the media was prepared using 10% fbs ( 16140 - 071- gibco ) in dmem / f-12 ( 1x ) , liquid 1:1 ( 10565 - 042- invitrogen ) with 0.6 % agar solution purified , granulated ( 1.01614.1000- emd ) .
for the preparation of media 5ml of fbs ( 16140 - 071- gibco ) was added to 45ml of dmem / f-12 ( 1x ) , liquid 1:1 ( 10565 - 042- invitrogen ) making final volume of 50ml .
the agar solution was cool down to 37c temperature by keeping it into the water bath .
we used equal volume of media from step 1.2 and 1.3 and prepared stock solution .
the ratio of 0.6 % agar ( 0.5ml ) and 10% fbs - d - mem / f-12 media ( 0.5ml ) was 1:1 , making the total volume of 1 ml in each well of 6 well plates .
glioblastoma multiforme ( gbm ) tissues were received immediately after the surgery from department of pathology .
the tissue was transferred to patri - plate using forceps carefully and washed with ice - cold 5ml 1xpbs for 3 times .
serial sections of the specimens were cut from the dissected samples to create tumor blocks ( approximate 10 mm in diameter ) with a surgical blade ( feather-2976#10 ) and forcep ( fisher scientific ) .
tumor blocks were then injected with either dmso ( 5% ) or tmz ( 2.5 nm ) and incubated for 16 hours at 37 c in humidified air containing 5% co2 .
( 5% ) or tmz was injected 3 times in tumor blocks at different places for consistent treatment .
the drug was injected using insulin syringe 1ml 0.37x12.7 mm 28g1/2 ( comfort point-26027 ) .
after washing the blocks were fixed with 10ml of 10 % v / v formalin for 24 hours ( ricca chemical company ) in 50 ml tubes ( basix-5539802 ) and processed for paraffin embedded 4m sections .
the formalin fixed section was placed in beaker on the stir plate with " no heat " and processed through the following solutions for 30 minutes in each solution .
30% ethanol , 50% ethanol , 75% ethanol , 80% ethanol , 95% ethanol , 100% ethanol , and xylene .
the tissue was blotted on paper towels and placed in melted paraffin ( 58 to 60 c , fisher paraplast paraffin ) for 30 minutes .
the tissue was embedded in molds using surgipath blue ribbon paraffin , embedded tissue was cooled to room temperature .
xylene for 3x5 minutes ( 3 times for 5 minutes ) , 100% ethanol for 3x5 minutes , 95% ethanol for 1x5 minutes , 70% ethanol for 1x5 minutes , rinse in running tap water for 3 minutes .  
antigen retrieval with heat induced epitope retrieval immerse slides in 1x citrate buffer ( thermo scientific - ap9003 - 500 ) diluted in distilled water in a glass beaker .
boil for 15 minutes on a hot plate ( make sure sections don"t fall off the slide ) . cool the solution for 30 minutes at room temperature . rinse the slides with 1x pbs for 3x5 minutes .  
inhibition of internal peroxide immerse the slides in methanol ( fisher scientific - a-452 - 4 ) with 0.3% hydrogen peroxide ( fisher scientific- bp2633 - 500- diluted in distilled water ) in a glass beaker for 15 minutes .
rinse in 1x pbs for 3x5 minutes .   blocking cover the tissues with 10% normal goat serum .
transfer the slides in a humid box and incubate for 1 hour at room temperature .
primary antibody sections were incubated overnight at 4c with primary antibody at following concentrations : human specific caspase-3 ( 1:1,000 , r&d systems , af835 ) , ki67 ( 1:1 , dako , 15626 ) diluted with 1x pbs .
secondary antibody reaction cover the tissues with 2 - 3 drops of hrp labeled secondary antibody ( envision systems ) .
detection develop for the chromogen dab kit ( vector laboratories - sk-4100 ) for detection of primary antibody following the manufacturer 's protocol .  
counter staining immerse the slides with hematoxylin ( 10 - 15 seconds , make sure don"t overrun dab signal ) .
dehydration immerse the slides in following order , 70% ethanol for 5 minutes , 95% ethanol for 5 minutes , 100 % ethanol for 3x5 minutes , xylene for 3x5 minutes .  
cover slip the slides with permount mounting reagent ( fisher scientific - sp-15 - 100 ) .  
images were taken using olympus fluorescence microscope ( dp-72).3.11 ) . in all experiments , specific labeling
the patients underwent the first surgery without prior chemotherapies including temozolomide ( tmz ) . the t1-weighted image of mri with gadolinium enhancement in figure-1a demonstrated an enhanced tumor in the right frontal lobe before surgery .
the surgical tissues were sent to the department of pathology for the clinical diagnosis purpose , and the remaining specimens were processed to the tissue procurement under the approved institutional review board ( irb ) protocol at the ohio state university medical center .
hematoxylin and eosin ( h&e ) staining demonstrated the presence of necrosis , pseudopallisading cells , and microvascular proliferation ( figure 1-c ) .
of note , tumor explants following incubation without tmz up to 48 hours maintained the cytoarchitecture of gbm .in contrast , with incubation for 72 hours or longer , we noticed the increased number of condensed nuclei in the specimens , suggesting some of the tumor cells starting to undergo apoptosis .
as a result , tumor tissues following longer incubation contained patchy regions that lost tumor cells , which were preferentially observed at and around necrotic areas ( data not shown ) .   in order to investigate if these tumor explant samples can reliably be utilized for the purpose of drug efficacy test , we tested the effect of tmz treatment
a recent study indicated that intratumoral injection of tmz has more potent effect on growth control of malignant glioma than that of the systemic administration of this drug .
following incubation for 16 hours , these explants were embedded with paraffin and serial sections were prepared for staining .
immunohistochemistry of tmz - treated gbm tissues demonstrated a substantial reduction of ki-67-positive tumor cells in comparison with the control samples .
in turn , immunohistochemistry with an apoptosis marker , caspase-3 , did not yield any significant difference between tmz - treated glioma specimens and the control samples ( figure-3 ) .
treatment effect with tmz was further characterized by combining this explant assay together with either in vitro culture or flow cytometry .
specifically , we sought to determine the effect on stem cell - like tumor cells ( scltc ) .
therefore , following intratumoral treatment with tmz , we performed sphere forming assay and flow cytometry of these tumor explants .
the tmz - treated specimens were dissociated into individual single cells and these single cells were seeded in a low density ( 1 cell per microliter or lower ) in 96-well plates in serum - free medium supplemented with bfgf and egf . in the control group ,
given that sphere formation is a property of scltc , alterations in the number of tumor spheres by a drug treatment indicates the effect on scltc .
similarly , flow cytometry of the dissociated tumor explants with a cell surface marker , cd133 , was carried out to verify the effect on scltc .
if scltc in heterogeneous tumor cells in gbm are selectively eradicated by a drug treatment , one might predict that flow cytometry should detect decrease of the cd133-positive fraction by treatment ( data not shown ) .
figure-2b shows dissection of tumor block into 10 mm small pieces with the help surgical blade .
immunohistochemistry of gbm tissue blocks injected with dmso or tmz with activated caspase-3 and ki67 .
the media was prepared using 10% fbs ( 16140 - 071- gibco ) in dmem / f-12 ( 1x ) , liquid 1:1 ( 10565 - 042- invitrogen ) with 0.6 % agar solution purified , granulated ( 1.01614.1000- emd ) .
for the preparation of media 5ml of fbs ( 16140 - 071- gibco ) was added to 45ml of dmem / f-12 ( 1x ) , liquid 1:1 ( 10565 - 042- invitrogen ) making final volume of 50ml .
the agar solution was cool down to 37c temperature by keeping it into the water bath .
we used equal volume of media from step 1.2 and 1.3 and prepared stock solution .
the ratio of 0.6 % agar ( 0.5ml ) and 10% fbs - d - mem / f-12 media ( 0.5ml ) was 1:1 , making the total volume of 1 ml in each well of 6 well plates .
glioblastoma multiforme ( gbm ) tissues were received immediately after the surgery from department of pathology .
the tissue was transferred to patri - plate using forceps carefully and washed with ice - cold 5ml 1xpbs for 3 times .
serial sections of the specimens were cut from the dissected samples to create tumor blocks ( approximate 10 mm in diameter ) with a surgical blade ( feather-2976#10 ) and forcep ( fisher scientific ) .
tumor blocks were then injected with either dmso ( 5% ) or tmz ( 2.5 nm ) and incubated for 16 hours at 37 c in humidified air containing 5% co2 .
0.1ml of dmso ( 5% ) or tmz was injected 3 times in tumor blocks at different places for consistent treatment .
the drug was injected using insulin syringe 1ml 0.37x12.7 mm 28g1/2 ( comfort point-26027 ) .
after washing the blocks were fixed with 10ml of 10 % v / v formalin for 24 hours ( ricca chemical company ) in 50 ml tubes ( basix-5539802 ) and processed for paraffin embedded 4m sections .
the formalin fixed section was placed in beaker on the stir plate with " no heat " and processed through the following solutions for 30 minutes in each solution .
30% ethanol , 50% ethanol , 75% ethanol , 80% ethanol , 95% ethanol , 100% ethanol , and xylene .
the tissue was blotted on paper towels and placed in melted paraffin ( 58 to 60 c , fisher paraplast paraffin ) for 30 minutes .
the tissue was embedded in molds using surgipath blue ribbon paraffin , embedded tissue was cooled to room temperature .
xylene for 3x5 minutes ( 3 times for 5 minutes ) , 100% ethanol for 3x5 minutes , 95% ethanol for 1x5 minutes , 70% ethanol for 1x5 minutes , rinse in running tap water for 3 minutes .  
antigen retrieval with heat induced epitope retrieval immerse slides in 1x citrate buffer ( thermo scientific - ap9003 - 500 ) diluted in distilled water in a glass beaker .
boil for 15 minutes on a hot plate ( make sure sections don"t fall off the slide ) . cool the solution for 30 minutes at room temperature . rinse the slides with 1x pbs for 3x5 minutes .  
inhibition of internal peroxide immerse the slides in methanol ( fisher scientific - a-452 - 4 ) with 0.3% hydrogen peroxide ( fisher scientific- bp2633 - 500- diluted in distilled water ) in a glass beaker for 15 minutes .
rinse in 1x pbs for 3x5 minutes .   blocking cover the tissues with 10% normal goat serum .
transfer the slides in a humid box and incubate for 1 hour at room temperature .
primary antibody sections were incubated overnight at 4c with primary antibody at following concentrations : human specific caspase-3 ( 1:1,000 , r&d systems , af835 ) , ki67 ( 1:1 , dako , 15626 ) diluted with 1x pbs .
secondary antibody reaction cover the tissues with 2 - 3 drops of hrp labeled secondary antibody ( envision systems )
. put slides in a humid box and incubate for 1 hour at room temperature .
detection develop for the chromogen dab kit ( vector laboratories - sk-4100 ) for detection of primary antibody following the manufacturer 's protocol .  
counter staining immerse the slides with hematoxylin ( 10 - 15 seconds , make sure don"t overrun dab signal )
dehydration immerse the slides in following order , 70% ethanol for 5 minutes , 95% ethanol for 5 minutes , 100 % ethanol for 3x5 minutes , xylene for 3x5 minutes .  
cover slip the slides with permount mounting reagent ( fisher scientific - sp-15 - 100 ) .  
images were taken using olympus fluorescence microscope ( dp-72).3.11 ) . in all experiments , specific labeling
the patients underwent the first surgery without prior chemotherapies including temozolomide ( tmz ) . the t1-weighted image of mri with gadolinium enhancement in figure-1a demonstrated an enhanced tumor in the right frontal lobe before surgery .
the surgical tissues were sent to the department of pathology for the clinical diagnosis purpose , and the remaining specimens were processed to the tissue procurement under the approved institutional review board ( irb ) protocol at the ohio state university medical center .
hematoxylin and eosin ( h&e ) staining demonstrated the presence of necrosis , pseudopallisading cells , and microvascular proliferation ( figure 1-c ) .
thus , these tumors were histopathologically diagnosed as gbm . of note , tumor explants following incubation without tmz up to 48 hours maintained the cytoarchitecture of gbm .in contrast , with incubation for 72 hours or longer , we noticed the increased number of condensed nuclei in the specimens , suggesting some of the tumor cells starting to undergo apoptosis .
as a result , tumor tissues following longer incubation contained patchy regions that lost tumor cells , which were preferentially observed at and around necrotic areas ( data not shown ) .   in order to investigate if these tumor explant samples can reliably be utilized for the purpose of drug efficacy test , we tested the effect of tmz treatment
a recent study indicated that intratumoral injection of tmz has more potent effect on growth control of malignant glioma than that of the systemic administration of this drug .
following incubation for 16 hours , these explants were embedded with paraffin and serial sections were prepared for staining .
immunohistochemistry of tmz - treated gbm tissues demonstrated a substantial reduction of ki-67-positive tumor cells in comparison with the control samples . in turn , immunohistochemistry with an apoptosis marker , caspase-3 , did not yield any significant difference between tmz - treated glioma specimens and the control samples ( figure-3 ) .
treatment effect with tmz was further characterized by combining this explant assay together with either in vitro culture or flow cytometry .
specifically , we sought to determine the effect on stem cell - like tumor cells ( scltc ) . therefore , following intratumoral treatment with tmz , we performed sphere forming assay and flow cytometry of these tumor explants .
the tmz - treated specimens were dissociated into individual single cells and these single cells were seeded in a low density ( 1 cell per microliter or lower ) in 96-well plates in serum - free medium supplemented with bfgf and egf . in the control group ,
given that sphere formation is a property of scltc , alterations in the number of tumor spheres by a drug treatment indicates the effect on scltc .
similarly , flow cytometry of the dissociated tumor explants with a cell surface marker , cd133 , was carried out to verify the effect on scltc .
if scltc in heterogeneous tumor cells in gbm are selectively eradicated by a drug treatment , one might predict that flow cytometry should detect decrease of the cd133-positive fraction by treatment ( data not shown ) .
figure-2b shows dissection of tumor block into 10 mm small pieces with the help surgical blade .
immunohistochemistry of gbm tissue blocks injected with dmso or tmz with activated caspase-3 and ki67 .
  there is a gap between the drug efficacy in the current pre - clinical experimental models and the efficacy in patients .
more specifically , in the brain tumor research field , novel and reliable methods are required that would help filling the existing gap between the drug evaluation with the current methods and the efficacy in patients . the described assay in this study is an additional asset , if not a solution , to facilitate filling the discrepancy of the experimental data and outcome of affected patients .   in vitro cell cultures
preferentially expand certain tumor cell types regardless of the culture conditions , and represent only a subpopulation of the entire tumor .  
in addition , genetic and phenotypic transformation of the artificial cell expansion occurs and is inevitable with the long - term cell cultures .
one of the major hurdles to treat malignant glioma is the heterogeneity of tumor cells , both within a single tumor and between tumor samples .
therefore , selective enrichment of a certain population of tumor cells , regardless of one type or another , may not be an appropriate means to determine efficacy of any chemotherapeutic agents on heterogeneous tumor cells .  
any animal model of brain tumors derived from a subpopulation of tumor cells shed lights on drug efficacy on a subpopulation , but not the entire tumor .   
several limitations , on the other hand , still remain in this tumor explant method .
malignant tumor cells are considered to acquire resistance to most , if not all , therapies over time , initial control of the short - term tumor cell growth may not be reflected by the long - term patient prognosis , as was recently reported with an anti - angiogenic agent , bevacizumab .
thus , improvement of the protocol for this explant assay to preserve tissues for a longer period is required with further investigations .
another question is a specific effect of a tested drug on scltc in the tumor . given that scltc are relatively resistant to the current therapies for malignant glioma , identification of novel anti - cancer drugs that potently eradicate scltc
we currently seek to combine this explant assay with the subsequent in vitro experiments , such as neurosphere forming assay ( data not shown ) .
we expect to learn more about the effects , if any , of a drug candidate on scltc through these combined assays .
lastly , using small blocks of tumor samples may not reflect the entire tumor cell populations , as is the case with the conventional tumor cell lines or primary cultures from surgical specimens .
these issues aside , preserving tumor stroma , including the vascular niche , is helpful in characterizing the environmental factors for tumor cells .
therefore , this assay may have potential to evaluate any therapeutic strategies under a more physiologically relevant condition .    here , we established an assay for drug efficacy testing with explants of surgical specimens of gbm .
in fact , with the tested surgical specimens , we found that a direct injection of tmz reduces proliferation in the gbm samples .
this tissue explant method is theoretically applicable to other solid cancers and provides asset to determine drug efficacy on a patient 's tumor , which will hopefully help us avoiding another failure in clinical trials for cancers .
   the current therapies for malignant glioma have only palliative effect . for therapeutic development , 
 one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients . 
 thus , novel and reliable methods for evaluating drug efficacy are warranted in pre - clinical phase . in vitro culture of tumor tissues , 
 including cell lines , has substantial phenotypic , genetic , and epigenetic alterations of cancer cells caused by artificial environment of cell culture , which may not reflect the biology of original tumors in situ . 
 xenograft models with the immunodeficient mice also have limitations , i.e. , the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment . here 
 , we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects . to validate this method , 
 data with the current first - line chemotherapeutic agent , temozolomide ( tmz ) , are described .   
 we used the freshly - removed surgical specimen of malignant glioma for our experiments . 
 we performed intratumoral injection of tmz or other drug candidates , followed by incubation and analysis on surgical specimens . here 
 , we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti - cancer therapies so that we may be able to overcome , at least , some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient 's tumor . 
 this method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment . 
Protocol
1. Preparation of media
2. Tissue processing
3. Immunohistochemistry 
4. Representative Results:
Discussion
Disclosures