New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

The reciprocal interaction: Chemotherapy and tumor microenvironment

Despite significant improvements in ways to treat cancer, numerous patients still die from this disease. One of the reasons for this inability to cure cancer is the lack of ability of drugs to penetrate target cells properly. While studies on drug resistance have focused on the molecular mechanisms of single cells, there has been little attention on drug penetration or distribution in solid tumor tissues. It is reported that the factors that obstruct the penetration and distribution of drugs in solid tumors are closely related to the microenvironment of solid tumors. This review paper aims to discuss the microenvironment that hinders drug penetration in solid tumors and to investigate whether or not changes in the microenvironment can improve drug penetration. This review also introduces in vitro 3D multicellular culture systems that can reproduce the characteristics of solid tumors in vivo and that are required for such studies

Co-Culture of Tumor Spheroids and Fibroblasts in a Collagen Matrix-Incorporated Microfluidic Chip Mimics Reciprocal Activation in Solid Tumor Microenvironment

Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a ‘more clinically relevant’ tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation

Microfluidic co-culture of pancreatic tumor spheroids with stellate cells as a novel 3D model for investigation of stroma-mediated cell motility and drug resistance

Abstract Background Pancreatic stellate cells (PSCs), a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. Culturing various cells in microfluidic (microchannel) devices has proven to be a useful in studying cellular interactions and drug sensitivity. Here we present a microchannel plate-based co-culture model that integrates tumor spheroids with PSCs in a three-dimensional (3D) collagen matrix to mimic the tumor microenvironment in vivo by recapitulating epithelial-mesenchymal transition and chemoresistance. Methods A 7-channel microchannel plate was prepared using poly-dimethylsiloxane (PDMS) via soft lithography. PANC-1, a human pancreatic cancer cell line, and PSCs, each within a designated channel of the microchannel plate, were cultured embedded in type I collagen. Expression of EMT-related markers and factors was analyzed using immunofluorescent staining or Proteome analysis. Changes in viability following exposure to gemcitabine and paclitaxel were measured using Live/Dead assay. Results PANC-1 cells formed 3D tumor spheroids within 5 days and the number of spheroids increased when co-cultured with PSCs. Culture conditions were optimized for PANC-1 cells and PSCs, and their appropriate interaction was confirmed by reciprocal activation shown as increased cell motility. PSCs under co-culture showed an increased expression of α-SMA. Expression of EMT-related markers, such as vimentin and TGF-β, was higher in co-cultured PANC-1 spheroids compared to that in mono-cultured spheroids; as was the expression of many other EMT-related factors including TIMP1 and IL-8. Following gemcitabine exposure, no significant changes in survival were observed. When paclitaxel was combined with gemcitabine, a growth inhibitory advantage was prominent in tumor spheroids, which was accompanied by significant cytotoxicity in PSCs. Conclusions We demonstrated that cancer cells grown as tumor spheroids in a 3D collagen matrix and PSCs co-cultured in sub-millimeter proximity participate in mutual interactions that induce EMT and drug resistance in a microchannel plate. Microfluidic co-culture of pancreatic tumor spheroids with PSCs may serve as a useful model for studying EMT and drug resistance in a clinically relevant manner

Three-Dimensional Imaging for Multiplex Phenotypic Analysis of Pancreatic Microtumors Grown on a Minipillar Array Chip

Three-dimensional (3D) culture of tumor spheroids (TSs) within the extracellular matrix (ECM) represents a microtumor model that recapitulates human solid tumors in vivo, and is useful for 3D multiplex phenotypic analysis. However, the low efficiency of 3D culture and limited 3D visualization of microtumor specimens impose technical hurdles for the evaluation of TS-based phenotypic analysis. Here, we report a 3D microtumor culture-to-3D visualization system using a minipillar array chip combined with a tissue optical clearing (TOC) method for high-content phenotypic analysis of microtumors. To prove the utility of this method, phenotypic changes in TSs of human pancreatic cancer cells were determined by co-culture with cancer-associated fibroblasts and M2-type tumor-associated macrophages. Significant improvement was achieved in immunostaining and optical transmission in each TS as well as the entire microtumor specimen, enabling optimization in image-based analysis of the morphology, structural organization, and protein expression in cancer cells and the ECM. Changes in the invasive phenotype, including cellular morphology and expression of epithelial&ndash;mesenchymal transition-related proteins and drug-induced apoptosis under stromal cell co-culture were also successfully analyzed. Overall, our study demonstrates that a minipillar array chip combined with TOC offers a novel system for 3D culture-to-3D visualization of microtumors to facilitate high-content phenotypic analysis

Design of microfluidic chip for tumor spheroid-fibroblast co-culture.

<p>Structure and organization of a microfluidic chip used for 3D co-culture of human colorectal cancer cells (HT-29) and normal colorectal fibroblasts (CCD-18Co). One chip contained 4 units and one unit consisted of 7 channels for either cell loading or media fill. Channel designation for co-culture: cancer cells and fibroblast cells were loaded in channel 4 and 2, respectively, and other channels (1 and 3) were used for media fill. A cell loading channel is shown with detailed structure and dimension (left-bottom).</p

Activation of fibroblasts under co-culture with 3D tumor spheroids.

<p>(A) Fluorescence images of fibroblasts stained for F-actin and α-SMA showing differential expression levels under mono- and co-culture conditions. (B) Increased migration ability of fibroblasts towards 3D tumor compartment. A picture for a representative regions showing fibroblast migrated out of the designated channel and comparison of migration distance. The migration distance of fibroblasts was measured between the nucleus of fibroblast in medium channel and end of fibroblast culture channel. Cells were grown for 6 days before all measurements. Optical sections were acquired at 3 μm intervals and stacked into a z-projection from which fluorescence intensity was calculated. Student t-test was used to test the statistical significance. Data are expressed as the mean ± SE of 3 replicates. (Scale bars 200 μm, 100 μm). * p<0.05.</p

Effect of co-culture on growth and size distribution of spheroids.

<p>Cells were grown in collagen-supported microfluidic channels with or without fibroblast co-cultures and number and size distribution was determined at day 5. (A) Growth of HT-29 spheroids in size and number (scale bar = 200 μm). (B) Comparison of mean diameter of HT-29 spheroids over 5 days. (C) Comparison of size distribution of tumor spheroids on day 5. Diameter of spheroids was calculated using bright field images and Image J program. Cell aggregates of diameter larger than 50 μm were considered as spheroids. Student t-test as well as χ² test were used for the statistical significance. Data are expressed as the mean ± SE of 3 replicates. * p<0.05, **p<0.01.</p

Differential protein expressions in HT-29 tumor spheroids with or without fibroblast co-cultures.

<p>When co-cultured with fibroblasts, TS showed up-regulation of 7 angiogenesis-related proteins with 1.5-fold or greater changes (A) and down-regulation of 5 apoptosis-related proteins with greater than 30% changes (B). HT-29 TSs were grown for 6 days with or without fibroblasts in microfluidic channels and harvested for analysis using Proteome Profiler™ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159013#sec002" target="_blank">Materials and Methods</a> for details).</p