Christopher B. Raub

Directed cell migration is central to the early stages of breast cancer metastasis, when cancer cells detach from the primary tumor and migrate toward distant sites. Here, we use magnetic fields to manipulate this process and examine directional migration and morphology. Magnetic alginate-chitosan (MAC) beads were embedded within a collagen hydrogel to create force-driven micro-topography, and we compared the migration and morphology of MDA-MB-231 breast cancer cells on these constructs. Using video microscopy, we quantified changes in cell shape, velocity, and migration direction in response to intratissue strain. The magnetically actuated cancer metastasis model (MACMM) provides a controllable platform for regulating the magnitude and direction of applied forces to mimic tumor-stroma forces. Highly invasive MDA-MB-231 cells migrated more slowly near the beads, where strain was minimal. A computational model was developed to map magnetic field gradients and stress-strain profiles within the system. Simulations showed that a field gradient of ~159 T/m induces a compressive stress of ~122.8 Pa at 850 µm, decreasing to ~39.4 Pa at 1500 µm, a 3.11-fold difference that correlates with the transition from rounded to elongated cells. Magnetic actuation enables precise control of ECM deformation, strain, and applied force, facilitating the collagen fibers alignment and providing insight into cancer cell responses.

Digital holographic microscopy and other quantitative phase imaging methods provide accurate reconstructions of the microscale features of cells, related to cell shape, height, and index of refraction, useful for phenotypic profiling. The following section details a typical cell imaging experiment by digital holographic microscopy, image analysis, and machine learning steps directed toward phenotypic profiling of cells from one or more populations. The analysis results in single and multi-class classification and identification of typical and atypical cells.

Immunocytochemistry (IHC) and immunofluorescence (IF) offer crucial diagnostic insights in clinical settings. Most IF assays are performed using fluorophore-conjugated antibodies, but these fluorophores can be subject to issues, such as photobleaching and autofluorescence, that result in lower signal-to-noise ratios (SNR). Gold nanoparticles provide greater signal stability and scatter light well, making them easily separable from the background of biological tissues and improving SNR. In this study, we sought to determine the labeling efficiency, signal quality, and image artifacts of 2.2, 10, and 40 nm diameter gold nanoparticle probes conjugated to antibodies in IF applications on fixed, permeabilized cells in comparison to traditional fluorophores. Overall, micrographs of nanoparticle labels had higher SNR due to lower background signal, and punctate appearances as compared with the continuously distributed signal of the immunofluorescent label. Signal-to-noise ratios varied with nanoparticle diameter, and signal fidelity was worse for keratin versus epithelial growth factor receptor (EGFR). Labeling of EGFR was successful using both extracellular and intracellular epitopes, while poor labeling of keratin 19 with 10 nm diameter nanoparticles was improved by pretreatment with heat and sonication, suggesting hindrance of nanoparticle labels within the fixed, permeabilized cell.

Active mechanical stresses in and around tumors affect cancer cell behavior and independently regulate cancer progression. To investigate the role of mechanical stress in breast cancer cell invasion, magnetic alginate beads loaded with iron oxide nanoparticles were coated with MDA-MB-231 breast cancer cells and embedded in a three-dimensional extracellular matrix (ECM) model subjected to an external magnetic field during culture. Bead displacement, cell shape and patterns of invasion of the collagen gel, and cell proliferation were assessed over 7 days of culture. The alginate beads swelled over the first 24 h in culture, creating circumferential stress akin to that created by tumor growth, while bead magnetic properties enabled local mechanical loading (compression, tension, and relaxation) and motion within the in vitro tissue constructs upon exposure to an external magnetic field. Beads displaced 0.2–1.6 mm through the collagen gels, depending on magnet size and distance, compressing the collagen network microstructure without gel mechanical failure. Invading cells formed a spatulate pattern as they moved into the compressed ECM region, with individual cells aligned parallel to the bead surface. During the first 24 hours of compressive magnetic force loading, invading cancer cells became round, losing elongation and ability to invade out from the bead surface, while still actively dividing. In contrast, cell invasion in unloaded constructs and in loaded constructs away from the compression region invaded as single cells, transversely outward from the bead surface. Finally, cell proliferation was 1.3× higher only after external magnet removal, which caused relaxation of mechanical stress in the collagen network. These findings indicate effects on breast cancer invasion of mechanical loading of ECM, both from compressive loading and from load relaxation. Findings point to the influence of mechanical stress on cancer cell behavior and suggest that relaxing mechanical stress in and around a tumor may promote cancer progression through higher proliferation and invasion.

Simple methods to detect biomolecules including specific nucleic acid sequences have received renewed attention since the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus pandemic. Notably, biomolecule detection that uses some form of signal amplification will have some form of amplification-related error, which in the polymerase chain reaction involves mispriming and subsequent signal amplification in the no template control, ultimately providing a limit of detection. To demonstrate the feasibility of the detection of a DNA target sequence without molecular or chemical signal amplification that avoids amplification errors, a gold nanoparticle aggregation assay was developed and tested. Two primers bracketing a 94 base pair target sequence from SARS-CoV-2 were conjugated to 10 nm diameter gold nanoparticles by the salt aging method, with conjugation and primer-target hybridization confirmed by agarose gel electrophoresis and nanospectrophotometry. Upon mixing of both conjugated nanoparticles with target, a surface plasmon resonance shift of 6 nm was observed, and lower electrophoretic mobility of a band containing both DNA fluorescence and gold absorption signals. This did not occur in the presence of a control DNA molecule of the same size and composition as the target but with a randomly scrambled base position. Nanoparticle tracking at 30 frames per second using a sensitive darkfield microscope revealed a lower measured diffusion coefficient of scattering objects in the target mixture than in the control mixture or with bare gold nanoparticles.

Oral mucositis (OM) is a severe complication of cancer therapies caused by off‐target cytotoxicity. Palifermin, which is recombinant human keratinocyte growth factor (KGF), is currently the only mitigating treatment available to a subset of OM patients. This study used a previously established model of oral mucositis on a chip (OM‐OC) comprised of a confluent human gingival keratinocytes (GIE) layer attached to a basement membrane‐lined subepithelial layer consisting of human gingival fibroblasts (HGF) and human dermal microvascular endothelial cells (HMEC) on a stable collagen I gel. Cisplatin, radiation, and combined treatments are followed by a recovery period in the OM‐OC to determine possible cellular and molecular mechanisms of OM under effects of KGF. Cancer treatments affected the keratinocyte layer, causing death and epithelial barrier loss. Both keratinocytes and subepithelial cells died rapidly, as evidenced by propidium iodide staining. In response to radiation exposure, cell death occurred in the apical epithelial layer, predominantly, within 24h. Cisplatin exposure predominantly promoted death of basal epithelial cells within 32‐36h. Presence of KGF in OM‐OC protected tissues from damage caused by cancer treatments in a dose‐dependent manner, being more effective at 10 ng/mL. As verified by F‐actin staining and the Alamar Blue assay, KGF contributed to epithelial survival and induced proliferation of GIE and HGF as well as HMEC within 120h. When the expression of eighty inflammatory cytokines is evaluated at OM induction (Day 12) and resolution (Day 18) stages in OM‐OC, some cytokines are identified as potential novel therapeutic targets. In comparison with chemoradiation exposure, KGF treatment showed a trend to decrease IL‐8 and TNF‐a expression at Day 12 and 18, and TGF‐β1 at Day 18 in OM‐OC. Taken together, these findings support the utility of OM‐OC as a platform to model epithelial damage and evaluate molecular mechanisms following OM treatment.

Optical diffraction tomography (ODT) solves an inverse scattering problem to obtain label-free, 3D refractive index (RI) estimation of biological specimens. This work demonstrates 3D RI retrieval methods suitable for partially-coherent ODT systems supported by intensity-only measurements consisting of axial and angular illumination scanning. This framework allows for access to 3D quantitative RI contrast using a simplified non-interferometric technique. We consider a traditional iterative tomographic solver based on a multiple in-plane representation of the optical scattering process and gradient descent optimization adapted for focus-scanning systems, as well as an approach that relies solely on 3D convolutional neural networks (CNNs) to invert the scattering process. The approaches are validated using simulations of the 3D scattering potential for weak phase 3D biological samples.

Digital holographic microscopy is an imaging technique particularly well suited to the study of living cells in culture, as no labeling is required and computed phase maps produce high contrast, quantitative pixel information. A full experiment involves instrument calibration, cell culture quality checks, selection and setup of imaging chambers, a sampling plan, image acquisition, phase and amplitude map reconstruction, and parameter map post-processing to extract information about cell morphology and/or motility. Each step is described below, focusing on results from imaging four human cell lines. Several post-processing approaches are detailed, with an aim of tracking individual cells and dynamics of cell populations. Key words Cell motility Holography Microscopy Quantitative phase Cell lines Cell morphology Image processing Machine learning

Microfluidic-integrated freestanding membranes with suitable biocompatibility and tunable physicochemical properties are in high demand for a wide range of life science and biological studies. However, there is a lack of facile and rapid methods to integrate such versatile membranes into microfluidics. A recently invented interfacial electrofabrication of chitosan membranes offers an in-situ membrane integration strategy that is flexible, controllable, simple, and biologically friendly. In this follow-up study, we explored the ability to program the physical properties of these chitosan membranes by varying the electrofabrication conditions (e.g., applied voltage and pH of alginate). We found a strong association between membrane growth rate, properties, and fabrication parameters: high electrical stimuli and pH of alginate resulted in high optical retardance and low permeability, and vice versa. This suggests that the molecular alignment and density of electrofabricated chitosan membranes could be actively tailored according to application needs. Lastly, we demonstrated that this interfacial electrofabrication could easily be expanded to produce chitosan membrane arrays with higher uniformity than the previously well-established flow assembly method. This study demonstrates the tunability of the electrofabricated membranes’ properties and functionality, thus expanding the utility of such membranes for broader applications in the future.