This work was supported by National Institutes of Health/National Institute of Neurological Disorders &#38; Stroke (NIH/NINDS) Grants R37-NS094804, R01-NS074387, R21-NS091555 to M.G.C.; NIH/NINDS Grants R01-NS076991, R01-NS082311, and R01-NS096756 to P.R.L.; NIH/NINDS R01-EB022563; 1UO1CA224160; the Department of Neurosurgery; Leah&#39;s Happy Hearts to M.G.C. and P.R.L.; and RNA Biomedicine Grant F046166 to M.G.C. F.M.M. is supported by an F31 NIH/NINDS-F31NS103500. J.P. was supported by a Fulbright-Argentinian Ministry of Sports and Education Fellowship.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.
1. Generation of Brain Tumor Neurospheres Derived from a Mouse Model
<ol>
	<li>Before the dissection procedure, prepare neural stem cell media (NSC Media: DMEM/F12 with 1x B27 supplement, 1x N2 supplement, 1x normocin, and 1x antibiotic/antimycotic supplemented with human recombinant EGF and basic-FGF at concentrations of 20 ng/mL each). Fill a 1.5 mL tube with 300 &#181;L of NSC media and reserve for later.</li>
	<li>Select a mouse with a large tumor. 
	NOTE: The size of a tumor can be monitored by bioluminescence if the plasmid or virus injected encodes luciferin. Usually, a large tumor has a bioluminescence of &#62; 106 photons/s/cm2/sr.</li>
	<li>Euthanize the mouse with an overdose of isoflurane: infuse a tissue paper with isoflurane and introduce the tissue in a euthanization chamber, avoiding direct contact with the mice to prevent irritation. Wait until the mice do not show a pedal reflex (usually 5-10 min). Decapitate the mouse and dissect the brain as described in Calinescu et al.13.</li>
	<li>If the tumors are fluorescent, use a dissecting microscope equipped with a fluorescent lamp to identify the tumor mass within the brain. With fine forceps, dissect the tumor (usually 5-7 mm in diameter) from the normal brain tissue.</li>
	<li>Place the dissected tumor in the 1.5 mL tube with NSC media (step 1.1). Homogenize the tumor with a disposable plastic pestle that fits into the walls of the microcentrifuge tube by applying gentle pressure.</li>
	<li>To dissociate the cell clumps, add 1 mL of enzyme-free dissociation media and incubate for 5 min at 37 &#176;C.</li>
	<li>Filter the cell suspension through a 70 &#181;m cell strainer and wash the strainer with 20 mL of NSC media.</li>
	<li>Centrifuge at 300 x g for 5 min, decant the supernatant, and re-suspend the pellet into 6 mL or 15 mL of NSC media, depending on the size of the pellet.</li>
	<li>Plate the tumor cell suspension into a T-25 or T-75 culture flask and culture at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2.</li>
	<li>After 3 days, transfer the healthy suspended NS and re-plate them into a T-25 or T-75 tissue culture flask. If necessary, add more NSC media to the culture. 
	NOTE: There will usually be a mixed population of cells, some will be dead, some will have adhered, and some will be forming NS that will be floating in the media</li>
</ol>
2. Maintaining and Passaging the NS
NOTE: Prevent overgrowth and maintain cells at relatively high density. Confluence is determined by spheres size (black centers of large spheres mean overgrowth). The growth rate of the NS will depend on the oncogenes used to generate tumors.
<ol>
	<li>Once the NS culture has reached confluency, collect the culture in a sterile centrifuge tube and pellet the NS at 300 x g for 5 min.</li>
	<li>Discard the supernatant, add 1 mL of the cell detachment solution, and pipette to re-suspend the pellet.</li>
	<li>Incubate at 37 &#176;C for 5 min, flicking the tube every 2 min to help cell dissociation. 
	NOTE: When NS reach confluency, they can be usually seen macroscopically as small white spheres. To assure cell dissociation, at least all the visible NS must have disappeared.</li>
	<li>Add 5 mL of HBSS 1x and pipette several times. Pellet cells at 300 x g for 5 min. Discard the supernatant. Re-suspend the pellet in 5 mL of NSC media supplemented with recombinant EGF and basic-FGF.</li>
	<li>Add 1 mL of cells to 15 mL of NSC media and culture in a T-75 flask&#160;at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2.</li>
	<li>Add growth factors (EGF and basic-FGF) every 3 days. If the media turns light orange and the cells are not yet confluent, change the media. To do this, centrifuge the NS at 300 x g for 4 min and re-suspend in NSC media supplemented with growth factors. 
	NOTE: Depending on the size of the pellet formed, cells may need to be split into more flasks.</li>
</ol>
3. Freezing NS for Long-term Storage
<ol>
	<li>When the NS culture becomes confluent, dissociate the cells as described in steps 2.1-2.4. Once the pellet has been re-suspended in HBSS, remove an aliquot and count the cells.</li>
	<li>Centrifuge cells at 300 x g for 4 min and remove as much as possible of the supernatant.</li>
	<li>Re-suspend the pellet in freezing media (heat-inactivated FBS, 10% DMSO). It is recommended to freeze between 1 x 106 and 3 x 106 cells per vial per mL.</li>
	<li>Divide the re-suspended cells in as many cryovials as needed and slowly cool down the vials by first transferring them to ice, then to -20 &#176;C, then to -80 &#176;C, and finally the next day to a liquid nitrogen storage container.</li>
</ol>
4. Fixation of Tumor NS
<ol>
	<li>Culture tumor NS in a T-25 flask for 2-3 days until cells are confluent (~ 3 x 106 cells). Collect tumor NS in a 15 mL conical tube from a least one T-25 confluent flask. Pellet at 300 x g for 5 min.</li>
	<li>Re-suspend the pellet in 1 mL of 10% formalin and keep at room temperature (RT) for 10 min. Add 5 mL of 1x HBSS and pellet the cells as done in step 3.2. Repeat this step one more time.</li>
	<li>Place the 15 mL conical tube containing the pellet on ice.</li>
</ol>
5. Paraffin Embedding of Tumor NS
<ol>
	<li>Re-suspend the washed NS pellet in 500 &#181;L of 2% warm liquid agarose and mix gently by pipetting.</li>
	<li>Immediately place the agarose-embedded NS on ice until the agarose polymerizes. Remove the agarose piece harboring the NS. Place the polymerized agarose piece with the NS into the cassette for tissue processing (Figure 2).</li>
	<li>Continue with tissue processing (using an automatic paraffin processor) and paraffin embedding (using an embedding station).</li>
</ol>
6. Sectioning of Paraffin-embedded NS
<ol>
	<li>Set the water bath to 42 &#176;C and insert the blade on the microtome carefully using two paint brushes to make sure it is leveled. Use this blade only for trimming.</li>
	<li>Place the paraffin block on the microtome. With the non-dominant hand, press down on the lever located on left-hand side that controls the thickness of each section. Press to the second stop that indicates a 30 &#181;m thickness.</li>
	<li>Begin trimming the block (i.e., removing excess paraffin) by turning the coarse hand wheel with the right hand until the surface of the sample is reached. At this point, release the lever that controls the trim thickness to either 10 &#956;m or 5 &#956;m. Stop trimming when the surface of the sample is reached.</li>
	<li>Fill an ice box with ice and add just enough water so that when the paraffin block is placed on ice, the water acts as a film around the paraffin block, protecting it from directly touching the ice. Incubate the paraffin block on ice for 20 min.</li>
	<li>Discard the old blade and insert a new one for sectioning. Dry block using gauze, then place it on the microtome.</li>
	<li>With the non-dominant hand, obtain a pair of curved forceps that will be used to pull the paraffin ribbon. Keep a damp paint brush near the right hand, which will be used to transfer the paraffin ribbon to the water bath. Begin to section by turning the coarse hand wheel with the right hand at a fast pace. Use the forceps in the left hand to hold the paraffin ribbon.</li>
	<li>Use the damp paint brush to transfer the paraffin ribbon to the water bath.</li>
	<li>Use the curve of the forceps to break apart the sections by superficially placing the forceps in between two paraffin sections, then pushing the two sections away gently. 
	NOTE: This motion should be entirely on the surface of the paraffin section. Do not press down on the section.</li>
	<li>Use the damp paint brush to push the separated paraffin sections onto positively charged adhesion microscope slides.</li>
	<li>Allow the slides to dry overnight inside a chemical hood before storing them in slide boxes. The storage of slides depends on the type of stain performed.</li>
</ol>
7. Immunohistochemistry (IHC) of Paraffin-embedded NS
<ol>
	<li>Melt paraffin by placing the slides in a metal rack and incubating them at 60 &#176;C for 20 min.</li>
	<li>Deparaffinize the slides by incubation in a rehydration train at RT: 3x xylene for 5 min, 100% 2x ethanol for 2 min, 95% 2x ethanol for 2 min, 70% 1x ethanol for 2 min, and 1x deionized water for 2 min. Hereon, keep the slides in tap water until antigen retrieval, as drying out will cause non-specific antibody binding.</li>
	<li>Incubate the slides in citrate buffer (10 mM citric acid, 0.05% non-ionic detergent, pH 6) at 125 &#176;C for 30 s and at 90 &#176;C for 10 s. Let them cool down, then rinse the slides 5 times with distilled water.</li>
	<li>Incubate slides at RT in 0.3% H2O2 for 30 min.</li>
	<li>Wash the slides 3 times in washing buffer (0.025% detergent in TBS) for 5 min with gentle agitation.</li>
	<li>Incubate the slides at RT with blocking solution (10% horse serum, 0.1% BSA in TBS) for 30 min.</li>
	<li>Incubate the slides overnight at 4 &#176;C in a humidified chamber with primary antibody prepared in dilution solution (0.1% BSA in TBS). Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li>
	<li>Incubate the slides at RT for 1 h with biotinylated secondary antibody prepared in dilution solution. Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li>
	<li>Incubate the slides at RT in the dark for 30 min with avidin-biotinylated horseradish peroxidase complex. Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li>
	<li>Incubate the slides with DAB-H2O2 substrate brands here for 2-5 min at RT.</li>
	<li>Rinse 3 times with tap water. Dip (1-2 s) the slides in hematoxylin solution to counterstain, and rinse them thoroughly in tap water until clearing.</li>
	<li>Dehydrate the slides as follows: incubate in distilled water for 2 min, dip 3 times in 80% ethanol, incubate in 95% ethanol 2 times for 2 min each, incubate in 100% ethanol 2 times for 2 min each, and finally in xylene 3 times for 3 min each.</li>
	<li>Coverslip the slides with a xylene-based mounting medium and let them dry on a flat surface at RT until they are ready for imaging.&#160; &#160; &#160; &#160;NOTE: If performing 3,3&#39;-diaminobenzidine (DAB) staining, the slides can be stored at RT. However, if immunofluorescence staining is performed, slides should be stored in the dark at -20 &#176;C to reduce photo-bleaching.</li>
</ol>

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The authors have nothing to disclose.

In this article, we detail a versatile and reproducible method to perform immunohistochemistry on paraffin-embedded glioma NS, which maintain the characteristic 3D structure of tumor cells growing in the brain in situ. This technique possesses the following advantages over using cells plated onto glass or plastic surfaces: i) preservation of the 3D structure of NS; ii) avoiding of stress of cell dissociation before analysis of protein expression; iii) quenching of any endogenous fluorophore expression from genet...

Gliomas are primary solid tumors of the central nervous system classified by their phenotypic and genotypic characteristics according to the World Health Organization1. This classification incorporates the presence or absence of driver mutations, which represent an attractive source of biomarkers2. Biomarkers are biological characteristics that can be measured and evaluated to indicate normal and pathological processes, as well as pharmacological responses to a therapeutic intervention3. Biomarkers can be detected in tumor tissue and cells derived from glioma, enabling its biological characterization in different aspects. Some examples of glioma biomarkers include mutated isocitrate dehydrogenase 1 (IDH1), a mutant enzyme characteristic of lower-grade gliomas associated with better prognosis4. Mutated IDH1 is frequently expressed in combination with TP53 and alpha-thalassemia/mental retardation syndrome X-linked (ATRX)-inactivating mutations, defining a specific glioma subtype4,5. ATRX-inactivating mutations also occur in 44% of pediatric high-grade gliomas2,6 and have been associated with aggressive tumors and genomic instability6,7. In addition, pediatric diffuse intrinsic pontine gliomas (DIPG) are differentiated in subgroups according to the presence or absence of a K27M mutation in histone H3 gene H3F3A, or in the HIST1H3B/C gene1,6. Therefore, the introduction of molecular characterization permits the subdivision, study, and treatment of gliomas as separate entities, which will more allow the development of more targeted therapies for each subtype. In addition, the analysis of biomarkers can be used to evaluate different biological processes such as apoptosis8, autophagy9, cell cycle progression10, cell proliferation11, and cell differentiation12.
Genetically engineered animal models that harbor the genetic lesions present in human cancers are critical for the study of signaling pathways that mediate disease progression. Our laboratory has implemented the use of the Sleeping Beauty (SB) transposase system to develop genetically engineered mouse models of glioma harboring specific mutations that recapitulate human glioma subtypes13,14. These genetically engineered mouse models are used for generating tumor-derived NS, which enable in vitro studies in a 3D system, mirroring salient features present in the tumors in situ15. Also, the orthotopical transplantation of NS into mice can generate secondary tumors that retain features of the primary tumor and mimic the histopathological, genomic, and phenotypic properties of the corresponding primary tumor15.
In serum-free culture, brain tumor cells with stem cell properties can be enriched, and in the presence of epidermal growth factors (EGF) and fibroblast growth factors (FGF), they can be grown as single cell-derived colonies such as NS cultures. In this selective culture system, most differentiating or differentiated cells rapidly die, whereas stem cells divide and form the cellular clusters. This allows the generation of a NS culture that maintains glioma tumor features16,17,18. Glioma NS can be used to evaluate several aspects of tumor biology, including the analysis of biomarkers that have applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. Here, we detail a protocol to generate glioma NS and embed the 3D cultures in paraffin to be used for IHC staining. One advantage of fixing and embedding glioma NS is that the morphology of NS is maintained better compared to the conventional cytospin method19, in which glioma NS are subjected to stressful manipulation for cell dissociation and become flattened. In addition, embedding quenches any endogenous fluorophore expression from genetically engineered tumor NS, enabling staining across the fluorescent spectra. The overall goal of this method is to preserve the 3D structure of glioma cells through a paraffin embedding process and enable characterization of glioma neurospheres using immunohistochemistry.

<table border="0" cellpadding="0" cellspacing="0" style="width:884px;" width="884">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Coated microtome blade HP35</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:221px;">3150734</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtome RM 2135</td>
			<td style="width:191px;">Leica</td>
			<td style="width:221px;">MR2135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:191px;">Sigma-aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rabbit polyclonal anti-ATRX,</td>
			<td style="width:191px;">Santa Cruz Biotechnology</td>
			<td style="width:221px;">sc-15408</td>
			<td>IHC, 1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Rabbit polyclonal anti-Ki-67</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:221px;">Ab15580</td>
			<td>IHC, 1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit polyclonal anti-OLIG2</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>IHC, 1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat polyclonal anti-rabbit biotin-conjugated</td>
			<td style="width:191px;">Dako</td>
			<td style="width:221px;">E0432</td>
			<td>IHC, 1:1000 dilution</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:305px;">Vectastain Elite ABC HRP kit</td>
			<td style="width:191px;">Vector Laboratories Inc</td>
			<td style="width:221px;">PK-6100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">BETAZOID DAB CHROMOGEN KIT</td>
			<td style="width:191px;">Biocare medical</td>
			<td style="width:221px;">BDB2004 L/price till 12/18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">N-2 Supplement</td>
			<td style="width:191px;">ThermoFisher</td>
			<td style="width:221px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">N-27 Supplement</td>
			<td style="width:191px;">ThermoFisher</td>
			<td style="width:221px;">A3582801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Accutase&#174; Cell Detachment Solution</td>
			<td style="width:191px;">Biolegend</td>
			<td style="width:221px;">423201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">AGAROSE LE</td>
			<td style="width:191px;">GoldBio</td>
			<td style="width:221px;">A-201-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">Olympus 15 ml</td>
			<td style="width:221px;">21-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">TC-75 treated Flask</td>
			<td style="width:221px;">25-209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">TC-25 treated Flask</td>
			<td style="width:221px;">25-207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">DMEM/F12</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:221px;">11330-057</td>
			<td>NS media</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:305px;">HBSS</td>
			<td style="width:191px;">GibcoTM</td>
			<td style="width:221px;">14175-103</td>
			<td>balanced salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">C57BL/6</td>
			<td style="width:191px;">Taconic</td>
			<td style="width:221px;">B6-f</td>
			<td>C57BL/6 mouse</td>
		</tr>
	</tbody>
</table>

evaluation of biomarkers in glioma by immunohistochemistry on paraffin-embedded 3d glioma neurosphere cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

To monitor tumor development and evaluate if the tumor has reached the size necessary to generate NS, we analyzed the luminescence emitted by tumors using bioluminescence. This allows the study of transduction efficiency in the pups and tumor progression by the luminescence signal of luciferase (Figure 1A and 1B). When the tumor size reaches a signal of 1 x 107 photons/s/cm2/sr (Figure 1</stron...

Watch this Scientific Journal Video about Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures at JoVE.com

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

evaluation-biomarkers-glioma-immunohistochemistry-on-paraffin

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Cancer Research

7.8K Views.  University of Michigan Medical School. Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Video: Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been considered the leading cause of death in lung cancer patients. The complexity of the tumor microenvironment requires effective methods to understand cell-to-cell relationships in tumor tissues. The multiplex immunohistochemistry (mIHC) technique has become a key tool for inferring the relationship between the expression of proteins upstream and downstream of signaling pathways in tumor tissues and developing clinical diagnoses and treatment plans. mIHC is a multi-label immunofluorescence staining method based on Tyramine Signal Amplification (TSA) technology, which can simultaneously detect multiple target molecules on the same tissue section sample to achieve different protein co-expression and co-localization analysis. In this experimental protocol, paraffin-embedded tissue sections of lung squamous carcinoma of clinical origin were subjected to multiplex immunohistochemical staining. By optimizing the experimental protocol, multiplex immunohistochemical staining of labeled target cells and proteins was achieved, solving the problem of autofluorescence and channel crosstalk in lung tissues. In addition, multiplex immunohistochemical staining is widely used in the experimental validation of tumor-related, high-throughput sequencing, including single-cell sequencing, proteomics, and tissue space sequencing, providing intuitive and visual pathology validation results.

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

This experimental protocol describes and optimizes a multiplex immunohistochemistry (IHC) staining method, mainly by optimizing single-channel antibody incubation conditions and adjusting the settings of antibodies and channels to solve the problems of autofluorescence and channel crosstalk in lung cancer tissues of clinical origin.

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...