lentiviral infection of mpnst cells and genomic dna isolation for genome-scale shrna screens

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

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.

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

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

We are striving to understand the pathogenesis of sporadic and NF1-associated malignant peripheral nerve sheath tumors. Our goal is to logically develop new treatments for these highly aggressive neoplasms. Technologies we are currently implementing to advance our research include unbiased methods like genome-scale shRNA screens, as well as single-cell sequencing, BaseScope in situ probes, and bioinformatics.Using the various current approaches, we have identified multiple receptors that are essential for malignant peripheral nerve sheath tumors'proliferation and survival, as well as being therapeutically targetable. These receptors include the neuregulin receptor, ErbB3, and lysophosphatidic acid receptor 3. The advantage of using this approach lies in its capacity to examine the proliferative significance of over 15, 000 genes in MPNST cancer cell lines.This is an unbiased approach that identifies unexplored targets in the treatment of MPNSTs. Moving forward, our laboratory's focus will concentrate on validating the targets identified in our screen. This includes using inhibitors on cell lines and culture and ultimately, translating that to animal work.

Watch this Scientific Journal Video about Lentiviral Infection of MPNST Cells and Genomic DNA Isolation for Genome-Scale shRNA Screens at JoVE.com

Lentiviral Infection of MPNST Cells and Genomic DNA Isolation for Genome-Scale shRNA Screens  

For lentiviral infection, plate the trypsinized MPNST target cells after counting them, maintaining 2.5 million cells per 15 centimeter dish. Transduce the cells with the thawed virus at a 0.5 multiplicity of infection, in the presence of five micrograms per milliliter of the cationic polymer. Trypsinize the cells three days after the addition of puromycin.Centrifuge half of the cell population at 200 G for five minutes in a tabletop centrifuge. After re-plating the other half of the cell population, grow them for approximately seven population doublings before harvesting and centrifuging as previously demonstrated. Divide the re-suspended cell pellet corresponding to the desired time into two 15 milliliter polymethylpentene tubes.Add 500 microliters of 10%SDS per tube. Mix and incubate at room temperature for five minutes. Place the tubes in a DNA shearing device to sonicate the DNA.Following the addition of phenol and chloroform. Mix well by vortexing vigorously at the maximum possible speed for 45 to 60 seconds. Transfer three milliliters of the resulting clear upper phase from each tube to another fresh 15 milliliter tube.Add 0.5 milliliters of three molar sodium acetate and four milliliters of isopropanol and mix well. After centrifuging the tubes for 30 minutes at 7, 200 G and 20 degrees Celsius and discarding the supernatant, add 0.5 milliliters of 70%ethanol to the pellet and dislodge it by pipetting up and down. Combine the re-suspended pellets from both tubes into a 1.5 milliliter centrifuge tube.Perform the PCR reactions using the conditions shown on the screen. After the second PCR reaction, combine the four samples into one micro-centrifuge tube. And add 80 microliters of 6X loading dye to it.Visualize the gel and confirm a band at approximately 250 base pairs. Human MPNST cells transduced with a non-target control and lentivirus, expressing four different Bcl-6 short hairpin RNA, revealed that several of the Bcl-6 short hairpin RNAs markedly reduced cell numbers. The immuno blot showed that the degree of decrease in cell numbers correlated with the degree of Bcl-6 knockdown.

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Research

JoVE Journal

Cancer Research

97 visualizações.  Medical University of South Carolina. Para a infecção lentiviral, plaquear as células-alvo do MPNST tripsinizado após contá-las, mantendo 2,5 milhões de células por placa de 15 centímetros. Transduzir as células com o vírus descongelado a uma multiplicidade de 0,5 de infecção, na presença de cinco microgramas por mililitro do polímero catiônico. Tripsinizar as células três dias após a adição de puromicina.Centrifugar metade da população celular a 200 G por cinco minutos em uma centrífuga de mesa. Dep...