Early-stage diagnosis of bladder cancer using surface-enhanced Raman spectroscopy combined with machine learning algorithms in a rat model.

Early diagnosis and accurate assessment of tumor development facilitate early bladder cancer resection and initiation of drug therapy. This study enabled an early, accurate, label-free, noninvasive diagnosis of bladder tumors by analyzing nano-biomarkers in a single drop of urine through surface-enhanced Raman spectroscopy (SERS). In a standard N-butyl-N-4-hydroxybutyl nitrosamine-induced rat model of bladder cancer, cancer stage and polyp tumor development were monitored using a small endoscope with a diameter of 1.2 mm in a minimally invasive manner without the need to kill the rats. Samples were divided into cancer-free, early-stage, and polyp-form cancer. Training data were classified according to micro-cystoscopic 5-aminolevulinic acid fluorescence diagnosis, and specimens were postmortem verified through histopathological analysis. A drop of urine from each sample group was placed on an Au-coated zinc oxide nanoporous chip to filter nano-biomaterials and selectively enhance the Raman signals of nanoscale analytes via SERS. Principal component analysis was used to reduce the dimensionality of the collected Raman spectra, and partial least squares discriminant analysis was used to find diagnostic clusters based on the labeled samples. The combination of SERS and machine learning achieved an accuracy ≥99.6% in diagnosing both early- and polyp-stage bladder tumors. With an area under the receiver operating characteristic curve greater than 0.996, the accuracy of the diagnosis in the rat model suggests that SERS-based diagnostic methods are promising when coupled with machine learning. Low-cost, label-free, and noninvasive surface-enhanced Raman spectra are ideal for developing clinically relevant point-of-care diagnostic techniques.

Mobility of Nucleostemin in Live Cells Is Specifically Related to Transcription Inhibition by Actinomycin D and GTP-Binding Motif.

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.

SERS Effect on Spin-Coated Seeding of Tilted Au-ZnO Nanorods for Low-Cost Diagnosis.

Uniformly parallel Au-coated ZnO nanorods have previously been shown to amplify local Raman signals, providing increased sensitivity to disease markers in the detection of inflammation and cancer. However, practical and cost-effective fabrication methods of substrates for surface-enhanced Raman spectroscopy (SERS) fail to produce highly uniform surfaces. Here, the feasibility of Raman enhancement on less-uniform substrates is assessed. ZnO nanorod structures were fabricated by hydrothermal synthesis, starting from spin-coated seed substrates. Following analysis, the nanostructures were coated with Au to create stochastically variant substrates. The non-uniformity of the fabricated Au-coated ZnO nanorod structures is confirmed morphologically by FE-SEM and structurally by X-ray diffraction, and characterized by the angular distributions of the nanorods. Monte Carlo finite element method simulations matching the measured angular distributions and separations predicted only moderate increases in the overall Raman enhancement with increasing uniformity. Highly variant substrates exhibited approximately 76% of the Raman enhancement of more uniform substrates in simulations and experiments. The findings suggest that, although highly inhomogeneous Au-coated ZnO nanorod substrates may not attain the same Raman enhancement as more uniform substrates, the relaxation of fabrication tolerances may be economically viable.

Longitudinal micro-endoscopic monitoring of high-success intramucosal xenografts for mouse models of colorectal cancer.

Colorectal cancer (CRC) is one of the most frequently lethal forms of cancer. Intramucosal injection allows development of better mouse models of CRC, as orthotopic xenografts allow development of adenocarcinoma in the submucosa of the mouse colon wall. In this paper, a method of orthotopic injection is monitored longitudinally using cellular-resolution real-time in vivo fluorescence microendoscopy, following the injection of three different cell lines: 3T3-GFP to confirm immunosuppression and HCT116-RFP cells to model CRC. Adenoma formation is first observable after 7 to 10 days, and by use of 33 G needles a tumor induction rate of greater than 85% is documented. An additional experiment on the injection of rapamycin reveals drug efficacy and localization between 24 and 48 hours, and suggests the promise of real-time cellular-resolution fluorescence micro-endoscopy for developing longitudinal therapy regimes in mural models of CRC.

Micro-endoscopic In Vivo Monitoring in the Blood and Lymphatic Vessels of the Oral Cavity after Radiation Therapy.

Radiotherapy, although used worldwide for the treatment of head, neck, and oral cancers, causes acute complications, including effects on vasculature and immune response due to cellular stress. Thus, the ability to diagnose side-effects and monitor vascular response in real-time during radiotherapy would be highly beneficial for clinical and research applications. In this study, recently-developed fluorescence micro-endoscopic technology provides non-invasive, high-resolution, real-time imaging at the cellular level. Moreover, with the application of high-resolution imaging technologies and micro-endoscopy, which enable improved monitoring of adverse effects in GFP-expressing mouse models, changes in the oral vasculature and lymphatic vessels are quantified in real time for 10 days following a mild localized single fractionation, 10 Gy radiotherapy treatments. Fluorescence micro-endoscopy enables quantification of the cardiovascular recovery and immune response, which shows short-term reduction in mean blood flow velocity, in lymph flow, and in transient immune infiltration even after this mild radiation dose, in addition to long-term reduction in blood vessel capacity. The data provided may serve as a reference for the expected cellular-level physiological, cardiovascular, and immune changes in animal disease models after radiotherapy.

Longitudinal intravital imaging of transplanted mesenchymal stem cells elucidates their functional integration and therapeutic potency in an animal model of interstitial cystitis/bladder pain syndrome.

Rationale: Mesenchymal stem cell (MSC) therapy may be a novel approach to improve interstitial cystitis/bladder pain syndrome (IC/BPS), an intractable disease characterized by severe pelvic pain and urinary frequency. Unfortunately, the properties of transplanted stem cells have not been directly analyzed in vivo, which hampers elucidation of the therapeutic mechanisms of these cells and optimization of transplantation protocols. Here, we monitored the behaviors of multipotent stem cells (M-MSCs) derived from human embryonic stem cells (hESCs) in real time using a novel combination of in vivo confocal endoscopic and microscopic imaging and demonstrated their improved therapeutic potency in a chronic IC/BPS animal model. Methods: Ten-week-old female Sprague-Dawley rats were instilled with 10 mg of protamine sulfate followed by 750 µg of lipopolysaccharide weekly for 5 weeks. The sham group was instilled with phosphate-buffered saline (PBS). Thereafter, the indicated dose (0.1, 0.25, 0.5, and 1×106 cells) of M-MSCs or PBS was injected once into the outer layer of the bladder. The distribution, perivascular integration, and therapeutic effects of M-MSCs were monitored by in vivo endoscopic and confocal microscopic imaging, awake cystometry, and histological and gene expression analyses. Results: A novel combination of longitudinal intravital confocal fluorescence imaging and microcystoscopy in living animals, together with immunofluorescence analysis of bladder tissues, demonstrated that transplanted M-MSCs engrafted following differentiation into multiple cell types and gradually integrated into a perivascular-like structure until 30 days after transplantation. The beneficial effects of transplanted M-MSCs on bladder voiding function and the pathological characteristics of the bladder were efficient and long-lasting due to the stable engraftment of these cells. Conclusion: This longitudinal bioimaging study of transplanted hESC-derived M-MSCs in living animals reveals their long-term functional integration, which underlies the improved therapeutic effects of these cells on IC/BPS.

In Vivo Fluorescence Microendoscopic Monitoring of Stent-Induced Fibroblast Cell Proliferation in an Esophageal Mouse Model.

PURPOSE: To evaluate the feasibility of self-expanding metal stent (SEMS) placement and fluorescence microendoscopic monitoring for determination of fibroblast cell proliferation after stent placement in an esophageal mouse model. MATERIALS AND METHODS: Twenty fibroblast-specific protein (FSP)-1 green fluorescent protein (GFP) transgenic mice were analyzed. Ten mice (Group A) underwent SEMS placement, and fluoroscopic and fluorescence microendoscopic images were obtained biweekly until 8 weeks thereafter. Ten healthy mice (Group B) were used for control esophageal values. RESULTS: SEMS placement was technically successful in all mice. The relative average number of fibroblast GFP cells and the intensities of GFP signals in Group A were significantly higher than in Group B after stent placement. The proliferative cellular response, including granulation tissue, epithelial layer, submucosal fibrosis, and connective tissue, was increased in Group A. FSP-1-positive cells were more prominent in Group A than in Group B. CONCLUSIONS: SEMS placement was feasible and safe in an esophageal mouse model, and proliferative cellular response caused by fibroblast cell proliferation after stent placement was longitudinally monitored using a noninvasive fluorescence microendoscopic technique. The results have implications for the understanding of proliferative cellular response after stent placement in real-life patients and provide initial insights into new clinical therapeutic strategies for restenosis.

Enhancement of local surface plasmon resonance (LSPR) effect by biocompatible metal clustering based on ZnO nanorods in Raman measurements.

The development of size-selective and non-destructive detection techniques for nanosized biomarkers has many reasons, including the study of living cells and diagnostic applications. We present an approach for Raman signal enhancement on biocompatible sensing chips based on surface enhancement Raman spectroscopy (SERS). A sensing chip was fabricated by forming a ZnO-based nanorod structure so that the Raman enhancement occurred at a gap of several tens to several hundred nanometers. The effect of coffee-ring formation was eliminated by introducing the porous ZnO nanorods for the bio-liquid sample. A peculiarity of this approach is that the gold sputtered on the ZnO nanorods initially grows at their heads forming clusters, as confirmed by secondary electron microscopy. This clustering was verified by finite element analysis to be the main factor for enhancement of local surface plasmon resonance (LSPR). This clustering property and the ability to adjust the size of the nanorods enabled the signal acquisition points to be refined using confocal based Raman spectroscopy, which could be applied directly to the sensor chip based on the optimization process in this experiment. It was demonstrated by using common cancer cell lines that cell growth was high on these gold-clad ZnO nanorod-based surface-enhanced Raman substrates. The porosity of the sensing chip, the improved structure for signal enhancement, and the cell assay make these gold-coated ZnO nanorods substrates promising biosensing chips with excellent potential for detecting nanometric biomarkers secreted by cells.