Ex vivo validation of a real-time multispectral endoscopic system for the detection and biopsy of bladder tumors.

BACKGROUND: Real-time multispectral imaging (rMSI) simultaneously provides white light (WL), photodynamic diagnosis (PDD) images, and a real-time fusion of both. It may improve the detection of bladder tumors. However, rMSI has not been used for transurethral biopsy or resection so far. The aim of this ex vivo study was to test the feasibility of bladder tumor biopsies using the rMSI system and compare it to a conventional endoscopic system. METHODS: A 3D printed rigid bladder phantom was equipped with small and flat (5 mm × 1 mm) mock-bladder-tumors made of silicone and fluorescent Qdots655 (Thermo Fisher Scientific, Germany). Urologists (n=15) were asked to perform a rigid cystoscopy and biopsy of all identified lesions (n=6) using a prototype rMSI system and the Image1 S system (Karl Storz, Tuttlingen). Success rate and completion time were measured. The image quality of both systems and the usability of the rMSI system according to the system usability scale (SUS) were evaluated with a task-specific questionnaire. RESULTS: Tumor detection and biopsy rate were 100% (90/90) for the rMSI system and 98.9% (89/90) for the Image1 S system (P=0.3). The biopsy completion time did not differ significantly between the systems (P=0.48). Differentiation between healthy and suspect mucosa with the rMSI system was rated as comparable to the Image1 S system by 53% of surgeons and as better by 33% of the surgeons. The median SUS score for the rMSI system was 87.5%. CONCLUSIONS: Accurate transurethral biopsies are feasible with the rMSI system. Furthermore, the rMSI system has an excellent SUS. This study paves the way to the first in-human transurethral resections of bladder tumors (TUR-B) using rMSI technology.

Multiparametric Cystoscopy for Detection of Bladder Cancer Using Real-time Multispectral Imaging.

BACKGROUND: Various imaging modalities can be used in addition to white light (WL) to improve detection of bladder cancer (BC). OBJECTIVE: To use real-time multispectral imaging (rMSI) during urethrocystoscopy to combine different imaging modalities to achieve multiparametric cystoscopy (MPC). DESIGN, SETTING, AND PARTICIPANTS: The rMSI system consisted of a camera with a spectral filter, a multi-LED light source, a microcontroller, and a computer for display and data acquisition. MSI with this system was achieved via temporal multiplexing. SURGICAL PROCEDURE: MPC was performed in ten patients with a diagnosed bladder tumor. MEASUREMENTS: We gathered evidence to prove the feasibility of our approach. In addition, experienced urologists performed post-interventional evaluation of images of individual lesions. Images were independently rated in a semiquantitative manner for each modality. A statistical model was built for pairwise comparisons across modalities. RESULTS AND LIMITATIONS: Overall, 31 lesions were detected using the rMSI set-up. Histopathology revealed malignancy in 27 lesions. All lesions could be visualized simultaneously in five modalities: WL, enhanced vascular contrast (EVC), blue light fluorescence, protoporphyrin IX fluorescence, and autofluorescence. EVC and photodynamic diagnosis images were merged in real time into one MP image. Using the recorded images, two observers identified all malignant lesions via MPC, whereas the single modalities did not arouse substantial suspicion for some lesions. The MP images of malignant lesions were rated significantly more suspicious than the images from single imaging modalities. CONCLUSIONS: We demonstrated for the first time the application of rMSI in endourology and we established MPC for detection of BC. This approach allows existing imaging modalities to be combined, and it may significantly improve the detection of bladder cancer. PATIENT SUMMARY: Real-time multispectral imaging was successfully used to combine different imaging aids for more comprehensive illustration of bladder tumors for surgeons. In the future, this technique may allow better detection of bladder tumors and more complete endoscopic resection in cases of cancer.

Differential tumor biological role of the tumor suppressor KAI1 and its splice variant in human breast cancer cells.

The tetraspanin and tumor suppressor KAI1 is downregulated or lost in many cancers which correlates with poor prognosis. KAI1 acts via physical/functional crosstalk with other membrane receptors. Also, a splice variant of KAI1 (KAI1-SP) has been identified indicative of poor prognosis. We here characterized differential effects of the two KAI1 variants on tumor biological events involving integrin (αvß3) and/or epidermal growth factor receptor (EGF-R). In MDA-MB-231 and -435 breast cancer cells, differential effects were documented on the expression levels of the tumor biologically relevant integrin αvß3 which colocalized with KAI1-WT but not with KAI1-SP. Cellular motility was assessed by video image processing, including motion detection and vector analysis for the quantification and visualization of cell motion parameters. In MDA-MB-231 cells, KAI1-SP provoked a quicker wound gap closure and higher closure rates than KAI1-WT, also reflected by different velocities and average motion amplitudes of singular cells. KAI1-SP induced highest cell motion adjacent to the wound gap borders, whereas in MDA-MB-435 cells a comparable induction of both KAI1 variants was noticed. Moreover, while KAI1-WT reduced cell growth, KAI1-SP significantly increased it going along with a pronounced EGF-R upregulation. KAI1-SP-induced cell migration and proliferation was accompanied by the activation of the focal adhesion and Src kinase. Our findings suggest that splicing of KAI1 does not only abrogate its tumor suppressive functions, but even more, promotes tumor biological effects in favor of cancer progression and metastasis.

Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells.

Plasma membrane tension is responsible for a variety of cellular functions such as motility, cell division, and endocytosis. Since membrane tension is dominated by the attachment of the actin cortex to the inner leaflet of the plasma membrane, we investigated the importance of ezrin, a major cross-linker of the membrane-cytoskeleton interface, for cellular mechanics of confluent MDCK II cells. For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface. Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction. PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively. We observed substantial stiffening of cells and an increase in membrane tension after addition of PIP2 micelles. In contrast, reduction of active ezrin led to a decrease of membrane tension accompanied by loss of excess surface area, increase in cortical tension, remodelling of actin cytoskeleton, and reduction of cell height. The data confirm the importance of the ezrin-mediated connection between plasma membrane and cortex for cellular mechanics and cell morphology.

Cytoskeleton remodelling of confluent epithelial cells cultured on porous substrates.

The impact of substrate topography on the morphological and mechanical properties of confluent MDCK-II cells cultured on porous substrates was scrutinized by means of various imaging techniques as well as atomic force microscopy comprising force volume and microrheology measurements. Regardless of the pore size, ranging from 450 to 5500 nm in diameter, cells were able to span the pores. They did not crawl into the holes or grow around the pores. Generally, we found that cells cultured on non-porous surfaces are stiffer, i.e. cortical tension rises from 0.1 to 0.3 mN m(-1), and less fluid than cells grown over pores. The mechanical data are corroborated by electron microscopy imaging showing more cytoskeletal filaments on flat samples in comparison to porous ones. By contrast, cellular compliance increases with pore size and cells display a more fluid-like behaviour on larger pores. Interestingly, cells on pores larger than 3500 nm produce thick actin bundles that bridge the pores and thereby strengthen the contact zone of the cells.

Atomic force microscopy-based microrheology reveals significant differences in the viscoelastic response between malign and benign cell lines.

Mechanical phenotyping of cells by atomic force microscopy (AFM) was proposed as a novel tool in cancer cell research as cancer cells undergo massive structural changes, comprising remodelling of the cytoskeleton and changes of their adhesive properties. In this work, we focused on the mechanical properties of human breast cell lines with different metastatic potential by AFM-based microrheology experiments. Using this technique, we are not only able to quantify the mechanical properties of living cells in the context of malignancy, but we also obtain a descriptor, namely the loss tangent, which provides model-independent information about the metastatic potential of the cell line. Including also other cell lines from different organs shows that the loss tangent (G″/G') increases generally with the metastatic potential from MCF-10A representing benign cells to highly malignant MDA-MB-231 cells.

Tension monitoring during epithelial-to-mesenchymal transition links the switch of phenotype to expression of moesin and cadherins in NMuMG cells.

Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final mesenchymal-like state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT.

Toxicity of gold-nanoparticles: synergistic effects of shape and surface functionalization on micromotility of epithelial cells.

Nanoparticle exposure is monitored by a combination of two label-free and non-invasive biosensor devices which detect cellular shape and viscoelasticity (quartz crystal microbalance), cell motility and the dynamics of epithelial cell-cell contacts (electric cell-substrate impedance sensing). With these tools we have studied the impact of nanoparticle shape on cellular physiology. Gold (Au) nanoparticles coated with CTAB were synthesized and studied in two distinct shapes: Spheres with a diameter of (43 ± 4) nm and rods with a size of (38 ± 7) nm × (17 ± 3) nm. Dose-response experiments were accompanied by conventional cytotoxicity tests as well as fluorescence and dark-field microscopy to visualize the intracellular particle distribution. We found that spherical gold nanoparticles with identical surface functionalization are generally more toxic and more efficiently ingested than rod-shaped particles. We largely attribute the higher toxicity of CTAB-coated spheres as compared to rod-shaped particles to a higher release of toxic CTAB upon intracellular aggregation.