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

Summary

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.

Abstract

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.

Introduction

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 HNC^2,3,4 (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 therapy^8,9.

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 developed^10,11,12. 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.

Protocol

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

1. Day 1: Cell culture
   1. 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 µg/µL).
2. Day 2: Transfection of HEK293T cells using PEI
   1. Remove media and refeed with warm DMEM 1 h before the transfection.
   2. Prewarm transfection reagents and DMEM.
   3. Use 10 µg of the plasmid of interest containing AAV ITR (AAV pCM109 EFS Cre sg APC sg KRAS sg P53- KRAS HDR), 10 µg of the AAV 2/9n capsid plasmid (with the rep gene of AAV2 and the cap gene of AAV9, and 10 µg of the helper plasmid (pAdDelta F5 helper) per plate (See Table of Materials).
      NOTE: A new generation of helper plasmid - pAdDeltaF6^14,15,16 that can also be used for AAV production is available.
   4. Mix the plasmids in 1 mL of plain DMEM per plate. Then add 90 µL of polyethylenimine (total plasmid: PEI concentration = 1:3) to the plasmid mix and vortex briefly.
   5. Incubate the PEI-plasmid DNA mix for 20 min at room temperature (RT).
   6. Add the mix dropwise on top of the HEK293T cells and transfer the plates to the cell culture incubator at 37 °C with 5% CO₂ for 24 h.
3. Day 3: Medium change after transfection
   1. Remove the medium completely and add 15 mL of fresh DMEM.
4. Day 5: Harvesting the virus
   1. Prepare a dry ice/ethanol bath.
   2. Collect the cells with a cell scraper and transfer them into 50 mL tubes (2 plates per 50 mL tube).
   3. Spin tubes at 800 x g for 15 min at room temperature.
   4. 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.
   5. 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.
   6. Subject the cell suspension to three rounds of ~10 min freeze/thaw cycles between a dry ice/ethanol bath and a 37 °C water bath. Vortex briefly after each thawing.
   7. If the purification is carried out on the same day, set the equilibration and elution buffer (see Table 1 for the recipe) at RT.
   8. Add 50 units of benzonase per plate and incubate at 37 °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.
   9. Spin the tubes at 3,000 x g for 15 min at 4 °C.
   10. Collect the supernatant in a syringe using a 18 G steel needle and push the solution through a 0.45 µm filter into a 15 mL tube to obtain the crude lysate.
   11. Store the crude lysate at 4 °C for a few weeks until purification or continue purification.

2. AAV purification

1. Day 5 or later
   1. Place the equilibration and elution buffer at RT.
   2. Wash chromatography columns with 10 mL of the equilibration buffer.
   3. 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.
   4. Elute the equilibration buffer from the column by gravity.
      NOTE: Leave some equilibration buffer to prevent the agarose from drying out.
   5. Load the crude lysate onto the column and incubate for 2 h at 4 °C with constant agitation. Bring the column into an upright position and allow the agarose to sediment.
   6. Elute the crude lysate by gravity.
   7. Wash the column with equilibration buffer (4x the volume of the heparin-agarose).
   8. 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.
   9. Centrifuge the filter at 3,000 x g for ~30 min until less than 1 mL is left in the filter.
   10. 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.
   11. 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 µL).
   12. Aspirate the virus solution with a needle and syringe and push the solution through a 0.22 µm filter into a tube.
   13. Make aliquots of concentrated viral particles for storage.
       NOTE: The aliquots should be ~20 µL for short-term storage at 4 °C and ~5 µL for long-term storage at -80 °C.
   14. Determine the viral titer and viral transduction efficiency as described previously^14,18,19,20.

3. Isolation and culture of primary cells

1. Day 1
   1. Euthanize a 6-week-old male or female B6;129-Gt(ROSA)26Sor^{tm1(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.
   2. Dissect the tongue from the euthanized mice using surgical scissors.
   3. 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).
   4. Add the triple enzyme mix (200 µl) (see Table 1 for the recipe) to the tissue fragments.
   5. Incubate the tissue-enzyme mix at 37 °C for 30 min and tap the tube every 10 min to enhance enzymatic dissociation of the tissue.
   6. Add 5% fetal bovine serum (FBS) containing HBSS/PBS to the tissue-enzyme mix to stop the enzyme action.
   7. Filter the above cell suspension through a sterile 70 µm nylon mesh to separate the dispersed cells and larger tissue fragments.
   8. Wash the filtered cell suspension by centrifugation for 300 x g for 5 min in HBSS/PBS at RT.
   9. Resuspend the pellet in culture medium (10% FBS in RPMI/DMEM) and grow in 60 mm culture dishes until distinct cell colonies are formed.
      1. 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.
   10. 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 °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.

4. AAV transduction of primary cells

1. Day 10
   1. Seed 2 x 10⁵ primary cells per well in 6 well plates in 2 mL of complete media (10% FBS in RPMI/DMEM).
   2. 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 °C.
   3. 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.
   4. 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.

5. Validating the transformation of normal cells to tumorigenic cells using immunofluorescence and western blotting

1. Immunofluorescence
   1. Seed 2 x 10⁵ cells on 12 mm glass coverslips and place them in an incubator overnight.
   2. Fix cells in 4% paraformaldehyde in PBS (pH = 7.4) and process for immunofluorescence labeling.
   3. 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 °C. The primary antibodies used are the rabbit monoclonal anti-KRT 14, rabbit monoclonal anti-E-cadherin antibody, and Cas9 mouse mAb.
   4. Remove the primary antibody solution from the coverslips by washing the cells 3x for 5 min with 1x PBS.
   5. 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.
   6. Remove the secondary antibody solution from the coverslips and wash the cells 3x as described in step 5.1.4.
   7. Mount the coverslips with a drop of mounting medium containing DAPI (DAPI Fluoromount).
   8. Store in the dark at 4 °C until the slides are imaged using fluorescence microscopy.
2. Western blotting
   1. Seed 1 x 10⁶ transformed cells in a 100 mm culture dish and place them in an incubator overnight.
   2. Wash and scrape the transformed cells into 200 µl ice cold PBS.
   3. Centrifuge the tube containing the cell suspension for 10 min at 2,000 x g at 4 °C to pellet the cells.
   4. 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 °C. Centrifuge the lysates for 10 min at 10,000 x g and 4 °C and collect the cleared lysates.
   5. Use a commercially available Bradford assay kit to determine the protein concentration following the manufacturer'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 µg/µL. Boil at 96 °C for 5 min.
      NOTE: The samples can be stored at -80 °C or until a Western blot analysis is performed.
   6. Separate equal amounts of total lysate (30 µg) 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.
   7. 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-β catenin, anti p53, anti-phospho-ERK, and anti-β actin diluted in 5% BSA TBST) at 4 °C overnight.
   8. 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.
   9. 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.
3. 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.
   1. Use 6-8 week-old female NOD.CB17-Prkdc-scid/NCr Hsd (NOD.SCID) and C57BL/6 mice for the study.
   2. Trypsinize the AAV-Cas9 transformed cells. Stop the trypsinization using DMEM prewarmed at 37 °C and collect in a 50 mL tube.
   3. 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.
   4. 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 × 10⁶ primary cells or the AAV-Cas9 transformed cells into the tongue of C57BL/6 immunocompetent mice.
   5. Euthanize the animals 2 weeks postinjection by CO₂ asphyxiation, and dissect the tumors from the euthanized mice for immunohistochemistry analysis.

Results

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-containi...

Discussion

Several methods have previously been used to transform primary cells in culture with variable degrees of success^25,26,27,28. Most of these methods use mouse fibroblast cells for transformation^14,17,18,19 or use carcinogens such as 4-nitroquinoline-1-oxide (4-NQO)

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibodies
Anti mouse HRP	Jackson ImmunoResearch	115-035-146
Anti rabbit HRP	Jackson ImmunoResearch	711-035-152
Cas9 Mouse mAb	Cell Signaling Technology	14697
Cre	BioLegend	900901
Cy3-AffiniPure Goat Anti-Mouse IgG	Jackson ImmunoResearch	115-165-062
Cy-AffiniPure Goat Anti-Rabbit IgG	Jackson ImmunoResearch	111-165-144
GFP	Santa Cruz Biotechnology	sc-9996
Phospho-p44/42 MAPK (Erk1/2)	Cell Signaling Technology	4370
Rabbit monoclonal anti E cadherin	Cell Signaling Technology	3195S
Rabbit monoclonal anti-KRT 14	Abcam	AB-ab181595
β actin	MP Biomedicals	691001
β catenin	Cell Signaling Technology	9582S
Cell lines
HEK93T	ATCC	CRL-3216
Culture Media, Chemicals and Reagents
Bradford Reagent	Bio-Rad	30015484
BSA	Amresco	0332-TAM-50G
DAPI fluoromount	Southern Biotech	0100-20
DMEM	Biological Industries Israel Beit-Haemek Ltd.	01-055-1A
ECL (Westar Supernova and Westar Nova 2.0)	Cyanagen	XLS3.0100 and XLS071.0250
FBS	Biological Industries Israel Beit-Haemek Ltd.	04-127-1A
HBSS	Sigma	H6648
Heparin - Agarose	Sigma	H6508
Isolate II Genomic DNA Kit	Bioline	BIO-52066
MgCl2	Panreac AppliChem	300283
NaCl	Bio Lab Ltd	1903059100
PBS	Biological Industries Israel Beit-Haemek Ltd.	02-023-1A
PEI	Polysciences	23966-1
Pen Strep Solution	Biological Industries Israel Beit-Haemek Ltd.	03-031-1B
PFA	Santa Cruz Biotechnology	30525-89-4
Phosphatase inhibitor cocktail	Biotool	B15001A/B
Protease inhibitor cocktail	MilliporeSigma	P2714-1BTL
Tris buffer	MERCK Millipore	648311-1KG
Enzymes
Benzonase	Sigma	E1014
Collagenase IV	Thermo Fisher Scientific	17104019
DNAse	Thermo Fisher Scientific	18047019
Hyaluronidase	MilliporeSigma	H3506
Trypsin	Biological Industries Israel Beit-Haemek Ltd.	03-050-1B
Glass wares
Cover slips	Bar Naor	BNCB00130RA1
Slides	Bar Naor	BN9308C
Mouse strains
C57BL/6	Envigo
B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J	Jackson labs	24857
NOD.CB17-Prkdc-scid/NCr Hsd (Nod.Scid)	Envigo
Plasmids
AAV pCM109 EFS Cre sg APC sg Kras sg P53- Kras HDR	Broad Institute of MIT		Kind gift from Dr Randall J Platt and Dr. Joseph Rosenbluh, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
AAV 2/9 capsid vector	Addgene	112865
pAD Delta F5 helper	Ben Gurion University of the Negev		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.
Plastic wares
Amicon-ULTRA filter 100 KDa	Millipore	UFC910024
0.22 µm sterile filters, 4 mm	Millex	SLGV004SL
0.45 µm sterile filters, 13 mm	Millex	SLHV013SL
Culture plates	Greiner Bio-One
Falcon tubes	Greiner Bio-One