This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01 NS048353 and R01 NS109655 to S.L.C.; R01 NS109655-03S1 to D.P.J.), the National Cancer Institute (R01 CA122804 to S.L.C.), and the Department of Defense (X81XWH-09-1-0086 and W81XWH-12-1-0164 to S.L.C.).

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RASopathies:+Biology,+genetics+and+therapeutic+options.">The RASopathies: Biology, genetics and therapeutic options.</a> Adv Cancer Res. 153, 305-341 (2022).</li><li>Birindelli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rb+and+TP53+pathway+alterations+in+sporadic+and+NF1-related+malignant+peripheral+nerve+sheath+tumors.">Rb and TP53 pathway alterations in sporadic and NF1-related malignant peripheral nerve sheath tumors.</a> Lab Invest. 81 (6), 833-844 (2001).</li><li>Legius, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TP53+mutations+are+frequent+in+malignant+NF1+tumors.">TP53 mutations are frequent in malignant NF1 tumors.</a> Genes Chromosomes Cancer. 10 (4), 250-255 (1994).</li><li>Menon, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+17p+deletions+and+p53+gene+mutations+associated+with+the+formation+of+malignant+neurofibrosarcomas+in+von+Recklinghausen+neurofibromatosis.">Chromosome 17p deletions and p53 gene mutations associated with the formation of malignant neurofibrosarcomas in von Recklinghausen neurofibromatosis.</a> Proc Natl Acad Sci U S A. 87 (14), 5435-5439 (1990).</li><li>Upadhyaya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+and+somatic+NF1+gene+mutation+spectrum+in+NF1-associated+malignant+peripheral+nerve+sheath+tumors+(MPNSTs).">Germline and somatic NF1 gene mutation spectrum in NF1-associated malignant peripheral nerve sheath tumors (MPNSTs).</a> Hum Mutat. 29 (1), 74-82 (2008).</li><li>Kourea, H. P., Orlow, I., Scheithauer, B. W., Cordon-Cardo, C., Woodruff, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletions+of+the+INK4A+gene+occur+in+malignant+peripheral+nerve+sheath+tumors+but+not+in+neurofibromas.">Deletions of the INK4A gene occur in malignant peripheral nerve sheath tumors but not in neurofibromas.</a> Am J Pathol. 155 (6), 1855-1860 (1999).</li><li>Nielsen, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+transformation+of+neurofibromas+in+neurofibromatosis+1+is+associated+with+CDKN2A/p16+inactivation.">Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation.</a> Am J Pathol. 155 (6), 1879-1884 (1999).</li><li>Gregorian, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+dosage+is+essential+for+neurofibroma+development+and+malignant+transformation.">PTEN dosage is essential for neurofibroma development and malignant transformation.</a> Proc Natl Acad Sci U S A. 106 (46), 19479-19484 (2009).</li><li>Lee, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRC2+is+recurrently+inactivated+through+EED+or+SUZ12+loss+in+malignant+peripheral+nerve+sheath+tumors.">PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors.</a> Nat Genet. 46 (11), 1227-1232 (2014).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+mutations+of+SUZ12+in+malignant+peripheral+nerve+sheath+tumors.">Somatic mutations of SUZ12 in malignant peripheral nerve sheath tumors.</a> Nat Genet. 46 (11), 1170-1172 (2014).</li><li>Varela, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+structural+rearrangements+in+genetically+engineered+mouse+mammary+tumors.">Somatic structural rearrangements in genetically engineered mouse mammary tumors.</a> Genome Biol. 11 (10), 100 (2010).</li><li>Johnson, R. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+mutagenesis+in+p19(ARF)-+and+p53-deficient+mice+identifies+cancer+genes+and+their+collaborative+networks.">Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks.</a> Cell. 133 (4), 727-741 (2008).</li><li>Starr, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transposon-based+genetic+screen+in+mice+identifies+genes+altered+in+colorectal+cancer.">A transposon-based genetic screen in mice identifies genes altered in colorectal cancer.</a> Science. 323 (5922), 1747-1750 (2009).</li><li>Dupuy, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+sleeping+beauty+transposon+system+that+can+be+used+to+model+a+wide+variety+of+human+cancers+in+mice.">A modified sleeping beauty transposon system that can be used to model a wide variety of human cancers in mice.</a> Cancer Res. 69 (20), 8150-8156 (2009).</li><li>Carroll, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenge+of+Cancer+Genomics+in+Rare+Nervous+System+Neoplasms:+Malignant+Peripheral+Nerve+Sheath+Tumors+as+a+Paradigm+for+Cross-Species+Comparative+Oncogenomics.">The Challenge of Cancer Genomics in Rare Nervous System Neoplasms: Malignant Peripheral Nerve Sheath Tumors as a Paradigm for Cross-Species Comparative Oncogenomics.</a> Am J Pathol. 186 (3), 464-477 (2016).</li><li>Huijbregts, R. P., Roth, K. A., Schmidt, R. E., Carroll, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+neuropathies+and+malignant+peripheral+nerve+sheath+tumors+in+transgenic+mice+overexpressing+glial+growth+factor+beta3+in+myelinating+Schwann+cells.">Hypertrophic neuropathies and malignant peripheral nerve sheath tumors in transgenic mice overexpressing glial growth factor beta3 in myelinating Schwann cells.</a> J Neurosci. 23 (19), 7269-7280 (2003).</li><li>Kazmi, S. 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The authors have no conflicts of interest to disclose.

The detailed methods presented here were developed to study peripheral nervous system neoplasia and MPNST pathogenesis. Although we have found these methods to be effective, it should be recognized that there are some potential limitations to the methods we describe here. Below, we discuss some of those limitations and potential strategies for overcoming them in other model systems.
We have found that whole exome sequencing effectively identifies mutations of interest in P0-GGF&#946...

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive spindle cell neoplasms that arise in association with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), sporadically in the general population and at sites of previous radiotherapy1,2,3. NF1 patients are born with a wild-type copy of the NF1 tumor suppressor gene and a second NF1 allele with a loss-of-function mutation. This state of haploinsufficiency renders NF1 patients susceptible to a second loss-of-function mutation in their wild-type NF1 gene, which triggers tumorigenesis. When this &#34;second hit&#34; NF1 mutation occurs in a cell in the Schwann cell lineage, the resulting tumor is either a dermal neurofibroma arising in the skin or a plexiform neurofibroma that develops in large nerves or nerve plexuses. Although the pathology of dermal and plexiform neurofibromas is identical, their biologic behavior is quite different-although both dermal and plexiform neurofibromas are benign, only plexiform neurofibromas can undergo transformation and give rise to MPNSTs. In addition to the loss of neurofibromin, the Ras GTPase-activating protein encoded by the NF1 gene, MPNSTs carry mutations of multiple other tumor suppressor genes, including TP534,5,6,7, CDKN2A8,9, and PTEN10, mutations of genes encoding components of&#160;polycomb repressive complex 211,12 (PRC2; the SUZ12 and EED genes) and aberrant expression of receptor tyrosine kinases1,2. Mutations of NF1 and the other genes noted above are also present in sporadic and radiation-induced MPNSTs11,12.
While these advances in our understanding of the genomic abnormalities in MPNSTs have been invaluable for understanding their pathogenesis, they have not yet resulted in the development of effective new therapies for MPNSTs. A major barrier impeding the development of new treatments is the fact that MPNSTs are rare cancers. Because of this, it is difficult to obtain the large number of patient samples that are required for global analyses defining key driver mutations such as those undertaken by The Cancer Genome Atlas (TCGA). In our experience, accumulating even a modest number of human MPNST specimens can take years. To overcome such limitations, many investigators studying other rare cancer types have turned to the use of cross-species comparative oncogenomics to identify essential driver gene mutations, define the essential cytoplasmic signaling pathways in their tumor of interest, and identify new therapeutic targets. Since the signaling pathways that are essential for tumorigenesis are highly conserved between humans and other vertebrate species, applying functional genomics approaches such as genome-scale shRNA screens can be an effective means of identifying these new driver mutations, signaling pathways, and therapeutic targets13,14,15,16,17,18,19, particularly when studying rare human tumor types that are available in limiting numbers20.
In the methodologies presented here, we describe this approach to performing genomic profiling in human MPNST cell lines and early passage MPNST cultures derived from P0-GGF&#946;3 mice, a genetically engineered mouse model (GEM) in which Schwann cell-specific overexpression of the growth factor neuregulin-1 (NRG1) promotes the pathogenesis of plexiform neurofibromas and their subsequent progression to MPNSTs21,22,23. The first step in this approach is to identify candidate driver genes in P0-GGF&#946;3 MPNSTs, human MPNST cell lines, and surgically resected human MPNSTs. To functionally validate the signaling pathways affected by these mutations, we then use genome-scale shRNA screens to identify the genes required for proliferation and survival in human and mouse MPNST cell lines. After identifying the genes required for proliferation and survival, we then identify the druggable gene products within the collection of &#34;hits&#34; using the Drug Gene Interaction Database. We also compare the &#34;hits&#34; in human and mouse MPNST cells, to determine whether the GEM model and human MPNSTs demonstrate similar dependence on the same genes and signaling pathways. Identifying overlaps in the genes required for proliferation and survival and the affected signaling pathways serves as a means of validating the P0-GGF&#946;3 mouse model at a molecular level. This approach also emphasizes the effectiveness of combining human and mouse screens to identify novel therapeutic targets, where the mouse model can serve as a complement to the human screens. The value of this cross-species approach is particularly apparent when looking for therapeutic targets in rare tumors, where human tumors and cell lines are difficult to obtain.

<table><tbody><tr><td>Bioruptor Sonication System</td><td>Diagenode&amp;nbsp;</td><td>UCD-600</td><td></td></tr><tr><td>CASAVA 1.8.2</td><td></td><td></td><td></td></tr><tr><td>Cbot</td><td>Illumina, San Diego, CA</td><td>N/A</td><td></td></tr><tr><td>Celigo Image Cytometer</td><td>Nexcelom</td><td>N/A</td><td></td></tr><tr><td>Cellecta Barcode Analyzer and Deconvoluter software</td><td></td><td></td><td></td></tr><tr><td>Citrisolve Hybrid</td><td>Decon Laboratories</td><td>5989-27-5</td><td></td></tr><tr><td>Corning 96-well Black Microplate</td><td>Millipore Sigma</td><td>CLS3603</td><td></td></tr><tr><td>Diagenode Bioruptor 15ml conical tubes</td><td>Diagenode&amp;nbsp;</td><td>C30010009</td><td></td></tr><tr><td>dNTP mix</td><td>Clontech</td><td>639210</td><td></td></tr><tr><td>Eosin Y</td><td>Thermo Scientific</td><td>7111</td><td></td></tr><tr><td>Elution buffer</td><td>Qiagen&amp;nbsp;</td><td>19086</td><td></td></tr><tr><td>Ethanol (200 Proof)</td><td>Decon Laboratories</td><td>2716</td><td></td></tr><tr><td>Excel&amp;nbsp;</td><td>Microsoft&amp;nbsp;</td><td></td><td></td></tr><tr><td>FWDGEX 5&amp;rsquo;-CAAGCAGAAGACGGCATACGAGA-3&amp;rsquo;</td><td></td><td></td><td></td></tr><tr><td>FWDHTS 5&amp;rsquo;-TTCTCTGGCAAGCAAAAGACGGCATA-3&amp;rsquo;</td><td></td><td></td><td></td></tr><tr><td>GexSeqS (5&amp;rsquo; AGAGGTTCAGAGTTCTACAGTCCGAA-3&amp;rsquo;</td><td></td><td></td><td>HPLC purified</td></tr><tr><td>GraphPad Prism</td><td>Dotmatics</td><td></td><td></td></tr><tr><td>Harris Hematoxylin</td><td>Fisherbrand</td><td>245-677</td><td></td></tr><tr><td>Illumina HiScanSQ</td><td>Illumina, San Diego, CA</td><td>N/A</td><td></td></tr><tr><td>Paraformaldehyde (4%)</td><td>Thermo Scientific</td><td>J19943-K2</td><td></td></tr><tr><td>PLUS Transfection Reagent</td><td>Thermo Scientific</td><td>11514015</td><td></td></tr><tr><td>Polybrene Transfection Reagent</td><td>Millipore Sigma</td><td>TR1003G</td><td></td></tr><tr><td>PureLink Quick PCR Purification Kit</td><td>Invitrogen</td><td>K310001</td><td></td></tr><tr><td>Qiagen Buffer P1</td><td>Qiagen&amp;nbsp;</td><td>19051</td><td></td></tr><tr><td>Qiagen Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>RevGEX 5&amp;rsquo;-AATGATACGGCGACCACCGAGA-3&amp;rsquo;</td><td></td><td></td><td></td></tr><tr><td>RevHTS1 5&amp;rsquo;-TAGCCAACGCATCGCACAAGCCA-3&amp;rsquo;</td><td></td><td></td><td></td></tr><tr><td>Titanium Taq polymerase</td><td>Clontech</td><td>639210</td><td></td></tr><tr><td>Trimmomatic software</td><td></td><td>www.usadellab.org</td><td></td></tr></tbody></table>

genetic profiling and genome-scale dropout screening to identify therapeutic targets in mouse models of malignant peripheral nerve sheath tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Figure 5&#160;plots display depletion scores of core essential genes (CEGs) labeled as TRUE compared to non-CEGs (labeled as FALSE) in each human cell line that was screened. Points represent log2 of fold depletion scores for individual genes, which are plotted over a boxplot representation of the overall score distribution. Student&#39;s t-test was used to test for a significant difference in the mean of depletion scores between the two groups in each cell line. The resulting p...

Watch this Scientific Journal Video about Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens at JoVE.com

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

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Cancer Research

1.1K Views.  Medical University of South Carolina. We streven ernaar om de pathogenese van sporadische en NF1-geassocieerde kwaadaardige perifere zenuwschedetumoren te begrijpen. Ons doel is om logischerwijs nieuwe behandelingen te ontwikkelen voor deze zeer agressieve neoplasmata. Technologieën die we momenteel implementeren om ons onderzoek vooruit te helpen, zijn onder meer onbevooroordeelde methoden zoals shRNA-schermen op genoomschaal, evenals sequencing van één cel, BaseScope in situ-sondes en bio-informatica.Met behulp van de...

Video: Auteur in de schijnwerpers: nieuwe therapeutische doelen vinden voor kwaadaardige perifere zenuwschedetumoren door middel van shRNA-schermen op genoomschaal 

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

Pathology Assessment of PN-MPNST Progression in the P0-GGFβ3 Mouse Model

Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that subsequently transform into highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). Understanding the process by which a PN transforms into an MPNST would be facilitated by the availability of genetically engineered mouse (GEM) models that accurately replicate the PN-MPNST progression seen in humans with NF1. Unfortunately, GEM models with Nf1 ablation do not fully recapitulate this process. This led us to develop P0-GGF&#946;3 mice, a GEM model in which overexpression of the Schwann cell mitogen neuregulin-1 (NRG1) in Schwann cells results in the development of PNs that progress to become MPNSTs with high frequency. However, to determine whether tumorigenesis and neoplastic progression in P0-GGF&#946;3 mice accurately model the processes seen in NF1 patients, we had to first prove that the pathology of P0-GGF&#946;3 peripheral nerve sheath tumors recapitulates the pathology of their human counterparts.
Here, we describe the specialized methodologies used to accurately diagnose and grade peripheral nervous system neoplasms in GEM models, using P0-GGF&#946;3 and P0-GGF&#946;3;Trp53+/- mice as an example. We describe the histologic, immunohistochemical, and histochemical methods used to diagnose PNs and MPNSTs, how to distinguish these neoplasms from other tumor types that mimic their pathology, and how to grade these neoplasms. We discuss the establishment of early-passage cultures from GEM MPNSTs, how to characterize these cultures using immunocytochemistry, and how to verify their tumorigenicity by establishing allografts. Collectively, these techniques characterize the pathology of PNs and MPNSTs that arise in GEM models and critically compare the pathology of these murine tumors to their human counterparts.

Author Spotlight: Genetically Engineered Mouse Models and Pathological Characterization of Neurofibromatosis Type 1 Associated Tumors

We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P0-GGF&#946;3 mouse model.

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that ...

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

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