Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer

Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 μM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 μM) and Imatinib (10 μM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance

Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10–35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression

A Case of Severe Panuveitis Associated with Psoriasis Vulgaris Successfully Treated with Infliximab

Purpose: Uveitis associated with psoriasis vulgaris is usually seen as an anterior segment inflammation, and it is very rare that the inflammation extends to the posterior segment. We herein report a case of severe panuveitis associated with psoriasis vulgaris presenting as retinal neovascularization, leading to vitreous hemorrhages that were successfully treated with infliximab (IFX). Case Report: A 27-year-old male with psoriasis vulgaris was referred to our hospital due to prolonged severe uveitis OU. He showed a severe anterior chamber inflammation with fibrin formation and total posterior iris synechia OU. With topical corticosteroid treatment, these conditions were relieved for a short time; however, the intraocular inflammation was exacerbated with vitreous hemorrhages caused by retinal neovascularization OS. After the administration of IFX therapy, the intraocular inflammation and retinal neovascularization was resolved, and so far, no severe recurrences have been seen for 3 years with the therapy. Conclusion: When we see patients with severe panuveitis associated with psoriasis extending to the posterior segment, IFX treatment may be a good therapeutic option

Two speed factors of visual recognition independently correlated with fluid intelligence.

Growing evidence indicates a moderate but significant relationship between processing speed in visuo-cognitive tasks and general intelligence. On the other hand, findings from neuroscience proposed that the primate visual system consists of two major pathways, the ventral pathway for objects recognition and the dorsal pathway for spatial processing and attentive analysis. Previous studies seeking for visuo-cognitive factors of human intelligence indicated a significant correlation between fluid intelligence and the inspection time (IT), an index for a speed of object recognition performed in the ventral pathway. We thus presently examined a possibility that neural processing speed in the dorsal pathway also represented a factor of intelligence. Specifically, we used the mental rotation (MR) task, a popular psychometric measure for mental speed of spatial processing in the dorsal pathway. We found that the speed of MR was significantly correlated with intelligence scores, while it had no correlation with one's IT (recognition speed of visual objects). Our results support the new possibility that intelligence could be explained by two types of mental speed, one related to object recognition (IT) and another for manipulation of mental images (MR)

Quantitation of sevoflurane in whole blood and aqueous solutions by volatile organic compound sensing

Introduction: It is difficult to quantify poorly soluble volatile anesthetics in aqueous solutions; this necessitates the development of alternative prompt methods to analyze the in vivo blood concentrations of anesthetics for the clinical assessment of anesthesia depth. In this study, we demonstrated that the difficulties can be overcome by using volatile organic compound (VOC) sensors, which allow the levels of vaporized VOCs to be quantified in several seconds and obviate the need for conventional techniques such as gas chromatography or nuclear magnetic resonance (NMR). Methods: The concentrations of a volatile general anesthetic (sevoflurane) in aqueous solutions containing human blood components and rabbit blood were measured using a VOC sensor and those in distilled water and phosphatidylcholine suspension were compared to those determined by NMR. Results: For all aqueous solutions with concentrations of up to 5 mM, the relationship between the VOC content and sevoflurane concentration was represented by a straight line passing through the origin. The concentration of sevoflurane determined by VOC sensing was well correlated with the values obtained by NMR at <1 mM, which is within the clinically relevant concentration levels. Discussion: Considering the results from this study, we can conclude that VOC sensing may be useful for measuring intraoperative blood anesthetic concentrations

A matrix of correlation coefficients among the APM score, inspection time (IT), and behavioral measures for mental rotation (MR) task.

<p>*<i>p</i><0.05.</p

Example stimuli and data analyses in mental rotation (MR) and inspection time (IT) tasks.

<p>In the MR task (panel A), subjects were instructed to answer whether two images of three-dimensional (3D) objects (each consisted of eleven cubes attached face-to-face, see Methods for details) were the same or different in their 3D structures. We plotted reaction times as a function of angular differences between the two shapes and estimated a slope and intercept with a linear fitting. The smaller slope of the linear function represents a higher speed of mental rotation. In the IT task (panel B), target and mask stimuli were successively presented near a fixation point. Subjects judged whether the left or right vertical line of the target was longer. In the figure above, a correct answer is left. A stimulus onset asynchrony (SOA) between the target and mask started from 80 ms, being changed in a step of 10 ms based on an accuracy of the last two trials. We plotted the accuracy as a function of SOA, estimating a 75% threshold with a linear fitting as an index of the IT. The smaller IT represents a faster speed of object recognition.</p

The relationship among three psychological measures (APM scores, MR slope, and IT) depicted over a template brain image in Statistical Parametric Mapping (SPM, available online athttp://www.fil.ion.ucl.ac.uk/spm/software/spm8/).

<p>Previous studies have indicated a close relationship between intelligence and a function of the frontal cortex. A speed of object recognition (IT) reflects a function of the ventral pathway (from the occipital to temporal regions), while the rotation of mental images is mainly performed in the dorsal pathway (from the occipital to parietal regions). Our study found that the MR slope (not intercept) and the IT were individually correlated with the APM scores, suggesting that the speed of mental rotation and object recognition reflect different factors of fluid intelligence.</p