The HPV + OPC cell line, UDSCC2 (SCC2), used in this study was kindly provided by Professor Hoffmann and Dr S Schulz (University of Ulm, Germany). SCC2 and HEK293T cells (ATCC, CRL-1573) were cultured in complete media containing DMEM (Gibco-Invitrogen, Waltham, MA) supplemented with 10% heat inactivated foetal bovine serum (FBS) and 1% antibiotic/glutamine preparation (100 U/ml penicillin G, 100 U/ml streptomycin sulphate, and 2.9 mg/ml L-glutamine) (Gibco-Invitrogen, Waltham, MA). SCC2 cells constitutively expressing Cas9 (SCC2 Cas9) were developed as previously described (11) and maintained in media supplemented with blasticidin.
Plasmids used in this study include, envelope VSV-G containing plasmid (pMDG2.G, Addgene #12259), packaging plasmids, pRSV-Rev (Addgene #12253) and pMDLg/pRRE (Addgene #12251), and the E7 gRNA containing FgH1tUTG plasmid. The doxycycline (dox)-inducible gRNA expression plasmid vector, FgH1tUTG (Addgene #70183), was kindly provided by Professor Marco Herold (Walter and Eliza Hall Institute of Medical Research, Australia) (17). CRISPick design tool was used to design the gRNA targeting HPV 16 E7 oncogene and cross-checked against other gRNA selection tools (ChopChop and Crispor). The E7 targeting gRNA (5′-GCAAGTGTGACTCTACGCTT-3’) was then cloned into the FgH1tUTG plasmid, purified (#12362, Endofree Plasmid Maxi Kit, QIAGEN, Hilden, Germany) and confirmed by PCR and Sanger sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems 2002).
Lentivirus production and transducing SCC2 Cas9 cells
Low passage HEK293T cells (human embryo kidney cell line) were co-transfected with pMDG2.G, pRSV-Rev, pMDLg/pRRE, and the E7 gRNA containing FgH1tUTG plasmid using the Lipofectamine 3000 transfection reagent (#L3000015, Thermo Fisher Scientific, Waltham, MA). After 48h post-transfection, viral supernatant was concentrated in Amicon ® Ultra-15 Centrifugal Filter units (Merck, Germany) and viral titre determined by flow cytometry (BD LSR FORTESSA cell analyser (BD bioscience, San Jose, CA)) as previously described (18). SCC2 Cas9 cells were then infected with lentivirus bearing the E7 targeting gRNA sequence at the multiplicity of infection (MOI) of 10 for 24 h, before cells were subjected to cell sorting on the BD FACSAria™ III Cell Sorter (BD bioscience, San Jose, CA).
Chemicals and animal fodder
To promote sgRNA expression in cell lines, doxycycline hyclate (dox) (#D9891, Sigma-Aldrich, St Louis, MI) was dissolved in sterile water at a stock concentration of 10mg/ml and added to cell culture media at a final concentration of 10µg/ml. Standard rodent food was supplemented with doxycycline (600mg/kg body weight) (Dox fodder) for in vivo work (#SF08-026, Specialty Feeds, WA, Australia). Alisertib was purchased from Selleck Chemicals (Houston, TX) and powder dissolved in sterile-grade DMSO (Sigma-Aldrich, St Louis, MI). For in vivo work, alisertib was prepared in 10% 2-hydroxypropyl-cyclodextrin (Sigma-Aldrich, St Louis, MI)/10% sodium bicarbonate (Sigma-Aldrich, St Louis, MI).
Cell viability determination
Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT reagent (Sigma Aldrich, St Louis, MI) was added into cell meadia at a final concentration of 0.5 mg/ml for 2 h. MTT crystals were dissolved in DMSO before measuring colorimetric absorbance at 544 nm using a FLUOstar OPTIMA microplate reader (BMG LabTech, Germany).
SCC2 Cas9 xenografts and treatment protocol
Female nude mice (aged 6–8 weeks) were purchased from the animal resource centre (ARC), Perth, Australia. All animal experiments were performed in accordance with the Australian and New Zealand Council for the Care and Use of Animals in Research standards and were approved by the Griffith University Animal Ethics committee (MHIQ/14/20). Tumours were established by subcutaneously injecting 2×106 SCC2 Cas9 cells (100 µl/injection in 50% PBS:50% Corning ® Matrigel® solution (vehicle) (Sigma-Aldrich, St Louis, MI) in the right flank of mice as done previously (6). Mice were monitored for tumour growth and when the tumours reached a size of approximately 100 mm3 (22 days after transplantation), mice were either vehicle administered (untreated) or oral gavage (o.g.) administered with 100µl of Alisertib at 30 mg/kg once per day for ten consecutive days as previously described (19) (Alisertib). For dox treatment, fed dox fodder was given at day 32 to induce the dox-inducible CRISPR system (DOX) as previously described (17). Mice in the untreated and alisertib group were fed standard non-dox fodder. For the combination treatment regimen, mice first received daily doses of Alisertib for ten days before feeding mice with dox fodder. Tumours volumes were measured using digital calipers. Tumour size was measured every two days and mice health monitored daily. Mice were euthanized and culled at the end of designated monitoring period or when tumour mass reached 1000 mm3 or reached clinical endpoint (> 20% weight loss, lack of grooming, reduce activity and appetite). Mice deaths: Untreated (Day 49 - All 6 mice culled based on endpoint criteria).
Tumour proteins were extracted using AllPrep DNA/RNA/Protein Kit (#80004, QIAGEN, Hilden, Germany), according to manufacturer’s protocol. Immunoblots were probed with antibodies against HPV 16E7 (NM2) (Santa Cruz Technologies Biotechnologies, Dallas, TX), PARP (Cell Signaling Technologies, Danvers, MA) and S6 (Cell Signaling Technologies, Danvers, MA). Rabbit and mouse secondary antibodies (Cell Signaling Technologies, Danvers, MA) and ECL was used to detect protein signals on a Chemidoc XRS Visualiser (BioRad, Hercules, CA).
Determining gene editing efficiency
Cell genomic DNA was isolated using the QIAamp DNA Mini Kit (#51306 QIAGEN, Hilden, Germany) according to manufacturers’ instructions. Tumor DNA and RNA were extracted using AllPrep DNA/RNA/Protein Kit (# 80004, QIAGEN, Hilden, Germany), according to manufacturers’ protocol. The E7 gene was amplified by PCR using primers previously designed (6) and purified by ultrafiltration before sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems 2002 (Part# 4337035A) following manufacturers’ protocol. Sanger sequencing results were analyzed using the online tool, TIDE (Tracking Indels by DEcomposition https://tide.nki.nl) as previously described (20).
Differences between treatment groups were done using a two-way ANOVA followed by Tukey's multiple comparisons test on GraphPad Prism v9.