Name: quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections
Uploaded: 2017-07-31T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

The authors thank Drs. Fahad Bafakih and Jim Mandell for acquisition and identification of patient samples, Garrett F. Beeghly for assistance with immunohistochemistry, and the Biorepository and Tissue Research Facility, the Cardiovascular Research Center Histology Core, and the Biomolecular Analysis Facility at the University of Virginia for assistance with sample acquisition, immunohistochemistry, and imaging.

This protocol identifies cellular components in formalin-fixed paraffin embedded (FFPE) samples, as is typical for banked clinical patient samples. Paraffin embedding allows for the best maintenance of cellular and tissue morphology as well as has better longevity of sections. The samples used for this analysis were accessed through the University of Virginia Biorepository and Tissue Research Facility. Patient samples were selected by a neuropathologist based on a definitive diagnosis of glioblastoma (astrocytoma, WHO gr...

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Our method proposed here is a quantitative approach to analyzing histological samples stained using traditional chromogenic immunohistochemistry. Current methodology for this type of analysis includes similar staining protocols followed by grading by independent pathologists. This method has been reliable, yet for a number of applications, a more precise understanding of the cellular make-up is required, such as better understanding of the heterogeneity associated with tumors and accurate recapitulation of tumors for in ...

Glioblastoma (GBM), the most common and malignant brain cancer, is characterized by highly diffuse invasion from the primary tumor bulk into the surrounding healthy brain parenchyma1,2. This diffuse invasion makes the tumor particularly difficult to resect fully, and the invading cancer cells that remain post-therapy is the most common reason for inevitable recurrence2,3,4. Previously, we found inhibiting the diffuse glioma cell invasion to be therapeutically beneficial5, however little is known about the complex mechanisms contributing to GBM invasion. The tumor microenvironment, or tissue surrounding the cancer, has been implicated in the progression of tumors in multiple cancers6,7. The glioblastoma tumor microenvironment, in particular, is relatively under-characterized and is uniquely complex, composing of multiple glial cells, such as astrocytes, microglia, and oligodendrocytes, as well as extracellular matrix, soluble factors, and biophysical factors. Experimentally, astrocytes and microglia have been shown to increase glioma progression and invasion8,9,10, but the composition of all glial cells in the native human brain microenvironment is unknown.
We previously showed microenvironmental components can predict patient survival by quantitatively analyzing cellular components of the glioblastoma microenvironment and incorporating our analyses into a proportional hazards model11. Here, we describe the quantitative analysis method for identifying the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations and ImageJ to analyze percent coverage of staining for each glial population. Assessing percent coverage creates a simple measurement for determining the morphological differences of cells, particularly those affected by interactions with cancer cells. Previous studies for quantifying histopathological staining use standard staining such as hematoxylin and eosin12 or Masson&#39;s trichrome13, which do not take advantage of the specificity of antibody-based immunohistochemistry staining. Our method was developed to directly quantify the glial populations within glioblastoma patient tumor resections, which we aim to use to elucidate the complex glioblastoma microenvironment.

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			<td height="42" style="height:42px;width:216px;">Xylene</td>
			<td style="width:71px;">Fisher Chemical</td>
			<td style="width:119px;">X3P</td>
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			<td height="21" style="height:21px;width:216px;">Ethanol</td>
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			<td height="42" style="height:42px;width:216px;">High pH antigen unmasking solution</td>
			<td style="width:71px;">Vector Labs</td>
			<td>H-3301</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">TBS</td>
			<td style="width:71px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;width:216px;">Triton-X</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">9002-93-1</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Horse serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Anti-ALDH1L1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab56777</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Anti-Iba1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab5076</td>
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			<td height="42" style="height:42px;width:216px;">Anti-Oligodendrocyte Specific Protein1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab53041</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">ImmPRESS anti-goat</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">MP-7405</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImmPRESS Universal (anti-mouse/rabbit)</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">MP-7500</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrogen peroxide</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">216763</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImmPACT DAB substrate</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">SK-4105</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hematoxylin counterstain</td>
			<td style="width:71px;">ThermoScientific</td>
			<td style="width:119px;">72404</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Histochoice Mounting Media</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">H157-475</td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Aperio Scanscope</td>
			<td style="width:71px;">Leica Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Image Scanscope</td>
			<td style="width:71px;">Leica Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Super HT PAP Pen</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">195506</td>
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quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

For this analysis, two regions of interests within our tumor resections - the primary tumor bulk and the adjacent regions, primarily composed of healthy tissue with diffuse invading cancer cells (Figure 1A, 1B) - were identified by collaborating neuropathologists on hematoxylin and eosin stained patient samples. Within each patient sample, positive staining for astrocytes (Figure 1C), microglia (<strong class="xf...

Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

The overall goal of this protocol is to quantitively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and image J.This method can help answer key questions in the tumor microenvironment field, such as how these components look in patient samples and how we can determine physiologically relevant ratios of cells. The main advantage of this technique is that it is straightforward for analysis in patient samples. The implications of this technique extend toward elucidating the undercharacterized, complex glioblastoma tumor microenvironment.Generally, individuals new to this method will struggle because timing for chromogenic development needs to be closely monitored to prevent overdevelopment and saturation of the tissue. Begin the protocol by performing the washes specified in the accompanying text protocol. Dilute tris-based high pH antigen unmasking solution and distilled water using the manufacturers recommendations.Move on to heat mediated antigen retrieval by adding the diluted unmasking solution to a non-sealed microwaveable vessel. Place slides into the vessel. Then place the vessel slides into the microwave.Next, boil the vessel for 20 minutes at high power. Monitor the liquid levels for evaporation and replenish the vessel with distilled water as necessary. Allow samples to cool in solution for one hour at room temperature.Outline the tissue sample with a hydrophobic to minimize the volume of reagents necessary to cover the sample. Then, pipe in enough permeabilization solution, 100 to 200 microliters to cover the tissue sample. Remove and discard the solution and cover the tissue sample with permeabilization solution again.Incubate the samples at room temperature with blocking solution for one hour. Then, incubate the samples overnight in four degrees Celsius with primary antibodies diluted in blocking solution. The next day, detect primary antibodies using a horseradish peroxidase polymer reagent corresponding with the primary antibody host animal.Pipe in enough permeabilization solution to cover the tissue sample and incubate the sample for five minutes. Remove and discard solution and cover the tissue sample in permeabilization solution one more time for five minutes. Incubate the slides in 0.3%hydrogen peroxide and IX TBS for 15 minutes.Develop the samples with a peroxidase diaminobenzidine or dab substrate for two to 10 minutes until the desired stain intensity is achieved. Counterstain the samples to identify cell nuclei, such as with hematoxylin following the manufacturers protocol. Dehydrate samples with 100%ethanol and xylene.Finally, mount the samples permanently with mounting media. Two regions of interest within our tumor resections, the primary tumor Bulk and the Adjacent regions, primarily composed of healthy tissue with diffused invading cancer cells were identified by collaborating neuropathologist on hematoxylin and eosin stained patient samples. Chromogenic immunohistochemistry identified positive staining for astrocytes, microglia, and oligodendrocytes within each patient sample.We performed staining with no primary antibody for a negative control. The manufacturer recommended and further dilutions before settling on the one to 200 dilution due to optimal positive staining of cell body and process with minimizing background for the anti ALDH 1l1 antibody. Using Image J, the percent coverage of OSP-1 positive oligodendrocyte staining was quantified to eventually compare differences in our regions of interest.Once the appropriate threshold has been applied to the image, the percent coverage from the threshold can be measured and compared for multiple patients. Five adjacent and bulk regions of glioblastoma or GBM patient samples, along with healthy brain tissue were compared for oligodendrocyte-specific protein one or OSP1 percent coverage. Once mastered, the staining technique can be done in four hours on the first day and two hours the second day if it is performed properly.While attempting this procedure, it's important to remember to closely monitor the tissue staining intensity and hydration.

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Research

JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...