Microfluidic-based human prostate-cancer-on-chip.

Lack of adequate models significantly hinders advances in prostate cancer treatment, where resistance to androgen-deprivation therapies and bone metastasis remain as major challenges. Current in vitro models fail to faithfully mimic the complex prostate physiology. In vivo animal models can shed light on the oncogenes involved in prostate cancer development and progression; however, the animal prostate gland is fundamentally different from that of human, and the underlying genetic mechanisms are different. To address this problem, we developed the first in vitro microfluidic human Prostate-Cancer-on-Chip (PCoC) model, where human prostate cancer and stromal fibroblast cells were co-cultivated in two channels separated by a porous membrane under culture medium flow. The established microenvironment enables soluble signaling factors secreted by each culture to locally diffuse through the membrane pores affecting the neighboring culture. We particularly explored the conversion of the stromal fibroblasts into cancer-associated fibroblasts (CAFs) due to the interaction between the 2 cell types. Immunofluorescence microscopy revealed that tumor cells induced CAF biomarkers, αSMA and COL1A1, in stromal fibroblasts. The stromal CAF conversion level was observed to increase along the flow direction in response to diffusion agents, consistent with simulations of solute concentration gradients. The tumor cells also downregulated androgen receptor (AR) expression in stromal fibroblasts, while an adequate level of stromal AR expression is maintained in normal prostate homeostasis. We further investigated tumor invasion into the stroma, an early step in the metastatic cascade, in devices featuring a serpentine channel with orthogonal channel segments overlaying a straight channel and separated by an 8 µm-pore membrane. Both tumor cells and stromal CAFs were observed to cross over into their neighboring channel, and the stroma's role seemed to be proactive in promoting cell invasion. As control, normal epithelial cells neither induced CAF conversion nor promoted cell invasion. In summary, the developed PCoC model allows spatiotemporal analysis of the tumor-stroma dynamic interactions, due to bi-directional signaling and physical contact, recapitulating tissue-level multicellular responses associated with prostate cancer in vivo. Hence, it can serve as an in vitro model to dissect mechanisms in human prostate cancer development and seek advanced therapeutic strategies.

Mantle cell lymphoma polarizes tumor-associated macrophages into M2-like macrophages, which in turn promote tumorigenesis.

Tumor-associated macrophages (TAMs) are recognized as a hallmark of certain solid cancers and predictors of poor prognosis; however, the functional role of TAMs in lymphoid malignancies, including B-cell lymphoma, has not been well defined. We identified infiltration of F4/80+ TAMs in a syngeneic mouse model using the recently generated murine mantle cell lymphoma (MCL) cell line FC-muMCL1. Multicolor flow cytometric analysis of syngeneic lymphoma tumors showed distinct polarization of F4/80+ TAMs into CD206+ M2 and CD80+ M1 phenotypes. Using human MCL cell lines (Mino, Granta, and JVM2), we further showed that MCL cells polarized monocyte-derived macrophages toward an M2-like phenotype, as assessed by CD163+ expression and increased interleukin-10 (IL-10) level; however, levels of the M1 markers CD80 and IL-12 remained unaffected. To show that macrophages contribute to MCL tumorigenesis, we xenografted the human MCL cell line Mino along with CD14+ monocytes and compared tumor growth between these 2 groups. Results showed that xenografted Mino along with CD14+ monocytes significantly increased the tumor growth in vivo compared with MCL cells alone (P < .001), whereas treatment with liposomal clodronate (to deplete the macrophages) reversed the effect of CD14+ monocytes on growth of MCL xenografts (P < .001). Mechanistically, IL-10 secreted by MCL-polarized M2-like macrophages was found to be responsible for increasing MCL growth by activating STAT1 signaling, whereas IL-10 neutralizing antibody or STAT1 inhibition by fludarabine or STAT1 short hairpin RNA significantly abolished MCL growth (P < .01). Collectively, our data show the existence of a tumor microenvironmental network of macrophages and MCL tumor and suggest the importance of macrophages in interventional therapeutic strategies against MCL and other lymphoid malignancies.

Inhibition of DUX4 expression with antisense LNA gapmers as a therapy for facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD), characterized by progressive muscle weakness and deterioration, is genetically linked to aberrant expression of DUX4 in muscle. DUX4, in its full-length form, is cytotoxic in nongermline tissues. Here, we designed locked nucleic acid (LNA) gapmer antisense oligonucleotides (AOs) to knock down DUX4 in immortalized FSHD myoblasts and the FLExDUX4 FSHD mouse model. Using a screening method capable of reliably evaluating the knockdown efficiency of LNA gapmers against endogenous DUX4 messenger RNA in vitro, we demonstrate that several designed LNA gapmers selectively and effectively reduced DUX4 expression with nearly complete knockdown. We also found potential functional benefits of AOs on muscle fusion and structure in vitro. Finally, we show that one of the LNA gapmers was taken up and induced effective silencing of DUX4 upon local treatment in vivo. The LNA gapmers developed here will help facilitate the development of FSHD therapies.

PARP1 Differentially Interacts with Promoter region of DUX4 Gene in FSHD Myoblasts.

OBJECTIVE: The goal of the study is to identity proteins, which interact with the promoter region of double homeobox protein 4 (DUX4) gene known to be causative for the autosomal dominant disorder Facioscapulohumeral Muscular Dystrophy (FSHD). METHODS: We performed a DNA pull down assay coupled with mass spectrometry analysis to identify proteins that interact with a DUX4 promoter probe in Rhabdomyosarcomca (RD) cells. We selected the top ranked protein poly (ADP-ribose) polymerase 1 (PARP1) from our mass spectrometry data for further ChIP-qPCR validation using patients' myoblasts. We then treated FSHD myoblasts with PARP1 inhibitors to investigate the role of PARP1 in the FSHD myoblasts. RESULTS: In our mass spectrometry analysis, PARP1 was found to be the top ranked protein interacting preferentially with the DUX4 promoter probe in RD cells. We further validated this interaction by immunoblotting in RD cells (2-fold enrichment compared to proteins pulled down by a control probe, p<0.05) and ChIP-qPCR in patients' myoblasts (65-fold enrichment, p<0.01). Interestingly, the interaction was only observed in FSHD myoblasts but not in the control myoblasts. Upon further treatment of FSHD myoblasts with PARP1 inhibitors, we showed that treatment with a PARP1 inhibitor, 3-aminobenzamide (0.5 mM), for 24 h had a suppression of DUX4 (2.6 fold, p<0.05) and ZSCAN4, a gene previously shown to be upregulated by DUX4, (1.6 fold, p<0.01) in FSHD myoblasts. Treatment with fisetin (0.5 mM), a polyphenol compound with PARP1 inhibitory property, for 24 h also suppressed the expression of DUX4 (44.8 fold, p<0.01) and ZSCAN4 (2.2 fold, p<0.05) in the FSHD myoblasts. We further showed that DNA methyltransferase 1 (DNMT1), a gene regulated by PARP1 was also enriched at the DUX4 promoter in RD cells through immunoblotting (2-fold, p<0.01) and immortalized FSHD myoblasts (42-fold, p<0.01) but not control myoblasts through ChIP qPCR. CONCLUSION: Our results showed that PARP1 and DNMT1 interacted with DUX4 promoter and may be involved in modulating DUX4 expression in FSHD.

Association of BRAF V600E Mutation and MicroRNA Expression with Central Lymph Node Metastases in Papillary Thyroid Cancer: A Prospective Study from Four Endocrine Surgery Centers.

BACKGROUND: Studies have demonstrated an association of the BRAF(V600E) mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAF(V600E) mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM). METHODS: Between January 2012 and June 2013, 237 consecutive patients underwent total thyroidectomy and prophylactic central lymph node dissection (CLND) at four endocrine surgery centers. All tumors were tested for the presence of the BRAF(V600E) mutation and miR-21, miR-146b-3p, miR-146b-5p, miR-204, miR-221, miR-222, and miR-375 expression. Bivariate and multivariable analyses were performed to examine associations between molecular markers and aggressive clinicopathologic features of PTC. RESULTS: Multivariable logistic regression analysis of all clinicopathologic features found miR-146b-3p and miR-146b-5p to be independent predictors of CLNM, while the presence of BRAF(V600E) almost reached significance. Multivariable logistic regression analysis limited to only predictors available preoperatively (molecular markers, age, sex, and tumor size) found miR-146b-3p, miR-146b-5p, miR-222, and BRAF(V600E) mutation to predict CLNM independently. While BRAF(V600E) was found to be associated with CLNM (48% mutated in node-positive cases vs. 28% mutated in node-negative cases), its positive and negative predictive values (48% and 72%, respectively) limit its clinical utility as a stand-alone marker. In the subgroup analysis focusing on only classical variant of PTC cases (CVPTC), undergoing prophylactic lymph node dissection, multivariable logistic regression analysis found only miR-146b-5p and miR-222 to be independent predictors of CLNM, while BRAF(V600E) was not significantly associated with CLNM. CONCLUSION: In the patients undergoing prophylactic CLNDs, miR-146b-3p, miR-146b-5p, and miR-222 were found to be predictive of CLNM preoperatively. However, there was significant overlap in expression of these miRs in the two outcome groups. The BRAF(V600E) mutation, while being a marker of CLNM when considering only preoperative variables among all histological subtypes, is likely not a useful stand-alone marker clinically because the difference between node-positive and node-negative cases was small. Furthermore, it lost significance when examining only CVPTC. Overall, our results speak to the concept and interpretation of statistical significance versus actual applicability of molecular markers, raising questions about their clinical usefulness as individual prognostic markers.

MicroRNA Expression and Association with Clinicopathologic Features in Papillary Thyroid Cancer: A Systematic Review.

BACKGROUND: Studies have suggested that microRNAs (miR) may be useful prognostic markers and are associated with aggressive clinicopathologic features in papillary thyroid cancer (PTC). This systematic review examined associations between miRs and aggressive clinicopathologic features in PTC. METHODS: A literature search was performed within the PubMed, Embase, Cochrane, Web of Science, and Scopus databases for papers published prior to November 24, 2014. The search was performed by combining the concepts "thyroid tumor" with "microRNA" and by using "and" as the Boolean operator. Upon retrieval of candidate studies, full-text publications were reviewed in their entirety and selected if they examined the prognostic significance between miR expression and established aggressive clinicopathologic features of PTC. RESULTS: Fifteen studies from 13 unique groups that included 807 patients were reviewed. Most of the studies were retrospective, and none included patients who had undergone routine central lymph node dissection. Expression levels of miRs-21, -34b, -130b, -135b, -146b, -151, -181b, -199b-5p, -221, -222, -451, -623, -1271, -2861, and let-7e showed significant association with at least one aggressive feature, such as large tumor size, extrathyroidal extension, multifocality, lymphovascular invasion, lymph node metastases, distant metastasis, advanced American Joint Cancer Committee stage, and presence of the BRAF(V600E) mutation. Herein we summarize the literature with regard to these associations. CONCLUSION: Further studies are needed to investigate whether miRs are independent predictors of aggressive clinicopathologic features before it can be recommended that miR expression levels should be incorporated into the management algorithm for patients with PTC. A well-designed prospective study is needed to assess these potential associations.

Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts.

Facioscapulohumeral muscular dystrophy (FSHD) is believed to be caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR) on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT) primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01) and primary (4.7 fold, p < 0.01) FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.