ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 1 Establishment of Glioblastoma Multiforme Cell Line Bank Rachel Patten University of Florida Dr. Brent Reynolds
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 2 Abstract Glioblastoma Multiforme (GBM) is a grade IV astrocytoma and is therefore, one of the most aggressive and lethal tumors. Currently th e treatments available are not very successful in maintaining the longevity of a person who receives this diagnosis However, t here is a great deal of rese arch being done on GBM and much of the success is being s een at the molecular level. Due to the se positive results, it will be important f or further molecular research to continue The areas that will likely lead to the most successful results involve studying microRNAs, various proteins and enzymes, as well as a plethora of other gene structures. With the importance of GBM research increasing the collection and analysis of GBM cell lines will be vital Therefore, through this research the growth rate s as well as morphology of different GBM cell lines will be found. Once each of the lines has reached the appropriate amount (>10 0 m illion cells ) they will then be suspended in TRIzol TM and sent to have an RNA sequence anal ysis conducted By the end of the research project the cell lines will have been placed in cry ostore for long term preservation to be used by researchers to continue studying GBM Keywords: glioblastoma multiforme, cell bank, tumor, growth rates, TRIzol TM RNA sequence
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 3 Acknowledgments I would like to ident ify the people who have played a vital role in my academic success that ultimately lead to this thesis being written First, I would like to thank Dr. Brent Reynolds. He provided me with my fi rst research assistant position which was an unbelievable learning experience. I was able to work on various cancer r elated projects, specifically ones involving Glioblastoma Multiforme which holds a special meaning to me He constantly made himself avail able to meet and provided continuous guidance in the preparation of this project. I would also like to thank my parents for always supporting each of my endeavors throughout my undergraduate career. I would not have developed such a strong work ethic and love for learning had it not been for their inspiration. Their unwavering support is the reason I have achieved each of my goals and have grown into then person I am today. I also would like to thank Zachary Tucker. Through his diagnosis with Glioblasto ma Multiforme I have been shown what a true hero l ooks like. Knowing the research I am involved in could potentially help patients like him continually motivates me in my search for answers. Finally, I would like to thank Tyler Peterson. Throughout my ent ire undergraduate career, he has been there to continuously support every decision I have made. His unwavering encouragement allowed me to follow my passions and continually achieve my goals
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 4 Literature Review Due to the more recent advances in te chnology the diagnosis of cancer is not accompanied with as grim a prognosis at it would have been several years ago. Although, e ven with these advances in technology one malignant glioma tumor glioblastoma m ultiforme (GBM) still remains excee dingly difficult to treat (Parsons et al, 2008 ). This tumor makes up about 60% of the bra in tumors found in adults and u pon diagnosis, the median survival is about 14 to 15 months (Hanif, Farina et al, 2017 ). Malignant gliomas are cancers of the central nervou s system and are the most lethal and frequently occurring in this system (Parsons et al, 2008 ). These types of tumors arise from astrocytes found in the brain which are a type of glial cell ( ). Astrocytes give rise to GBM tumors thus gi ving them the categorization of an astrocytoma (Hanif, Farina et al, 2017 ). Furthermore, the World Health Organization (WHO) has classified tumors by grade on a scale of I to IV, with grade IV being the most lethal and aggressive (Hanif, Farina et al, 2017 ). GBM has been given the classification of grade IV ( Hanif, Farina et al, 2017 ). Upon further examination, GBM has been found to consist of two tumor types pri mary and secondary ( Ohgaki, Hiroko, and Paul Kleihues 2007) These two subtypes of GBM have different genetic markers, effect different age groups, and develop differently ( Ohgaki, Hiroko, and Paul Kleihues 2007) Primary GBM tumors constitute the majority of the cases seen, arou nd 90% ( Ohgaki, Hiroko, and Paul Kleihues 2007 ). These tumor s rapidly develop de novo, meaning they arise without the presence of any other can c er or lesion in the body ( Ohgaki, Hiroko, and Paul Kleihues 2007 ). The genetic markers of a primary GBM tumor are as follows: loss of het erozygosity on 10q, EGFR amplification, p16 INK4a del etion, and PTEN mutations ( Ohgaki, Hiroko, and Paul Kleihues 2007 ). Secondary
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 5 GBM tumors only constitute about 10% of the GBM tumors seen but are still aggressive and ). Th is type of GBM develops from the metastases of a The first genetic marker of this tumor type is the mutation seen in the TP53 gene, this is then characterized further by G:C A :T mutations at CpG sites (Ohgaki & Kleihues, 2007 ). Secondary tumor s also are more likely to be seen in younger patients while primary tumors are more likely to be seen in adult patients (Ohgaki & Kleihues, 2007 ). While these is a great number of differenc es between the tumor types similarities exist as well. The most frequently observed genetic alteration, the loss of heterozygosity 10q25 qter, is seen in both tumor types (Ohgaki & Kleihues, 2007 ). Currently, it is extremely difficult to treat GBM. This difficultly is due to a variety of facto rs such as the challenge of crossing the blood brain barrier age of patient at the onset of the disease and the lack of understanding of the pat hophysiology of the tumor (Carlsson et al, 2014 ). Treatment currently used is fractionated rad iotherapy with temozolomide paired wi th adjuvant temozolomide ( Yi, Yang, et al 2016 ). Although, t his therapy is often ineffective due to the i nvasiveness of the tumor. Additionally, GBM has also been seen to be resistant to radi oth erapy and chemotherapy ( Yi, Yang, et al 2016 ). This resistance is thou ght to come from cells in GBM called glioblastoma stem like cells (GSCs) ( Yi, Yang, et al 2016 ). These cells are found to exhibit characteristics similar to those found in stem cells T hese characteristics are: self renewal, drug and radiation resistance, differentiation into different cell lineages high tumorigenicity, and the pathways similar to normal stem cell s ( Yi, Yang, et al, 2016 ). A marker that is frequently found on the surfa ce of normal stem cells is also found on the GSCs. ( Yi, Yang, et al 2016 ) Transcription factors that deal with the maintenance of stem cells are also found to be expressed in some subpopulations of GSCs these are c Myc, SOX2, OCT4,
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 6 NANOG, SALL4, STAT3, B mil, and KLF4 ( Yi, Yang, et al 2016 ). Each of these transcriptional factors has a different role in giving stem cells the properties they are known to possess ( Yi, Yang, et al 2016) The discovery of these factors shows the importance of continuing the research on the microscopic features of GBM in order to better understand the pathophysiology Another factor that contributes to the a ggression and threat of GBM is the microRNA (miRN A) alterations found within the tumor tissue. When compared to healthy brain tissue it was seen that there is both more upregu lation and downregulation of miRNA s ( Shea, Amanda, et al 2016 ). The miRNAs seen in cancer have a strong tendency to target the de velo pmental genes, thus making them a strong factor in the regulation of proliferation, differentiatio n, and apoptosis of cells ( Shea, Amanda, et al 2016 ). These miRNAs have even been noted to have a role in the evasion of growth suppressors, be key regul ators in drug resistance, and induce the replica tive immortality of the cells ( Shea, Amanda, et al 2016 ). Therefore, the understanding of these RNA sequences will be vital to better understanding GBM While GBM is not the most prevalent t umor, it is th e most lethal ( Whiteman, Honor 2017 ). Lengthening the median survival of patients with GBM is what fuels the desire to continually learn more about this vicious brain tumo r New research has emerged from scientists from the Massachusetts Institute of Technology dealing with a specific gene, the PRMT5 ( Whiteman, Honor 2017 ). This gene creates an enzyme that plays a role in gene splicing, removing introns from messenger RNA (mRNA) because they do not code for a protein ( Whiteman, Honor 2017 ). When the se introns remain in the mRNA strand, the strand cannot be exported outside the nucleus and begins to accumulate ( Whiteman, Honor 2017 ). This process causes genes involved in expression of proliferation to be retained in the nucleus, and thus the cell doe s not continually divide ( Whiteman, Honor 2017 ). The group investing hypothesized
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 7 that GBM has higher levels of PRMT5 throughout its entire life cycle allowing for the continued prolife ration of the cells ( Whiteman, Honor 2017 ). This theory was proven co rrect for when they delivered PRMT5 inhibitors to mice models with GBM and human GBM cells in vitro the growth and di vision of cells was stopped ( Whiteman, Honor 2017 ) This information as well as other studies conducted demonstrate the importance of cont inuin g to study at the molecular level The difficulty that comes in treating this disease is the availably of GBM cell line s available to use for research ( NIH R24 NS086554 01 ). GBM cells can be cultured in the same manner as adult neural stem cells (NSC) through a NeuroSphere Assay (NSA) but the GBM cells are difficult for labs to obtain ( NIH R24 NS086554 01 ). The difficulty in obtaining GBM cells comes from the fact that many in stitutions cannot get fresh samples because they are not n ear a hospital performi ng surgeries on GBM patients ( NIH R24 NS086554 01 ). Although, once the GBM cells are obtained they can often be easier to maintain than NSC cultures because once established t hey are more forgiving ( NIH R24 NS086554 01 ). That being said the skills required to handle the culture of GBM are still difficult and require the researcher to pay close attention to the culture ( NIH R24 NS086554 01 ) Methodology Passage of Primary Tum or We received p rimary GBM tumor samples fresh in a vial promptly following their removal from the patient. The tiss ue was then transferred to the lab on ice and placed in the hood. We then transferred t he tumor piece onto a sterile plastic surface on which the blood vessels were removed with a scalpel. We diced the sample with the scalpel and once the pieces became small enough we added 500 L of 0.5% trypsin and the dicing continued. We moved t he
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 8 soluti on of tissue and trypsin into a 15mL conical tube and another added 500 L of 0.5% of trypsin The conical tube was then placed in a water bath at 37 C for 10 minutes. After the 10 minutes passed we added 1 mL of trypsin inhibitor and DNAse and th en titrat ed the mixture five times. We then passed the solution through a 40 m straine r into a 50 mL conical tube. The liquid collected was transferred to a 15 mL conical tube and spun down in a centrifuge at 800 rpm for five minutes. The pellet formed from this centrifugation was red. We removed the supernatant liquid, added 1 mL of re d blood cell (RBC) lysis buffer, and titrated the solution The tube was again placed in a water bath for 10 minutes. After 10 minutes we added 2 mL of complete media to the conica l tube and the n centrifuged the tube for five minutes at 800 rpm This pellet created was no longer red. We removed t he supernatant liquid and added 1 mL of complete media All of the liquid was then placed in a T25 flask with 5 mL of complete media and placed in an incubator. Passaging of Cell Lines Twice a week the T25 flaks containing the cell lines were checked to see if the cells had become confluent and should be passaged To passage the cell lines, we brought the T25 flaks to a sterile hood. The cell solution was moved to a 15 mL conical tube labeled with that line s identification number If the cells were attached to the bottom of the T25 flask we hit the flask gently to detach the cells, or we added a small amoun t of trypsin to facilitate the movement of the cells. The cell solution was then spun down at 800 rpm for five minut es. We removed the supernatant, added 1 mL of trypsin to the pell et, and titrated the solution five times We then placed the conical tube i n a water bath at 37 C for two minutes. After two minutes in the water
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 9 bath we brought the conical tube back to the hood and added 1 mL of trypsin inhibitor, titrated the solution five times and then it was spun down for five minutes at 800 rpm Once a pe llet was observed we removed the supernatant Then we added 1 mL of phosphate buffer solution (PBS) and titrated five times Once the cells were suspended in PBS we counted the number of cells present using a hemo cytometer. We brought an Eppendorf tube containing 90 L of trypan blue to the hood and 10 L of the cell suspension w as added and mixed in well. We loaded the hemocytometer with 10 L of the trypan blu e cell suspension and then took it to the microscope to be counted. We counted each of the four quadrants in the hemocytometer. To find the total number of cells present in the solution we divide d the number counted on the hemocytometer by four. Next, we multiplied by the dilution factor of 10 and then by the constant for the hemoc y tometer which is 10,000. Next we set up a proportion to solve for the amount of cell solution we put into the flask for incubation : The number of cells to be plated depends on the size of the flask being used. A T25 flask is plated with 250,000 cells a T80 is plated with 1,000,000 cells, and a T175 is plated with 2,000,000 cells. Once X has been found the appropriate flask has the appropriate amount of media added. A T25 gets 5 mL of complete media, a T80 gets 20 mL of complete media, and a T175 ge ts 40 mL of c omplete media. Once the correct amount of media was added then we then added the
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 10 correct amount of the cell s olution (X) Afterwards, we looked at the flask under the microscope to ensure that cells were added to the flask. The flask was then placed in the incubator. Banking the Cell Lines Banking cells requires that 100 Million cells are present in the flasks in the incubator. The 100 million cells should be present in ten T175 flas ks with 10 million cells per flask Once the cells in the ten T175 flasks reached confluence, we took 2 mL from one flask to count the cells present. The number of cells we obtained from the 2 mL was the n multiplied by the total number of milliliters from each T175 flask to determine the total number of cells from all the flasks The total number of milliliters in each T175 flask was found when the cell suspension was moved into 50 mL conical tube s The 2 mL removed and counted previously was then plated in a T25 flask an d labeled with the cell line identification number, the date We spun down e ach of the 50 mL conical tubes for 5 minutes at 800 rpm. The supernatant was then removed. Using the total number of cells calculated we added the appropriate amount of cryostore to each 50 mL concial to make it so each milliliter of cry o store had one million cells. One milliliter of each cell suspension was placed in a c ry ovial and the c ry ovials were placed i n a Mr. Frosty. The Mr. Frosty allows the cell suspension t o freeze slowly over 24 hours in the 80C freeze r Once the 24 hours ha d passed we took the c ry o vials out of the Mr. Frosty and placed them in a container for long t erm storage in the 80C freezer Cell Line Creation Once the GBM cells were expanded to 10 0 mil lion cells and banked in the 80C freezer they were said to be a cell line.
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 11 Defrosting Cell Lines Before removing the vial from the 80C freezer a T25 flask with 5 mL of complete media needed to be equilibrated for one hour. Once the hour passed, we r emove d the cryovial containing the cryostore cell suspension from the freezer and place d it in a 37C water bath for no more than two minutes. Next, we m ove d the cryosto re cell suspension t o a 15 mL conical tube in the hood using a pipette. Dropwise, we slowly add ed 10 mL of warm complete media. The conical tube was then spun down for five minutes at 8 00 rpm. Once the five minutes had passed we brought the conical tube ba ck to the hood, remove d the supernatant, and suspend ed the pellet in 1 mL of complete media from the T25 flask that was equilibrated. We then a dd ed the 1 mL back to the T25 and place d it in the incubator. TRIzol TM Protocol The cells being suspended in TRIzol TM were removed from the freezer in small groups because the defrosting of the cells needed to occur in a time sensitive manner. Once we had removed the vials from the 80C we brought them into the hood to twist the cap just enough to the reduce any pressure that could have accumulated. With the cap retightened the vials were then placed in a water bath at 37C for no more than two minutes. Under no circumstance should the cell suspension be vortexed. After two minutes the vials were removed, wiped with ethanol, and brought into the hood. We titrated the mixture to homogenize the cells in the cryostore The solution was then transferred to a 15 mL conical tube. Next, dropwise we added 5 mL of warm media to the ce ll suspension to dilute the cry ostore. The 15 mL conical tube was then centrifuged at 800 rpm for five minutes. After the five minutes, the conical tube was brought back to the hood and the supernatant was removed. We added the TRIzol T M reagent at a ratio of 0.75 mL of
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 12 TRIzol TM to 5 10x10 6 cells (ratio for an animal subject) The solution was pipetted five times to h omogenize the solution. Once the cells were suspended in the TRIzol TM we transferred the solution to an Eppendorf tube. Th e Eppendorf tubes can be stored in the 80C for up to one year. Results Growth Rate The numbers used in the calculation of the growth rate s were taken after 4 to 6 passages in order for the cells to reach a complex equilibrium ( Deleyrolle, Loic P., et al. 2011 ). Within the culture of the neural c ells there exist two types of cells long term proliferating (LTP) cells and short term proliferating (STP) cells ( Deleyrolle, Loic P., et al. 2011 ). e cel ls being measured, they are defined as infinite in their replication and assumed to be stem cell like ( Deleyrolle, Loic P., et al. 2011 ) s have a finite life and are thought to be more similar to progenitor cells ( Deleyrolle, Loic P., et al. 2011 ). The equation used to calculate the rate of LTP cell symmetric division (LTPCSD) is: The F present in the equation is the fold expansion of the cell line. It is taken by dividing the cells present at the start of the passage, T i by the cells counted at the end of th e passage, T f The t f present in the equation represents the amount of time between the beginning of the passage and the end of the passage. The unit for the t f variable is days. When cal culating the LTP
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 13 cell symmetric division rate the F and t f were the average numbers calculated from five rounds of passaging ( Deleyrolle, Loic P., et al. 2011 ). Line = R24 37 Start Date End Date tf (days) Beginning Cell Count End Cell Count Expansion 11 Jul 17 1 Aug 17 15 50,000 950,000 19 1 Aug 17 17 Aug 17 16 50,000 950,000 19 17 Aug 17 18 Sep 17 13 50,000 1.150Mil 23 18 Sep 17 2 Nov 17 22 50,000 300,000 6 2 Nov 17 28 Nov 17 26 50,000 150,000 3 Mean tf: 18.4 Mean Expansion (F): 14 LTP: 0.143 Line = R24 63 Start Date End Date tf (days) Beginning Cell Count End Cell Count Expansion (F) 31 May 17 27 Jun 17 27 250,000 1.6Mil 6.4 27 Jun 17 7 Aug 17 41 50,000 375,000 7.5 7 Aug 17 7 Sep 17 31 50,000 2.05Mil 41 7 Sep 17 25 Oct 17 48 50,000 700,000 14 25 Oct 17 13 Dec 17 49 50,000 375,000 7.5 Mean tf : 39.2 Mean Expansion (F): 1 5.3 LTP: 0.070 Line = L2 Start Date End Date tf (days) Beginning Cell Count Cell Count Expansion 10 Aug 17 23 Aug 17 13 50,000 3.075Mil 61.5 23 Aug 17 5 Sep 17 13 50,000 1.05Mil 21 5 Sep 17 18 Sep 17 13 50,000 3.225Mil 64.5 18 Sep 17 3 Oct 17 15 50,000 7.6Mil 152 3 Oct 17 17 Oct 17 14 50,000 2.725Mil 54.5 Mean tf : 13.6 Mean Expansion (F): 70.7 LTP: 0.313 Line = CA7 Start Date End Date tf (days) Beginning Cell Count End Cell Count Expansion (F) 3 Oct 17 23 Oct 17 21 50,000 2.375Mil 47.5 23 Oct 17 31 Oct 17 8 50,000 166,666 3.33 31 Oct 17 16 Nov 17 16 50,000 2Mil 40 16 Nov 17 30 Nov 17 14 50,000 1.8Mil 36 30 Nov 17 13 Dec 17 13 50,000 8.5Mil 170 Mean tf : 14.4 Mean Expansion (F): 59.4 LTP: 0.2835 Line = CA9 Start Date End Date tf (days) Beginning Cell Count End Cell Count Expansion 6 Jul 17 27 Jul 17 21 50,000 425,000 8.5 27 Jul 17 7 Aug 17 11 50,000 675,000 13.5 7 Aug 17 23 Aug 17 16 50,000 400,000 8 23 Aug 17 7 Sep 17 15 50,000 400,000 8 7 Sep 17 22 Sep 17 15 50,000 650,000 13 Mean tf : 15.6 Mean Expansion (F): 10.2 LTP: 0.148
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 14 Photographs Line R24 37 Figure 1. Photo of cells from Line R24 37. Magnification 10x. Shows neurospheres. The darkened center of the larger neurosphere marks cell death. Figure 2. Photo of cell from Line R24 37. Magnification 20x. Picture of larger neurosphere. Figure 3. Photo of cells from Line R24 37. Picture of smaller neurosphere. Magnification 20x. This is a conglomeration of multiple cells.
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 15 Line R24 63 Line L2 Figure 6. Photo of cells from Line L2. Magnification 10x. These are neural progenitor cells and they will come together to form a neurosphere. Figure 7. Photo of cells from Line L2. Magnification 20x. These are neural progenitor cells. Figure 4. Photo of cells from Line R24 63. Magnification 10x. Neurosphere present. Neural progenitor cells can be seen i n the neurospheres. Figure 5. Photo of cells from Line R24 63. Magnification 20x. Small neurosphere.
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 16 Line CA7 Line CA9 Figure 8. Photo of cells from Line CA7. Magnification 10x. Beginning of the neural progenitor cells forming the neurosphere. Figure 9. Photo of cells from Line CA7. Magnification 20x. Beginning of the neurosphere formation. Figure 10. Photo of cells from Line CA9. Magnification 10x. Beginning of neural progenitor cells forming the neurosphere. Figure 11. Photo of cells from Line CA9. Magnification 20x. Individual neural progenitor cells.
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 17 Discussion From the study conducted it can be seen that Glioblastoma Multiforme cells lines can be established in culture when obtained from a human subject. It was also shown that it is possible for the GBM cells lines to expand enough to obtain the high volume of cells (>10 0 million) needed to create a cell line bank. The time re quired to bank each of the lines differed depending on the growth rate. The growth rates, which were based on the Long Term Proliferating (LTP) cell symmetric division, were dece nt for each of the cell lines. The growth rates found will allow for researchers to predict the amount of ti me required to grow the cells This will allow researchers to cre ate a more accurate time line for their experiment. The differences seen in the growth rates most likely occur because the of the genetic variation be tween GBM tumor cells. The GBM could also differ because the samples came from patients at different stages in their life and th e tumor s could have been at a different stage in their life cycle. These seemingly small differences can lead to significant changes in morphology and growth rate. Furthermore, a fter the cryopreservation process, s ome of the cell lines were defrosted, plat ed, and have been successfully grow n. Thus, demonstrating the cryopreservation process does not destroy the cells. All of this information together will allow for researchers to make an educated decision when deciding what GBM cell line to use in their exp eriment This research will also allow for potential further cla ssification of GBM tumors Limitations The success of this experiment and data collection relied heavily on the rel ationship our lab has with the Brain B ank at the University of Florida. If any other researchers wish to establish a bank such this one, they would have a difficult time without such a relationship.
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 18 Therefore, the labs hoping to replicate this would need to create a relationship such a this be fore beginning. Future Research The creation of the cell bank opens the doors for a plethora of future research. The first piece of research that could be found is the RNA sequence of each of the cell lines. This information will determine if any specifi c genes are over or under expressed in the GBM line. It could also lead to the potential determination of what genetic markers signal a primary or secondary GBM tumor. Fu rthermore, researchers could obtain different lines of the GBM cells from our bank to conduct research on them. Such projects that could be perform ed are testing the effectiveness of a new treatment implanting cell s in vivo to test treatments or conduct ing further genetic analys is. The research that could be done on these cells is endless making this bank vital in furthering research on this cancer.
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 19 References AANS www.aans.org/Patients/Neurosurgical Conditions and Treatments/Astrocytoma Tumors EMBO Molecular Medicine BlackWell Publishing Ltd, 13 Oct. 2014, www.ncbi.nlm.nih.gov/pmc/articles/PMC4237465/ PMC National Center for Biotechnology Information U.S. National Library o f Medicine, 1 Oct. 2016, www.ncbi.nlm.nih.gov/pmc/ Renewing PLOS ONE Public Library of Science, 5 Jan. 2011, journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0015844. Glioblastoma (GBM) A merican Brain Tumor Association 2014, www.abta.org/brain tumor information/types of tumors/glioblastoma.html?referrer=https%3A%2F%2Fwww.google .com%2F. Asian Pacific Journal of Cancer Prevention : APJCP West Asia Organization for Cancer Prevention, 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5563115/ National Cancer Institute National Institute of Health, www.cancer.gov/publications/dictionaries/cancer ter ms?cdrid=44277. The American Journal of Pathology American Society for Investigative Pathology, May 2007, www.ncbi.nlm.nih.gov/pmc/articles/PMC1854940/.
ESTABLISHMENT OF GLIOBLASTOMA MULTIFORME CELL LINE BANK 20 Parsons, Science (New York, N.Y.) U.S. National Library of Medicine, 26 Sept. 2008, www.ncbi.nlm.nih.gov/pmc/articles/PMC2820389/ omo Sapiens (Human)] Gene National Center for Biotechnology Information U.S. National Library of Medicine, 31 Dec. 2017, www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=DetailsSearch&Term=10419 athogenesis and Cancer Medicine John Wiley and Sons Inc., 10 June 2016, www.ncbi.nlm.nih.gov/pmc/articles/PMC4971921/ Medical News Today MediLexicon International, 30 Sept. 2017, www.medicalnewstoday.com/articles/319601.php. Like Cells: Characteristics, Microenvironment, and Frontiers in Pharmacology Frontiers Media S.A., 7 Dec. 2016, www.ncbi.nlm.nih.gov /pmc/articles/PMC5141588/ NIH R24 NS086554 01