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Real-time quantitative and qualitative analyses of metastasis-associated proteases are critical for precise diagnosis and novel therapeutic treatment of advanced cancers. However, conventional methods based on DNA, peptides, and proteins require sophisticated chemistry and additional processes to expose detection moieties, and they lack elements of temporal control, which limit their applicability. We designed unique protease-activatable polymersomes (PeptiSomes) for high sensitivity, in situ quantitative analysis of activating membrane-type 1 matrix metalloproteinases (MT1-MMP, MMP14). To do this, we first synthesized an amphiphilic block polymer–peptide and a copolypeptide based on mPEG-b-pLeu and MT1-peptide-b-pLeu, respectively. Amphiphilic self-assembled PeptiSomes in water were capable of disassembling and releasing the encapsulated self-quenched fluorescence dye (calcein) via enzymatic activation by MT1-MMP. Our PeptiSome system may potentially prevent the initiation and progression of cancer metastasis. Furthermore, the PeptiSome approach described here is likely to facilitate the development of rapid protease assay techniques and further extend the role of proteases as metastasis indicators and therapeutic targets.

This study aims to evaluate the performance of a radiomic signature-based model for predicting recurrence-free survival (RFS) of locally advanced gastric cancer (LAGC) using preoperative contrast-enhanced CT. This retrospective study included a training cohort (349 patients) and an external validation cohort (61 patients) who underwent curative resection for LAGC in 2010 without neoadjuvant therapies. Available preoperative clinical factors, including conventional CT staging and endoscopic data, and 438 radiomic features from the preoperative CT were obtained. To predict RFS, a radiomic model was developed using penalized Cox regression with the least absolute shrinkage and selection operator with ten-fold cross-validation. Internal and external validations were performed using a bootstrapping method. With the final 410 patients (58.2 ± 13.0 years-old; 268 female), the radiomic model consisted of seven selected features. In both of the internal and the external validation, the integrated area under the receiver operating characteristic curve values of both the radiomic model (0.714, P P = 0.010 [external validation]) and the merged model (0.719, P P = 0.014) were significantly higher than those of the clinical model (0.616; 0.594). The radiomics-based model on preoperative CT images may improve RFS prediction and high-risk stratification in the preoperative setting of LAGC.

Background Recently, many potential non-invasive biomarkers have been developed using exosome-based liquid biopsy for early detection, diagnosis, and treatment of cancer. However, exosome analyses from small liquid biopsy samples have been limited with regard to sensitivity and efficiency. Methods We used a breast cancer model mouse with ERBB2 overexpression and collected liquid biopsy samples. We performed two-stage PCR for ERBB2, PPP1R1B, GRB7, and STARD3 genes. We compared conventional PCR methods with our two-stage PCR method. Both methods had same step of primer and cycle and amplification condition for RT-qPCR. PCR amplicons quality was validated by using the Sanger method. Results Quantitative analysis of all samples by conventional PCR compared with two-stage PCR template dilution (1/200) showed similar Ct values in all sample groups (cell, supernatant, urine, and plasma). Sequencing of the two-stage PCR-derived products revealed no changes in product sequence compared to that of conventional PCR. The obtained sequences showed 98-99% match of target gene sequences in the databases according to BLAST searches. Conclusion Two-stage PCR has better sensitivity and efficiency than conventional PCR methods for small-volume samples. Additionally, this method is useful for monitoring, as many genes can be assayed at once.

Manganese oxide (MnO) nanocubes were fabricated and their surface were modified by ligand encapsulation or ligand exchange, to render them water-soluble. And then, MnO formed the hollow structure by etching using acidic solution (phthalate buffer, pH 4.0). Depending on the ligand of the MnO surface, it increases the interaction between MnO and water molecules. Also, the hollow structure of MnO, as well as the ligand, can greatly enhance the accessibility of water molecules to metal ions by surface area-to-volume ratio. These factors provide high R1 relaxation, leading to strong T1 MRI signal. We have confirmed T1-weighted MR contrast effect using 4-kinds of MnO nanocubes (MnOEn, MnOEnHo, MnOEx and MnOExHo). They showed enough a MR contrast effect and biocompatibility. Especially, among them, MnOExHo exhibited high T1 relaxivity (r1) (6.02 mM−1 s−1), even about 1.5 times higher sensitivity than commercial T1 MR contrast agents. In vitro/in vivo studies have shown that MnOExHo provides highly sensitive T1-weighted MR imaging, thereby improving diagnostic visibility at the disease site.

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We investigated molecular structure change of d - and l -glucose by proton beam irradiation of 7.8 MeV, using terahertz time-domain spectroscopy. Both glucose pellets exposed to the irradiation of 1013, 4 × 1013, 6 × 1013, and 8 × 1013 particles/cm2 of the particle’s fluence, were characterized using complex refractive indices in terahertz frequency region. We found that fingerprints of two types of glucose in terahertz frequency were disappeared above 8 × 1013 particles/cm2. Crystallinity breaking and molecular structure change by proton irradiation was reaffirmed using X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy.

Summary Background Adjuvant chemotherapy after surgery improves survival of patients with stage II–III, resectable gastric cancer. However, the overall survival benefit observed after adjuvant chemotherapy is moderate, suggesting that not all patients with resectable gastric cancer treated with adjuvant chemotherapy benefit from it. We aimed to develop and validate a predictive test for adjuvant chemotherapy response in patients with resectable, stage II–III gastric cancer. Methods In this multi-cohort, retrospective study, we developed through a multi-step strategy a predictive test consisting of two rule-based classifier algorithms with predictive value for adjuvant chemotherapy response and prognosis. Exploratory bioinformatics analyses identified biologically relevant candidate genes in gastric cancer transcriptome datasets. In the discovery analysis, a four-gene, real-time RT-PCR assay was developed and analytically validated in formalin-fixed, paraffin-embedded (FFPE) tumour tissues from an internal cohort of 307 patients with stage II–III gastric cancer treated at the Yonsei Cancer Center with D2 gastrectomy plus adjuvant fluorouracil-based chemotherapy (n=193) or surgery alone (n=114). The same internal cohort was used to evaluate the prognostic and chemotherapy response predictive value of the single patient classifier genes using associations with 5-year overall survival. The results were validated with a subset (n=625) of FFPE tumour samples from an independent cohort of patients treated in the CLASSIC trial (NCT00411229), who received D2 gastrectomy plus capecitabine and oxaliplatin chemotherapy (n=323) or surgery alone (n=302). The primary endpoint was 5-year overall survival. Findings We identified four classifier genes related to relevant gastric cancer features ( GZMB, WARS, SFRP4 , and CDX1 ) that formed the single patient classifier assay. In the validation cohort, the prognostic single patient classifier (based on the expression of GZMB, WARS , and SFRP4 ) identified 79 (13%) of 625 patients as low risk, 296 (47%) as intermediate risk, and 250 (40%) as high risk, and 5-year overall survival for these groups was 83·2% (95% CI 75·2–92·0), 74·8% (69·9–80·1), and 66·0% (60·1–72·4), respectively (p=0·012). The predictive single patient classifier (based on the expression of GZMB, WARS , and CDX1 ) assigned 281 (45%) of 625 patients in the validation cohort to the chemotherapy-benefit group and 344 (55%) to the no-benefit group. In the predicted chemotherapy-benefit group, 5-year overall survival was significantly improved in those patients who had received adjuvant chemotherapy after surgery compared with those who received surgery only (80% [95% CI 73·5–87·1] vs 64·5% [56·8–73·3]; univariate hazard ratio 0·47 [95% CI 0·30–0·75], p=0·0015), whereas no such improvement in 5-year overall survival was observed in the no-benefit group (72·9% [66·5–79·9] in patients who received chemotherapy plus surgery vs 72·5% [65·8–79·9] in patients who only had surgery; 0·93 [0·62–1·38], p=0·71). The predictive single patient classifier groups (chemotherapy benefit vs no-benefit) could predict adjuvant chemotherapy benefit in terms of 5-year overall survival in the validation cohort (p interaction =0·036 in univariate analysis). Similar results were obtained in the internal evaluation cohort. Interpretation The single patient classifiers validated in this study provide clinically important prognostic information independent of standard risk-stratification methods and predicted chemotherapy response after surgery in two independent cohorts of patients with resectable, stage II–III gastric cancer. The single patient classifiers could complement TNM staging to optimise decision making in patients with resectable gastric cancer who are eligible for adjuvant chemotherapy after surgery. Further validation of these results in prospective studies is warranted. Funding Ministry of ICT and Future Planning; Ministry of Trade, Industry, and Energy; and Ministry of Health and Welfare.

This paper presents the development of an oscillator and a detector based on InP HBT technology operating near 300 GHz, and their application to terahertz reflection-mode imaging. The fabricated fundamental-mode common-base (CB) cross-coupled oscillator exhibits a peak output power of 5.3 dBm at 305.8 GHz, and the CB direct detector shows a responsivity higher than 40 kV/W and noise equivalent power lower than ${\text{35 pW/}}\sqrt {{\text{Hz}}} $ for a frequency range of 270–345 GHz. The imaging system employing the fabricated circuits as the signal source and detector was characterized for the resolution and dynamic range, and then, successfully applied for imaging various biological samples. The results show that low-power terahertz reflection-mode imaging based on solid-state electronic sources and detectors based on a commercial semiconductor technology is promising for various biomedical imaging applications.

Multifunctional nano-systems are an enticing approach toward the design of nanoparticles with desired characteristics because of interactions between various components of the system. The combination of various imaging modalities often augments the advantages and simultaneously overcomes restrictions encountered by the individual techniques. This report describes the development of multifunctional gold core/iron oxide porous-shell nanoparticles (AuFe NPs) functionalized with matrix metalloproteinase peptide for targeted multi-mode magnetic resonance and computed tomography imaging. This study demonstrates that AuFe NPs simultaneously possess both T2-based contrast effect and X-ray attenuation properties in vitro. The drug loading capacity of porous iron oxide shell is also proposed.

The use of drug-eluting stents (DESs) is a promising strategy for non-vascular diseases, especially human biliary cancer. However, the implementation of DESs suffers from two major obstacles: the side effects of drugs and the difficulty of controlling the drug release. These problems can be overcome if the stent elutes targeting nanoparticles that release drugs at time intervals that are dictated by the mechanisms of those drugs. We designed temporally controlled polymeric multi-prodrug nanoparticles (TCMPNs) that can be eluted from stents comprising polyurethane (PU) nanofiber as a polymeric matrix and paclitaxel (PTX)-loaded, CD44-targeting, hyaluronic acid-conjugated poly(lactic-co-glycolic acid) and gemcitabine (GEM) (P-H-G). TCMPNs enable two different types of drugs to be released temporally; PTX is released first owing to the collapse of the structure in the endosomes, and GEM, which induces synergistic anticancer activities, is hydrolyzed from P-H-G later in response to low pH. Embedded in the PU nanofiber, the TCMPNs demonstrate low initial burst behavior and sustainable release of the prodrug in vitro. Furthermore, TCMPN-eluting stents (TESs) exhibit continuous synergistic efficacy as available targeted cellular uptake prodrug delivery systems in tumor-bearing mice. These results demonstrate that this technology will open up cancer therapy by combining localized delivery and functional multi-drug-loaded nanoparticles.

We detected keratinizing squamous cell carcinoma (SCC) of the tongue using terahertz reflection imaging and reported unexpected characteristics that THz reflection intensity of tumor was low in normal regions.

A portable and nonlabeled plasmonic biosensor was advanced to enable the sensitive and selective detection of microRNA (miRNA) in a biological sample. miRNAs can act on several key cellular processes, including cell differentiation, cell cycle progression, and function as oncogenes. Detection of circulating miRNAs, especially in blood or urine samples, allows noninvasive and simple diagnosis of diseases. Herein, we report a localized surface plasmon resonance sensor (LSPR) based on an enzyme-assisted target recycling system and a developed LSPR probe for the detection of gastric cancer relevant miRNAs, miR-10b. The sensitivity of the sensor was improved by increasing the concentration of the signal-amplifying agent using the duplex-specific nuclease and by strongly binding the developed LSPR probe, tannic acid capping gold nanoparticles, to the DNA. Under optimal conditions, miR-10b detection could be realized in the range of 5 pM-10 nM with a detection limit of 2.45 pM. This integrated detection system represents an approach to sensitive detection of miRNAs and offers great applications in personalized medicine and monitoring of cancer.

We have proposed a novel strategy for miRNA detection through enzyme-free signal amplification by self-circulation of the hybridization between the miRNAs and molecular beacon (MB) circuits. Unlike general MB-based miRNA detection based on the one-to-one (1 : 1) hybridization between MBs and miRNA, our system consists of four species of MBs (MBs A, B, C and D) (MB circuits) and is activated by a hybridization chain reaction. MBs stably coexist as hairpin structures that hardly show fluorescence signals in the absence of target miRNA. After miRNA detection, this MB circuit is able to generate fluorescence signals and amplify the fluorescence signal, contributing to improvement in detection sensitivity under iso-thermal conditions without an enzyme. Furthermore, in vitro and in vivo studies have proven that MB circuits can detect low levels of miRNA with high sensitivity, compared to when only one MB alone is used. Therefore, the MB circuits can provide a useful platform for target miRNA detection.

Small animal positron emission tomography (PET) is a noninvasive imaging modality that enables in vivo imaging and quantification of the biological processes of small experimental animals. We have developed a small animal PET that utilizes a high-resolution multiplexed readout and charge signal transmission (CST) method. The small animal PET was composed of six detector blocks consisting of SiPMs and LYSO arrays. Six detector blocks were mounted on a PET gantry having an inner diameter of 76 mm, outer diameter of 112 mm, and axial length of 40.8 mm. The charge signals of SiPM output were transmitted to the input of multiplexed readout using 4 m flexible flat cables. The multiplexed readout was composed of six main boards, each of which included 36 detector boards, to reduce the number of readout channels by a factor of 36, with a multiplexing ratio of 144:4. The performance of the small animal PET was evaluated using NEMA NU 4-2008 standards, and its imaging capability was demonstrated by in vivo mouse imaging studies. The average energy and time resolutions were 13.2% ± 0.3% and 3.8 ns, respectively. The spatial resolution at the center of the transaxial FOV was 1.1 mm, and the peak sensitivity at the center of the axial FOV was 1.5%. The peak noise equivalent count (NEC) rate and scatter fraction were 21.1 kcps at 18.2 MBq and 21%, respectively. The acquired images demonstrated high quality tracer uptake patterns of small experimental animals. The results of performance evaluation and animal imaging indicate that the small animal PET developed in this study can provide high-quality small animal imaging with cost-effectiveness and compactness.

MicroRNAs (miRNAs) are important post-transcriptional gene regulators and can serve as potential biomarkers for many diseases. Most of the current miRNA detection techniques require purification from biological samples, amplification, labeling, or tagging, which makes quantitative analysis of clinically relevant samples challenging. Here we present a new strategy for the detection of miRNAs with uniformity over a large area based on signal amplification using enzymatic reactions and measurements using time-of-flight secondary ion mass spectrometry (ToF-SIMS), a sensitive surface analysis tool. This technique has high sequence specificity through hybridization with a hairpin DNA probe and allows the identification of single-base mismatches that are difficult to distinguish by conventional mass spectrometry. We successfully detected target miRNAs in biological samples without purification, amplification, or labeling of target molecules. In addition, by adopting a well-known chromogenic enzymatic reaction from the field of biotechnology, we extended the use of enzyme-amplified signal enhancement ToF (EASE-ToF) to protein detection. Our strategy has advantages with respect to scope, quantification, and throughput over the currently available methods, and is amenable to multiplexing based on the outstanding molecular specificity of mass spectrometry (MS). Therefore, our technique not only has the potential for use in clinical diagnosis, but also provides evidence that MS can serve as a useful readout for biosensing to perform multiplexed analysis extending beyond the limitations of existing technology.

The cancer stem cell (CSC) hypothesis postulates that cancer cells overexpressing CD44 are marked as CSCs that cause tumorigenesis and recurrence. This hypothesis suggests that CD44 is a potential therapeutic target that can interfere with CSCs qualities. MicroRNA-34a (miR-34a) is a promising candidate for CD44 repression-based cancer therapy as it has been reported to inhibit proliferation, metastasis, and survival of CD44-positive CSCs. Here, we used nanovesicles containing PLI/miR complexes (NVs/miR) to systemically deliver miR-34a and induce miR-34a-triggered CD44 suppression in orthotopically and subcutaneously implanted tumors in nude mice. Poly(l-lysine-graft-imidazole) (PLI) condenses miRs and is functionally modified to deliver miRs to the site of action by buffering effect of imidazole residues under endosomal pH. Indeed, NVs/miR consisting of PEGylated lipids enveloping PLI/miR complexes greatly reduced inevitable toxicity of polycations by compensating their surface charge and markedly improved their in vivo stability and accumulation to tumor tissue compared to PLI/miR polyplexes. Our NVs-mediated miR-34a delivery system specifically increased endogenous target miR levels, thereby attenuating proliferation and migration of gastric cancer cells by repressing the expression of CD44 with decreased levels of Bcl-2, Oct 3/4 and Nanog genes. Our strategy led to a greater therapeutic outcome than PLI-based delivery with highly selective tumor cell death and significantly delayed tumor growth in CD44-positive tumor-bearing mouse models, thus providing a fundamental therapeutic window for CSCs.

// Cheol Keun Park 1 , Ji Soo Park 2 , Hyo Song Kim 2 , Sun Young Rha 2 , Woo Jin Hyung 3 , Jae-Ho Cheong 3 , Sung Hoon Noh 3 , Sang Kil Lee 4 , Yong Chan Lee 4 , Yong-min Huh 5 , Hyunki Kim 1 1 Department of Pathology, Yonsei University College of Medicine, Seoul, Republic of Korea 2 Division of Medical Oncology, Yonsei Cancer Center, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea 3 Department of Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea 4 Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea 5 YUMS-KRIBB Medical Convergence Research Institute, Department of Radiology, Yonsei University College of Medicine, Seoul, Republic of Korea Correspondence to: Hyunki Kim, email: kimhyunki@yuhs.ac Keywords: gastric cancer, receptor tyrosine kinase, amplification, screening algorithm Received: June 17, 2016 Accepted: September 17, 2016 Published: September 27, 2016 ABSTRACT Although targeted therapy for receptor tyrosine kinases (RTKs) of advanced gastric cancers (AGCs) has been in the spotlight, guidelines for the identification of RTK-amplified gastric cancers (RA-GCs) have not been established. In this study, we investigate clinicopathologic characteristics of RA-GCs and propose a screening algorithm for their identification. We performed immunohistochemistry (IHC) for MLH1, MSH2, PMS2, MSH6, key RTKs (EGFR, HER2, MET), and p53, in situ hybridization for Epstein-Barr virus encoding RNA, and silver in situ hybridization (SISH) for EGFR , HER2 , and MET using tissue microarrays of 993 AGCs. On IHC, 157 (15.8%) 61, (6.15%), and 85 (8.56%) out of 993 cases scored 2+ or 3+ for EGFR, HER2, and MET, respectively. On SISH, 31.2% (49/157), 80.3% (49/61), and 30.6% (26/85) of 2+ or 3+ cases on IHC showed amplification of the corresponding genes. Of the 993 cases, 104 were classified as RA-GCs. RA-GC status correlated with older age ( P < 0.001), differentiated histology ( P = 0.001), intestinal or mixed type by Lauren classification ( P < 0.001), lymphovascular invasion ( P = 0.026), and mutant-pattern of p53 ( P < 0.001). The cases were divided into four subgroups using two classification systems, putative molecular classification and histologic-molecular classification , based on Lauren classification, IHC, and SISH results. The histologic-molecular classification showed higher sensitivity for identification of RA-GCs and predicted patient prognosis better than the putative molecular classification . In conclusion, RA-GCs show unique clinicopathologic features. The proposed algorithm based on histologic-molecular classification can be applied to select candidates for genetic examination and targeted therapy.

T1/T2 dual-mode magnetic resonance (MR) contrast agents (DMCAs) have gained much attention because of their ability to improve accuracy by providing two pieces of complementary information with one instrument. However, most of these agents are "always ON" systems that emit MR contrast regardless of their interaction with target cells or biomarkers, which may result in poor target-to-background ratios. Herein, we introduce a rationally designed magnetic relaxation switch (MGRS) for an activatable T1/T2 dual MR imaging system. Redox-responsive heteronanocrystals, consisting of a superparamagnetic Fe3O4 core and a paramagnetic Mn3O4 shell, are synthesized through seed-mediated growth and subsequently surface-modified with polysorbate 80. The Mn3O4 shell acts as both a protector of Fe3O4 in aqueous environments to attenuate T2 relaxation and as a redoxable switch that can be activated in intracellular reducing environments by glutathione. This simultaneously generates large amounts of magnetically decoupled Mn(2+) ions and allows Fe3O4 to interact with the water protons. This smart nanoplatform shows an appropriate hydrodynamic size for the EPR effect (10-100 nm) and demonstrates biocompatibility. Efficient transitions of OFF/ON dual contrast effects are observed by in vitro imaging and MR relaxivity measurements. The ability to use these materials as DMCAs is demonstrated via effective passive tumor targeting for T1- and T2-weighted MR imaging in tumor-bearing mice.

A novel method (i.e., continuous magnetic cell separation in a microfluidic channel) is demonstrated to be capable of inducing multifractionation of mixed cell suspensions into multiple outlet fractions. Here, multicomponent cell separation is performed with three different distinguishable magnetic nanoclusters (MnFe2O4, Fe3O4, and CoFe2O4), which are tagged on A431 cells. Because of their mass magnetizations, which can be ideally altered by doping with magnetic atom compositions (Mn, Fe, and Co), the trajectories of cells with each magnetic nanocluster in a flow are shown to be distinct when dragged under the same external magnetic field; the rest of the magnetic characteristics of the nanoclusters are identically fixed. This proof of concept study, which utilizes the magnetization-controlled nanoclusters (NCs), suggests that precise and effective multifractionation is achievable with high-throughput and systematic accuracy for dynamic cell separation.

Porous noble metal nanoparticles exhibit many attractive nanoplasmonic features, and these structures have potential applications in many fields. However, such applications have been hindered by a lack of synthetic methods with the ability to mass-produce mono-disperse nanoparticles. Current synthetic approaches to porous gold nanostructure fabrication involve galvanic replacement approaches or electrochemical deposition methods that are generally limited by stringent multi-step protocols and relatively low yields. Here, we introduce the facile synthesis of scalable, mono-disperse, mesoporous gold nanoparticles (MPGNs) using an acidic emulsification method. This method facilitates high synthetic yields (>93%) and tunable particle sizes (130–400 nm). MPGNs exhibit enhanced payloads of gadolinium (Gd), a contrast agent for magnetic resonance imaging. Additionally, they permit photo-thermal conversion under near-infrared light (NIR) irradiation due to the increased surface area to volume ratio and the unique, structure-mediated LSPR effect. Specifically, MPGNs fabricated using our method provided Gd payloads 2–4 orders of magnitude greater than previously reported theranostic nano-probes. We believe that our novel synthetic technique will not only contribute to large-scale production of homogeneous porous gold nanoparticles, but will also promote further research in porous noble metal nanostructures.

Exosomal messenger RNA (mRNA) has emerged as a valuable biomarker for liquid biopsy-based disease diagnosis and prognosis due to its stability in body fluids and its biological regulatory function. Here, we report a rapid one-step isothermal gene amplification reaction based on three-way junction (3WJ) formation and the successful detection of urinary exosomal mRNA from tumor-bearing mice. The 3WJ structure can be formed by the association of 3WJ probes (3WJ-template and 3WJ-primer) in the presence of target RNA. After 3WJ structure formation, the 3WJ primer is repeatedly extended and cleaved by a combination of DNA polymerase and nicking endonuclease, producing multiple signal primers. Subsequently, the signal primers promote a specially designed network reaction pathway to produce G-quadruplex probes under isothermal conditions. Finally, G-quadruplex structure produces highly enhanced fluorescence signal upon binding to thioflavin T. This method provides a detection limit of 1.23 pM (24.6 amol) with high selectivity for the target RNA. More importantly, this method can be useful for the sensing of various kinds of mRNA, including breast cancer cellular mRNA, breast cancer exosomal mRNA, and even urinary exosomal mRNA from breast cancer mice. We anticipate that the developed RNA detection assay can be used for various biomedical applications, such as disease diagnosis, prognosis, and treatment monitoring.

Superparamagnetic iron oxide nanoparticles (SPIONs) are actively used as highly sensitive imaging probes to provide contrast in MRI. In this study, we propose the use of SPIONs encapsulated with antibody-conjugated poly(lactic-co-glycolic acid) (PLGA) as a potent theragnostic agent. The SPIONs were synthesized by a chemical co-precipitation method of ferric and ferrous ions, and subsequently encapsulated with PLGA by using an emulsification-diffusion method. Herceptin was chemically conjugated to the SPION-encapsulating PLGA nanoparticles to target the human epidermal growth factor receptor 2 (Her2/neu) overexpressing breast cancers. FACS and MR molecular imaging revealed that the Her2/neu overexpressing cell line showed a stronger contrast enhancement than the Her2/neu non-expressing cell lines, and the signal intensity of in vivo MR imaging decreased as the concentration of Herceptin increased. This strategy of encapsulating SPIONs with PLGA will be highly useful in functionalizing magnetic nanoparticles and improving the diagnostic and therapeutic efficacy of a wide array of cancer treatments.

Localized surface plasmon resonance (LSPR) biosensors have attracted much interest due to their capacity for multiplexing, miniaturization, and high performance, which offers the potential for their integration into lab-on-a-chip platforms for point-of-care (POC) diagnostics. The need for microRNA (miRNA)-sensing platforms is particularly urgent because miRNAs are key regulators and biomarkers in numerous pathological processes and diseases. Unfortunately, however, development of such miRNA-sensing platforms has not yet been achieved. In order to realize the detection of these important biomarkers, there has been an increasing demand for POC-sensing platforms that enable label-free quantification with low sample consumption, good sensitivity, real-time responsiveness, and high throughput. Here, we developed a highly specific, sensitive LSPR miRNA-sensing platform on a flexible, scalable plasmonic nanostructure to enable single-base mismatch discrimination and attomole detection of miRNAs in clinically relevant samples. The hairpin probe contained a locked nucleic acid (LNA) that enabled the discrimination of single base mismatches based on differences in melting temperatures of perfectly matched or single base mismatched miRNAs when they formed base pairs with probes. In addition, through hybridization induced signal amplification based on precipitate formation on the gold surface through the enzyme reaction, we observed a dramatic LSPR peak shift, which enabled attomole detection. Additionally, our LSPR miRNA sensor enabled the detection of miR-200a-3p in total RNA extracts from primary cancer cell lines without purification or labeling of the miRNA. This label-free and highly specific miRNA sensing platform may have applications in POC cancer diagnostics without the need for gene amplification.

We have developed pyrene-modified magnetic polyaniline nanoparticles (MPANs), a hybridization of MnFe2O4 magnetic nanoparticles (MNPs) and polyaniline (PANI) with pyrene as a cross-linker, for diagnosis by simultaneous magnetic resonance (MR) imaging and photothermal therapy. In general, the strong affinity between PANI molecules, owing to interchain hydrogen bonding and the π–π interactions mediated by their aromatic composition, hinders the successful synthesis of complexes of PANI with inorganic materials. To overcome this obstacle, we used pyrene as a cross-linker to synthesize a stable complex of PANI with MNPs. We first modified the hydrophobic MNPs with pyrene through a hydrophobic interaction and then formed a complex with PANI. The pyrene-induced π–π interactions between the pyrene molecules on the MNPs and PANI reduced the affinity between the PANI molecules, and the strong affinity between the PANI molecules and MNPs rendered stable nanocomplexes of MPANs. Finally, Cetuximab-conjugated MPANs were fully tested for simultaneous diagnosis by magnetic resonance imaging (MRI) from superparamagnetic MnFe2O4 and photothermal ablation therapy from the near-infrared absorbing characteristics of PANI in epidermal growth factor receptor (EGFR) overexpressed cancer cells (A-431 cells) in vitro.

Study of the absorption spectrum in film dosimetry is important because the spectral absorbance of the film is related to the film’s total absorption dose. We investigated the absorption spectra of Gafchromic EBT2 film at various rotational angles in a visible wavelength band. The film was irradiated with 6- MV photon beams for a total dose of 300 cGy. The absorption spectra for different angles of rotation were obtained 24 h after the irradiation and we fitted the spectra by using Lorentzian functions. Two dominant absorption peaks, one at approximately 586 nm (green) and the other at 634 nm (red) were observed. The measured spectrum was decomposed into peaks at 542 nm, 558 nm, 578 nm, 586 nm, 626 nm, 634 nm, and 641 nm. The maximum total area of the red absorption band occurred at 45° (225°) and the minimum at 90° (270°). As the angle of rotation changed, the intensities and integrated areas of the blue and the green peaks also changed with a 180° period, with minima at 90° and 270°, and maxima at 0° and 180°, although the overall absorbance was very low. The spectral peak wavelengths remained constant within ±1.2 nm for all angles. The absorption spectrum of the film should no hysteresis of; spectra taken at 0° and 360° were substantially the same and showed similar behaviors for all rotational angles. The change in the absorbance with the rotational angle of the film affected the dosimetric properties, resulting in rotational variations of the film’s dosimetry in each red-green-blue channel.

To access smart optical theragnosis for cancer, an easily processable heterocyclic conjugated polymer (poly(sodium3-((3-methyl-3,4-dihydro-2H-thieno[3,4-b][1,4]dioxepin-3-yl)methoxy)propane-1-sulfonate), PPDS) nanoassembly is fabricated by a surfactant-free one-step process, without the laborious ordinary multicoating process. The conjugated nanoassembly, with a self-doped structure, provides strong absorbance in the near-infrared (NIR) range even in a neutral pH medium and exhibits excellent stability (>six months). In addition, the prepared PPDS nanoassembly shows a high photothermal conversion efficiency of 31.4% in organic photothermal nanoparticles. In particular, the PPDS nanoassembly is stably suspended in the biological medium without any additives. Through a simple immobilization with the anti-CD44 antibody, the prepared biomarker-targetable PPDS nanoassembly demonstrates specific targeting toward CD44 (expressed in stem-like cancer cells), allowing NIR absorbance imaging and the efficient targeted photothermal damaging of CD44-expressing cancer cells, from in vitro 3D mammospheres (similar to the practical structure of tumor in the body) to in vivo xenograft mice tumor models (breast cancer and fibrosarcoma). In this study, the most simplified preparation method is for this organic conjugated polymer-based nanoassembly by a molecular approach is reported, and demonstrated as a highly promising optical nanoagent for optical cancer theragnosis.

A decade ago, cancer stem cells (CSCs) were introduced as target cells for an innovative cancer treatment. Particularly, there have been a lot of biological researches on glioblastoma (GBM) CSCs. However, as there is a comprehensive change in the concept of CSCs, it is required to review how the different CSCs for patients can be clinically used, or clinical credentialing, and summarize the possibilities of clinical credentialing. In this regard, this review aims to introduce the tumorsphere obtained from GBM specimen and summarize the clinical dilemma and clinically applicable areas.

Although statins, or hydroxymethylglutaryl coenzyme A (HMG-Co A) reductase inhibitors, are generally used to decrease levels of circulating cholesterol, they have also been reported to have neuroprotective effects through various mechanisms. However, recent results have indicated that they may be harmful in patients with amyotrophic lateral sclerosis (ALS). In this study, we investigate whether atorvastatin protects motor neuron-like cells (NSC-34D) from oxidative stress. To evaluate the effects of atorvastatin or hydrogen peroxide or both on NSC-34D cells, the cells were treated with various combinations of these agents. To evaluate the viability of the cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and trypan blue staining were performed. Levels of free radicals and intracellular signaling proteins were evaluated using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. Atorvastatin protected NSC-34D cells against oxidative stress in a concentration-dependent manner. This neuroprotective effect of atorvastatin was blocked by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor and by FR180204, a selective extracellular signal-related kinase (ERK) inhibitor. Atorvastatin treatment increased the expression levels of p85αPI3K, phosphorylated Akt, phosphorylated glycogen synthase kinase-3β, phosphorylated ERK, and Bcl-2, which are proteins related to survival. Furthermore, atorvastatin decreased the levels of cytosolic cytochrome C, Bax, cleaved caspase-9, and cleaved caspase-3, which are associated with death in oxidative stress-injured NSC-34D cells. We conclude that atorvastatin has a protective effect against oxidative stress in motor neurons by activating the PI3K and ERK pathways as well as by scavenging free radicals. These findings indicate that statins could help protect motor neurons from oxidative stress.

In this study, T2- and T*2-weighted imaging potential of polysorbate-coated magnetic nanocrystals (P-MNCs) was investigated as a blood-pool contrast agent using a 9L-rat glioma model after intravenous injection via 3.0T MRI. Magnetic nanocrystals (MNCs, Fe3O4) synthesized by the thermal decomposition method were coated with polysorbate 80 using a nanoemulsion method to generate a water-stable MRI contrast agent. The physical properties and MR imaging capability of P-MNCs were verified. The orthotopic tumor models were established by implanting 9L-rat glioma cells into the rat brain. After tail-vein injection of P-MNCs, T2- and T*2-weighted imaging of tumor sites was performed. Blood clearance and biodistribution studies were also performed. The hydrodynamic diameter of P-MNCs was 10.5 ± 0.8 nm and a spherical magnetic core was confirmed. The r2 value of P-MNCs was calculated to be 114.1 mM−1 s−1. Heterogeneous contrast T2-weighted MRI images of the 9L-rat glioma model were visualized at the tumor site before injecting the MRI contrast agent. In particular, T*2-weighted images demonstrated more obvious signal intensity changes than did T2-weighted images. Neovasculature in the tumor tissue was clearly observed in T*2 images compared with T2-weighted images. The blood half-life of P-MNCs was 2 h and the Fe ion concentration of blood had returned to the baseline after 16 h. Well-tailored P-MNCs can be effectively used as a novel MRI contrast agent for visualizing of vasculatures for solid tumors via T2- and T*2-weighted imaging.

The integration of contrast-enhanced diagnostic imaging and therapy could utilize image guided therapy to plan treatment strategy. Toward this goal, a unique construction and operation of a pseudo metal based photothermal therapeutic agent (PPAM) is introduced by polyaniline (PANI) generation templated on iron oxide metal nanoclusters (MNCs). Notably, MNC core interferes as a catalytic agent and enables aniline polymerization under ambient acidic conditions. The intrusion of transition metal enhanced the proton sensitivity of PANI, which led to pH responsive conversion even at dilute proton concentrations (pH 5, 6) compared to the PANI particles prepared by conventional methods. Under physiological pH, PPAM reveals no special features; however, under low pH conditions, which is a notable characteristic of the cancer microenvironment, PPAM automatically converts into its emeraldine salt (ES) state and thus activates as a photothermal therapeutic agent. Utilizing this specific redox responsive switched off-on behavior of PPAM, precise and systemized photothermal therapy is demonstrated, proving itself as a smart and efficient photothermal therapeutic agent.

Noninvasive investigation of microRNAs (miRNAs) expression, which is deeply related to biological phenomena such as stem cell differentiation, in culture soup is particularly useful for monitoring of stem cell differentiation without phototoxicity of living cells, especially when cell morphologies remain unchanged during differentiation. However, real-time detection of miRNA in culture soup is not recommended because of insufficient miRNA amounts in culture soup. In this study, a convenient method is introduced for real-time assessing intracellular miRNA in culture soup by using lipovesicular miRNA beacon (Lipo-mB) and mechanical stimulus-mediated exocytosis. Pipetting-harvest of culture soup induces exocytosis-secretion of fluorescence signal of Lipo-mB from cytoplasm into culture soup. To demonstrate this method, Lipo-mB is applied for monitoring of adipogenesis by analyzing the expression levels of various intracellular miRNAs, which are related to adipogenesis regulators. The fluorescence intensity profile of the culture soup is correlated with the quantitative reverse-transcription-polymerase chain reaction data and absorbance of Oil Red O staining. These results demonstrate that Lipo-mB can successfully monitor stem cell differentiation by sensing changes in miRNA expression from culture soup of living cells. Lipo-mB can be further developed as an accurate sensing system for analyzing subtle differences in genotype, even when changes in phenotype cannot be observed.

Various types of nanoprobes have recently been utilized to monitor living organisms by detecting and imaging intracellular biomarkers, such as microRNAs (miRs). We here present a simple one-pot method to prepare stellate gold nanoparticles functionalized with miR-detecting molecular beacons (SGNP-MBs); low pH conditions permitted the rapid-high loading of MBs on the surface of SGNPs. Compared to the conventional gold nanoparticle-based MBs, SGNPs carried a 4.5-fold higher load of MBs and exhibited a 6.4-fold higher cellular uptake. We demonstrated that SGNP-MBs were successfully internalized in human gastric cancer cell lines and could be used to accurately detect and image intracellular miRs in an miR-specific manner. Furthermore, the relative levels of intracellular miRs in three different cell lines expressing miR-10b (high, moderate, and low levels) could be monitored using SGNP-MBs. Consequently, these results indicated that SGNP-MBs could have applications as highly potent, efficient nanoprobes to assess intracellular miR levels in living cells.

Vesicular pH modulates the function of many organelles and plays a pivotal role in cell metabolism processes such as proliferation and apoptosis. Here, we introduce a simple colorimetric redox-polyaniline nanoindicator, which can detect and quantify a broader biogenic pH range with superior sensitivity compared to pre-established trafficking agents employing one-dimensional turn-on of the fluorescence resonance-energy-transfer (FRET) signal. We fabricated polyaniline-based nanoprobes, which exhibited convertible transition states according to the proton levels, as an in situ indicator of vesicular transport pH. Silica-coated Fe3O4-MnO heterometal nanoparticles were synthesised and utilised as a metal oxidant to polymerise the aniline monomer. Finally, silica-coated polyaniline nanoparticles with adsorbed cyanine dye fluorophores Cy3 and Cy7 (FPSNICy3 and FPSNICy7) were fabricated as proton-sensitive nanoindicators. Owing to the selective quenching induced by the local pH variations of vesicular transport, FPSNICy3 and FPSNICy7 demonstrated excellent intracellular trafficking and provided sensitive optical indication of minute proton levels.

The development of a synchronized delivery and imaging system for small interfering RNA (siRNA) is required for the clinical application of RNA interference (RNAi) in cancer treatment. Herein, we report a pH-responsive, magnetic nanoparticle-based siRNA delivery system that can facilitate the safe and efficient delivery and visualization of therapeutic siRNA by high-resolution magnetic resonance (MR) imaging. Cationic poly-l-lysine-graft-imidazole (PLI) with a reactive silane moiety was stably immobilized onto the surface of the assembled manganese ferrite nanoparticles (MFs) through an emulsion process, ensuring high water solubility, enhanced MR contrast effect, and endosome-disrupting functionality. The synthesized nanovectors were then complexed with siRNA targeting the CD44 gene via electrostatic interactions to verify the specific gene-silencing effect. The imidazolized magnetic nanovector (ImMNV) architectures developed here facilitated improved cellular internalization and exhibited a high level in vitro downregulation compared to non-imidazolized MNVs in metastatic breast cancer cells.

Here, wire-framed Au nanobundles (WNBs), which consist of randomly oriented and mutually connected Au wires to form a bundle shape, are synthesized. In contrast to conventional nanoparticles (spheres, rods, cubes, and stars), which exhibit nanostructure only on the surface, cross-sectional view image shows that WNBs have nanostructures in a whole volume. By using this specific property of WNBs, an externally controllable multistep photothermic-driven drug release (PDR) system is demonstrated for in vivo cancer treatment. In contrast to conventional nanoparticles that encapsulate a drug on their surface, WNBs preserve the drug payload in the overall inner volume, providing a drug loading capacity sufficient for cancer therapy. An improved in vivo therapeutic efficacy of PDR therapy is also demonstrated by delivering sufficient amount of drugs to the target tumor region.

We describe the development of biomarker-sensitive nanoprobes based on nanoparticle surface energy transfer (NSET) effect that enabling recognition of the expression of membrane type-1 matrix metalloproteinase (MT1-MMP) anchored on invasive cancer cells and its proteolytic activity simultaneously. First of all, we confirmed invasiveness of cancer cell lines (HT1080 and MCF7) via migration and invasion assay. We also prepared gold nanoparticle (GNP) acts as a quencher for fluorescein isothiocyanate (FITC). This FITC is conjugated in end-terminal of activatable fluorogenic peptide (ActFP). The ActFP attach to surface of GNP (GNP-ActFP) for a targeting moiety and proteolytic activity ligand toward MT1-MMP. The GNP-ActFP can generate fluorescence signal when ActFP is cleaved by proteolytic activity after targeting toward MT1-MMP. In order to study specificity for MT1-MMP, GNP-ActFP is treated to HT1080 and MCF7 cells, and then, we determine the in vitro targeting potential and fluorogenic activity of GNP-ActFP for MT1-MMP via fluorescence multi-reader. We also confirmed fluorogenic activity of GNP-ActFP via confocal microscopic imaging, and finally, endocytosis of GNP-ActFP is observed via cellular transmission electron microscopic imaging.

Oleyl dextran-coated magnetic nanoclusters (ODMCs) are fabricated for the accurate detection of macrophage-rich atherosclerotic plaques using magnetic resonance (MR) imaging. Dextran is introduced to the cluster surface of magnetic nanocrystals (MNCs) through self-assembly using amphiphilic oleic acid-conjugated dextran (ODex) to provide not only hydrophilicity but also a high affinity to macrophages. Enhanced magnetism of the ODMCs is engineered by optimizing the degree of substitution (DS) of the oleyl group in ODex and the concentration of ODex used for the synthesis of ODMC. Consequently, ODMCs exhibit significantly increased T2 relaxivity and excellent macrophage-targeting ability without cytotoxicity, in vitro. Moreover, in vivo T2-weighted MR imaging after intravenous injection of ODMCs into a rat artery balloon injury model demonstrates considerable MR contrast strength efficacy in the plaques of the injured carotid artery. These novel ODMCs may offer a highly efficient MR imaging nanoprobes for the selective diagnosis of atherosclerosis.

By loading Gd(III) inside NIR-absorbing polyaniline nanostructures, a novel diagnostic and photothermal agent with enhanced MR sensitivity, targeting ability, and photothermal ability to treat epithelial cancer is developed.

We have formulated hexagonal-shaped gold nanoplates in a single-step for photothermal therapy that gold ions to gold particles using pyrenyl dextran as reducible stabilizer and template. They exhibit anisotropic structure with broad surface plasmon resonance (SPR) band into near-infrared (NIR) spectrum enabling photothermal therapy. These gold nanoplates are also confirmed biocompatibility and high uptake efficiency due to binding with dextran molecules on the surface of gold nanoplates and cells. From

Purpose Clinical implications of single patient classifier (SPC) and microsatellite instability (MSI) in stage II/III gastric cancer have been reported. We investigated SPC and the status of MSI and Epstein-Barr virus (EBV) as combinatory biomarkers to predict the prognosis and responsiveness of adjuvant chemotherapy for stage II/III gastric cancer. Materials and Methods Tumor specimens and clinical information were collected from patients enrolled in CLASSIC trial, a randomized controlled study of capecitabine plus oxaliplatin-based adjuvant chemotherapy. The results of nine-gene based SPC assay were classified as prognostication (SPC-prognosis) and prediction of chemotherapy benefit (SPC-prediction). Five quasimonomorphic mononucleotide markers were used to assess tumor MSI status. EBV-encoded small RNA in situ hybridization was performed to define EBV status. Results There were positive associations among SPC, MSI, and EBV statuses among 586 patients. In multivariate analysis of disease-free survival, SPC-prognosis [hazard ratio (HR): 1.879 (1.101–3.205), 2.399 (1.415–4.067), p=0.003] and MSI status (HR: 0.363, 95% confidence interval: 0.161–0.820, p=0.015) were independent prognostic factors along with age, Lauren classification, TNM stage, and chemotherapy. Patient survival of SPC-prognosis was well stratified regardless of EBV status and in microsatellite stable (MSS) group, but not in MSI-high group. Significant survival benefit from adjuvant chemotherapy was observed by SPC-Prediction in MSS and EBV-negative gastric cancer. Conclusion SPC, MSI, and EBV statuses could be used in combination to predict the prognosis and responsiveness of adjuvant chemotherapy for stage II/III gastric cancer.

Poly(ethylene glycol)-coated cross-linked iron oxide nanoparticles (PCIONs) are developed for therapeutic engineering of mesenchymal stem cells (MSCs) and their monitoring via magnetic resonance (MR) imaging at a time. PCIONs successfully combine with plasmid DNA (pDNA) via ionic interaction. Accordingly, PCION/pDNA complexes mediate superior translocations of vascular endothelial growth factor (VEGF) pDNA into intracellular regions of MSCs under external magnetic field, which significantly elevate production of VEGF from MSCs. Genetically engineered MSCs are also clearly visualized via MR imaging after administration to rat cerebrovascular ischemia models, which enable tracking of MSCs migration from injected sites to injured ischemic area.

Cells resembling bone marrow mesenchymal stem cells (BM-MSCs) have been isolated from glioma specimens; however, little is known about the existence of mesenchymal stem-like cells (MSLCs) in meningioma. Here, we hypothesized that cells similar to BM-MSCs exist in meningioma specimens and sought to investigate whether these putative meningioma stroma MSLCs (MS-MSLCs) could be isolated. To this end, we cultured fresh meningioma specimens using the same protocols as used previously to isolate BM-MSC. Cultured cells were analyzed for surface markers associated with BM-MSCs by fluorescence-activated cell sorting (FACS) and candidate cells were exposed to mesenchymal differentiation conditions. Possible locations of MS-MSLCs were determined by immunohistochemical analysis of sections of meningioma specimens. Spindle-shaped and, adherent cells similar to BM-MSCs were isolated in 2 of 20 meningioma specimens. FACS analysis showed that the surface markers of MS-MSLCs were similar to those of BM-MSCs and the chosen cells demonstrated an ability to differentiate into osteogenic, adipogenic and chondrogenic cells. The tumorigenicity of MS-MSLCs was tested by injection of these cells into the brain of athymic nude mice; no tumors were subsequently discovered. Immunohistochemical analyses indicated that CD105+ cells were closely associated with endothelial cells and pericytes in meningioma specimens. Our results established for the first time that cells similar to BM-MSCs exist in meningioma specimens. These cells, termed MS-MSLCs, could be one component of the meningioma cellular microenvironment.

Neurogenesis in the adult brain is important for memory and learning, and the alterations in neural stem cells (NSCs) may be an important part of Alzheimer's disease pathogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway has been suggested to play an important role in neuronal cell survival and is highly involved in adult neurogenesis. Recently, coenzyme Q10 (CoQ10) was found to affect the PI3K pathway. We investigated whether CoQ10 could restore amyloid β (Aβ)25-35 oligomer-inhibited proliferation of NSCs by focusing on the PI3K pathway. To evaluate the effects of CoQ10 on Aβ25-35 oligomer-inhibited proliferation of NSCs, NSCs were treated with several concentrations of CoQ10 and/or Aβ25-35 oligomers. BrdU labeling, Colony Formation Assays, and immunoreactivity of Ki-67, a marker of proliferative activity, showed that NSC proliferation decreased with Aβ25-35 oligomer treatment, but combined treatment with CoQ10 restored it. Western blotting showed that CoQ10 treatment increased the expression levels of p85α PI3K, phosphorylated Akt (Ser473), phosphorylated glycogen synthase kinase-3β (Ser9), and heat shock transcription factor, which are proteins related to the PI3K pathway in Aβ25-35 oligomers-treated NSCs. To confirm a direct role for the PI3K pathway in CoQ10-induced restoration of proliferation of NSCs inhibited by Aβ25-35 oligomers, NSCs were pretreated with a PI3K inhibitor, LY294002; the effects of CoQ10 on the proliferation of NSCs inhibited by Aβ25-35 oligomers were almost completely blocked. Together, these results suggest that CoQ10 restores Aβ25-35 oligomer-inhibited proliferation of NSCs by activating the PI3K pathway.

It has been reported that cancer stem cells (CSCs) can be isolated from primitive neuroectodermal tumor (PNET) specimens. Moreover, mesenchymal stem-like cells (MSLCs) have been isolated from Korean glioma specimens. Here, we tested whether tumor spheres and MSLCs can be simultaneously isolated from a single PNET specimen, a question that has not been addressed.We isolated single-cell suspensions from PNET specimens, then cultured these cells using methods for MSLCs or CSCs. Cultured cells were analyzed for surface markers of CSCs using immunocytochemistry and for surface markers of bone marrow-derived mesenchymal stem cells (BM-MSCs) using fluorescence-activated cell sorting (FACS). Tumor spheres were exposed to neural differentiation conditions, and MSLCs were exposed to mesenchymal differentiation conditions. Possible locations of MSLCs within PNET specimens were determined by immunofluorescence analysis of tumor sections.Cells similar to tumor spheres and MSLCs were independently isolated from one of two PNET specimens. Spheroid cells, termed PNET spheres, were positive for CD133 and nestin, and negative for musashi and podoplanin. PNET spheres were capable of differentiation into immature neural cells and astrocytes, but not oligodendrocytes or mature neural cells. FACS analysis revealed that adherent cells isolated from the same PNET specimen, termed PNET-MSLCs, had surface markers similar to BM-MSCs. These cells were capable of mesenchymal differentiation. Immunofluorescence labeling indicated that some CD105(+) cells might be closely related to endothelial cells and pericytes.We showed that both tumor spheres and MSLCs can be isolated from the same PNET specimen. PNET-MSLCs occupied a niche in the vicinity of the vasculature and could be a source of stroma for PNETs.