1. GlioBlastoma Multiforme (GBM) WHO classification name "glioblastotoma,also known as Grade IV Astrocytoma “ WHO classification name Most common and most aggressive malignant primary brain tumor in humans.malignantbrain tumor Involve glial cells. It presents two variants: giant cell glioblastoma and gliosarcoma.giant cell glioblastomagliosarcoma The Central Brain Tumor Registry of the United States (http://www.cbtrus.org) states that the 5-year survival rate from the time of diagnosis is only 3.3 %. Standard-of-care treatment involves radiation, chemotherapy, and maximal surgical re-section of the tumor; however, as the poor survival rate indicates, these treatments have not been effective in preventing disease progression. One of the hallmarks of GBM is its invasiveness. Infiltrating cells extend from the tumor body into the surrounding brain parenchyma, making complete surgical removal of tumor nearly impossible and recurrent tumors often appear within 2 cm of the initial lesion. Direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment.

2. Protocol Summary Preparing the mouse 1. The mice are anesthetized using an injection mix of Ketamine/Xylazine/Saline. The mixture is 120 mg/kg of Ketamine, 10 mg/kg of Xylagine and Saline. The mice are weighed individually and given 0.1 ml per 10 g based on their weight. It will take approximately 15 min for the mice to reach a sleeping phase for no pain response. 2. After the mouse is fully anesthetized, eye ointment is applied to their eyes to prevent them from drying out. 3. The mouse is placed on the bed of the stereotaxic with the front upper teeth in the notch on the mouth bar. The bed is then placed in the stereotaxic. 4. The nose guard is placed over the mouse's nose to prevent movement of the head, and the ear bars are gently place in the ear canals of the mice. Be careful not to do either placement of the nose bar or ear bars too tight, as the skull may fracture and/or prevent the mouse from breathing. 5.Alcohol and providine iodine is applied on the head of the mouse alternating for a total of 3 application of alcohol and 3 applications of iodine. One final application of an alcohol pad with 70% is applied to the head.

3. Surgical procedure 1.All instruments in the procedure should be sterilized either by autoclaving or soaking instruments in 70% ethanol before beginning the procedure. 2.An incision is made on top of the skull behind the right eye diagonally back to the left posterior of the skull. The incision should be about 10 mm to 15 mm long. 3.Using a Q-tip, slowly separate the incision to expose the skull and wipe away the membrane that is between the skull and the skin. Use a Q-tip to blot any blood and watch for excessive bleeding. 4.Expose the top of the skull to visualize the bregma. Using the Hamilton syringe point, position it over the bregma. The correct position of the hole to be drilled in the skull is 2 mm right and 1 mm anterior of bregma. A surgeon's felt-tipped marker is used to mark the location of the hole to be drilled. Please note that the hole is located close to the anterior suture line. 5.Next, drill a small hole in the skull at the marked position using the point of a 22G needle or 18G needle. 6.Load the 10 μl glass Hamilton syringe (with a blunt 30G to 33G fixed needle) with 5 μl of cells. Be sure to mix the cells before loading them, either by pipetting them or using a 1ml syringe with a 25G needle. 7.Lower the bunt end of the needle to the surface of the skull, once the needle is level with the skull; lower the needle until it is at the depth of 3 mm below the surface of the skull. 8.After the needle is lowered, it shall remain there for 2 minutes to avoid any major trauma from the needle affecting the brain. Implants the cells at a rate of 1 μl per minute for a total of 6-8 minutes, be careful of any backflow. After the last of the cells have been implanted, leave the needle in place for another 2 minutes, to allow all the cells to settle in. 9.Remove the needle from the brain, and clean the hole with a Q-tip. Proceed to clean the needle and syringe with 50% Actone, 100% EtOH, and PBS, 3x in each solution and in that order. 10.The skin on the skull is ready to be sutured at this point. Close the incision with about 3 stitches. 11.Allow the mouse to wake up on a heat pad, and reapply eye ointment if needed. Total time to complete the whole process - around 30 min for one animal.

4. S. No.AuthorCell Line Cell density / mouse- Injection rate Animal/Age /sex Point of injection & CoordinateSyringe & Needle Gauge Tumor development period End point Parameters 1 Casey A Maguire,2008 U87 and Gli36 (ATCC) Fluroscence Tag 5 X 10 5 /1-2 µL of MEM Medium- 0.2 μl/min 6-8-week-old male nude mice Striatum From bregma in mm: anterior-posterior +0.5, medio-lateral +2.0,dorso-ventral −2.5 ( 3 mm depth) Micro 4 Microsyringe Pump Controller attached with Hamilton syringe with a 33-gauge needle (Hamilton, Rena, NV). 14-21 days ( Tumor growth was monitored by Bioluminiscence on day 4,14 & 21) (1) Body wt ( weekly) (2) At day 21 histopathology of brain (3) Survival ( end point defined by b. wt loss >20 %) Median Survival - 22 day for U87 2 Chen Hua--- 2.5 mm right to the sagittal suture, 1.0mm to the cranial coronal suture ( 3 mm depth) -- - 3 Tamalee R. 2012U87 1 X 106 /5 µL of PBS -1 μl/min- Location of Craniotomy - 2 mm to the right and 1 mm rostral to Bregma Hamilton syringe is 10 μl with a blunt 30G to 33G fixed needle attached to it 14-28 days ( Tumor growth was monitored by BLI, MRI or CT on day 14 & 28) (1) Body wt ( weekly) (2) At day 28 histopathology of brain (3) Survival ( end point defined by b. wt loss >20 %) Median Survival (4) Physical signs- hunched posture, Loss of B. Wt. and inactivity 4 Sharareh Gholamin, 2013 Patient Derived Tumor -Mechanical dissociation 5-7.5 X 104 / µL of PBS containing 1 % BSA ( IgG free) - 1 μl/min 6- 8 weeks old NOD - SCID gamma mice Supratentorial injections (i.e.,glioblastoma), coordinates of 2 millimeters posterior to the bregma, 1 millimeter laterally, and 2 millimeters deep to the dura Hamilton syringes and needles (26G) 14 day -6 months ( Tumor growth was monitored by BLI weekly) (1) Body wt ( weekly) (2) Histopathology of brain (3) Survival ( end point defined by b. wt loss >20 %)Median Survival (4) Physical signs-hunched posture, Loss of B. Wt. and inactivity

5. S. No.AuthorCell LineCell densityAnimal/Age/sex Point of injection & Coordinate Tumor development period End point Parameters 5 Jann N. Sarkaria, 2006 Patient Derived Tumor - S.C xenograft - Mechanical dissociation - Culture in Vitro- intracranial 5 X 10 5 /1-2 µL of MEM Medium Nude MiceRight basal gangliatwo weeks (1)Body wt ( weekly) (2)Histopathology of brain (3)Survival ( end point defined by b. wt loss >20 %)- Median Survival (4)Physical signs- hunched posture, Loss of B. Wt. and inactivity 6 Hiroaki Uchida, 2012 GBM30 cells 2 X 10 5 /4 µL of HBSS 6- 8 weeks old female nude mice A burr hole was drilled 2 mm lateral and 0.5 mm anterior to the bregma to a depth of 3 mm 21-35 days ( Tumor growth was monitored by MRI ) (1)(Body wt ( weekly) (2)Histopathology of brain (3)Survival ( end point defined by b. wt loss >20 %) -Median Survival (4)Physical signs- hunched posture, Loss of B. Wt. and inactivity 7 Hippokratis Kiaris, 2000 U-87 MG- SC Xenograft- orthotopic 5X 10 4 /15 µL of Normal saline 5- 6 weeks old Male nude mice A midline incision was made over the anterior aspect of the cranium and the scalp retracted to the right. A guarded 26-gauge needle was used to drill a hole 3–4 mm deep in the skull, 3 mm to the right of the midline, just anterior to the coronal suture. 21-35 days (1)(Body wt ( weekly) (2)Histopathology of brain (3)Survival ( end point defined by b. wt loss >20 %) -Median Survival (4)Physical signs- hunched posture, Loss of B. Wt. and inactivity 8 Qian Xie, 2008 DBTRG-05MG, U87, and U118 -ELM 2 times- Orthotopic 5 X 10 5 /5 µL of PBS 6 weeks old nude mice A burr hole was created through the skull 2 mm posterior to the bregma, 3 mm deep in the Skull 21-35 days (1)Body wt ( weekly) (2)Histopathology of brain (3)Survival ( end point defined by b. wt loss >20 %) - Median Survival (4)Physical signs- hunched posture, Loss of B. Wt. and inactivity

6. S. No. AuthorCell Line Cell density / mouse Animal/Age/sex Point of injection & Coordinate Tumor development period End point Parameters 9 Kyeung Min Joo, 2012 Patient derived GBM cell ( primarily cultured GBM cells that had short term in vitro culture in the NBE condition (in vitro passage <6, 2.0 X 10 5 cells for each animal) 2.5 X 10 4 -5 X 10 5 / 10 µL of HBSS NOG mice 2mm left and 1mm anterior to the bregma, 2mm deep from the dura - (1)(Body wt ( weekly) (2)Histopathology of brain (3)Survival ( end point defined by b. wt loss >20 %)- Median Survival (4)Physical signs- hunched posture, Loss of B. Wt. and inactivity 10 Sameer Agnihotri, 2014 A172 cells expressing EV, APNG, MGMT, or both APNG and MGMT 4.5 X 10 5 / 10 µL of HBSS NOD-SCID coordinates, x = –1.0, y = 1.5, z = 2.4; Bregma serving as the 0 point for x and y) 14-21 days ( Tumor growth was monitored by MRI) (1)(Body wt ( weekly) (2)Histopathology of brain (3)Survival ( end point defined by b. wt loss >20 %)- Median Survival (4)Physical signs- hunched posture, Loss of B. Wt. and inactivity 11Avadhut D. Joshi GBM stem Cell-20913 5 X 10 6 /1-2 µL of MEM Medium female athymic nude Using a surgical drill, a hole was made 1 mm lateral of midline and 1 mm lateral of Bregma over the parietal lobe 58 day median Survival (1)Body wt ( weekly) (2)Histopathology of brain (3)Survival ( end point defined by b. wt loss >20 %)- Median Survival (4)Physical signs- hunched posture, Loss of B. Wt. and inactivity 12Xi Feng Fei, 2010 Patient derived tumor tissue ( typical glioblastoma multiforme & brain metastasis from lung adenocarcinoma ) Tumor tissue, 2 mm 3 per mouse 4-6 male/female athymic nude A small burr hole, 2 mm in diameter was made 2 mm to the midline and 0.5 mm anterior to bregma using microskull dril to depth of 3.5 mm from the skull by 24# trochar 2-3 weeks ( Tumor growth was monitored by MRI on day 10, 15, 20, 25 and day 30 post tumor implantation ) (1)Body wt ( weekly) (2)Histopathology of brain (3)Survival ( end point defined by b. wt loss >20 %)- Median Survival (4)Physical signs- hunched posture, Loss of B. Wt. and inactivity