation, survival, and metastatic ability of cancer cells. The tumor stroma has an active biological role in the transmission of signals, such as growth factors and chemokines that activate oncogenic signaling pathways by autocrine and paracrine mechanisms. Moreover, the constituents of the stroma define the mechanical properties and the physical features of solid tumors, which influence cancer progression and response to therapy. Inspired by the emerging importance of tumor-stroma crosstalk and oncogenic physical forces, numerous biosensors, or advanced imaging and analysis techniques have been developed and applied to investigate complex and challenging questions in cancer research. These techniques facilitate measurements and biological readouts at scales ranging from subcellular to tissue-level with unprecedented level of spatial and temporal precision. Here we examine the application of biosensor technology for studying the complex and dynamic multiscale interactions of the tumor-host system.

The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a three-dimensional (3D) microenvironment can inform detection and understanding of cell processes and the development of effective therapeutic agents. DNA origami technology allows for the design and fabrication of biocompatible and 3D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3D tissue model using a microfluidic device. We used a DNA origami cell sensing platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3D environments under controlled biomolecular transport.

DNA origami nanotechnology is a rapidly developing field that shows promise in scientific applications such as mechanically aided drug delivery, molecular sensing, force sensing, and probing of single molecule dynamics. Complex and dynamic 3-dimensional structures can perform a prescribed function through controlled actuation making their use precise and reproducible. The device in our study, called a nanodyn, acts as a force sensor. Consisting of two origami bundles linked by six crossover strands, the device can exist in either an open or closed configuration.

Mechanical forces due to fluid shear stress can promote the metastatic capabilities of cancer cells. However, measuring shear flow in native 3-D tissue environments can be challenging due to the high inhomogeneity of the composition and microarchitecture of the tumor extracellular matrix (ECM). DNA-based molecular sensors have been of particular interest for studying cell interactions due to their high tunability, sensitivity and easy integrability into various biological systems. However, measuring cell-ECM interactions due to fluid flow using these sensors is yet to be achieved.

The extracellular matrix (ECM) enveloping cells in living tissue is comprised of signaling and structural support molecules. The ECM is vital to maintenance of normal tissue as it controls cell signaling and is a semiporous barrier to interstitial fluid flow. Two important types of ECM molecules are fibrillar collagen (e.g. type I) and glycosaminoglycans (GAGs). In certain solid tumors, hyaluronic acid (HA) and chondroitin sulfate (CS) are two types of GAG molecules that are known to be unusually abundant. These GAGs are negatively charged with the propensity to imbibe aqueous fluid and promote tissue swelling. While many studies have described the effect each molecule has on shaping ECM properties, studies describing how the CS and HA interact with each other are much less common. This study uses an integrated approach to characterize the effects of HA and CS on fluid transport (hydraulic permeability), stiffness (indentation modulus), collagen polymerization kinetics (turbidity), and matrix microarchitecture (pore size and fiber radius). Utilizing a microfluidic approach to study hydraulic permeability observed that the addition of GAGs decreased convective fluid transport. Indenting the hydrogels with a macroscale indenter demonstrated no changes in stiffness when adding CS, while HA, and CS/HA increased hydrogel stiffness. Analysis of confocal reflectance microscopy images showed increases in both pore size and fiber radius with the addition of both GAG molecules. Finally, turbidity measurements concluded that adding GAGs to a collagen gel accelerated its polymerization, with the change in fiber radius being the primary determinant in enhanced fibril formation. This study provides additional evidence that GAGs play an important role in defining physical properties of the ECM and demonstrates further ways into controllably modifying collagen based hydrogels via the addition of native ECM molecules.

Multicellular organisms depend on cell-cell and cell-environment interactions to guide spatiotemporal patterning and organization of tissue. Dysregulation of these interactions underlie disease progression, such as metabolic disorders, autoimmune diseases, and cancer. Thus, the ability to externally monitor cellular interactions is of a great significance. Conventional assays such as ELISA only focus on the end point cell respond and fail to provide real-time information of single cells interaction with its microenvironment. More recent studies have focused on engineering cell surface proteins or incorporating synthetic protein constructs into the cell membrane, which are only capable of understanding cell interaction with one single target molecule and cannot monitor the interaction of cell membrane with multiple biomolecules. Moreover, cell interactions are highly affected by physical properties of the surrounding extracellular matrix and its biochemical properties. Hence, it is of a great significant to study intercellular interaction within a 3D tissue model which leads to elucidating on underlying mechanisms of disease progression mediated by those interactions. In this study, we established a method to sense multiple biomolecules on the membrane of living cells in the tissue model. We designed a DNA origami Cell Sensing Platform (CSP) capable of detecting the presence of two specific DNA nucleic acid sequences with fluorescence-based reporting and successfully mounted the structures on the membrane of suspension and adherent cell types, and seeded those into collagen matrix. Using microfluidics to precisely control the ECM structure formation and the flow of target molecules, we show how multifunctional DNA origami devices can be used to elucidate on temporal interactions of cells and their local environment with subcellular resolution in a model tissue system.

Cancer is a complex and dynamic disease that is aberrant both biologically and physically. There is growing appreciation that physical abnormalities with both cancer cells and their microenvironment that span multiple length scales are important drivers for cancer growth and metastasis. The scope of this review is to highlight the key advancements in micro- and nano-scale tools for delineating the cause and consequences of the aberrant physical properties of tumors. We focus our review on three important physical aspects of cancer: 1) solid mechanical properties, 2) fluid mechanical properties, and 3) mechanical alterations to cancer cells. Beyond posing physical barriers to the delivery of cancer therapeutics, these properties are also known to influence numerous biological processes, including cancer cell invasion and migration leading to metastasis, and response and resistance to therapy. We comment on how micro- and nanoscale tools have transformed our fundamental understanding of the physical dynamics of cancer progression and their potential for bridging towards future applications at the interface of oncology and physical sciences.

In this paper we study the dynamics of single cells encapsulated in water-in-oil emulsions in a microchannel. The flow field of a microfluidic channel is coupled to the internal flow field of a droplet through viscous traction at the interface, resulting in a rotational flow field inside the droplet. An encapsulated single cell being subjected to this flow field responds by undergoing multiple orbits, spins, and deformations that depend on its physical properties. Monitoring the cell dynamics, using a high-speed camera, can lead to the development of new label-free methods for the detection of rare cells, based on their biomechanical properties. A sheath flow microchannel was proposed to strengthen the rotational flow field inside droplets flowing in Poiseuille flow conditions. A numerical model was developed to investigate the effect of various parameters on the rotational flow field inside a droplet. The multi-phase flow model required the tracking of the fluid–fluid interface, which deforms over time due to the applied shear stresses. Experiments confirmed the significant effect of the sheath flow rate on the cell dynamics, where the speed of cell orbiting was doubled. Doubling the cell speed can double the amount of extracted biomechanical information from the encapsulated cell, while it remains within the field of view of the camera used.