ﻘﺤﺗ ﻪﻟﺎﻘﻣ ﻴ
ﻘ ﻲ
ﺍ ﻳ ﻝﺪﻣ ﺩﺎﺠ ﺎﻫ
ﻱ ﻣﻮﺑ ﺖﻓﺍﺮﮔﻮﻧﺯ ﻲ
ﻟﻮﻠﺳ ﻩﺩﺭ ﺯﺍ ﻩﺪﺷ ﻖﺘﺸﻣ ﻭ ﻲ
ﻠﮔ ﺩﺭﺍﺪﻧﺎﺘﺳﺍ ﻴ
ﺘﻟﺎﻣ ﺎﻣﻮﺘﺳﻼﺑﻮ ﻲ
ﺩ ﻪﻣﺭﻮﻓ ﺍ ﺭ
ﻳ ﻥﺍﺮ
ﻠﻋ ﻲ ﻮﻨﺷﻮﺧ ﺎﺿر ﻳ
1نﺎﺴ داﮋﻧﺪﻤﺤﻣ ﺪﺣا ، داﮋﻧﺪﻤﺤﻣ ﺪﻤﺻ ،2
ﺮﻫﺎﻈﻣ هﺮﻫز ،2 2ي
ﺮﻣ ، ﻳ ﻢﻇﺎﻛ ﻢ ﻘﺣ ﻴﻘ
2ﻲ خﺮﻓ ،
ﺗﻴ ﺮﮔﺮ
2ي ﻠﻋﺪﻤﺤﻣ ، ﻲ ﺑﺎﻘﻋ ﻴ
3نﺎ
، ﻌﺳ ﻴ رﻮﭙﻧﺎﻣا ﺪ
* 1
هﺪﻴﻜﭼ
فاﺪﻫا : ﻲﺘﻟﺎﻣ ﺎﻣﻮﺘﺳﻼﺑﻮﻴﻠﮔ مرﻮﻓ
ﻢﻴﺧﺪﺑ و ﻦﻳﺮﺘﻌﻳﺎﺷ نﺎـﻣرد ﻪـﺑ ﺖﺒﺴﻧ و هدﻮﺑ ﻲﺘﻴﺳوﺮﺘﺳآ رﻮﻣﻮﺗ ﻦﻳﺮﺗ
ﺞـﻳار يﺎـﻫ
ﻲﻣ نﺎﺸﻧ ﺖﻣوﺎﻘﻣ ﻲـﻣ رادرﻮـﺧﺮﺑ ﻲﻨﻴﺋﺎـﭘ ءﺎـﻘﺑ ناﺰـﻴﻣ زا رﻮـﻣﻮﺗ ﻦﻳا ﻪﺑ ءﻼﺘﺒﻣ نارﺎﻤﻴﺑ ﻦﻳاﺮﺑﺎﻨﺑ ،ﺪﻫد
ﺪﻨـﺷﺎﺑ . ﺎـﻴﻧد رد
تﺎﻌﻟﺎﻄﻣ ﺰﻴﻧ ناﺮﻳا رد و هدﻮﺑ مﺎﺠﻧا لﺎﺣ رد رﻮﻣﻮﺗ ﻦﻳا يور ﺮﺑ يدﺎﻳز تﺎﻌﻟﺎﻄﻣ in vitro
ﺮﺑ هدر يور ﻦـﻳا ﻲﻟﻮﻠﺳ
ترﻮﺼﺑ نﻮﻨﻛﺎﺗ ﺎﻣا ﺖﺳا ﻪﺘﻓﺮﮔ ترﻮﺻ رﻮﻣﻮﺗ in vivo
ﺖﺳا هﺪﺸﻧ ﻪﻌﻟﺎﻄﻣ .
شور ﺎﻫ شﻮﻣ: يﺎﻫ Athymic Nude Mice 6
- 4 دراﺪﻧﺎﺘـﺳا ﻲﻟﻮﻠـﺳ هدر ﻪﺘـﺳد ﻚـﻳ ﻪـﺑ و بﺎـﺨﺘﻧا ﻪـﺘﻔﻫ U-
زا هﺪﻳدﺮﮔ اﺪﺟ87MG شﻮﻣ ﻪﺑ ﻚﻴﭘﺎﺗوﺮﺘﻫ ﻞﻜﺷ ﻪﺑ ﻲﻧﺎﺴﻧاGBM
ﺪﻳدﺮﮔ ﻖﻳرﺰﺗ ﺎﻫ .
ﻢﻴﻘﺘـﺴﻣ رﻮﻣﻮﺗ ﺲﭙﺳ ﻪـﺳ زا
شﻮﻣ ﺪﻠﺟ ﺮﻳز رد ﻲﺣاﺮﺟ ﻖﻳﺮﻃ زا و ﺬﺧأ رﺎﻤﻴﺑ ﺪﺷ ﻪﺘﺷﺎﻛ ﺎﻫ
. شﻮـﻣ ﺪـﻌﺑ هﺎﻣ ود ﻖـﻳﺮﻃ زا ﺎـﻫرﻮﻣﻮﺗ و ﻲﻧﺎـﺑﺮﻗ ﺎـﻫ
ﮓﻧر يﺰﻴﻣآ H&E ، GFAP و Ki-67 ﺪﻨﺘﻓﺮﮔ راﺮﻗ ﻲﻜﻳژﻮﻟﻮﺗﺎﭘ ﻪﻌﻟﺎﻄﻣ درﻮﻣ .
ﺎﻫ ﻪﺘﻓﺎﻳ رد: H&E و تﺎﻴﺻﻮﺼﺧGFAP ﻲﻟﻮﻠـﺳ هدر رد نﻮﻴـﺳاﺮﻔﻴﻟوﺮﭘ ناﺰـﻴﻣ و ﺪـﻳدﺮﮔ زوﺮﺑGBM
6 % رد و
رﻮﻣﻮﺗ ﻢﻴﻘﺘﺴﻣ ﺖﻓاﺮﮔﻮﻧز 9
% ﻢﻫ رﺎﻤﻴﺑ ﻲﻜﻳژﻮﻟﻮﺗﺎﭘ ﺞﻳﺎﺘﻧ ﺎﺑ ﻪﻛ ﺪﻳدﺮﮔ مﻼﻋا ﺖﺷاد ﻲﻧاﻮﺧ
.
ﻪﺠﻴﺘﻧ يﺮﻴﮔ ﺖﻓاﺮﮔﻮﻧز لﺪﻣ: تﺎﻌﻟﺎﻄﻣ ياﺮﺑ لﺪﻣ ﻦﻳﺮﺘﺒﺳﺎﻨﻣGBM
in vivo يرﻮـﻣﻮﺗ لﺪﻣ ﻦﻳا ﺎﺑ نﺎﻘﻘﺤﻣ و هدﻮﺑ
ﻲﻣ نﺎﻣرد ﺪﻨﻧاﻮﺗ ﻦﻳا ﺎﺑ ﻦﻴﻨﭽﻤﻫ و ﺪﻨﻫﺪﺑ راﺮﻗ ﻪﻌﻟﺎﻄﻣ درﻮﻣ ار نﺎﻃﺮﺳ ﻦﻳا ﺪﻳﺪﺟ يﺎﻫ
لﺪـﻣ ﻲـﻣ ﺎـﻫ تﺎـﻓﻼﺘﺧا ناﻮـﺗ
ﺦﺳﺎﭘ رد ار ﻲﻧاﺮﻳا داﮋﻧ ﺲﻜﻴﻣﻮﻨﻴﺟﻮﻛﺎﻣرﺎﻓ دﺮﻛ ﻲﺑﺎﻳزرا نﺎﻣرد ﻪﺑ ﻲﻫد
.
تﺎﻤﻠﻛ ﺪﻴﻠﻛ :ي شﻮﻣ ،ﺖﻓاﺮﮔﻮﻧز رﻮﻣﻮﺗ ،ﺎﻣﻮﺘﺳﻼﺑﻮﻴﻠﮔ ﻲﺑ يﺎﻫ
سﻮﻤﻴﺗ ﺪﻗﺎﻓ يﻮﻣ
1 . يرﺎﻤﻴﺑ تﺎﻘﻴﻘﺤﺗ ﺰﻛﺮﻣ و ﺰﻐﻣ يﺎﻫ
ﺎﺼﻋا و ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ،ب
ناﺮﻬﺗ ﻲﻧﺎﻣرد ﻲﺘﺷاﺪﻬﺑ تﺎﻣﺪﺧ 2 . مﻮﻠﻋ هﺎﮕﺸﻧاد ،نﺎﻃﺮﺳ تﺎﻘﻴﻘﺤﺗ ﺰﻛﺮﻣ ﻲﻧﺎﻣرد ﻲﺘﺷاﺪﻬﺑ تﺎﻣﺪﺧ و ﻲﻜﺷﺰﭘ
ناﺮﻬﺗ 3 . رد يژﻮﻟﻮﻨﻜﺗ و مﻮﻠﻋ تﺎﻘﻴﻘﺤﺗ ﺰﻛﺮﻣ
ﻲﻜﺷﺰﭘ لﻮﺌﺴﻣ هﺪﻨﺴﻳﻮﻧ :
رﻮﭙﻧﺎﻣا ﺪﻴﻌﺳ ﺮﺘﻛد
مﻮﻠﻋ هﺎﮕﺸﻧاد ،نﺎﻃﺮﺳ تﺎﻘﻴﻘﺤﺗ ﺰﻛﺮﻣ ﺮﻬﺗ ﻲﻧﺎﻣرد ﻲﺘﺷاﺪﻬﺑ تﺎﻣﺪﺧ و ﻲﻜﺷﺰﭘ نا
ﻦﻔﻠﺗ : 02166940021
ﻲﻜﻴﻧوﺮﺘﻜﻟا ﺖﺴﭘ :
.
52
BCCR
Basic &Clinical Cancer Research
ORIGINAL ARTICLE
Establishment Establishment Establishment
Establishment o o of Autochthonous & o f Autochthonous & f Autochthonous & f Autochthonous &
Standard Cell Line Derived Xenograft Standard Cell Line Derived Xenograft Standard Cell Line Derived Xenograft Standard Cell Line Derived Xenograft
Models Models Models
Models o o o of Glioblastoma Multiform f Glioblastoma Multiform f Glioblastoma Multiform iiiin f Glioblastoma Multiform n n n Iran Iran Iran
Iran
A. Khoshnevisan 1, A. Muhammadnejad 2, S. Muhammadnejad 2, Z.Mazaheri 2, M. Kazem haghighi 2, F.Tirgari 2, MA. Oghabian 3
S. Amanpour 2*
A B S T R A C T
Background: GBM is the most common and malignant astrocity tumor and it is persistent to common treatment so, these patients have a very low survival.
Several researchers around the world, including Iran, have been investigated GBM-cell line in vitro. However in vivo studies have not been fulfilled.
Methods: As standard cell line (U-87MG) derived from human GBM and total GBM tumor derived from 3 patients were heterotypic ally injected into 4-6 weeks old athymic nude mice. Pathologic investigation by H&E, GFAP and Ki-67 were examined 2 months post implantation.
Results: GBM characteristics appeared in H&E and GFAP and the rate of proliferation was 6% and in direct xenograft tumor was 9% which was consistent with the pathologic result of patient.
Conclusion: GBM Xengraft is the most suitable model for in vivo investigation and researcher can evaluate new treatments for this tumor. On the other hands, Pharmacogenomics differences in treatment response could be indicated among Iranians.
Keywords : Glioblastom, Xengraft, Tumor
1- Iranian Center of Neurological Research, Tehran University of Medical Sciences
2- Cancer Research Center, Tehran University of Medical Sciences
3- Research Center Sciences Technology in Medicine, Tehran University of Medical Sciences
*Corresponding Author:
Saeid Amanpour, Cancer Research Center, Tehran University of Medical Sciences
Tel: (+9821) 66940021 E mail:
[email protected] Date Receive: 12.21.2010
Date Acceptance:01.15.2010
53
BCCR
INTRODUCTION
lioblastoma multiform (GBM) is the most prevalent and malignant primary brain tumor and its incidence increases significantly among adults above 50 [1]. Virchow described this tumor for the first time in 1963 and then, Bailey and Cushing named it “Glioblastoma and spongioblastoma”. According to World Health Organization (WHO), astrocytic tumors are classified into four categories including GBM and grade IV [2]. Due to indeterminate boundary between tumoral und healthy brain tissues, the complete removal of the glioma high grade tumor is impossible. On the other hand, the sensitivity of these tumoral cells to chemotherapeutic drugs is low. There are also many limitations regarding the application of radiotherapy, as cerebral cells have a low endurance threshold for radiation.
Therefore, the prognosis of this disease, despite significant developments in prognostic tools and using modern treatment equipments, is poor and the survival time of these patients is about one year [3].
As GBM has a high incidence in the world and does not respond properly to the treatment, several basic studies, both in vitro and in vivo, have been carried out in internationally recognized cancer research centers. In in vitro studies, cell lines derived from human GBM are cultured and proliferated in laboratory and the resultant cells are used in pharmacological research in order to investigate the effects of anti-cancer drugs. These cell lines are also marked with some drugs and then irradiated by various ray doses. Thereafter, an animal model is developed and the treatment factors used in in vitro phase of the study are evaluated in vivo. The xenograft models are accepted for the present time as the best animal models of cancer types. Nude mice without immune cells are used in basic cancer investigations and preclinical studies of new anti- cancer drugs.
Regarding the possibility of raising nude mice inside the country, it seemed necessary that first a GBM model of standard cell lines is developed
and then the autochthonous cell lines of GBM are derived. The objective of this study was to develop a Glioma High grade xenograft model using simultaneously both standard cell lines and fresh tumoral tissue graft from native patients in order to facilitate basic research on central nervous system cancers with the help of these models. In addition the study aimed to study the sensitivity of both models versus routine chemotherapeutic drugs. The latter would be helpful in determination of pharmacogenomic differences in Iranian races.
METHODS
Animals
Male athymic nude mice of the age 4-6 weeks were used in this study. The mice were kept in microisolator cages in experimental tumor implantation laboratory of Imam Hospital, which is under supervision of Iran University of Medical Sciences (IUMS). These animals were fed ad libitum with autoclaved food and water. All phases of this investigation were planned and performed according to the internationally accepted ethical principles of working with laboratory animals.
Cell line
U-87MG, which is one of the cell lines of Glioblastoma multiform (GBM) was provided by the National Cell bank of Iran (NCBI) affiliated to Pasteur Institute. It was then cultured in RPMI 1640 culture medium containing 10% FBS.
Development of tumoral xenograft model from U-87MG cell line
To develop a xenograft model, 5×106 cells from the cell line of U-87MG in 200µl of RPMI 1640 culture medium without FBS were heterotopically and subcutaneously injected into the right flank region of nude mouse.
Development of tumoral xenograft model from primary human tumor
The primary GBM tumor specimens were obtained from three patients in the Shariati
G
Hospital of Tehran University of Medical Sciences, after they had signed the consent form.
It is noteworthy that the nature of tumor was confirmed through MRI before and after surgery.
The tumor specimens in RPMI 1640 sterile culture medium were transported to the experimental tumor implantation laboratory while they were stored in ice box, and there they were cut into pieces of the dimension 1×1×0.5 mm. For each patient’s tumor, five nude mice were anesthetized, according to the anesthesia protocol, by injecting the following dosages of drugs: 100 mg/kg of Ketamine 10% (Alfasan; Netherlands) and 10 mg/kg of Xylazxine 2% (Alfasan; Netherlands).
Thereafter, a 5mm long incision was made on the skin of right and left flank region and a cut piece of the tumoral specimen was implanted subcutaneously in above-mentioned regions.
Finally, the incision site was sutured with nylon 0- 4 suture thread
Figure 1 Tumor of GBM xenograft model
Pathological studies
Two months later, euthanasia was conducted on the mice in a human manner. The resultant tumors were removed from the animal skin, fixed in 10%
buffered formalin and transported for further processing to pathology laboratory, where some slides were made of the specimens by H&E staining and also immunohistochemical staining with GFAP and Ki-67 (made of the Danish
company DAkO). A pathologist then studied the slides.
RESULTS
Histopathologic studies by applying H&E staining on U-87MG cell line and on the tumoral graft to be transferred directly to nude mouse indicated the increased cellularity with many mitochondria.
The nuclei showed also strong polymorphism.
vascular proliferation and numerous cases of necrosis were the other histopathologic characteristics observed. Necrotic zones were surrounded by anaplastic cells. In pathology, this histopathological scene is denominated
“pseudopalisading necrosis” and is the hallmark of Glioblastoma multiforme. The immunohistochemistry (IHC) results gave evidence to positive Glial fibrillary acidic protein (GFAP), which is a proper marker for astrocytic tumors. The proliferation level of tumoral cells was assessed through Ki-67 marker. This marker indicated the increase of tumoral cells in the cell line by 6% and in the direct graft of human tumoral tissue by 9%, which correlated with the proliferation level of patients’ real tumors.
55
DISCUSSION
GBM is the most prevalent astrocytic tumor and despite using standard surgical, chemotherapeutic and radiotherapeutic treatment methods, the survival time after beginning of treatment is between one and at most two years [4]. Therefore, developing animal models of this disease and conducting various treatment studies seem to be essential. GBM-contracted dogs are one of the suggested models for treatment studies [5]. As morphologic and histopathologic characteristics of GBM in dogs are in many aspects similar to human GBM, some researchers hope that they, by curing brain tumors in dogs, could find a treatment model for human GBM [6]. However, doing experimental researches on cancer- contracted dogs has some limitations due to observance of animal rights and protection rules and ethical principles of empirical experiments on domestic animals; and using this animal is only then allowable that the suggested treatment has passed all experimental studies successfully. Just before applying this new method of treatment in human clinical phase, the cancer-contracted dog must be treated with it [7].
Athymic nude mice were introduced in 1968 by Pantelouris [8]. These mice do not have the thymus cortex, so their T-lymphocytes haven’t become mature and they lack cell immunity. One year after the introduction of these mice, the first xenograft model of tumors was created [9].
Recognized associations like the Us National
Cancer Institute, have been using the xenograft models in their cancer studies and they conduct their preclinical phase of anti-cancer drugs on these models [10, 11, 12], as these models, due to use of human cancer cells, enjoy more validity comparing to other models [13].
Regarding the results of this study, the standard cell lines of GBM showed the same histopathological characteristics as human GBM and the new tumor graft to be transferred to mouse indicated the same characteristics as patient’s tumor. Researchers have suggested various treatment methods for GBM, however, to date, none of these offered methods, except for a few new ones, have reached the human clinical phase. Anyway, there is the hope that longer survival time can be achieved for GBM- contracted patients by continuing these studies. To date, limited in vitro pharmacological and radiotherapeutic studies have been conducted in Iran on the GBM cell lines [14, 15] but none of them have been assessed by in vivo tests due to the lack of animal models. Development of GBM xenograft models simultaneously from both standard cell line and human tumoral graft to be transferred directly to nude mice, is achieved in Iran for the first time and we hope that the investigators take positive steps in basic studies of central nervous system (CNS) primary tumors by using these models and can also determine the pharmacogenomic differences in Iranian races by evaluation of standard chemotherapeutic drugs used for both standard and autochthonous cell lines. We hope that these models make the evaluation of new anti-cancer drugs introduced for the treatment of this disease possible. At present Figure 2: a) the scene of pseudopalisading necrosis, in which the necrotic region in the middle is surrounded by tumoral cells. This is one of the most important diagnostic signs of GBM (H&E). b) Immunohistochemical staining using GFAP. c) Immunohistochemical staining using Ki-67.
anti-angiogenic drugs are being suggested for the treatment of this disease and Bevacizumab is being prescribed to human patients but, due to its side effects, there is disagreement over its efficiency to increase the patients’ survival time [16]. Now Iranian investigators will be able to evaluate the effectiveness of many drugs, which are expected to inhibit the angiogenesis of GBM tumors, through these models.
57
REFERENCES
1. Lopes MBS, B. H. Central Nervous System Tumors. In: Weidner N, Cote RJ, Suster S, eds WL, editors. Modern surgical pathology. 1 ed. Philadelphia:
W.B. Saunders 2003. p. 2063-9.
2. Kumar V, Fausto N, Abbas A, Aster J. The central Nervous system. In: Matthew P. Frosch, Douglas C. Anthony, Girolami UD, editors. Robbins and Cotran Pathologic Basis of Disease. New York:
W.B. Saunders 2010. p. 1464.
3. Charles B Stevenson, Thompson RC.
Neurosurgical oncology: neoplasms of the brain and meninges. In: Poston GJ, editor. Textbook of Surgical oncology. Distributed in North and South America:
Taylor & Francis; 2007. p. 393-410.
4. DeVita VT, Hellman S, Rosenberg SA.
Principles & Practice of Oncology. 8 ed. T DV, S H, Rosenberg SA, editors. Philadelphia: Lippincott Williams & Wilkins; 2008.
5. Dickinson PJ, LeCouteur RA, Higgins RJ, Bringas JR, Larson RF, Yamashita Y, et al. Canine spontaneous glioma: a translational model system for convection-enhanced delivery. Neuro Oncol. 2010 Sep;12(9):928-40.
6. Lipsitz D, Higgins RJ, Kortz GD, Dickinson PJ, Bollen AW, Naydan DK, et al. Glioblastoma multiforme: clinical findings, magnetic resonance imaging, and pathology in five dogs. Vet Pathol. 2003 Nov;40(6):659-69.
7. Conn PM. Canine and Feline Models for Cancer. In: Vico De Gionata MP, editor. Source Book of Models for Biomedical Research2008. p. 794 8. Pantelouris EM. Absence of thymus in a mouse mutant. Nature. 1968 Jan 27;217(5126):370-1.
9. Rygaard J, Povlsen CO. Heterotransplantation of a human malignant tumour to "Nude" mice. Acta Pathol Microbiol Scand. 1969;77(4):758-60.
10. Sarkaria JN, Carlson BL, Schroeder MA, Grogan P, Brown PD, Giannini C, et al. Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin Cancer Res. 2006 Apr 1;12(7 Pt 1):2264-71.
11. Zhao Y, Xiao A, diPierro CG, Carpenter JE, Abdel-Fattah R, Redpath GT, et al. An extensive invasive intracranial human glioblastoma xenograft model: role of high level matrix metalloproteinase 9.
Am J Pathol. 2010 Jun;176(6):3032-49.
12. Kerbel RS. Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived- but they can be improved. Cancer Biol Ther. 2003 Jul- Aug;2(4 Suppl 1):S134-9.
13. Aldape. K CH, James. C.D. Models of malignant glioma Drug Discovery Today: Disease Models. 2006;3(2):191-6.
ﺮﺒﻛا ﻲﻠﻋ ﻲﻗﺮﺷ ،ﺮﻴﻨﻣ يﺪﻴﻬﺷ ،ﻲﻠﻋ ﺰﻳر ﻪﺘﺳﺎﺸﻧ 14.
. شور ﻪـﺴﻳﺎﻘﻣ
ﺪـﻳ ﺎﺑ هﺪﺷ راد نﺎﺸﻧ ﻦﻳﺪﻳرﻮﻳ ﻲﺴﻛﺎﻳد يﺮﻴﮔرﺎﻛ ﻪﺑ ﺎﺑ ﺪﻨﻤﻓﺪﻫ ﻲﻧﺎﻣرد ﻮﺗﺮﭘ 125 ﻂﻳاﺮـﺷ رد ﺎﻣﻮﻴﻠﮔ نﺎﻣرد رد ،ﻲﺟرﺎﺧ ﻲﻧﺎﻣرد ﻮﺗﺮﭘ شور ﺎﺑ In vitro
ناﺮﻳا ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ﻪﻠﺠﻣ 159
. 1382 33; .
ﻲﻠﻋ ﺰﻳر ﻪﺘﺳﺎﺸﻧ ،هﺪﻴﻤﺳ ﻲﻳﻮﺧ ،ﻦﻳﺪﻟارﻮﻧ يﺮﺳ ﻪﺸﻴﺑ 15.
. ﻲـﺳرﺮﺑ
يﺎـﻬﻧ ﺎـﻣز ﻲـﻃ ناﺰﻴﻧﻮـﻳ يﺎـﻫﻮﺗﺮﭘ زا زا ﻲﺷﺎﻧ ﻲﻜﻴﺘﻧژﻮﺘﻴﺳ تﺎﻣﺪﺻ ناﺰﻴﻣ ﺎﺑ ﺎﻬﻟ ﻮﻠﺳ نﻮﻴﺳﺎﺑﻮﻜﻧا ﻒﻠﺘﺨﻣ Iudr
يﺎـﻬﻟ ﻮﻠﺳ زا ﺪﻴﻳوﺮﻔﺳا ﺖﺸﻛ لﺪﻣ رد
ﻪﺑ ﺎﻣﻮﻴﻠﮔ ﺖﻣﺎﻛ شور ناﺮﻳا ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد 177
. 1387 ﻢﻫدﺰﻧﺎﭘ هرود; )
58 ).
16. Vredenburgh JJ, Desjardins A, Herndon JE, 2nd, Marcello J, Reardon DA, Quinn JA, et al.
Bevacizumab plus irinotecan in recurrent glioblastoma multiforme. J Clin Oncol. 2007 Oct 20;25(30):4722-9.