Epithelial p53 Status Modifies Stromal-Epithelial Interactions During Basal-Like Breast Carcinogenesis.

Basal-like breast cancers (BBC) exhibit subtype-specific phenotypic and transcriptional responses to stroma, but little research has addressed how stromal-epithelial interactions evolve during early BBC carcinogenesis. It is also unclear how common genetic defects, such as p53 mutations, modify these stromal-epithelial interactions. To address these knowledge gaps, we leveraged the MCF10 progression series of breast cell lines (MCF10A, MCF10AT1, and MCF10DCIS) to develop a longitudinal, tissue-contextualized model of p53-deficient, pre-malignant breast. Acinus asphericity, a morphogenetic correlate of cell invasive potential, was quantified with optical coherence tomography imaging, and gene expression microarrays were performed to identify transcriptional changes associated with p53 depletion and stromal context. Co-culture with stromal fibroblasts significantly increased the asphericity of acini derived from all three p53-deficient, but not p53-sufficient, cell lines, and was associated with the upregulation of 38 genes. When considered as a multigene score, these genes were upregulated in co-culture models of invasive BBC with increasing stromal content, as well as in basal-like relative to luminal breast cancers in two large human datasets. Taken together, stromal-epithelial interactions during early BBC carcinogenesis are dependent upon epithelial p53 status, and may play important roles in the acquisition of an invasive morphologic phenotype.

All-fiber probes for endoscopic optical coherence tomography of the large airways.

Endoscopic optical coherence tomography of large airways poses unique challenges. A hybrid lens is described that consists of a section of coreless fiber and graded index fiber (GIF), followed by a ball lens section. This design produces low numerical aperture beams better suited for large airway imaging. The performance of this lens is compared against conventional GIF and ball lens designs. Forward- and side-viewing probes were modeled, fabricated, and tested. The impact of a sheath on the beam profile was also investigated. Probes with working distances larger than 10 mm and depth-of-focus exceeding 12 mm are demonstrated with the proposed design.

Localized compliance measurement of the airway wall using anatomic optical coherence elastography.

We describe an elastographic method to circumferentially-resolve airway wall compliance using endoscopic, anatomic optical coherence tomography (aOCT) combined with an intraluminal pressure catheter. The method was first demonstrated on notched silicone phantoms of known elastic modulus under respiratory ventilation, where localized compliance measurements were validated against those predicted by finite element modeling. Then, ex vivo porcine tracheas were scanned, and the pattern of compliance was found to be consistent with histological identification of the locations of (stiff) cartilage and (soft) muscle. This quantitative method may aid in diagnosis and monitoring of collapsible airway wall tissues in obstructive respiratory disorders.

Probing biological nanotopology via diffusion of weakly constrained plasmonic nanorods with optical coherence tomography.

Biological materials exhibit complex nanotopology, i.e., a composite liquid and solid phase structure that is heterogeneous on the nanoscale. The diffusion of nanoparticles in nanotopological environments can elucidate biophysical changes associated with pathogenesis and disease progression. However, there is a lack of methods that characterize nanoprobe diffusion and translate easily to in vivo studies. Here, we demonstrate a method based on optical coherence tomography (OCT) to depth-resolve diffusion of plasmon-resonant gold nanorods (GNRs) that are weakly constrained by the biological tissue. By using GNRs that are on the size scale of the polymeric mesh, their Brownian motion is minimally hindered by intermittent collisions with local macromolecules. OCT depth-resolves the particle-averaged translational diffusion coefficient (DT) of GNRs within each coherence volume, which is separable from the nonequilibrium motile activities of cells based on the unique polarized light-scattering properties of GNRs. We show how this enables minimally invasive imaging and monitoring of nanotopological changes in a variety of biological models, including extracellular matrix (ECM) remodeling as relevant to carcinogenesis, and dehydration of pulmonary mucus as relevant to cystic fibrosis. In 3D ECM models, DT of GNRs decreases with both increasing collagen concentration and cell density. Similarly, DT of GNRs is sensitive to human bronchial-epithelial mucus concentration over a physiologically relevant range. This novel method comprises a broad-based platform for studying heterogeneous nanotopology, as distinct from bulk viscoelasticity, in biological milieu.

Imaging Extracellular Matrix Remodeling In Vitro by Diffusion-Sensitive Optical Coherence Tomography.

The mammary gland extracellular matrix (ECM) is comprised of biopolymers, primarily collagen I, that are created and maintained by stromal fibroblasts. ECM remodeling by fibroblasts results in changes in ECM fiber spacing (pores) that have been shown to play a critical role in the aggressiveness of breast cancer. However, minimally invasive methods to measure the spatial distribution of ECM pore areas within tissues and in vitro 3D culture models are currently lacking. We introduce diffusion-sensitive optical coherence tomography (DS-OCT) to image the nanoscale porosity of ECM by sensing weakly constrained diffusion of gold nanorods (GNRs). DS-OCT combines the principles of low-coherence interferometry and heterodyne dynamic light scattering. By collecting co- and cross-polarized light backscattered from GNRs within tissue culture, the ensemble-averaged translational self-diffusion rate, DT, of GNRs is resolved within ∼3 coherence volumes (10 × 5 µm, x × z). As GNRs are slowed by intermittent collisions with ECM fibers, DT is sensitive to ECM porosity on the size scale of their hydrodynamic diameter (∼46 nm). Here, we validate the utility of DS-OCT using pure collagen I gels and 3D mammary fibroblast cultures seeded in collagen/Matrigel, and associate differences in artificial ECM pore areas with gel concentration and cell seed density. Across all samples, DT was highly correlated with pore area obtained by scanning electron microscopy (R(2) = 0.968). We also demonstrate that DS-OCT can accurately map the spatial heterogeneity of layered samples. Importantly, DS-OCT of 3D mammary fibroblast cultures revealed the impact of fibroblast remodeling, where the spatial heterogeneity of matrix porosity was found to increase with cell density. This provides an unprecedented view into nanoscale changes in artificial ECM porosity over effective pore diameters ranging from ∼43 to 360 nm using a micron-scale optical imaging technique. In combination with the topical deposition of GNRs, the minimally invasive nature of DS-OCT makes this a promising technology for studying tissue remodeling processes.

High-speed and high-sensitivity parallel spectral-domain optical coherence tomography using a supercontinuum light source.

The three most important metrics in optical coherence tomography (OCT) are resolution, speed, and sensitivity. Because there is a complex interplay between these metrics, no previous work has obtained the best performance in all three metrics simultaneously. We demonstrate that a high-power supercontinuum source, in combination with parallel spectral-domain OCT, achieves an unparalleled combination of resolution, speed, and sensitivity. This system captures cross-sectional images spanning 4 mm×0.5 mm at 1,024,000 lines/s with 2×14 µm resolution (axial×transverse) at a sensitivity of 113 dB. Imaging using the proposed system is demonstrated on highly differentiated human bronchial epithelial cells to capture and spatially localize ciliary dynamics.

Magnetic and Plasmonic Contrast Agents in Optical Coherence Tomography.

Optical coherence tomography (OCT) has gained widespread application for many biomedical applications, yet the traditional array of contrast agents used in incoherent imaging modalities do not provide contrast in OCT. Owing to the high biocompatibility of iron oxides and noble metals, magnetic and plasmonic nanoparticles, respectively, have been developed as OCT contrast agents to enable a range of biological and pre-clinical studies. Here we provide a review of these developments within the past decade, including an overview of the physical contrast mechanisms and classes of OCT system hardware addons needed for magnetic and plasmonic nanoparticle contrast. A comparison of the wide variety of nanoparticle systems is also presented, where the figures of merit depend strongly upon the choice of biological application.

Effect of finishing technique on the occurrence and length of microcracks in resin-based materials.

PURPOSE: To evaluate the presence and length of microcracks in resin-based materials finished with different techniques, using optical coherence tomography (OCT). METHODS: Standardized Class V preparations (3x2x2mm) were made in the facial and lingual surfaces of 20 recently-extracted human third molars. 20 preparations were restored with a resin-based composite material (RBC; Filtek Supreme Ultra) and the other 20 with a resin-modified glass-ionomer material (RMGI; Ketac Nano). After final polymerization, specimens were further stratified by finishing system: aluminum oxide discs (Sof-Lex) or spiral fluted carbide bur series (H48L). By random allocation, each extracted tooth therefore received one RBC and one RMGI restoration, and equal numbers of restorations from each material were finished using each finishing system (n= 10). After 24 hours of storage in 100% humidity at room temperature, the specimens were evaluated at x20 to x600 under environmental SEM. Cross-sectional occlusal-cervical B-mode images were obtained in increments of 25 mm from the mesial margin to the distal margin of the restoration using a spectral-domain (SD) OCT system and analyzed using Image J software to identify and measure microcrack penetration into each restoration. The total length (mm) at the point of the deepest microcrack penetration in each specimen was recorded. Data were statistically analyzed using a t-test. RESULTS: No microcracks were observed in the RBC samples. However, microcrack presence was identified in all of the RMGI specimens. The t-test showed a statistically significant difference (P< 0.05) in mean microcrack length values based on the finishing technique used for the RMGI samples. [SofLex: 0.67 (± 0.28) mm; carbide: 1.26 (± 0.30)] mm. Two-way ANOVA showed significant differences in the factors "finishing technique" and "restorative material" (P< 0.001). The interaction of these two factors was also statistically significant (P< 0.001). For the tested RMGI, Tukey post-hoc test revealed that the finishing with aluminum oxide groups resulted in statistically significant lower mean microcrack length when compared to spiral fluted carbide burs (P< 0.001). CLINICAL SIGNIFICANCE: Resin-modified glass-ionomer (RMGI) is more susceptible to microcrack presence than resin-based composites. Also, aluminum oxide discs produced lower values of mean microcrack length than spiral fluted carbide burs after the finishing procedure of RMGI restorations.

Compressed intracellular motility via non-uniform temporal sampling in dynamic optical coherence tomography.

Significance: Optical coherence tomography has great utility for capturing dynamic processes, but such applications are particularly data-intensive. Samples such as biological tissues exhibit temporal features at varying time scales, which makes data reduction challenging. Aim: We propose a method for capturing short- and long-term correlations of a sample in a compressed way using non-uniform temporal sampling to reduce scan time and memory overhead. Approach: The proposed method separates the relative contributions of white noise, fluctuating features, and stationary features. The method is demonstrated on mammary epithelial cell spheroids in three-dimensional culture for capturing intracellular motility without loss of signal integrity. Results: Results show that the spatial patterns of motility are preserved and that hypothesis tests of spheroids treated with blebbistatin, a motor protein inhibitor, are unchanged with up to eightfold compression. Conclusions: The ability to measure short- and long-term correlations compressively will enable new applications in (3+1)D imaging and high-throughput screening.

Longitudinal tracking of perfluorooctanoic acid exposure on mammary epithelial cell spheroids by dynamic optical coherence tomography.

We investigated the morphology and intracellular motility of mammary epithelial cell (MCF10DCIS.com) spheroids cultured in 3D artificial extracellular matrix under perfluorooctanoic acid (PFOA) exposure. Dynamic optical coherence tomography (OCT) was employed for real-time, non-invasive imaging of these spheroids longitudinally over 12 days under PFOA exposures up to 500 µM. Despite no significant changes in volume or asphericity of spheroids, morphological alterations were observed in OCT images of spheroids at 100 µM on Day 12 and from Day 4 at 500 µM. Intracellular motility was assessed by the inverse-power-law exponent of the speckle fluctuation spectrum (α), and an autocorrelation-based motility amplitude (M). Linear regression indicated that both PFOA concentration and culture time are highly significant predictors for both α and M (p < 0.001 for all). Both PFOA concentration and culture time have positive associations with α and negative association with M, where increased α indicates suppression of higher frequency fluctuations (∼> 2 Hz) relative to those at lower frequencies, and decreased M indicates overall suppression of intracellular motility. This study can lead to the future development of biomarkers for per- and polyfluoroalkyl substances (PFAS) exposure using dynamic OCT and its associated toolkit of quantitative metrics.

In situ pulmonary mucus hydration assay using rotational and translational diffusion of gold nanorods with polarization-sensitive optical coherence tomography.

Significance: Assessing the nanostructure of polymer solutions and biofluids is broadly useful for understanding drug delivery and disease progression and for monitoring therapy. Aim: Our objective is to quantify bronchial mucus solids concentration (wt. %) during hypertonic saline (HTS) treatment in vitro via nanostructurally constrained diffusion of gold nanorods (GNRs) monitored by polarization-sensitive optical coherence tomography (PS-OCT). Approach: Using PS-OCT, we quantified GNR translational (DT) and rotational (DR) diffusion coefficients within polyethylene oxide solutions (0 to 3 wt. %) and human bronchial epithelial cell (hBEC) mucus (0 to 6.4 wt. %). Interpolation of DT and DR data is used to develop an assay to quantify mucus concentration. The assay is demonstrated on the mucus layer of an air-liquid interface hBEC culture during HTS treatment. Results: In polymer solutions and mucus, DT and DR monotonically decrease with increasing concentration. DR is more sensitive than DT to changes above 1.5 wt. % of mucus and exhibits less intrasample variability. Mucus on HTS-treated hBEC cultures exhibits dynamic mixing from cilia. A region of hard-packed mucus is revealed by DR measurements. Conclusions: The extended dynamic range afforded by simultaneous measurement of DT and DR of GNRs using PS-OCT enables resolving concentration of the bronchial mucus layer over a range from healthy to disease in depth and time during HTS treatment in vitro.

Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ.

INTRODUCTION: Basal-like and luminal breast cancers have distinct stromal-epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal-epithelial interactions evolve in benign and pre-invasive lesions. METHODS: To study epithelial-stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis. RESULTS: Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basallike subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival. CONCLUSIONS: Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.

Motility-, autocorrelation-, and polarization-sensitive optical coherence tomography discriminates cells and gold nanorods within 3D tissue cultures.

We propose a method for differentiating classes of light scatterers based upon their temporal and polarization properties computed from time series of polarization-sensitive optical coherence tomography (PS-OCT) images. The amplitude (motility) and time scale (autocorrelation decay time) of the speckle fluctuations are combined with the cross-polarization pixel-wise to render Motility-, autocorrelation-, and polarization-sensitive (MAPS) OCT contrast images. This combination of metrics provides high specificity for discriminating diffusive gold nanorods and mammary epithelial cell spheroids within 3D tissue culture, based on their unique MAPS signature. This has implications toward highly specific contrast in molecular (nanoparticle-based) and functional (cellular activity) imaging using standard PS-OCT hardware.

In vivo magnetomotive optical molecular imaging using targeted magnetic nanoprobes.

Dynamic magnetomotion of magnetic nanoparticles (MNPs) detected with magnetomotive optical coherence tomography (MM-OCT) represents a new methodology for contrast enhancement and therapeutic interventions in molecular imaging. In this study, we demonstrate in vivo imaging of dynamic functionalized iron oxide MNPs using MM-OCT in a preclinical mammary tumor model. Using targeted MNPs, in vivo MM-OCT images exhibit strong magnetomotive signals in mammary tumor, and no significant signals were measured from tumors of rats injected with nontargeted MNPs or saline. The results of in vivo MM-OCT are validated by MRI, ex vivo MM-OCT, Prussian blue staining of histological sections, and immunohistochemical analysis of excised tumors and internal organs. The MNPs are antibody functionalized to target the human epidermal growth factor receptor 2 (HER2 neu) protein. Fc-directed conjugation of the antibody to the MNPs aids in reducing uptake by macrophages in the reticulo-endothelial system, thereby increasing the circulation time in the blood. These engineered magnetic nanoprobes have multifunctional capabilities enabling them to be used as dynamic contrast agents in MM-OCT and MRI.

Elastography of soft materials and tissues by holographic imaging of surface acoustic waves.

We use optical interferometry to capture coherent surface acoustic waves for elastographic imaging. An inverse method is employed to convert multi-frequency data into an elastic depth profile. Using this method, we image elastic properties over a 55 mm range with <5 mm resolution. For relevance to breast cancer detection, we employ a tissue phantom with a tumor-like inclusion. Holographic elastography is also shown to be well-behaved in ex vivo tissue, revealing the subsurface position of a bone. Because digital holography can assess waves over a wide surface area, this constitutes a flexible new platform for large volume and non-invasive elastography.

Elastometry of clot phantoms via magnetomotive ultrasound-based resonant acoustic spectroscopy.

Objective.An ultrasound-based system capable of both imaging thrombi against a dark field and performing quantitative elastometry could allow for fast and cost-effective thrombosis diagnosis, staging, and treatment monitoring. This study investigates a contrast-enhanced approach for measuring the Young's moduli of thrombus-mimicking phantoms.Approach.Magnetomotive ultrasound (MMUS) has shown promise for lending specific contrast to thrombi by applying a temporally modulated force to magnetic nanoparticle (MNP) contrast agents and measuring resulting tissue displacements. However, quantitative elastometry has not yet been demonstrated in MMUS, largely due to difficulties inherent in measuring applied magnetic forces and MNP densities. To avoid these issues, in this work magnetomotive resonant acoustic spectroscopy (MRAS) is demonstrated for the first time in ultrasound.Main results.The resonance frequencies of gelatin thrombus-mimicking phantoms are shown to agree within one standard deviation with finite element simulations over a range of phantom sizes and Young's moduli with less than 16% error. Then, in a proof-of-concept study, the Young's moduli of three phantoms are measured using MRAS and are shown to agree with independent compression testing results.Significance.The MRAS results were sufficiently precise to differentiate between thrombus phantoms with clinically relevant Young's moduli. These findings demonstrate that MRAS has potential for thrombus staging.

Magnetic Alignment for Plasmonic Control of Gold Nanorods Coated with Iron Oxide Nanoparticles.

Plasmonic nanoparticles that can be manipulated with magnetic fields are of interest for advanced optical applications, diagnostics, imaging, and therapy. Alignment of gold nanorods yields strong polarization-dependent extinction, and use of magnetic fields is appealing because they act through space and can be quickly switched. In this work, cationic polyethyleneimine-functionalized superparamagnetic Fe3 O4 nanoparticles (NPs) are deposited on the surface of anionic gold nanorods coated with bovine serum albumin. The magnetic gold nanorods (MagGNRs) obtained through mixing maintain the distinct optical properties of plasmonic gold nanorods that are minimally perturbed by the magnetic overcoating. Magnetic alignment of the MagGNRs arising from magnetic dipolar interactions on the anisotropic gold nanorod core is comprehensively characterized, including structural characterization and enhancement (suppression) of the longitudinal surface plasmon resonance and suppression (enhancement) of the transverse surface plasmon resonance for light polarized parallel (orthogonal) to the magnetic field. The MagGNRs can also be driven in rotating magnetic fields to rotate at frequencies of at least 17 Hz. For suitably large gold nanorods (148 nm long) and Fe3 O4 NPs (13.4 nm diameter), significant alignment is possible even in modest (<500 Oe) magnetic fields. An analytical model provides a unified understanding of the magnetic alignment of MagGNRs.

Anatomic Optical Coherence Tomography (aOCT) for Evaluation of the Internal Nasal Valve.

OBJECTIVES/HYPOTHESIS: To establish the utility of anatomic optical coherence tomography (aOCT) in evaluating internal nasal valve (INV). STUDY DESIGN: Anatomic specimen imaging study. METHODS: Fresh-harvested human specimen heads were evaluated using both computed tomography (CT) imaging as well as using aOCT. Scans were performed at three time points: 1) After septoplasty for cartilage harvest, 2) after placement of butterfly graft (BFG), and 3) after placement of bilateral spreader grafts (SG). Imaging data were then converted into 3D models of the nasal airway. CT- and aOCT-generated models were compared by both static volumetric analysis and computational fluid dynamics (CFD) to predict nasal resistance and pressure. RESULTS: Scans using aOCT showed comparable results to CT in terms of volumetric parameters both before and after intervention. Analysis of aOCT data by CFD demonstrated decrease in pressure after SG or BFG intervention. No statistically significant difference was observed when comparing CT- and aOCT-generated calculations of pressure or resistance. CONCLUSION: The INV can be imaged in a static fashion using aOCT technology. Advantages over traditional CT imaging include lack of exposure to radiation and rapid scan time. In addition, in-office use is possible as aOCT technology develops. Further investigation will be necessary to define the role of aOCT in the dynamic evaluation of this vital component of the nasal airway. LEVEL OF EVIDENCE: 3 Laryngoscope, 132:2148-2156, 2022.

Long-range alignment of gold nanorods in electrospun polymer nano/microfibers.

In this study, a scalable fabrication technique for controlling and maintaining the nanoscale orientation of gold nanorods (GNRs) with long-range macroscale order has been achieved through electrospinning. The volume fraction of GNRs with an average aspect ratio of 3.1 is varied from 0.006 to 0.045 in aqueous poly(ethylene oxide) solutions to generate electrospun fibers possessing different GNR concentrations and measuring 40-3000 nm in diameter. The GNRs within these fibers exhibit excellent alignment with their longitudinal axis parallel to the fiber axis n. According to microscopy analysis, the average deviant angle between the GNR axis and n increases modestly from 3.8 to 13.3° as the fiber diameter increases. Complementary electron diffraction measurements confirm preferred orientation of the {100} GNR planes. Optical absorbance spectroscopy measurements reveal that the longitudinal surface plasmon resonance bands of the aligned GNRs depend on the polarization angle and that maximum extinction occurs when the polarization is parallel to n.

Imaging and Elastometry of Blood Clots Using Magnetomotive Optical Coherence Tomography and Labeled Platelets.

Improved methods for imaging and assessment of vascular defects are needed for directing treatment of cardiovascular pathologies. In this paper, we employ magnetomotive optical coherence tomography (MMOCT) as a platform both to detect and to measure the elasticity of blood clots. Detection is enabled through the use of rehydrated, lyophilized platelets loaded with superparamagnetic iron oxides (SPIO-RL platelets) that are functional infusion agents that adhere to sites of vascular endothelial damage. Evidence suggests that the sensitivity for detection is improved over threefold by magnetic interactions between SPIOs inside RL platelets. Using the same MMOCT system, we show how elastometry of simulated clots, using resonant acoustic spectroscopy, is correlated with the fibrin content of the clot. Both methods are based upon magnetic actuation and phase-sensitive optical monitoring of nanoscale displacements using MMOCT, underscoring its utility as a broad-based platform to detect and measure the molecular structure and composition of blood clots.