Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology

Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy is difficult, costly, and sometimes not even an option. Tumor-derived exosomes have attracted increasing interest in non-invasive cancer diagnosis and monitoring of treatment response. However, the biology and clinical value of exosomes remains largely unknown due in part to current technical challenges in rapid isolation, molecular classification and comprehensive analysis of exosomes. Here we developed a new microfluidic approach to streamline and expedite the exosome analysis pipeline by integrating specific immunoisolation and targeted protein analysis of circulating exosomes. Compared to the conventional methods, our approach enables selective subpopulation isolation and quantitative detection of surface and intravesicular biomarkers directly from a minimally invasive amount of plasma samples (30 μL) within ~100 min with markedly improved detection sensitivity. Using this device, we demonstrated phenotyping of exosome subpopulations by targeting a panel of common exosomal and tumor-specific markers and multiparameter analyses of intravesicular biomarkers in the selected subpopulation. We were able to assess the total expression and phosphorylation levels of IGF-1R in non-small-cell lung cancer patients by probing plasma exosomes as a non-invasive alternative to conventional tissue biopsy. We foresee that the microfluidic exosome analysis platform will form the basis for critically needed infrastructures for advancing the biology and clinical utilization of exosomes

Drug Repurposing for Gastrointestinal Stromal Tumor

Despite significant treatment advances over the past decade, metastatic gastrointestinal stromal tumor (GIST) remains largely incurable. Rare diseases, such as GIST, individually affect small groups of patients but collectively are estimated to affect 25–30 million people in the U.S. alone. Given the costs associated with the discovery, development and registration of new drugs, orphan diseases such as GIST are often not pursued by mainstream pharmaceutical companies. As a result, “drug repurposing” or “repositioning”, has emerged as an alternative to the traditional drug development process. In this study we screened 796 FDA-approved drugs and found that two of these compounds, auranofin and fludarabine phosphate, effectively and selectively inhibited the proliferation of GISTs including imatinib-resistant cells. One of the most notable drug hits, auranofin (Ridaura®), an oral, gold-containing agent approved by the FDA in 1985 for the treatment of rheumatoid arthritis (RA), was found to inhibit thioredoxin reductase (TrxR) activity and induce reactive oxygen species (ROS) production, leading to dramatic inhibition of GIST cell growth and viability. Importantly, the anti-cancer activity associated with auranofin was independent of IM resistant status, but was closely related to the endogenous and inducible levels of ROS, therefore is prior to IM response. Coupled with the fact auranofin has an established safety profile in patients, these findings suggest for the first time that auranofin may have clinical benefit for GIST patients, particularly in those suffering from imatinib-resistant and recurrent forms of this disease

In silico and in vitro drug screening identifies new therapeutic approaches for Ewing sarcoma.

The long-term overall survival of Ewing sarcoma (EWS) patients remains poor; less than 30% of patients with metastatic or recurrent disease survive despite aggressive combinations of chemotherapy, radiation and surgery. To identify new therapeutic options, we employed a multi-pronged approach using in silico predictions of drug activity via an integrated bioinformatics approach in parallel with an in vitro screen of FDA-approved drugs. Twenty-seven drugs and forty-six drugs were identified, respectively, to have anti-proliferative effects for EWS, including several classes of drugs in both screening approaches. Among these drugs, 30 were extensively validated as mono-therapeutic agents and 9 in 14 various combinations in vitro. Two drugs, auranofin, a thioredoxin reductase inhibitor, and ganetespib, an HSP90 inhibitor, were predicted to have anti-cancer activities in silico and were confirmed active across a panel of genetically diverse EWS cells. When given in combination, the survival rate in vivo was superior compared to auranofin or ganetespib alone. Importantly, extensive formulations, dose tolerance, and pharmacokinetics studies demonstrated that auranofin requires alternative delivery routes to achieve therapeutically effective levels of the gold compound. These combined screening approaches provide a rapid means to identify new treatment options for patients with a rare and often-fatal disease

Evaluating new therapies in gastrointestinal stromal tumor using in vivo molecular optical imaging

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the US. The majority (~85%) of GISTs possess gain-of-function mutations in KIT or PDGFRA, causing constitutive activation of the kinase receptor. GIST management has been transformed by the identification of tumor driver mutations leading to unprecedented disease control of advanced GIST with the introduction of imatinib mesylate (IM). Despite IM’s efficacy, most patients experience primary and/or secondary resistance within 2 y of treatment. Additional therapies and methods to optimize screening of novel approaches in preclinical studies are warranted. Clinically, treatment efficacy is typically assessed using Response Evaluation Criteria In Solid Tumors (RECIST) guidelines or Choi criteria. Both require a period of time on therapy before changes indicative of response can be observed. In addition, neither informs directly about cell death. We evaluated the use of molecular imaging technology in an animal model using near-infrared (NIR) imaging probes together with three-dimensional fluorescence molecular tomography (FMT) for assessing therapeutic response and ultimately optimizing our understanding of the biologic effects of these agents. We determined the potential of NIR probes (PSVue(TM)794 and cell-penetrating KcapQ647) for detecting distinct markers of apoptosis and compare this to tumor size measured by MRI in response to IM treatment in GIST-T1 xenografts. Our studies revealed statistically significant increases in apoptosis due to IM treatment using both probes as early as 24 h post IM treatment which was confirmed by IHC. Molecular imaging will allow for faster and more effective screening of novel therapies in preclinical GIST models

A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer.

Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents that have similarly shown poor clinical activity

Insulin-like growth factor binding protein-3 has dual effects on gastrointestinal stromal tumor cell viability and sensitivity to the anti-tumor effects of imatinib mesylate in vitro

<p>Abstract</p> <p>Background</p> <p>Imatinib mesylate has significantly improved survival and quality of life of patients with gastrointestinal stromal tumors (GISTs). However, the molecular mechanism through which imatinib exerts its anti-tumor effects is not clear. Previously, we found up-regulation of insulin-like growth factor binding protein-3 (IGFBP3) expression in imatinib-responsive GIST cells and tumor samples. Because IGFBP3 regulates cell proliferation and survival and mediates the anti-tumor effects of a number of anti-cancer agents through both IGF-dependent and IGF-independent mechanisms, we hypothesized that IGFBP3 mediates GIST cell response to imatinib. To test this hypothesis, we manipulated IGFBP3 levels in two imatinib-responsive GIST cell lines and observed cell viability after drug treatment.</p> <p>Results</p> <p>In the GIST882 cell line, imatinib treatment induced endogenous IGFBP3 expression, and IGFBP3 down-modulation by neutralization or RNA interference resulted in partial resistance to imatinib. In contrast, IGFBP3 overexpression in GIST-T1, which had no detectable endogenous IGFBP3 expression after imatinib, had no effect on imatinib-induced loss of viability. Furthermore, both the loss of IGFBP3 in GIST882 cells and the overexpression of IGFBP3 in GIST-T1 cells was cytotoxic, demonstrating that IGFBP3 has opposing effects on GIST cell viability.</p> <p>Conclusion</p> <p>This data demonstrates that IGFBP3 has dual, opposing roles in modulating GIST cell viability and response to imatinib <it>in vitro</it>. These preliminary findings suggest that there may be some clinical benefits to IGFBP3 therapy in GIST patients, but further studies are needed to better characterize the functions of IGFBP3 in GIST.</p

An RNA Interference Lethality Screen of the Human Druggable Genome to Identify Molecular Vulnerabilities in Epithelial Ovarian Cancer

Targeted therapies have been used to combat many tumor types; however, few have effectively improved the overall survival in women with epithelial ovarian cancer, begging for a better understanding of this deadly disease and identification of essential drivers of tumorigenesis that can be targeted effectively. Therefore, we used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (EOC) cell lines. The top 300 “hits” affecting the viability of A1847 cells were rescreened across additional EOC cell lines and non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly aberrantly overexpressed in serous adenocarcinomas. Overall, our functional genomics results integrated with the genomics data provide an important unbiased avenue towards the identification of prospective therapeutic targets for drug discovery, which is an urgent and unmet clinical need for ovarian cancer

Smoking and Drinking Activates NF-κB /IL-6 Axis to Promote Inflammation During Cervical Carcinogenesis

Background: High-risk strains of HPV are known to cause cervical cancer. Multiple clinical studies have emphasized that smoking and drinking are critical risk factors for cervical cancer and its high-grade precursors. In this study, we investigated the molecular mechanisms involved in the interplay of smoking and/or drinking with HPV infectivity and defined a systematic therapeutic approach for their attenuation in cervical cancer. Methods: The impact of benzo[a]pyrene (B[a]P) and/or ethanol (EtOH) exposure on cervical cancer cells was assessed by measuring changes in cell proliferation, clonogenicity, biophysical properties, cell migration, and invasion. Expression of HPV16 E6/E7, NF-κB, cytokines, cell cycle, and inflammation mediators was determined using qRT-PCR, immunoblotting, ELISA, luciferase reporter assay and confocal microscopy. Results: The exposure of cervical cancer cells to B[a]P and/or EtOH altered the expression of HPV16 E6/E7 oncogenes and EMT markers; it also enhanced cellular clonogenicity, migration, and invasion. In addition, B[a]P and/or EtOH exposure promoted inflammation pathways through TNF-α and NF-κB signaling, leading to IL-6 upregulation and activation of VEGFA. These molecular effects caused by B[a]P and/or EtOH exposure were effectively attenuated by Cur/PLGA-Cur. Conclusion: These data suggest a molecular link between smoking, drinking, and HPV infectivity in cervical carcinogenesis. However, these events were determined to be attenuated by treatment with Cur/PLGA-Cur treatment, implying its role in cervical cancer prevention/treatment