We wish to thank Dr. Daniel Gitler for providing us with the pAd Delta 5 Helper plasmid. This work was funded by the Israel Science Foundation (ISF, 700/16) (to ME), the United States-Israel Binational Science Foundation (BSF, 2017323) (to ME and MS), the Israel Cancer Association (ICA, 20170024) (to ME), the Israel Cancer Research Foundation (ICRF, 17-1693-RCDA) (to ME), and the Concern Foundation (#7895) (to ME). Fellowship: the Alon fellowship to ME and BGU Kreitman fellowships to SJ and MP.

This study was approved by the Ben Gurion University of the Negev Animal Care and Use Committee. Animal experiments were approved by the IACUC (IL.80-12-2015 and IL.29-05-2018(E)). All aspects of animal testing, housing, and environmental conditions used in this study were in compliance with The Guide for the Care and Use of Laboratory Animals13.
1. Adeno-associated virus production
<ol>
	<li>Day 1: Cell culture
	<ol>
		<li>Seed 4 x 106 HEK293T cells per 14.5 cm plate in 15 mL of DMEM. Prepare 10 plates for transfection with polyethylenimine (PEI) (1 &#181;g/&#181;L).</li>
	</ol>
	</li>
	<li>Day 2: Transfection of HEK293T cells using PEI
	<ol>
		<li>Remove media and refeed with warm DMEM 1 h before the transfection.</li>
		<li>Prewarm transfection reagents and DMEM.</li>
		<li>Use 10 &#181;g of the plasmid of interest containing AAV ITR (AAV pCM109 EFS Cre sg APC sg KRAS sg P53- KRAS HDR), 10 &#181;g of the AAV 2/9n capsid plasmid (with the rep gene of AAV2 and the cap gene of AAV9, and 10 &#181;g of the helper plasmid (pAdDelta F5 helper) per plate (See Table of Materials). 
		NOTE: A new generation of helper plasmid - pAdDeltaF614,15,16 that can also be used for AAV production is available.</li>
		<li>Mix the plasmids in 1 mL of plain DMEM per plate. Then add 90 &#181;L of polyethylenimine (total plasmid: PEI concentration = 1:3) to the plasmid mix and vortex briefly.</li>
		<li>Incubate the PEI-plasmid DNA mix for 20 min at room temperature (RT).</li>
		<li>Add the mix dropwise on top of the HEK293T cells and transfer the plates to the cell culture incubator at 37 &#176;C with 5% CO2 for 24 h.</li>
	</ol>
	</li>
	<li>Day 3: Medium change after transfection
	<ol>
		<li>Remove the medium completely and add 15 mL of fresh DMEM.</li>
	</ol>
	</li>
	<li>Day 5: Harvesting the virus
	<ol>
		<li>Prepare a dry ice/ethanol bath.</li>
		<li>Collect the cells with a cell scraper and transfer them into 50 mL tubes (2 plates per 50 mL tube).</li>
		<li>Spin tubes at 800 x g for 15 min at room temperature.</li>
		<li>Discard the supernatant from all the tubes. Add 0.5 mL of the lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH = 8.5) per plate (i.e., 5 mL for 10 plates) to the first tube only. Resuspend the cells and transfer the total volume to the next tube and continue until the last tube.</li>
		<li>Transfer the cell suspension to a fresh 50 mL tube. Wash the tubes with the same volume of lysis buffer (0.5 mL per plate) using the same transfer method as described in step 1.4.4.</li>
		<li>Subject the cell suspension to three rounds of ~10 min freeze/thaw cycles between a dry ice/ethanol bath and a 37 &#176;C water bath. Vortex briefly after each thawing.</li>
		<li>If the purification is carried out on the same day, set the equilibration and elution buffer (see Table 1 for the recipe) at RT.</li>
		<li>Add 50 units of benzonase per plate and incubate at 37 &#176;C for 1.5 h. 
		NOTE: Benzonase is used to digest residual nucleic acids from the host producer cells and the plasmid DNA present in the cell suspension.</li>
		<li>Spin the tubes at 3,000 x g for 15 min at 4 &#176;C.</li>
		<li>Collect the supernatant in a syringe using a 18 G steel needle and push the solution through a 0.45 &#181;m filter into a 15 mL tube to obtain the crude lysate.</li>
		<li>Store the crude lysate at 4 &#176;C for a few weeks until purification or continue purification.</li>
	</ol>
	</li>
</ol>
2. AAV purification
<ol>
	<li>Day 5 or later
	<ol>
		<li>Place the equilibration and elution buffer at RT.</li>
		<li>Wash chromatography columns with 10 mL of the equilibration buffer.</li>
		<li>Add 0.5 mL per plate of heparin-agarose to the column17 and then add the equilibration buffer (4x the volume of the heparin-agarose). Mix the solutions by inverting the column and let the agarose sediment.</li>
		<li>Elute the equilibration buffer from the column by gravity. 
		NOTE: Leave some equilibration buffer to prevent the agarose from drying out.</li>
		<li>Load the crude lysate onto the column and incubate for 2 h at 4 &#176;C with constant agitation. Bring the column into an upright position and allow the agarose to sediment.</li>
		<li>Elute the crude lysate by gravity.</li>
		<li>Wash the column with equilibration buffer (4x the volume of the heparin-agarose).</li>
		<li>Place a 100 kDa centrifugal protein filter below the column and elute the virus using an elution buffer (3x the volume of the heparin-agarose) into the filter. 
		NOTE: The elution buffer should not exceed 15 mL.</li>
		<li>Centrifuge the filter at 3,000 x g for ~30 min until less than 1 mL is left in the filter.</li>
		<li>Fill up the filter with PBS and centrifuge at 3,000 x g for ~30 min until less than 1 mL is left in the filter. Repeat this step 2x to remove all salts.</li>
		<li>Concentrate the virus solution in the filter by centrifugation at 3,000 x g to arrive at a volume as small as possible (less than 200 &#181;L).</li>
		<li>Aspirate the virus solution with a needle and syringe and push the solution through a 0.22 &#181;m filter into a tube.</li>
		<li>Make aliquots of concentrated viral particles for storage. 
		NOTE: The aliquots should be ~20 &#181;L for short-term storage at 4 &#176;C and ~5 &#181;L for long-term storage at -80 &#176;C.</li>
		<li>Determine the viral titer and viral transduction efficiency as described previously14,18,19,20.</li>
	</ol>
	</li>
</ol>
3. Isolation and culture of primary cells
<ol>
	<li>Day 1
	<ol>
		<li>Euthanize a 6-week-old male or female B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J by CO2 asphyxiation or any other IACUC approved protocol. 
		NOTE: These CRISPR/Cas9 knockin mice have Cre recombinase-dependent expression of cas9 endonuclease and EGFP directed by a CAG promoter. The upstream Lox-Stop-Lox (LSL) sequence present in the genome of these mice prevent the expression of Cas9 and EGFP in the absence of Cre recombinase. When used in combination with single guide RNAs (sgRNAs) and a Cre source, they allow editing of single or multiple mouse genes in vivo or ex vivo14.</li>
		<li>Dissect the tongue from the euthanized mice using surgical scissors.</li>
		<li>Manually dissociate the tissue by mincing the tongue tissue into very small fragments using a scalpel. Collect the tissue fragments in a 15 mL tube containing 4.5ml of RPMI plain medium (with out serum).</li>
		<li>Add the triple enzyme mix (200 &#181;l) (see Table 1 for the recipe) to the tissue fragments.</li>
		<li>Incubate the tissue-enzyme mix at 37 &#176;C for 30 min and tap the tube every 10 min to enhance enzymatic dissociation of the tissue.</li>
		<li>Add 5% fetal bovine serum (FBS) containing HBSS/PBS to the tissue-enzyme mix to stop the enzyme action.</li>
		<li>Filter the above cell suspension through a sterile 70 &#181;m nylon mesh to separate the dispersed cells and larger tissue fragments.</li>
		<li>Wash the filtered cell suspension by centrifugation for 300 x g for 5 min in HBSS/PBS at RT.</li>
		<li>Resuspend the pellet in culture medium (10% FBS in RPMI/DMEM) and grow in 60 mm culture dishes until distinct cell colonies are formed.
		<ol>
			<li>Culture cell aggregates retained on top of the filter in a 60 mm culture dish containing 3 mL of complete media (10% FBS in RPMI/DMEM) until cell colonies are formed.</li>
		</ol>
		</li>
		<li>Microscopically examine the primary cells for the presence of fibroblast contamination after 1 week of culture. Treat the primary cells developed from aggregates and cell suspensions with 0.25 trypsin 0.02% EDTA solution at 37 &#176;C for 1 min to remove fibroblasts. 
		NOTE: Usually the primary culture from cell aggregates produces more colonies compared to single-cell suspensions. The cells from these colonies provide better transduction efficiency with AAV transduction.</li>
	</ol>
	</li>
</ol>
4. AAV transduction of primary cells
<ol>
	<li>Day 10
	<ol>
		<li>Seed 2 x 105 primary cells per well in 6 well plates in 2 mL of complete media (10% FBS in RPMI/DMEM).</li>
		<li>The next day, transduce the cells with 1012 viral genome/mL (1010 transducing units/mL) and incubate the cells in viral particle-containing media for 48 h at 37 &#176;C.</li>
		<li>Remove the viral particle-containing media and feed the AAV-transduced cells with complete media. 
		NOTE: Only cells that underwent transduction will express GFP and start to proliferate. Cells that did not undergo transduction will eventually die within 2 weeks.</li>
		<li>After 2 weeks of culture expansion, seed the cells for validation and the in vivo tumorigenic experiments. 
		NOTE: Genomic DNA fromAAV-transduced cells and normal primary cells from Cas9 mice were extracted for validating specific genome editing using standard protocols. Sequencing of the extracted DNA and analysis of the sequenced data (Table 2) were performed using a hybridization capture-based next-generation sequencing assay (e.g., MSK-IMPACT platform) as described previously21.</li>
	</ol>
	</li>
</ol>
5. Validating the transformation of normal cells to tumorigenic cells using immunofluorescence and western blotting
<ol>
	<li>Immunofluorescence
	<ol>
		<li>Seed 2 x 105 cells on 12 mm glass coverslips and place them in an incubator overnight.</li>
		<li>Fix cells in 4% paraformaldehyde in PBS (pH = 7.4) and process for immunofluorescence labeling.</li>
		<li>Incubate the fixed cells with the first primary antibody in 1% BSA or 1% serum in PBST in a humidified chamber for 1 h at RT or overnight at 4 &#176;C. The primary antibodies used are the rabbit monoclonal anti-KRT 14, rabbit monoclonal anti-E-cadherin antibody, and Cas9 mouse mAb.</li>
		<li>Remove the primary antibody solution from the coverslips by washing the cells 3x for 5 min with 1x PBS.</li>
		<li>Incubate the cells with Cy3 donkey anti-rabbit IgG and/or Cy3 goat anti-mouse IgG secondary antibodies in 5% BSA in PBST for 1 h at RT in the dark.</li>
		<li>Remove the secondary antibody solution from the coverslips and wash the cells 3x as described in step 5.1.4.</li>
		<li>Mount the coverslips with a drop of mounting medium containing DAPI (DAPI Fluoromount).</li>
		<li>Store in the dark at 4 &#176;C until the slides are imaged using fluorescence microscopy.</li>
	</ol>
	</li>
	<li>Western blotting
	<ol>
		<li>Seed 1 x 106 transformed cells in a 100 mm culture dish and place them in an incubator overnight.</li>
		<li>Wash and scrape the transformed cells into 200 &#181;l ice cold PBS.</li>
		<li>Centrifuge the tube containing the cell suspension for 10 min at 2,000 x g at 4 &#176;C to pellet the cells.</li>
		<li>Aspirate the supernatant and lyse the cells using lysis buffer (see Table 1) containing phosphatase inhibitor cocktails and a protease inhibitor for 10 min at 4 &#176;C. Centrifuge the lysates for 10 min at 10,000 x g and 4 &#176;C and collect the cleared lysates.</li>
		<li>Use a commercially available Bradford assay kit to determine the protein concentration following the manufacturer&#39;s protocol. Use 4x sample buffer (500 mM Tris pH = 6.8, 40% glycerol, 8% SDS, 20% H2O, 0.02% bromophenol blue) to adjust the protein samples to 0.5 or 1 &#181;g/&#181;L. Boil at 96 &#176;C for 5 min. 
		NOTE: The samples can be stored at -80 &#176;C or until a Western blot analysis is performed.</li>
		<li>Separate equal amounts of total lysate (30 &#181;g) by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ensure that the dye reaches the bottom of the gel. Transfer the protein to the nylon membrane by semi-dry blotting at 25 V for 30 min.</li>
		<li>Pour 5% BSA in Tris-buffered saline (TBS)-0.1% Tween (TBST) over the membrane to cover it completely. Block the membrane for 1 h at RT. Incubate the mebrane with primary antibodies (anti-Cre, anti-Cas9, anti-GFP, anti-&#946; catenin, anti p53, anti-phospho-ERK, and anti-&#946; actin diluted in 5% BSA TBST) at 4 &#176;C overnight.</li>
		<li>After incubation, wash the membranes for 10 min with 1x TBST making sure that the solution covers the membrane completely. Perform this wash 3x, and then add horseradish peroxidase (HRP)-conjugated secondary antibody (diluted 1:10,000 in 5% BSA TBST) to the membrane. Incubate for 1 h at RT.</li>
		<li>After incubation, wash the membranes for 10 min with 1x TBST making sure that the solution covers the membrane completely. Perform the chemiluminescence imaging (see Table of Materials) to expose the bands, and capture images accordingly.</li>
	</ol>
	</li>
	<li>Validating the tumorigenic potential of transformed cells in immunocompetent mice 
	NOTE: Mice were maintained and treated according to the institutional guidelines of the Ben-Gurion University of the Negev. NOD.CB17-Prkdc-scid/NCr Hsd (NOD.SCID) and C57BL/6 mice were used for the in vivo studies. Mice were housed in air-filtered laminar flow cabinets with a 12 hour light/dark cycle and were fed food and water ad libitum.
	<ol>
		<li>Use 6-8 week-old female NOD.CB17-Prkdc-scid/NCr Hsd (NOD.SCID) and C57BL/6 mice for the study.</li>
		<li>Trypsinize the AAV-Cas9 transformed cells. Stop the trypsinization using DMEM prewarmed at 37 &#176;C and collect in a 50 mL tube.</li>
		<li>Centrifuge the tube at 800 x g for 10 min at room temperature. Discard the supernatant and resuspend the cell pellet in DMEM medium without FBS. Perform the centrifugation again and resuspend the cell pellet in 1x PBS. 
		NOTE: Do not keep the cells in 1x PBS for too long. Always keep the cell suspension on ice to prevent cell clumping.</li>
		<li>Use an automatic cell counter to count the cells and dilute the cells to the desired concentration (2.5 x 107 cells/mL) using 1x PBS. Generate tumors through a subcutaneous injection of the AAV-Cas9 transformed cell suspension in the right flank of each NOD.CB17-Prkdc-scid/NCr Hsd (NOD.SCID) mouse (2 x 106 cells/mouse). To generate an orthotropic model in syngeneic mice, inject 0.5 &#215; 106 primary cells or the AAV-Cas9 transformed cells into the tongue of C57BL/6 immunocompetent mice.</li>
		<li>Euthanize the animals 2 weeks postinjection by CO2 asphyxiation, and dissect the tumors from the euthanized mice for immunohistochemistry analysis.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+transformation+of+mouse+embryo+fibroblasts+in+the+absence+of+activator+E2Fs.">Cellular transformation of mouse embryo fibroblasts in the absence of activator E2Fs.</a> Journal of Virology. 89 (9), 5124-5133 (2015).</li><li>Leong, H., Blewitt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrovirus+Mediated+Malignant+Transformation+of+Mouse+Embryonic+Fibroblasts.">Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts.</a> BIO-PROTOCOL. 3 (15), (2013).</li><li>Parikh, N., Nagarajan, P., Sei-ichi, M., Sinha, S., Garrett-Sinha, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+an+immortalized+oral+keratinocyte+cell+line+of+mouse+origin.">Isolation and characterization of an immortalized oral keratinocyte cell line of mouse origin.</a> Archives of Oral Biology. 53 (11), 1091-1100 (2008).</li><li>Badarni, 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=Repression+of+AXL+expression+by+AP-1/JNK+blockage+overcomes+resistance+to+PI3Ka+therapy.">Repression of AXL expression by AP-1/JNK blockage overcomes resistance to PI3Ka therapy.</a> JCI Insight. 5, 125341 (2019).</li><li>Chen, Y. F., 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=Establishing+of+mouse+oral+carcinoma+cell+lines+derived+from+transgenic+mice+and+their+use+as+syngeneic+tumorigenesis+models.">Establishing of mouse oral carcinoma cell lines derived from transgenic mice and their use as syngeneic tumorigenesis models.</a> BMC Cancer. 19 (1), 281 (2019).</li><li>Hoover, A. 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=The+Role+of+Human+Papillomavirus+16+E6+in+Anchorage-Independent+and+Invasive+Growth+of+Mouse+Tonsil+Epithelium.">The Role of Human Papillomavirus 16 E6 in Anchorage-Independent and Invasive Growth of Mouse Tonsil Epithelium.</a> Archives of Otolaryngology-Head &amp; Neck Surgery. 133 (5), 495 (2007).</li><li>Smahel, 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=Metastatic+MHC+class+I-negative+mouse+cells+derived+by+transformation+with+human+papillomavirus+type+16.">Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16.</a> British Journal of Cancer. 84 (3), 374-380 (2001).</li><li>He, L., 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=Increased+Growth+of+a+Newly+Established+Mouse+Epithelial+Cell+Line+Transformed+with+HPV-16+E7+in+Diabetic+Mice.">Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice.</a> PloS One. 11 (10), e0164490 (2016).</li></ol>

M.S. is on the Advisory Board of the Bioscience Institute, and Menarini Ricerche has received research funds from Puma Biotechnology, Daiichi-Sankio, Targimmune, Immunomedics, and Menarini Ricerche. M.S. is also a co-founder of Medendi Medical Travel, and in the past 2 years, has received honoraria from Menarini Ricerche and ADC Pharma. All other authors have nothing to disclose.

Several methods have previously been used to transform primary cells in culture with variable degrees of success25,26,27,28. Most of these methods use mouse fibroblast cells for transformation14,17,18,19 or use carcinogens such as 4-nitroquinoline-1-oxide (4-NQO)<sup ...

HNC is a common malignancy worldwide1. Modeling the genesis of HNC neoplasia is currently at a scientific turning point2. While many genetic mutations have been identified in HNC2,3,4 (e.g., TP53, PIK3CA, NOTCH1, FAT1, and RAS) the specific combinations of genomic alterations required in concert to induce HNC remain unclear.
The current use of human HNC cell lines has significantly helped to elucidate the mechanisms associated with pathogenesis and treatment3. However, the study of human cell lines in immunocompromised murine systems has its limitations, because these systems do not address the in vivo neoplastic process, the role of specific gene mutations, and treatment responses in an immune microenvironment. Hence, the development and establishment of murine cell lines with specific genetic alterations are of primary importance to study how different genes contribute to the transformation process and to test novel molecule-based therapies in immunocompetent mice.
Gene function studies in biomedical research have been significantly affected by advances in DNA editing technologies, by introducing double-strand breaks (DSBs), for example5. These technologies, including the use of zinc finger nucleases, transcription activator-like effector nucleases, and clustered regulatory interspaced short palindromic repeats (CRISPR/Cas9), allow for the manipulation of any gene of interest at its locus. The latest CRISPR/Cas9 systems is comprised of a guide RNA (gRNA) that directs the Cas9 nuclease to generate a DSB at a specific site in the genome. This system has gained extensive recognition in modifying endogenous genes in any cell or target tissue, even in the most traditionally difficult-to-treat organisms5. It has multiple advantages over other systems due to its simplicity, speed, and efficiency.
In oncology, CRISPR/CAS9 technology has fulfilled the need to effectively mimic cancer cells. To establish this system in HNC, we manipulated the potent KRAS oncogene and two important tumor suppressor genes, APC and p536. According to the GENIE database7, this combination is rare in HNC. RAS mutations (HRAS, NRAS, and KRAS) are present in only ~7% of all HNC populations. These tumors tend to be resistant to therapy8,9.
Delivery of Cas9 and its gRNA is achieved through viral transduction using either AAVs or lentiviruses. Recombinant AAV is often the preferred method for delivering genes to target cells owing to its high titer, mild immune response, ability to transduce a broad range of cells, and overall safety. Using an AAV system, various tissue-specific mouse cell lines have been generated, and new cell lines are still being developed10,11,12. However, an efficient genomic editing system that can generate murine HNC cell line models cells remains to be developed. In this study, we sought to develop an in vitro AAV-Cas9-based system for transforming primary murine tongue cells into a tumorigenic state. This unique CRISPR/Cas9 transformation protocol and the established tumor cell line can be used to better understand the biology of HNC induced by a diversity of genomic alterations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
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		</tr>
		<tr>
			<td>Anti mouse HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-035-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti rabbit HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-035-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 Mouse mAb</td>
			<td>Cell Signaling Technology</td>
			<td>14697</td>
			<td></td>
		</tr>
		<tr>
			<td>Cre</td>
			<td>BioLegend</td>
			<td>900901</td>
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		</tr>
		<tr>
			<td>Cy3-AffiniPure Goat Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-165-062</td>
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			<td>Cy-AffiniPure Goat Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-165-144</td>
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			<td>GFP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9996</td>
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			<td>Phospho-p44/42 MAPK (Erk1/2)</td>
			<td>Cell Signaling Technology</td>
			<td>4370</td>
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			<td>Cell Signaling Technology</td>
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			<td>Rabbit monoclonal anti-KRT 14</td>
			<td>Abcam</td>
			<td>AB-ab181595</td>
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		</tr>
		<tr>
			<td>&#946; actin</td>
			<td>MP Biomedicals</td>
			<td>691001</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946; catenin</td>
			<td>Cell Signaling Technology</td>
			<td>9582S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
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			<td>HEK93T</td>
			<td>ATCC</td>
			<td><a href="https://www.atcc.org/en/Products/All/CRL-3216.aspx" target="_blank">CRL-3216</a></td>
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			<td>Culture Media, Chemicals and Reagents</td>
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			<td>30015484</td>
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			<td>BSA</td>
			<td>Amresco</td>
			<td>0332-TAM-50G</td>
			<td></td>
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			<td>DAPI fluoromount</td>
			<td>Southern Biotech</td>
			<td>0100-20</td>
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			<td>DMEM</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
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			<td>B15001A/B</td>
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			<td>Protease inhibitor cocktail</td>
			<td>MilliporeSigma</td>
			<td>P2714-1BTL</td>
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			<td>Tris buffer</td>
			<td>MERCK Millipore</td>
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			<td></td>
			<td></td>
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			<td>Sigma</td>
			<td>E1014</td>
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			<td>Thermo Fisher Scientific</td>
			<td>17104019</td>
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			<td>Thermo Fisher Scientific</td>
			<td>18047019</td>
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			<td>Hyaluronidase</td>
			<td>MilliporeSigma</td>
			<td>H3506</td>
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		<tr>
			<td>Trypsin</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>03-050-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass wares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Bar Naor</td>
			<td>BNCB00130RA1</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Bar Naor</td>
			<td>BN9308C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J</td>
			<td>Jackson labs</td>
			<td>24857</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.CB17-Prkdc-scid/NCr Hsd (Nod.Scid)</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV pCM109 EFS Cre sg APC sg Kras sg P53- Kras HDR</td>
			<td>Broad Institute of MIT</td>
			<td></td>
			<td>Kind gift from Dr Randall J Platt and Dr. Joseph Rosenbluh, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA</td>
		</tr>
		<tr>
			<td>AAV 2/9 capsid vector</td>
			<td>Addgene</td>
			<td>112865</td>
			<td></td>
		</tr>
		<tr>
			<td>pAD Delta F5 helper</td>
			<td>Ben Gurion University of the Negev</td>
			<td></td>
			<td>Provided by Dr Daniel Gitler, Department of Physiology and Cell Biology, Faculty of Health Sciences, and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.</td>
		</tr>
		<tr>
			<td>Plastic wares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon-ULTRA filter 100 KDa</td>
			<td>Millipore</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m sterile filters, 4 mm</td>
			<td>Millex</td>
			<td>SLGV004SL</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m sterile filters, 13 mm</td>
			<td>Millex</td>
			<td>SLHV013SL</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture plates</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro establishment of a genetically engineered murine head and neck cancer cell line using an adeno-associated virus-cas9 system

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Using the AAV system to transform normal Cas9 cells 
Figure 1 provides a detailed vector map of the AAV transgene plasmid used in this study. Figure 2 outlines the design and working of the AAV-Cas9 based-system. To produce viral particles, the HEK293T cells were transfected with the AAV transgene vector and other viral packaging vectors using the PEI transfection method. After transfection, the virus-contai...

Watch this Scientific Journal Video about In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System at JoVE.com

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

in-vitro-establishment-genetically-engineered-murine-head-neck-cancer

Research

JoVE Journal

Cancer Research

7.8K Views.  Ben-Gurion University of the Negev. Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

Video: In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Live Cell Imaging of the TGF-	&amp;beta;/Smad3 Signaling Pathway &lt;em&gt;In Vitro&lt;/em&gt; and &lt;em&gt;In Vivo&lt;/em&gt; Using an Adenovirus Reporter System

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...