Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer.

Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx.

The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.

MACC1 regulates clathrin-mediated endocytosis and receptor recycling of transferrin receptor and EGFR in colorectal cancer.

Metastasis Associated in Colon Cancer 1 (MACC1) is a novel prognostic, predictive and causal biomarker for tumor progression and metastasis in many cancer types, including colorectal cancer. Besides its clinical value, little is known about its molecular function. Its similarity to SH3BP4, involved in regulating uptake and recycling of transmembrane receptors, suggests a role of MACC1 in endocytosis. By exploring the MACC1 interactome, we identified the clathrin-mediated endocytosis (CME)-associated proteins CLTC, DNM2 and AP-2 as MACC1 binding partners. We unveiled a MACC1-dependent routing of internalized transferrin receptor towards recycling. Elevated MACC1 expression caused also the activation and internalization of EGFR, a higher rate of receptor recycling, as well as earlier and stronger receptor activation and downstream signaling. These effects are limited by deletion of CME-related protein interaction sites in MACC1. Thus, MACC1 regulates CME and receptor recycling, causing increased growth factor-mediated downstream signaling and cell proliferation. This novel mechanism unveils potential therapeutic intervention points restricting MACC1-driven metastasis.

Laboratory Soft X-Ray Microscopy with an Integrated Visible-Light Microscope-Correlative Workflow for Faster 3D Cell Imaging.

Laboratory transmission soft X-ray microscopy (L-TXM) has emerged as a complementary tool to synchrotron-based TXM and high-resolution biomedical 3D imaging in general in recent years. However, two major operational challenges in L-TXM still need to be addressed: a small field of view and a potentially misaligned rotation stage. As it is not possible to alter the magnification during operation, the field of view in L-TXM is usually limited to a few tens of micrometers. This complicates locating areas and objects of interest in the sample. Additionally, if the rotation axis of the sample stage cannot be adjusted prior to the experiments, an efficient workflow for tomographic imaging cannot be established, as refocusing and sample repositioning will become necessary after each recorded projection. Both these limitations have been overcome with the integration of a visible-light microscope (VLM) into the L-TXM system. Here, we describe the calibration procedure of the goniometer sample stage and the integrated VLM and present the resulting 3D imaging of a test sample. In addition, utilizing this newly integrated VLM, the extracellular matrix of cryofixed THP-1 cells (human acute monocytic leukemia cells) was visualized by L-TXM for the first time in the context of an ongoing biomedical research project.

Lissajous scanning magnetic particle imaging as a multifunctional platform for magnetic hyperthermia therapy.

The use of engineered nanoscale magnetic materials in healthcare and biomedical technologies is rapidly growing. Two examples which have recently attracted significant attention are magnetic particle imaging (MPI) for biological monitoring, and magnetic field hyperthermia (MFH) for cancer therapy. Here for the first time, the capability of a Lissajous scanning MPI device to act as a standalone platform to support the application of MFH cancer treatment is presented. The platform is shown to offer functionalities for nanoparticle localization, focused hyperthermia therapy application, and non-invasive tissue thermometry in one device. Combined, these capabilities have the potential to significantly enhance the accuracy, effectiveness and safety of MFH therapy. Measurements of nanoparticle hyperthermia during protracted exposure to the MPI scanner's 3D imaging field sequence revealed spatially focused heating, with a maximum that is significantly enhanced compared with a simple 1-dimensional sinusoidal excitation. The observed spatial heating behavior is qualitatively described based on a phenomenological model considering torques exerted in the Brownian regime. In vitro cell studies using a human acute monocytic leukemia cell line (THP-1) demonstrated strong suppression of both structural integrity and metabolic activity within 24 h following a 40 min MFH treatment actuated within the Lissajous MPI scanner. Furthermore, reconstructed MPI images of the nanoparticles distributed among the cells, and the temperature-sensitivity of the MPI imaging signal obtained during treatment are demonstrated. In summary, combined Lissajous MPI and MFH technologies are presented; demonstrating for the first time their potential for cancer treatment with maximum effectiveness, and minimal collateral damage to surrounding tissues.

Cellular uptake of magnetic nanoparticles imaged and quantified by magnetic particle imaging.

Magnetic particle imaging (MPI) is a non-invasive, non-ionizing imaging technique for the visualization and quantification of magnetic nanoparticles (MNPs). The technique is especially suitable for cell imaging as it offers zero background contribution from the surrounding tissue, high sensitivity, and good spatial and temporal resolutions. Previous studies have demonstrated that the dynamic magnetic behaviour of MNPs changes during cellular binding and internalization. In this study, we demonstrate how this information is encoded in the MPI imaging signal. Through MPI imaging we are able to discriminate between free and cell-bound MNPs in reconstructed images. This technique was used to image and quantify the changes that occur in-vitro when free MNPs come into contact with cells and undergo cellular-uptake over time. The quantitative MPI results were verified by colorimetric measurements of the iron content. The results showed a mean relative difference between the MPI results and the reference method of 23.8% for the quantification of cell-bound MNPs. With this technique, the uptake of MNPs in cells can be imaged and quantified directly from the first MNP cell contact, providing information on the dynamics of cellular uptake.