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.
On day zero plate 10 million 293T cells in 30 milliliters of antibiotic-free DMEM containing 10%FBS in a 15 centimeter dish. Prepare a total of 10 such dishes. On the next day, which is day one, confirm that the cells are 80%confluent and ready for transfection.
Then in a 50 milliliter conical tube sequentially add 600 microliters of 0.5 micrograms per microliter packaging plasmid mix, and 60 microliters plasmid barcode library. In a separate 15 milliliter conical tube mix 900 microliters of transfection reagent and 12 milliliters DMEM by vortexing. Add 12.9 milliliters of this mixture to the DNA mix and just flick to combine.
Without mixing any further, incubate the mix at room temperature for 15 minutes. Then dropwise add 2.5 milliliters of the incubated mixture to each 15 centimeter dish containing the 293T cells and incubate the cells overnight in a tissue culture incubator. On day three, harvest the virus by passing the media through a 0.2 micron filtration unit.
Aliquot the filtrate-containing viral particles into 15 milliliter conical tubes. Additionally, prepare five one milliliter aliquots of filtered virus in cryovials for viral titering. To titer the lentiviral pools add 65 microliters of cationic polymer to a sterile glass flask containing 65 milliliters of tumor cell growth media.
Pipette one milliliter of the polymer-containing media per well into 11 6-well tissue culture plates. Next trypsinize the early passage tumor cells. After counting the cells using a preferred method, transfer the cells into the 6-well plates.
Thaw the one milliliter aliquots of the 48 hour lentivirus from the freezer in a water bath at 37 degrees Celsius. Follow the table to prepare the infection for each viral module. 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 200g for five minutes in a tabletop centrifuge.
After replating the other half of the cell population, grow them for approximately seven population doublings before harvesting and centrifuging as previously demonstrated. Divide the resuspended 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, 200g 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 resuspended 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 microcentrifuge 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-targeting control and lentivirus expressing four different BCL6 short hairpin RNA revealed that several of the BCL6 short hairpin RNAs markedly reduced cell numbers. The immunoblot showed that the degree of decrease in cell numbers correlated with the degree of BCL6 knockdown. To begin, plate the 80%confluent malignant peripheral nerve sheath tumor, or MPNST cells, at a density of 1, 200 cells per well in black walled 96-well plates.
After preparing dilutions of the drug to be tested, add each dilution or vehicle to at least three replicate cells. To assess direct cell number, add Hoechst 33342 to a final concentration of one to five micrograms per milliliter to the plates and incubate the plates for 30 minutes at 37 degrees Celsius. Place the plates on a high-throughput imaging cytometer and scan the plates using the direct cell count for total cell number option with 100, 000 to 600, 000 milliseconds exposure times.
Analyze the reads using the software, export them into a spreadsheet and use appropriate software for statistical analyses.
