Name: method for novel anti-cancer drug development using tumor explants of surgical specimens
Uploaded: 2011-07-29T12:00:00
Duration: 9 min 26 s
Description: Watch this Scientific Journal Video about Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens at JoVE.com

The American Cancer Society (MRSG-08-108-01), Vincent J. Sgro/The American Brain Tumor Association, and the Khan Family Foundation for I Nakano.

1. Preparation of media
<ol>
<li>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). Sterile conditions were maintained throughout the experiment.</li>
<li>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 prepared media was kept in water bath at 37&deg;C temperature.</li>
<li>The agar solution was prepared using heat microwave method. 0.3 grams granulated agar was dissolved in 50mL distilled water using microwave. The agar solution was cool down to 37&deg;C temperature by keeping it into the water bath.</li>
<li>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.</li>
</ol>
2. Tissue processing
<ol>
<li>Glioblastoma Multiforme (GBM) tissues were received immediately after the surgery from Department of Pathology. The tissue was classified as GBM from Department of Pathology.</li>
<li>The tissue was transferred to patri-plate using forceps carefully and washed with ice-cold 5mL 1XPBS for 3 times.</li>
<li>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).</li>
<li>Blocks were transferred into a 6 well plate. Each well was containing 1mL of media mention in step 1.4. The tissue had enough exposure to air to keep tissue viable. </li>
<li>Tumor blocks were then injected with either DMSO (5%) or TMZ (2.5 nM) and incubated for 16 hours at 37 &deg;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 0.37X12.7mm 28G1/2 (Comfort point-26027).</li>
<li>After 16 hours blocks were washed with 5 mL 1XPBS for 3 times. 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 4&mu;m sections.</li>
<li>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.</li>
<li>The tissue was blotted on paper towels and placed in melted paraffin (58 to 60 &deg;C, Fisher Paraplast paraffin) for 30 minutes.</li>
<li>The tissue was embedded in molds using Surgipath Blue Ribbon paraffin, embedded tissue was cooled to room temperature.</li>
<li>Paraffin embedded 4&mu;m tissue sections were taken and placed on Fisher Plus slides.</li>
</ol>
3. Immunohistochemistry 
Immunohistochemistry was performed as described previously. 1 
<ol>
<li>Deparaffinization and rehydration
Place the slides in a rack and perform following steps. 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.
</li>
<li>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.
</li>
<li>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.
</li>
<li>Blocking
Cover the tissues with 10% normal goat serum. Transfer the slides in a humid box and incubate for 1 hour at room temperature. Wash the slides with 1X PBS for 3x5 minutes.
</li>
<li>Primary antibody
Sections were incubated overnight at 4&deg;C with primary antibody at following concentrations: human specific Caspase-3 (1:1,000, R&amp;D systems, AF835), Ki67 (1:1, Dako, 15626) diluted with 1X PBS. Rinse the slides in 1X PBS 3x10 minutes on the following day.
</li>
<li>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. Wash with 1X PBS for 3x10 minutes.
</li>
<li>Detection
Develop for the chromogen DAB kit (Vector laboratories-SK-4100) for detection of primary antibody following the manufacturer's protocol.
</li>
<li>Counter staining
Immerse the slides with hematoxylin (10-15 seconds, make sure don"t overrun DAB signal). Rinse with running tap water for 3 minutes.
</li>
<li>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.
</li>
<li>Cover slip the slides with permount mounting reagent (Fisher Scientific-SP-15-100). </li>
<li>Images were taken using Olympus fluorescence microscope (DP-72).3.11). In all experiments, specific labeling was confirmed by the negative control without primary antibody.</li>
</ol>
4. Representative Results:
We collected surgical specimens of glioblastoma multiforme (GBM). 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 postoperative image (Figure-1b) confirmed subtotal removal of the lesion after 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&amp;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. Figure-2 represents the flow of the experiments in this study. 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 2. 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, formation of tumor spheres from the tumor explants was observed after 7 day incubation. Given that sphere formation is a property of SCLTC3 4, 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).
<img src="/files/ftp_upload/2846/2846fig1.jpg" alt="Figure 1"/> Figure.1 Magnetic resonance images (MRI) of a patient GBM. Representative figures of pre-operation Figure-1a and post-operation Figure-1b. Figure-1c is H&amp;E staining of fresh GBM tissue sample. Original magnification, 40X.
<img src="/files/ftp_upload/2846/2846fig2.jpg" alt="Figure 2"/> Figure. 2 The experimental flow of block culture experiment. Figure-2a. Image of fresh surgical GBM received from department of pathology. Figure-2b shows Dissection of tumor block into 10mm small pieces with the help surgical blade. Figure-2c is the drug (TMZ) treatment of tumor blocks using insulin syringe.
<img src="/files/ftp_upload/2846/2846fig3.jpg" alt="Figure 3"/> Figure. 3 Immunohistochemistry. Immunohistochemistry of GBM tissue blocks injected with DMSO or TMZ with activated Caspase-3 and Ki67. Hematoxylin was used for nuclear staining. Original magnification, 40X.

<ol>
	<li>Hemmati, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancerous+stem+cells+can+arise+from+pediatric+brain+tumors.">Cancerous stem cells can arise from pediatric brain tumors.</a> Proc Natl Acad Sci U S A. 100, 15178-15183 (2003).</li><li>Heimberger, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temozolomide+delivered+by+intracerebral+microinfusion+is+safe+and+efficacious+against+malignant+gliomas+in+rats.">Temozolomide delivered by intracerebral microinfusion is safe and efficacious against malignant gliomas in rats.</a> Clin Cancer Res. 6, 4148-4153 (2000).</li><li>Nakano, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+embryonic+leucine+zipper+kinase+is+a+key+regulator+of+the+proliferation+of+malignant+brain+tumors,+including+brain+tumor+stem+cells.">Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors, including brain tumor stem cells.</a> J Neurosci Res. 86, 48-60 (2008).</li><li>Laks, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+formation+is+an+independent+predictor+of+clinical+outcome+in+malignant+glioma.">Neurosphere formation is an independent predictor of clinical outcome in malignant glioma.</a> Stem Cells. 27, 980-987 (2009).</li><li>Phillips, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+subclasses+of+high-grade+glioma+predict+prognosis,+delineate+a+pattern+of+disease+progression,+and+resemble+stages+in+neurogenesis.">Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis.</a> Cancer Cell. 9, 157-173 (2006).</li><li>He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoproteomic+Analysis+of+Glioblastoma+Stem+Cell+Differentiation.">Glycoproteomic Analysis of Glioblastoma Stem Cell Differentiation.</a> J Proteome Res. , (2010).</li><li>Ma, Y. H., Xu, Q. S., Zheng, J. S., Zhou, Y. Q., Zhan, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+stem+cell+markers+and+proliferative+labeling+on+glioma+cell+lines].">Expression of stem cell markers and proliferative labeling on glioma cell lines].</a> Zhonghua Bing Li Xue Za Zhi. 37, 333-334 (2008).</li><li>Vik-Mo, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+stem+cells+maintain+overall+phenotype+and+tumorigenicity+after+in+vitro+culturing+in+serum-free+conditions.">Brain tumor stem cells maintain overall phenotype and tumorigenicity after in vitro culturing in serum-free conditions.</a> Neuro Oncol. 12, 1220-1230 (2010).</li><li>Hovinga, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+notch+signaling+in+glioblastoma+targets+cancer+stem+cells+via+an+endothelial+cell+intermediate.+Stem+Cells.">Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate. Stem Cells.</a> 28, 1019-1029 (2010).</li><li>de Groot, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+invasion+after+treatment+of+glioblastoma+with+bevacizumab:+radiographic+and+pathologic+correlation+in+humans+and+mice.">Tumor invasion after treatment of glioblastoma with bevacizumab: radiographic and pathologic correlation in humans and mice.</a> Neuro Oncol. 12, 233-242 (2010).</li></ol>

No conflicts of interest declared.

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. 5 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 6, 7. 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. 8 9 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. One such limitation is the relatively short period of treatment. Since 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 10. 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 is warranted. In this regard, 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.

<table border="1">
	<tr>
		<td>
			Name of the reagent
		</td>
		<td>
			Company
		</td>
		<td>
			Catalogue number
		</td>
	</tr>
	<tr>
		<td>
			Fetal Bovine Serum, Qualified, Heat-Inactivated
		</td>
		<td>
			GIBCO
		</td>
		<td>
			16140-071
		</td>
	</tr>
	<tr>
		<td>
			D-MEM/F-12 (1X) Liquid 1:1 
		</td>
		<td>
			Invitrogen
		</td>
		<td>
			10565-042
		</td>
	</tr>
	<tr>
		<td>
			Agar-Agar Purified, Granulated 
		</td>
		<td>
			EMD
		</td>
		<td>
			1.01614.1000
		</td>
	</tr>
	<tr>
		<td>
			DPBS for staining 
		</td>
		<td>
			Invitrogen
		</td>
		<td>
			14190
		</td>
	</tr>
	<tr>
		<td>
			Hematoxyllin
		</td>
		<td>
			Richalrd
			allan sci
		</td>
		<td>
			7211
		</td>
	</tr>
	<tr>
		<td>
			10% w/v formalin Caspase-3 
		</td>
		<td>
			Ricca chemical company
		</td>
		<td>
			3810
		</td>
	</tr>
	<tr>
		<td>
			Caspase-3
		</td>
		<td>
			R&amp;D
			systems
		</td>
		<td>
			AF835
		</td>
	</tr>
	<tr>
		<td>
			Ki67
		</td>
		<td>
			Dako
		</td>
		<td>
			15626
		</td>
	</tr>
	<tr>
		<td>
			DMSO
		</td>
		<td>
			Sigma
		</td>
		<td>
			D2650
		</td>
	</tr>
	<tr>
		<td>
			Envision system- anti-mouse
		</td>
		<td>
			Dako
		</td>
		<td>
			2012-07
		</td>
	</tr>
	<tr>
		<td>
			Envision system- anti-rabbit
		</td>
		<td>
			Dako
		</td>
		<td>
			2011-12
		</td>
	</tr>
	<tr>
		<td>
			DAB-kit
		</td>
		<td>
			Vector
			Lab.
		</td>
		<td>
			SK-4100
		</td>
	</tr>
</table>
Table 1. Materials.
<table border="1">
	<tr>
		<td>
			Name of the equipment
		</td>
		<td>
			Company
		</td>
		<td>
			Catalogue number
		</td>
	</tr>
	<tr>
		<td>
			6 Well Plate
		</td>
		<td>
			Greiner-bio one
		</td>
		<td>
			657160
		</td>
	</tr>
	<tr>
		<td>
			Petri dish
		</td>
		<td>
			VWR
		</td>
		<td>
			25384-302
		</td>
	</tr>
	<tr>
		<td>
			Serological pipette
		</td>
		<td>
			Axygen
		</td>
		<td>
			&nbsp;
		</td>
	</tr>
	<tr>
		<td>
			Filter tips
		</td>
		<td>
			USA scientific
		</td>
		<td>
			&nbsp;
		</td>
	</tr>
	<tr>
		<td>
			500 &mu;m polyester mesh Netwell insert
		</td>
		<td>
			Costar
		</td>
		<td>
			3480
		</td>
	</tr>
	<tr>
		<td>
			Matrigel Matrix
		</td>
		<td>
			BD Biosciences
		</td>
		<td>
			354230
		</td>
	</tr>
	<tr>
		<td>
			BD 10 mL syringe
		</td>
		<td>
			BD biosciences
		</td>
		<td>
			309604
		</td>
	</tr>
	<tr>
		<td>
			Hypodermic needle Aluminum Hub. 
		</td>
		<td>
			Tyco Health Care
		</td>
		<td>
			2000029
		</td>
	</tr>
	<tr>
		<td>
			Tissue culture flask 
		</td>
		<td>
			Corning
		</td>
		<td>
			&nbsp;
		</td>
	</tr>
	<tr>
		<td>
			Centrifuge tubes
		</td>
		<td>
			Basix
		</td>
		<td>
			5539800
		</td>
	</tr>
	<tr>
		<td>
			Thin wall tubes 
		</td>
		<td>
			Eton
		</td>
		<td>
			1107A
		</td>
	</tr>
	<tr>
		<td>
			Surgical Blade stainless steel 
		</td>
		<td>
			Feather
		</td>
		<td>
			2976#10
		</td>
	</tr>
	<tr>
		<td>
			Insulin Syringe 
	 </td>
		<td>
			Comfort point
		</td>
		<td>
			26027
		</td>
	</tr>
	<tr>
		<td>
			50mL flat top screw cap tube
		</td>
		<td>
			Basix
		</td>
		<td>
			5539802
		</td>
	</tr>
	<tr>
		<td>
			Dissection microscope
		</td>
		<td>
			Tritech
		</td>
		<td>
			&nbsp;
		</td>
	</tr>
	<tr>
		<td>
			Fluorescence microscope
		</td>
		<td>
			Olympus
		</td>
		<td>
			DP-72
		</td>
	</tr>
	<tr>
		<td>
			Forcep
		</td>
		<td>
			Fisher Sci
		</td>
		<td>
			&nbsp;
		</td>
	</tr>
</table>
Table 2. Equipment.

method for novel anti-cancer drug development using tumor explants of surgical specimens

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.

Watch this Scientific Journal Video about Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens at JoVE.com

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

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Medicine

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.