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Purpose: We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase ovarian cancer chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4-driven ovarian cancer chemoresistance. Experimental Design: We used RNA sequencing to identify NFATC4-mediated differential gene expression. CRISPR-Cas9 and FST (follistatin)-neutralizing antibodies were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy. Results: We found that NFATC4 upregulates FST mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2–dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR knockout (KO) of FST in ovarian cancer cells or antibody-mediated neutralization of FST sensitizes ovarian cancer cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy-resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of patients with ovarian cancer significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression-free, post–progression-free, and overall survival. Conclusions: FST is a novel therapeutic target to improve ovarian cancer response to chemotherapy and potentially reduce recurrence rates.

Supplementary Figure 2. RNA-seq analysis. A. Schematic of the RNA-seq experimental design. B. Waterfall plot of RNA-seq data showing the 141 genes which were DE at all time points compared to luciferase control in HEY1 and SKOV3 cell lines. C. Unsupervised clustering analysis of the 141 DE genes. D. Box plots demonstrating MYC expression following NFATC4 activation. E. STRING analysis of the 141 DE genes.

Purpose:We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase ovarian cancer chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4-driven ovarian cancer chemoresistance.Experimental Design:We used RNA sequencing to identify NFATC4-mediated differential gene expression. CRISPR-Cas9 and FST (follistatin)-neutralizing antibodies were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy.Results:We found that NFATC4 upregulates FST mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2–dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR knockout (KO) of FST in ovarian cancer cells or antibody-mediated neutralization of FST sensitizes ovarian cancer cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy-resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of patients with ovarian cancer significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression-free, post–progression-free, and overall survival.Conclusions:FST is a novel therapeutic target to improve ovarian cancer response to chemotherapy and potentially reduce recurrence rates.

Supplementary Table 1. Primers used in this study.

Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid‐phase formats are widely used for high‐performance protein detection in medical research. However, the affinity reagents used, which are mainly poly‐ and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid‐phase proximity‐dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real‐time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid‐phase PLAs, which use NDV‐selective DNA aptamers, are more sensitive than the sandwich enzymatic‐linked aptamer assay (ELAA), and have a comparable sensitivity to real‐time reverse transcription PCR (rRT‐PCR) as the gold standard detection method. In addition, the solid‐phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper‐ and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT‐PCR. The specificity of PLA is shown to be concordant with rRT‐PCR., Newcastle disease virus is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real‐time PCR.

Extracellular vesicles (EVs) are continuously released by most cells, and they carry surface markers of their cells of origin. Found in all body fluids, EVs function as conveyers of cellular information, and evidence implicates them as markers of disease. These characteristics make EVs attractive diagnostic targets. However, detection and characterization of EVs is challenging due to their small size. We've established a method, called ExoPLA, that allows individual EVs to be detected and characterized at high specificity and sensitivity. Based on the in situ proximity ligation assay (in situ PLA), proximal oligonucleotide-conjugated antibodies bound to their targets on the surfaces of the EVs allow formation of circular products that can be fluorescently labeled by rolling circle amplification. The intense fluorescent signals produced in this assay allow detection and enumeration of individual EVs by flow cytometry. We describe the procedures for ExoPLA, along with expected results and troubleshooting. © 2017 by John Wiley & Sons, Inc. Keywords: ExoPLA; exosomes; extracellular vesicles; flow cytometry; proximity ligation assay

BACKGROUND Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. METHODS Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. RESULTS We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. CONCLUSIONS PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.

Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs) and the proteins present on their surfaces has been hampered by the small size of the EVs – in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification.

Proximity ligation assays are a group of protein detection techniques in which reagents with affinity for target proteins, typically antibodies, are coupled to short strands of DNA. DNA-modified affinity reagents are combined in assays constructed such that the coordinated binding of individual target molecules or complexes of interacting proteins by two or more of the reagents, followed by DNA ligation and/or polymerization reactions, gives rise to amplifiable DNA reporter strands. Proximity ligation assays have been shown to exhibit excellent sensitivity in single and multiplexed protein assays for individual or interacting proteins, both in solution and in situ. This unit describes procedures for developing solid-phase proximity ligation assays for soluble proteins using either real-time PCR or DNA sequencing as the readout. In addition, critical steps for assay optimization are discussed.