Summary Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex shRNA screens to identify a novel class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small molecule lead identification to target discovery and validation.
Isolating tumor exosomes (TEX) secreted by cancer cells can provide valuable information about the state of a tumor. Here, we present a method to rapidly isolate TEX using magnetic nanowires (MNWs). Specifically, two sets of Fe/Au segmented MNWs were used to isolate TEX released by canine osteosarcoma cell lines (OSCA 8,32,and 40). These MNWs were prepared by electrodeposition showcasing similar length (2.2(1) μm) and diameter (36(3) nm) but different Fe/Au segment thickness: 120(20)/30( 6) nm (sample A) and 28(7)/3(1) nm (sample B).Magnetic measurements indicate that we can effectively tune the magnetic response of the MNWs by changing their segment thickness, obtaining a more anisotropic behavior for sample A. The internalization of these MNWs by OSCA cells as a function of their concentration has been followed by fluorescence microscopy, and a concentration around 25 μg of Fe/Au MNWs per 3 × 10 5 cells has been defined as optimal. Electron microscopy images have revealed that, once internalized, these MNWs end up residing within lysosomes inside the cancer cells, where they tend to be degraded (especially the Fe segments) and fragmented into smaller pieces. Lower degradation for sample B has been observed and related to differences in the synthesis/functionalization process of both samples. We have hypothesized that these fragments of Fe/ Au MNWs are packaged into TEX released to the medium which can then be isolated via a magnetic stand. This has been tested by carrying out TEX isolation experiments on the OSCA cell and comparing the magnetically isolated TEX with those isolated by using conventional methods based on centrifugation. Nanoparticle tracking analysis (NanoSight) has confirmed that the TEX isolated with MNWs have a comparable size distribution and yield to those obtained by using conventional methods, indicating that our magnetic isolation method can consistently provide relatively high TEX yields in a low-cost and fast way.
We developed a novel, genome-wide method for biomarker discovery that is able to separate intrinsic markers of disease from markers of host response. Specifically, we implanted cell lines derived from two primary canine osteosarcoma tumors with different biological behavior into the tibiae of athymic nude mice. Both cell lines formed orthotopic tumors, and each recapitulated the biological behavior of the original primary tumor in terms of growth rate and metastatic potential (1,2). We also developed a cross-species hybrid genome that allowed us to identify separate canine and mouse transcripts in tumor xenografts (2). In other words, we were able to determine the contribution of canine mRNA sequences (derived from the implanted tumor cells) and mouse mRNA sequences (derived from infiltrating stroma) to define tumor-intrinsic features and host-specific features that contribute to osteosarcoma progression. When we applied this analysis method to exosome-derived mRNAs, we found canine-derived transcripts that were only present in exosomes of tumor-bearing mice that were associated with functions such as protein kinase A (PKA) and actin cytoskeleton signaling, and with upstream transcriptional regulators such as FOXF1, ESR1, and TP53. We also found tumor-specific, canine-derived transcripts that were associated with functions such as caveolae-derived endocytosis, energy utilization and metabolism, immune interactions, and G-coupled receptor signaling, and with upstream transcriptional regulators such as HNF1A, ESR1, NIPR1, and HNF4A. This method has the potential to reduce the statistical uncertainty that arises from the use of pre-selected molecules among the background of thousands of genomic features in patient samples. Using genome-wide analyses, we are able to combine multiple, highly correlated genes to manage this problem, increase markers' robustness, and avoid random correlations. In conclusion, this technology is useful to identify nucleic acids that serve as cancer biomarkers with high precision, and inter-species sequence conservation can be used to further improve its predictions. References 1. Scott MC, Sarver AL, Tomiyasu H, Cornax I, Van Etten J, Varshney J, et al. Aberrant Retinoblastoma (RB)-E2F Transcriptional Regulation Defines Molecular Phenotypes of Osteosarcoma. J Biol Chem 2015;290(47):28070-83. 2. Scott MC, Tomiyasu H, Garbe JR, Cornax I, Amaya C, O'Sullivan MG, et al. Heterotypic models of osteosarcoma recapitulate tumor heterogeneity and biological behavior. Disease Models & Mechanisms 2016, doi: 10.1242/dmm.026849. Citation Format: Milcah C. Scott, John R. Garbe, Hirotaka Tomiyasu, Alicia Donnelly, Brad A. Bryan, Subbaya Subramanian, Jaime F. Modiano. Unbiased discovery of exosome-associated biomarkers using xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 817. doi:10.1158/1538-7445.AM2017-817
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