Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Podsumowanie

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

Streszczenie

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Wprowadzenie

Malignant gliomas are primary glial tumors of the central nervous system that occur in the brain and occasionally the spinal cord. Gliomas are classified by the World Health Organization (WHO) according to histologic resemblance to astrocytes, oligodendrocytes or ependymal cells and then numerically graded (I to IV) for pathologic features of malignancy. The most common histologic subtypes are astrocytomas, oligodendrogliomas and mixed oligoastrocytomas. Malignant gliomas encompassing WHO grades II to IV are characterized by invasive growth and recalcitrance to current therapies. Each year in the United States, approximately 15,750 individuals are diagnosed with a malignant glioma and an estimated 12,740 patients succumb to this disease. These statistics highlight the particularly lethal nature of malignant gliomas and important need for enhanced therapeutic efficacy.

Cancer models are essential for investigating tumor biology and therapies. Human cancer cell lines represent an important first step for in vitro manipulations and in vivo xenografting studies (secondary xenografts)¹. However, standard cancer cell cultures undergo phenotypic and genotypic transformation^2-4 that may not be restored in secondary xenografts⁵. Furthermore, genetic alterations such as isocitrate dehydrogenase (IDH) mutations⁶, distinct stem cell populations⁷ and dependency on key signaling pathways⁸ can be lost in cancer cell cultures. Genomic profiles can be maintained to better extent in cancer sphere culture, though still fail to mirror fully the genotype of primary tumors^2,3. Direct orthotopic transplantation negates the influences of in vitro culture, provides a proper microenvironment, and preserves the integrity of tumor-initiating cells^9,10. Therefore, primary xenografts represent a powerful and relevant preclinical model for rigorously testing targeted agents to aid in the rational design of future clinical trials^5,11,12.

Protokół

1. Preparation of Tumor Cell Suspension

Note: Appropriate institutional approvals for the use of patient material and animals are required to establish and maintain primary orthotopic glioma xenografts. At Vanderbilt University Medical Center, resected tumor material that is in excess of that required for diagnostic purposes is collected with patient consent for a research tissue repository. Specimens are labeled with a randomized 5-digit REDcap database number and all patient-specific identifiers are removed. The REDcap database contains deidentified clinical data for each specimen in the tissue repository that includes gender, age (in years), race, survival status, pathology, cancer treatments, year of resection, and time to progression. To maintain sterility and optimize success of the primary xenograft method, all of the reagents, steam-autoclaved surgical instruments, and the surgical site should be assembled or prepared in advance.

Note: Glioma specimens often contain large amounts of myelin and debris. Depending on specimen size, it may be necessary to use more than 4 discontinuous gradient tubes for adequate removal of myelin and debris.

Note: The process is completed when there is no, or little, clearly visible undissociated tissue remaining. Avoid the introduction of bubbles during the trituration process.

Note: Cell viability following dissociation is typically >90%. Lower viabilities may reflect many variables including suboptimal tissue handling or transport time from the operating room, cryopreservation method, or papain dissociation technique. Lower cellular viabilities may still be adequate, however, as long as a sufficient number of viable may be transplanted in the appropriate volume. For each mouse, 50,000-200,000 viable cells are implanted in volume of 2 µl. Due to the beveled tip of the syringe needle, an additional 5 µl of cell suspension should be included in the final volume to ensure that adequate material can be drawn into the syringe for each injection. For example, to inject 2 µl/mouse for 5 mice the final volume should be 15 µl (15 µl = (5 x 2 µl) + 5 µl).

1. Place freshly resected, deidentified patient material in a sterile, capped container on ice for transport to the laboratory. Process the specimen directly for transplantation or cryopreserve the specimen in liquid nitrogen (see below) for storage in liquid nitrogen and later use.
2. Prepare the papain dissociation solutions (papain solution, wash/protease inhibitor solution and discontinuous gradient solution) in a sterile hood with the kit components and instructions supplied by the manufacturer. Label sterile 15 ml polystyrene conical tubes and distribute the solutions as follows:
   • Tube 1: 5 ml of papain solution
   • Tube 2: 3 ml of wash/protease inhibitor solution
   • Tubes 3-6: 5 ml each of discontinuous gradient solution
3. Place the glioma specimen in tube 1 with 5 ml of papain solution and triturate with a 5 ml pipette over a 10-20 min time period.
4. Pipette the material up and down 10 times and then incubate the material for 2-3 min at room temperature before initiating the next cycle of trituration.
5. Position the tip of the 5 ml pipette closer to the bottom of the conical tube with each cycle of trituration such that the tip is touching the bottom of the tube for the last 2 cycles.
6. Centrifuge at 300 x g for 5 min at room temperature. Remove the supernatant and pipette the pellet up and down 10x in 3 ml of the wash/protease inhibitor solution.
7. Carefully layer an equal volume of the suspension over each of the 5 ml discontinuous gradient solutions (tubes 3-6), with a pipettor set at the “gravity” dispense speed.
8. Place the tubes in a centrifuge equipped with a swinging-bucket rotor, with the acceleration speed reduced to the lowest setting and the brake turned off. Centrifuge at 76 x g for 12 min at room temperature. With the brake turned off, centrifugation is generally completed after 20 min.
9. Remove the supernatant, resuspend and combine each pellet in 5 ml of sterile balanced salt solution and place the cells on ice.
10. Remove a portion (10-100 µl) of the cell suspension and determine the density of viable cells by trypan blue exclusion with a hemocytometer.
11. Centrifuge at 300 x g for 5 min. Remove the supernatant and resuspend the cell pellet at a density of 25,000-125,000 viable cells per µl.
12. Transfer an adequate volume of the cell suspension to a 200 μl microcentrifuge tube (PCR tube) and place on ice.

2. Preparation of Surgical Site and Instruments

Note: Sterile surgical gloves are worn during the procedure. Depending upon requirements of each institution, surgical gowns, caps and face guards may be required.

1. Prepare a disinfected benchtop for rodent surgery with separated adjoining areas for animal preparation, operating field and animal recovery.
2. Sterilize surgical instruments in a steam autoclave. Subsequently, use a hot bead sterilizer to flash-sterilize instruments when transitioning from one animal subject to the next.

3. Induction, Anesthesia, and Analgesia

Note: Surgeries exceeding 15 min from the time the mice is anesthetized require a contact heat source. To prevent hypothermia, the mice are provided with a heat source (circulating hot water blanket) during the pre- and post-operative periods.

1. Prepare ketamine/xylazine cocktail for induction anesthesia such that each mouse receives ketamine 100 mg/kg and xylazine 10 mg/kg by injecting 10 µl of the cocktail per gram of body weight.
2. Table 1. Anesthesia cocktail.

   	Stock concentration	Volume to prepare for
   		one mouse	five mice	ten mice
   Ketamine	100 mg/ml	25 µl	125 µl	250 µl
   Xylazine	100 mg/ml	2.5 µl	12.5 µl	25 µl
   Isotonic saline		223 µl	1,113 µl	2,225 µl
   Total		250 ul	1,250 µl	2,500 µl

3. Prepare ketoprofen solution for analgesia by diluting the stock solution (10 mg/ml) 1:20 in sterile, pyogen-free water such that each mouse receives a dose of 5 mg/kg by injecting 10 µl of the solution per gram of body weight.
4. Weigh and record presurgical weight. Individual mouse weights vary, but generally range from 18-22 g.
5. Inject 10 µl of the ketamine/xylazine cocktail per gram of mouse body weight into the intraperitoneal space with an insulin syringe. For example, inject 200 µl for a 20 g mouse.
6. Determine whether mouse is fully anesthetized by toe pinch of the hind leg. It generally takes 3-5 min for the mouse to no longer withdraw from the pinch.
7. Inject 10 µl of the ketoprofen solution per gram of mouse body weight subcutaneously in the loose skin of the flank with an insulin syringe. For example, inject 200 µl for a 20 g mouse.
8. Shave hair over the skull with clippers, scrub the exposed scalp with povidone-iodine using a cotton tip applicator and then wipe an alcohol pad. Repeat these steps, povidone-iodine scrub and alcohol wipe, two more times.
9. Apply ophthalmic ointment to the eyes of the mouse.
10. Make a 1 cm midline incision extending from behind the ears to just in front of the eyes with a sterile scalpel blade.
11. Place the mouse under a dissecting scope and expose the skull to identify the suture lines. Using circular motions with a drill, center a burr hole 2.5 mm lateral to the bregma and 1 mm posterior to the coronal suture.

4. Intracranial Transplantation

Note: Injection volumes exceeding 2.5 µl may be lethal to mice.

1. Position the mouse on a stereotactic frame by hooking the upper incisors over the incisor bar and applying the nose clamp so that the skull is firmly held in a neutral position.
2. Draw 5-10 µl of the cells in a microcentrifuge tube up and down several times into a 10 µl Hamilton syringe clamped in the probe holder of the stereotactic manipulator to mix the suspension and eliminate bubbles. Depress the plunger so that the syringe is loaded with 2 µl (the adequate volume to inject one animal).
3. Pull the lateral edge of the skin incision to expose the burr hole and, with the micromanipulator, introduce the needle into the burr hole so that the beveled portion is just below the skull surface.
   1. Advance the needle 3 mm and then withdraw the needle 0.5 mm to create a space for injection.
   2. Advance the plunger slowly over 30 sec and then allow the needle to remain in the brain for an additional 2 min. Withdraw the needle gradually by ascending 0.5 mm and waiting for an additional 30 sec before removing the needle from the brain.
4. Remove the mouse from the stereotactic frame and close the wound with an autoclip.
5. Place the mouse on a warming pad to maintain body temperature and monitor continuously the respiration pattern and color of mucous membranes and exposed tissues (soles of feet).
6. Place the mouse in a sterile microisolator cage once it recovers fully from anesthesia (sternal and ambulatory), and return it to the animal housing facility.

5. Post-implantation Animal Care and Observation

Note: The most reliable indication of tumor engraftment and infiltrative growth is gradual, sustained weight loss accompanied by development of hunched posture and rough hair coat. On rare occasions, neurological deficits are noted that include ataxia, paresis, and seizures. Orthotopic primary xenografts are highly invasive and develop over a long period of time. Mice typically manifest symptoms of tumor growth 3-6 months after transplantation.

1. Observe mice with orthotopic xenografts daily for food and water intake, behavior, grooming, and signs of infection at the incision site.
2. Administer ketoprofen (5 mg/kg subcutaneously, 1-2x a day) if pain or distress is observed postoperatively.
3. Remove autoclips 8-10 days after surgery.
4. Weigh mice weekly.
5. Monitor for signs and symptoms of tumor engraftment.

6. Tumor Harvesting

Note: By this method, the first coronal slice (#1) contains the posterior aspect of the olfactory bulbs and the anterior aspect of the frontal lobes. Engrafted tumor is most abundant in the right hemisphere of the subsequent 3 coronal slices (slice #2, #3, and #4) and the injection sight is routinely contained in coronal slice #3. Depending upon experimental needs, the material may be used for tumor passage, frozen tissue sections, formalin fixed paraffin embedded tissue sections, flow cytometry of dissociated tissue, cell culture or lysates for DNA, RNA, or protein analysis. Portions of the tumor may be cryopreserved for future needs with cell viability ranging from of 80-95%.

1. Euthanize mice by prolonged exposure to carbon dioxide followed by cervical dislocation.
2. Decapitate euthanized mice at the base of the brain (foramen magnum level) with a larger pair of surgical scissors, and pull the skin from incision forward to fully expose the skull.
3. Remove the brain.
   1. Insert one tip of pair of fine surgical scissors into the lateral aspect of the foramen magnum to level of the left external auditory meatus to cut the occipital bone along the left side of the skull. Perform the same cut on the right side.
   2. Cut the occipital bone along a coronal plane from one external auditory meatus to the other and remove the bone to expose the cerebellum.
   3. Cut the nasal bone plates between the eyes.
   4. Cut the sagittal suture by cutting posteriorly along the sagittal suture from the nasal bone plates to the bregma. Complete the midline incision along the sagittal suture by cutting anteriorly from the lambda to the bregma.
   5. Carefully insert the tip of a fine pair of forceps along the sagittal opening on each side to tear any dural adhesion of the skull to the brain and then peel the two skull halves laterally to expose the brain and olfactory bulbs dorsally.
   6. Slide the forceps between the ventral aspect of the olfactory bulbs and the skull to free any adhesions.
   7. Tilt the skull back to allow the brain to be retracted so that the optic and other cranial nerves are exposed. Sever the cranial nerves to release the brain into a dish containing sterile PBS.
4. Transfer the brain with a weighing spatula to a matrix slicer and generate 2 mm coronal slices with razor blades.
5. Arrange the coronal slices in a dish containing sterile PBS for further visual inspection and further tissue processing.

7. Tumor Cryopreservation

1. Prepare a stock solution of cryopreservation medium.
   1. Add 50 ml of proliferation supplement, 5 ml of 100x penicillin and streptomycin solution, and 450 µl of 0.2% heparin to 450 ml of basal medium.
   2. Store stock solution at 4 °C protected from light.
2. Prepare a working solution of cryopreservation medium.
   1. Combine 47.5 ml of cryopreservation medium and 2.5 ml of sterile DMSO in a 50 ml polypropylene conical tube and mix well.
   2. Aliquot 1.0 ml of working solution to each cryovial and store at 4 °C protected from light.
3. Place one 2 mm coronal slice of xenografted brain into a cryovial containing cryopreservation medium on ice.
4. Tightly cap the vial and place it in a freezing container at -80 °C. Transfer the cryovial the following day to a liquid nitrogen tank for longer storage.

Wyniki

Dissociated glioma cells are transplanted directly into the brains of immunocompromised mice to obtain primary orthotopic xenograft lines. Each tumor specimen is assigned a randomized number prior to transplantation, as part of the deidentification process to remove protected health information. We use a 5-digit REDcap database number for this purpose. Figure 1 illustrates the process and nomenclature for establishing a xenograft line from a glioblastoma (GBM 17182) with isocitrate dehydrogenase 1 (IDH1)...

Dyskusje

Cultured cell lines, xenografts and genetically engineered mice are the most common methods for modeling gliomas, and there are distinct benefits and limitations for each model system^3,13,14. Relevant benefits of primary orthotopic glioma xenografts include infiltrative growth that typifies diffuse gliomas and the retention of genetic alterations and important signaling mechanisms that can be exceedingly difficult to maintain in cultured glioma cells. For example, isocitrate dehydrogenase mutations an...

Materiały

Name	Company	Catalog Number	Comments
Phosphate buffered saline	Life Technologies	14040-133
Papain dissociation system	Worthington Biochemical Corp.	LK003150
Trypan blue solution 0.4%	Life Technologies	15250061
Ketamine HCl	Obtained from institutional pharmacy or local veterinary supply company
Xylazine HCl
Ketoprofen
Ophthalmic ointment
Povidone-iodine	Fisher Scientific	190061617
Cryopreservation medium and proliferation supplement	StemCell Technologies	05751
0.2% Heparin sodium salt in PBS	StemCell Technologies	07980
Penicillin-streptomycin	Life Technologies	15140-122
Dimethyl sulfoxide	Sigma-Aldrich	D6250-5X10ML
NOD.Cg-Prkdcscid I/2rgtm1Wjl/SzJ mice	The Jackson Laboratory	005557	NSG mice
Anti-human vimentin antibody	Dako	M7020	Use 1:200 to 1:800
Anti-human IDH1 R132H antibody	Dianova	DIA-H09	Use 1:100 to 1:400
Centrifuge with swinging bucket rotor
Pipetter with dispensing speed control
Disposable hemocytometer	Fisher Scientific	22-600-100
Sterile surgical gloves	Fisher Scientific	11-388128
Disposable gown	Fisher Scientific	18-567
Surgical mask	Fisher Scientific	19-120-1256
Tuberculin syringe	BD	305620
Alcohol pads	Fisher Scientific	22-246-073
Portable electronic scale	Fisher Scientific	01-919-33
Zoom stereomicroscope
Surgical clipper	Stoelting	51465
Scalpel handle	Fine Science Tools	10003-12
Scalpel blades, #10
Stereotaxic instrument	Stoelting	51730
High-speed drill	Stoelting	51449
Drill bit, 0.6 mm	Stoelting	514552
Hamilton syringe	Hamilton	80336
Autoclip, 9 mm	BD	427630
Circulating water warming pad	Kent Scientific	TP-700 TP-1215EA
Hot bead dry sterilizer	Kent Scientific	INS300850
Surgical scissors	Fine Science Tools	14101-14
Fine scissors	Fine Science Tools	14094-11
Spring scissors	Fine Science Tools	15018-10
Dumont forceps	Fine Science Tools	11251-30
Semimicro spatulas	Fisher Scientific	14374
Mouse brain slicer matrix	Zivic Instruments	BSMAS002-1
Cryogenic storage vials	Fisher Scientific	12-567-501