Ignite Creation Date:
2024-05-06 @ 1:59 PM
Last Modification Date:
2024-10-26 @ 1:23 PM
Study NCT ID:
NCT04180046
Status:
UNKNOWN
Last Update Posted:
2021-04-12
First Post:
2019-06-26
Brief Title:
Utility of Primary Glioblastoma Cell Lines
Sponsor:
Neuromed IRCCS
Organization:
Neuromed IRCCS
Study Overview
Official Title:
Glioblastoma Lines as the Disease Model
Status:
UNKNOWN
Status Verified Date:
2021-04
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
In this study the characterization of human malignant glioma cell lines is described After mechanical and enzymatic digestion of glioblastoma human biopsies from Neuromed IRCCS Neurosurgery patients the investigators analyzed the established cell lines by immunohistochemistry The investigators have already characterized 10 cell lines and results revealed that not all cell lines are positive for glial fibrillary acidic protein GFAP but only one was positive the so-called COGI cell line Moreover the expression of Isocitrate Dehydrogenase 1IDH1 and alpha thalassemiamental retardation syndrome X-linked protein ATRX was investigated in all established cell lines COGI cell line was also positive for IDH1R132 mutation and for ATRX The results of characterization were summarized in table 1
Detailed Description:
Glioblastoma GBM is the most aggressive type of brain tumor arising from glial cells accounting for 52 of all parenchymal brain cancer cases and 20 of all intracranial tumors GBM has pronounced mitotic activity substantial tendency toward neoangiogenesis microvascular proliferation necrosis and high proliferative rates Because of their intrinsic infiltrative nature GBM has a highly aggressive malignant clinical course The adjuvant chemo-radiotherapy RT with temozolomide TMZ after maximal safe resection remains the standard of care
Brain cancer is a unique because of the blood-brain barrier which severely restricts the bloodstream of the brain While the blood brain barrier BBB is great for protecting the brain from danger when the brain has cancer cells the BBB can be a problem Therefore it is important to find new drug targets The mechanisms underlying radio- and chemo-resistance are poorly understood Recent studies suggest that up-regulation of the molecular target of rapamycin mTOR plays a pivotal role in determining resistance to treatment The upregulation of mTOR in GBM has been reported by multiple experimental and pathological findings Moreover the up-regulation of mTOR is also the key for cell growth and cell proliferation as demonstrated by in vivo studies In fact specific factors derived from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway The key to successful treatment of glioblastoma will be no doubt in the realization that this clinic is an entity in biologic terms more than one disease and is likely that specific targeted therapies will be effective in molecularly defined subsets In this way the molecular classification of these tumors will be defined in clinically relevant terms based on the identification of markers that define subsets and are predictive of response to promising agents Additional investigations and identifications of new biomarkers will help to better define the clinical and biologic subtypes of glioblastoma and an improved disease control In short the brain tumor has peculiar ad personam mutations This is why the investigators have decided to set up primary lines starting from the patients biopsy
Expected results of the scientific research project
In the first instance the primary outcome will be to establish cell cultures and stem cells that faithfully reproduce in vitro the physiology of the tumor maintaining the same characteristics of patients neoplasm
1 Evaluate the effect of new target drugs on the proliferation of primary and continuous human glioblastoma cell lines by setting up growth curves and methyl thiazolyl tetrazolium MTT toxicity assays
2 Screening of natural and synthetic drugs using patient-derived primary glioblastoma cell lines
3 Characterize the mechanisms and proteins involved in the apoptotic and or autophagic pathway with immunohistochemistry and western blot assays in control and treated cells
4 Validation of previously identified molecular targets in preclinical models of brain cancers The investigators are able to identify novel molecular determinants that can be targeted by pharmacological intervention to decrease or block the tumor growth
Materials and methods
Tumor Specimen Collection and Cryopreservation
Resection specimens of glioblastoma GBM tumors n 20 were received sterile and freshly from Neuromed Neurosurgery Tumor tissue samples were snap frozen in liquid nitrogen and stored in the gas phase above liquid nitrogen Additionally tumor tissue cubes 3 3 3 mm were frozen vitally For this procedure tumor pieces were cut with a sterile scalpel blade and 4 tumor pieces were transferred into a sterile cryo-tube in 15 ml freezing medium fetal calf serum containing 10 DMSO sealed in a freezing container Nalgene Rochester USA and placed immediately at -80 C Until thawing tubes were kept at -80 C for at maximum of 6 weeks or after overnight cooling transferred to nitrogen tank for longer storage periods
Patient Cohort
Clinical samples from 5 patients with WHO grade IV GBM and 3 patient with a relapsed Astrocytoma WHO grade III and one with oligodendroglioma grade III Table 1 were collected from the Neurosurgery department at Neuromed IRCCS Prior informed consent was obtained
Tissue Culture and Cell Line Establishment
With written consent from patients andor in accordance with institutional guidelines immediately after the resection collect tumor samples 200-500 mg of tumor is recommended into a tube containing cold sterile stem cell media without growth factors Transport the specimen immediately to the tissue culture hood for processing
For surgeries at a remote site cut the tumor sample into smaller fragments and place into a tube containing cold sterile stem cell media without growth factors keep on ice for transportation The tumor can be processed within 2-3 hours after the resection Tumor specimens from a pre-clinical animal model of human GBM tumor can also be collected and processed in the same way
In sterile BSL II laminar flow hood place the tumor into a 35 mm petri dish with 3 mL of Hanks balanced salt solution HBSS Wash tumor specimen 2 to 3 times by transferring them sequentially to new 35 mm dishes filled with 5 mL HBSS to remove blood and debris Aspirate excessive HBSS from the dish Immediately cut the tumor into small fragments and mince with a sterile scalpel blade into approximately 1 mm3 fragments The best yield can be achieved when tumors are minced to very small pieces Add 3 mL of enzymatic digestion mixture collagenaseDDNase 1 to the minced tissue and collect the minced tissue with 5 mL disposable pipet pipetting up and down a few times Then transfer the tumor fragments into 30 mL of pre-warmed enzymatic digestion mixtureThe final concentration of enzymes should be 1 mg collagenase D and 01 mg DNase I per milliliter of HBSS After digestion tissue single cells were washed and plated The cell lines were identified with the first letters of patients name and surname
Growth Kinetics
Cells 5105 cells were plated in 5 ml media in quintuplicate in T25 culture flasks per cell line and allowed to attach for 48 h vital cells were assessed by trypan blue staining and one flask was counted every 24 h for five consecutive days using a Neubauer chamber
O6-methylguanine-DNA-methyltransferase MGMT promoter methylation analysis
For analyzing the MGMT promoter concerning methylation the MethyLight method was applied Briefly genomic DNA gDNA was subject to bisulfite conversion using the Epitect Bisulfite Kit Qiagen Hilden Germany according to the manufacturers recommendations A primerprobe combination specific for methylated MGMT promoter sequence was used forward 5-GCGTTTCGACGTTCGTAGGT-3 reverse 5-CACTCTTCCGAAAACGAAACG-3 probe 5-6FAM-CGCAAACGATACGCACCGCGA-TMR-3 with SensiFast Probe Kit Bioline Luckenwalde Germany Cytosine-phosphate-guanosine CpG Methylase SssI treated DNA served as calibrator as it is considered as fully methylated The collagenase gene 2A1 COL2A1 was used as endogenous control forward 5-TCTAACAATTATAAACTCCAACCACCAA-3 reverse 5-GGGAAGATGGGATAGAAGGGAATAT-3 probe 5-6FAM-CCTTCATTCTAACCCAATACCTATCCCACCTCTAAA-TMR-3 The percentage of methylated reference PMR value was calculated by dividing the MGMTCOL2A1 ratio of the sample by the MGMTCOL2A1 ratio of the SssI-treated DNA and multiplying by 100 Samples with a PMR value 4 were considered as methylated All reactions were performed in triplicate
Mutation analyses
Samples underwent analyses for the following loci IDH1 R132 exon 4 IDH2 R172 exon 4 B-Raf V600 exon 15 K-Ras G12 G13 exon 2 and Q61 exon 3 and TP53 exons 5 to 8 The desired genomic regions were amplified by PCR using specific primers The polymerase chain reaction PCR was performed using MyTaqHS polymerase Bioline according to the manufacturers recommendations The PCR reaction was controlled by agarose gel electrophoresis and 15 µl of the products were purified using 3 units of FAST AP Alkaline Phosphatase Fermentas St Leon-Rot Germany and 30U of Exonuclease I Fermentas by incubation at 37C for 15 min and subsequent heat inactivation at 85C for 15 min
Success Rates
The investigator assessed attachment and outgrowth rates of 2 consecutive WHO grade IV GBM tumor samples and two relapsed Astrocytoma when prepared fresh directly after resection culture 4 After fresh preparation cells attached in 100 of the cases The four most rapidly and stable outgrowing pairs of cell cultures were subsequently characterized in detail In the following stable outgrowing cultures could be passaged 10 times are termed cell lines Cell lines derived from fresh material were marked with the initials of the patients name and surname to ensure anonymity while respecting the patients privacy
Immunohistochemistry
Representative cell line of each tumour were stained by Immunohistochemistry for Ki67proliferation index estimated as a percentage of positive cells in a field of 100 IDH1 ATRX markers of brain tumors and GFAP glial marker Ventana Tucson Ariz was performed automatically with a Nexes instrument Ventana Antibody detection was performed using a multilink streptavidin-biotin complex method and antibodies were visualized by a diaminobenzidine chromagen method Negative control samples were incubated with primary antibodies only
Results
Table 1
Cell line Vimentin GFAP Atrx IDH1 MET
COGI FE
CL - -
CG FE -
PAP
DNA FE - -
DRA FE -
ZAR 6719 FE - -
VEM
DA - -
IP FE - -
On these established cell cultures new substances including natural substances will be tested and used as adjuvant substances for traditional therapy with Temodal In this project the investigator intends to use coumaric acid
1 Evaluate the effect of coumaric acid on the proliferation of human glioblastoma cells primary and continuous lines
2 Investigate the mechanisms triggered by coumaric acid in the neoplastic cell to block its growth Analyze the expression of regulatory cell cycle proteins in human glioblastoma cells after treatment with coumaric acid at various concentrations
3 Evaluate the growth of human glioblastoma cells in an animal model naked CD1 mice inoculated with a continuous line of human glioblastoma U87MG cells and after coumaric acid treatment
Brain cancer is a unique because of the blood-brain barrier which severely restricts the bloodstream of the brain While the blood brain barrier BBB is great for protecting the brain from danger when the brain has cancer cells the BBB can be a problem Therefore it is important to find new drug targets The mechanisms underlying radio- and chemo-resistance are poorly understood Recent studies suggest that up-regulation of the molecular target of rapamycin mTOR plays a pivotal role in determining resistance to treatment The upregulation of mTOR in GBM has been reported by multiple experimental and pathological findings Moreover the up-regulation of mTOR is also the key for cell growth and cell proliferation as demonstrated by in vivo studies In fact specific factors derived from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway The key to successful treatment of glioblastoma will be no doubt in the realization that this clinic is an entity in biologic terms more than one disease and is likely that specific targeted therapies will be effective in molecularly defined subsets In this way the molecular classification of these tumors will be defined in clinically relevant terms based on the identification of markers that define subsets and are predictive of response to promising agents Additional investigations and identifications of new biomarkers will help to better define the clinical and biologic subtypes of glioblastoma and an improved disease control In short the brain tumor has peculiar ad personam mutations This is why the investigators have decided to set up primary lines starting from the patients biopsy
Expected results of the scientific research project
In the first instance the primary outcome will be to establish cell cultures and stem cells that faithfully reproduce in vitro the physiology of the tumor maintaining the same characteristics of patients neoplasm
1 Evaluate the effect of new target drugs on the proliferation of primary and continuous human glioblastoma cell lines by setting up growth curves and methyl thiazolyl tetrazolium MTT toxicity assays
2 Screening of natural and synthetic drugs using patient-derived primary glioblastoma cell lines
3 Characterize the mechanisms and proteins involved in the apoptotic and or autophagic pathway with immunohistochemistry and western blot assays in control and treated cells
4 Validation of previously identified molecular targets in preclinical models of brain cancers The investigators are able to identify novel molecular determinants that can be targeted by pharmacological intervention to decrease or block the tumor growth
Materials and methods
Tumor Specimen Collection and Cryopreservation
Resection specimens of glioblastoma GBM tumors n 20 were received sterile and freshly from Neuromed Neurosurgery Tumor tissue samples were snap frozen in liquid nitrogen and stored in the gas phase above liquid nitrogen Additionally tumor tissue cubes 3 3 3 mm were frozen vitally For this procedure tumor pieces were cut with a sterile scalpel blade and 4 tumor pieces were transferred into a sterile cryo-tube in 15 ml freezing medium fetal calf serum containing 10 DMSO sealed in a freezing container Nalgene Rochester USA and placed immediately at -80 C Until thawing tubes were kept at -80 C for at maximum of 6 weeks or after overnight cooling transferred to nitrogen tank for longer storage periods
Patient Cohort
Clinical samples from 5 patients with WHO grade IV GBM and 3 patient with a relapsed Astrocytoma WHO grade III and one with oligodendroglioma grade III Table 1 were collected from the Neurosurgery department at Neuromed IRCCS Prior informed consent was obtained
Tissue Culture and Cell Line Establishment
With written consent from patients andor in accordance with institutional guidelines immediately after the resection collect tumor samples 200-500 mg of tumor is recommended into a tube containing cold sterile stem cell media without growth factors Transport the specimen immediately to the tissue culture hood for processing
For surgeries at a remote site cut the tumor sample into smaller fragments and place into a tube containing cold sterile stem cell media without growth factors keep on ice for transportation The tumor can be processed within 2-3 hours after the resection Tumor specimens from a pre-clinical animal model of human GBM tumor can also be collected and processed in the same way
In sterile BSL II laminar flow hood place the tumor into a 35 mm petri dish with 3 mL of Hanks balanced salt solution HBSS Wash tumor specimen 2 to 3 times by transferring them sequentially to new 35 mm dishes filled with 5 mL HBSS to remove blood and debris Aspirate excessive HBSS from the dish Immediately cut the tumor into small fragments and mince with a sterile scalpel blade into approximately 1 mm3 fragments The best yield can be achieved when tumors are minced to very small pieces Add 3 mL of enzymatic digestion mixture collagenaseDDNase 1 to the minced tissue and collect the minced tissue with 5 mL disposable pipet pipetting up and down a few times Then transfer the tumor fragments into 30 mL of pre-warmed enzymatic digestion mixtureThe final concentration of enzymes should be 1 mg collagenase D and 01 mg DNase I per milliliter of HBSS After digestion tissue single cells were washed and plated The cell lines were identified with the first letters of patients name and surname
Growth Kinetics
Cells 5105 cells were plated in 5 ml media in quintuplicate in T25 culture flasks per cell line and allowed to attach for 48 h vital cells were assessed by trypan blue staining and one flask was counted every 24 h for five consecutive days using a Neubauer chamber
O6-methylguanine-DNA-methyltransferase MGMT promoter methylation analysis
For analyzing the MGMT promoter concerning methylation the MethyLight method was applied Briefly genomic DNA gDNA was subject to bisulfite conversion using the Epitect Bisulfite Kit Qiagen Hilden Germany according to the manufacturers recommendations A primerprobe combination specific for methylated MGMT promoter sequence was used forward 5-GCGTTTCGACGTTCGTAGGT-3 reverse 5-CACTCTTCCGAAAACGAAACG-3 probe 5-6FAM-CGCAAACGATACGCACCGCGA-TMR-3 with SensiFast Probe Kit Bioline Luckenwalde Germany Cytosine-phosphate-guanosine CpG Methylase SssI treated DNA served as calibrator as it is considered as fully methylated The collagenase gene 2A1 COL2A1 was used as endogenous control forward 5-TCTAACAATTATAAACTCCAACCACCAA-3 reverse 5-GGGAAGATGGGATAGAAGGGAATAT-3 probe 5-6FAM-CCTTCATTCTAACCCAATACCTATCCCACCTCTAAA-TMR-3 The percentage of methylated reference PMR value was calculated by dividing the MGMTCOL2A1 ratio of the sample by the MGMTCOL2A1 ratio of the SssI-treated DNA and multiplying by 100 Samples with a PMR value 4 were considered as methylated All reactions were performed in triplicate
Mutation analyses
Samples underwent analyses for the following loci IDH1 R132 exon 4 IDH2 R172 exon 4 B-Raf V600 exon 15 K-Ras G12 G13 exon 2 and Q61 exon 3 and TP53 exons 5 to 8 The desired genomic regions were amplified by PCR using specific primers The polymerase chain reaction PCR was performed using MyTaqHS polymerase Bioline according to the manufacturers recommendations The PCR reaction was controlled by agarose gel electrophoresis and 15 µl of the products were purified using 3 units of FAST AP Alkaline Phosphatase Fermentas St Leon-Rot Germany and 30U of Exonuclease I Fermentas by incubation at 37C for 15 min and subsequent heat inactivation at 85C for 15 min
Success Rates
The investigator assessed attachment and outgrowth rates of 2 consecutive WHO grade IV GBM tumor samples and two relapsed Astrocytoma when prepared fresh directly after resection culture 4 After fresh preparation cells attached in 100 of the cases The four most rapidly and stable outgrowing pairs of cell cultures were subsequently characterized in detail In the following stable outgrowing cultures could be passaged 10 times are termed cell lines Cell lines derived from fresh material were marked with the initials of the patients name and surname to ensure anonymity while respecting the patients privacy
Immunohistochemistry
Representative cell line of each tumour were stained by Immunohistochemistry for Ki67proliferation index estimated as a percentage of positive cells in a field of 100 IDH1 ATRX markers of brain tumors and GFAP glial marker Ventana Tucson Ariz was performed automatically with a Nexes instrument Ventana Antibody detection was performed using a multilink streptavidin-biotin complex method and antibodies were visualized by a diaminobenzidine chromagen method Negative control samples were incubated with primary antibodies only
Results
Table 1
Cell line Vimentin GFAP Atrx IDH1 MET
COGI FE
CL - -
CG FE -
PAP
DNA FE - -
DRA FE -
ZAR 6719 FE - -
VEM
DA - -
IP FE - -
On these established cell cultures new substances including natural substances will be tested and used as adjuvant substances for traditional therapy with Temodal In this project the investigator intends to use coumaric acid
1 Evaluate the effect of coumaric acid on the proliferation of human glioblastoma cells primary and continuous lines
2 Investigate the mechanisms triggered by coumaric acid in the neoplastic cell to block its growth Analyze the expression of regulatory cell cycle proteins in human glioblastoma cells after treatment with coumaric acid at various concentrations
3 Evaluate the growth of human glioblastoma cells in an animal model naked CD1 mice inoculated with a continuous line of human glioblastoma U87MG cells and after coumaric acid treatment
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None