Name: four-color fluorescence immunohistochemistry of t-cell subpopulations in archival formalin-fixed, paraffin-embedded human oropharyngeal squamous cell carcinoma samples
Uploaded: 2017-07-29T14:00:00
Description: Watch this Scientific Journal Video about Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinom

Simone Punt was supported by grant UL2010-4801 from the Dutch Cancer Society. We would like to thank all the co-authors of the original paper that this JoVE protocol is based on: Emilie A. Dronkers, Marij J. P. Welters, Renske Goedemans, Senada Koljenovi&#263;, Elisabeth Bloemena, Peter J. F. Snijders, Arko Gorter, and Sjoerd van der Burg.

Patient samples were handled according to the medical ethical guidelines described in the Code of Conduct for Proper Secondary Use of Human Tissue of the Dutch Federation of Biomedical Scientific Societies (www.federa.org).
1. Prepare Slides
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	<li>Obtain resected tissue material (any kind of tissue) from a pathology department after obtaining medical ethical committee permission to use resected material. 
	NOTE: For the current experiment, pretreatment tumor samples were obtained fr...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+papillomavirus+detection+and+comorbidity:+critical+issues+in+selection+of+patients+with+oropharyngeal+cancer+for+treatment+De-escalation+trials.">Human papillomavirus detection and comorbidity: critical issues in selection of patients with oropharyngeal cancer for treatment De-escalation trials.</a> Ann Oncol. 24 (11), 2740-2745 (2013).</li><li>Wallis, S. P., Stafford, N. D., Greenman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+relevance+of+immune+parameters+in+the+tumor+microenvironment+of+head+and+neck+cancers.">Clinical relevance of immune parameters in the tumor microenvironment of head and neck cancers.</a> Head Neck. 37 (3), 449-459 (2015).</li><li>Ye, J., Livergood, R. S., Peng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+regulation+of+human+Th17+cells+in+tumor+immunity.">The role and regulation of human Th17 cells in tumor immunity.</a> Am J Pathol. 182 (1), 10-20 (2013).</li><li>Amedei, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+analysis+of+pancreatic+cancer-infiltrating+T+lymphocytes+reveals+that+ENO-specific+Tregs+accumulate+in+tumor+tissue+and+inhibit+Th1/Th17+effector+cell+functions.">Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytes reveals that ENO-specific Tregs accumulate in tumor tissue and inhibit Th1/Th17 effector cell functions.</a> Cancer Immunol Immunother. 62 (7), 1249-1260 (2013).</li><li>Whiteside, T. 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Association of CD3+CD56+ cells with platinum resistance.</a> Gynecol Oncol. 106 (1), 75-81 (2007).</li><li>Gasparoto, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patients+with+oral+squamous+cell+carcinoma+are+characterized+by+increased+frequency+of+suppressive+regulatory+T+cells+in+the+blood+and+tumor+microenvironment.">Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment.</a> Cancer Immunol Immunother. 59 (6), 819-828 (2010).</li><li>Jordanova, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+leukocyte+antigen+class+I,+MHC+class+I+chain-related+molecule+A,+and+CD8+/regulatory+T-cell+ratio:+which+variable+determines+survival+of+cervical+cancer+patients?.">Human leukocyte antigen class I, MHC class I chain-related molecule A, and CD8+/regulatory T-cell ratio: which variable determines survival of cervical cancer patients?.</a> Clin Cancer Res. 14 (7), 2028-2035 (2008).</li><li>van Esch, E. 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For the protocol described, one of the most critical steps is to determine the correct dilution of the primary antibodies used. The dilution of the labeled secondary antibodies was 1:200, as recommended by the manufacturer of these specific Alexa-labeled antibodies. The dilution of the primary antibodies should then be determined by a serial dilution, preferably using two different samples of the intended tissue type (in this case, OPSCC). The optimal working dilution is the dilution at which a clear signal is obtained, ...

Oropharyngeal squamous cell carcinomas (OPSCCs) are a heterogeneous group of squamous cell cancers originating in the oropharynx. Risk factors for OPSCC include human papillomavirus (HPV) infection and alcohol and tobacco use1,2. The role of the immune response and how to use this in a clinical setting is just starting to be explored. The tumor microenvironment is infiltrated by immune cells that are attracted to the cancer site. Although a high CD8+ cytotoxic T-cell frequency has been correlated with improved survival in OPSCC patients3, the role of other T-cell subsets, including Tregs and Th17 cells, is still unclear4. Whereas Th1 and Th17 cells are supposed to aid in the immune response targeting tumor cells, Tregs are well known for their abilities to suppress the activity of other T cells5. However, the presence of Tregs has been found to correlate with both favorable and unfavorable responses in different tumor types6. Since not all immune cells present in the blood infiltrate the tumor to the same extent, studying the local tumor microenvironment provides the most reliable measure of the immune response directed against the tumor. The aim of this study is to determine the correlation between the numbers and types of immune cells and the clinical outcome. We used four-color fluorescence IHC imaging to analyze the number and localization of various T-cell subpopulations in human OPSCC.
We focused on total T lymphocytes (CD3+), Th17 cells, and immunosuppressive FoxP3+ Tregs, whose differentiation pathway is closely related to Th17 cells. Th17 cells are characterized by the combination of CD3 and IL-17. The cytokine IL-17 can also be produced by non-T cells7. We determined the distribution of intra-epithelial and stromal T cells, Tregs, Th17, and IL-17+ non-T cells in a large series of OPSCC cases and analyzed the correlations with patient survival. Multicolor fluorescence IHC was used to identify the expression of CD3, Foxp3, and IL-17, in combination with a DAPI counterstain. This assay allowed for the easy and clear identification of both tumor cells (using DAPI nuclear staining) and the infiltrating T-cell populations (using a combination of different markers). Following sample preparation and staining, a fluorescent microscope and imaging software were used to separate the different fluorescent colors used and to determine the number and type of cells present in both the tumor epithelium and the tumor-associated stroma.
An alternative assay to quantify and phenotype immune cell populations is flow cytometry analysis, or cytometry by time of flight (CyTOF) analysis, of tumor or peripheral samples (i.e., blood or ascites). Using this technique, all information about localization and relative distribution of the different cell types is lost. The use and analysis of peripheral samples also does not provide information about which cells are able to infiltrate the tumor microenvironment. Blood and ascite immune-cell analyses have been shown not to reflect the phenotype and frequency of immune cell infiltration of the tumor tissue8,9.
Another alternative is the use of bright-field microscopy. An advantage of this technique over fluorescence imaging is the absence of tissue autofluorescence. Although some samples contain more autofluorescence&#8212;particularly erythrocytes, but also other cell types, including neutrophilic granulocytes&#8212;these areas can easily be removed in the analysis of almost all samples. Immunofluorescence offers the advantage of analyzing multiple markers in one sample by using a panel of targeted fluorescent wavelengths. This is currently impossible to the same extent for bright-field microscopy due to the lack of a sufficient number of labels and commercially available antibody isotypes for a particular antigen.
The multicolor fluorescence IHC technique described here has been used in many different cancer types and antibody combinations to study different immune cell populations, as well as tumor cell-expressed molecules, such as human leukocyte antigens (HLA) and PD-L110,11,12. The protocol has been established and validated using many different types of samples and antibodies.

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			<td height="21" style="height:21px;width:375px;">Pathos Delta Ultra Rapid Tissue Processor</td>
			<td style="width:167px;">Milestone and Histostar</td>
			<td style="width:143px;"></td>
			<td style="width:696px;">Automated tissue processor</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Histostar</td>
			<td style="width:167px;">Thermo Scientific&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:696px;">Tissue block embedding machine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Formaldehyde</td>
			<td style="width:167px;">Baker</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Xylol</td>
			<td style="width:167px;">Merck</td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ethanol</td>
			<td style="width:167px;">Merck</td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">milliQ water</td>
			<td style="width:167px;">Elaga Purelab Chorus</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Paraffin wax/Paraclean</td>
			<td style="width:167px;">Klinipath</td>
			<td style="width:143px;">5079A</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Microtome tissue holder&#160;</td>
			<td style="width:167px;">Leica</td>
			<td style="width:143px;">RM225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flex IHC side</td>
			<td style="width:167px;">Dako</td>
			<td style="width:143px;"></td>
			<td>Tissue slide</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris</td>
			<td style="width:167px;">Merk-Milipore</td>
			<td style="width:143px;">1,083,821,000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">EDTA</td>
			<td style="width:167px;">Baker</td>
			<td style="width:143px;">1073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">PBS</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>BUF036A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BSA</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:143px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rabbit anti-CD3</td>
			<td style="width:167px;">Abcam</td>
			<td style="width:143px;">ab828</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Mouse IgG1 anti-FoxP3</td>
			<td style="width:167px;">Abcam</td>
			<td style="width:143px;">ab20034</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Goat IgG anti-IL-17</td>
			<td style="width:167px;">R&#38;D Systems</td>
			<td style="width:143px;">AF-317-NA</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rabbit Ig isotype control antibody</td>
			<td style="width:167px;">Abcam</td>
			<td>ab27472</td>
			<td>Use at same final concentration as anti-CD3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Mouse IgG1 isotype control antibody</td>
			<td style="width:167px;">Abcam</td>
			<td>ab91353</td>
			<td>Use at same final concentration as anti-FoxP3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Goat IgG isotype control antibody</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">02-6202</td>
			<td>Use at same final concentration as anti-IL-17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-rabbit IgG A546</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A10040</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-mouse-A647</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A31571</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-goat IgG A488</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A11055</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">VectaShield containing DAPI</td>
			<td style="width:167px;">Vector Laboratories</td>
			<td style="width:143px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM700 confocal laser scanning microscope&#160;</td>
			<td style="width:167px;">Zeiss</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27 objective</td>
			<td style="width:167px;">Zeiss</td>
			<td>420852-9972-720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM Zen Software</td>
			<td style="width:167px;">Zeiss</td>
			<td>version 2009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM Image Browser</td>
			<td style="width:167px;">Zeiss</td>
			<td style="width:143px;">version 4.2.0.121</td>
			<td>Available to download at www.zeiss.com/microscopy/int/website/downloads/lsm-image-browser.html.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">SPSS</td>
			<td style="width:167px;">IBM Corp.</td>
			<td style="width:143px;">version 20.0</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ImageJ</td>
			<td style="width:167px;"></td>
			<td style="width:143px;">version 1.50i</td>
			<td>Available to download at http://rsb.info.nih.gov/ij.</td>
		</tr>
	</tbody>
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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.

A series of FFPE pretreatment tumor samples obtained from primary oropharyngeal tumors diagnosed in the Leiden University Medical Center, Leiden, the Netherlands between 1970 and 2011, selected as described before (n = 162), was stained using the protocol described13. One to four random images of each slide were analyzed (Figure 1). Some autofluorescent cells are indicated, which can clearly be distinguished by their complete yellow ap...

Watch this Scientific Journal Video about Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinom

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

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 ...

The overall goal of this experiment is to perform multicolor immunohistochemical visualization and enumeration of various T lymphocytes subpopulations in archival formalin-fixed paraffin-embedded tissue sections from oropharyngeal squamous cell carcinoma samples. This method can help answer key questions in immuno-oncology field such as, is there a link between tumor tissue infiltrating immune cells subpopulations and patient prognosis. With this technique, you can visualize several biomarkers in one tissue section.The implications of this technique extend towards cancer therapy, as the simultaneous evaluation of multiple immune cells or immune related factors might help in patient stratification for specific immune therapies. Begin by using a micro tongue to obtain four micrometer thick formalin-fixed paraffin-embedded tissue sections, laying the tissue ribbons on the surface of a 45 degree celsius de-ionized water bath as they are acquired. To capture the sections, place a glass slide in the water, directly underneath the tissue sample and carefully move the slide up at an angle.This section will stick to the glass surface as the slide is lifted out of the water. After drying the samples overnight at 37 degrees celsius, de-paraffinize the tissues with three five minute total immersions in 100%xylol. When the sections have been completed de-paraffinized, rehydrate the tissue samples with three descending ethanol immersions.Followed by a five minute wash in de-ionized water. Then, submerge the samples in boiling EDTA buffer for 10 minutes in the microwave and let the slides cool in the buffer for two hours. For labeling of the antigens of interest, rinse the samples with two five minute PBS washes.After the second wash, incubate the tissues with the primary antibodies and interest diluted in 1%bovine serum albumin in PBS at room temperature, overnight. The next morning, rinse the sections with three five minute PBS washes and incubate the samples with the appropriate secondary antibodies diluted in 1%serum in PBS at room temperature for one hour. At the end of the incubation, wash the slides three times in PBS as just demonstrated and mount the slides using an aqueous antifade mounting medium containing a nuclear counter stain.To analyze the samples, first select the 20 to 25 times objective on a fluorescence microscope and capture four images per slide using the appropriate filters for the secondary antibodies and counter stain. Save the images as they are obtained. Next, in the appropriate image analysis software, open the first image of interest and click on the overlay tab.Select the closed poly line shape to demarquee the boundary of the region of interest. When the shape is closed, the surface area of measurement will appear next to the region of interest. Save all the surface area data in the spreadsheet file to calculate the average surface areas and sound numbers per slide.Export the image as a TIFF or JPG file to be opened in ImageJ. Then, in ImageJ, open the first image of interest and select the cell counter under the plugins menu. Then, click initialize, click on type one and click on every cell designated as type one.Select a different type for each cell type to count the number of different types of cells in the image according to the fluorochrome expression. Finally, record the number of cells analyzed in each counter bin for each image in the spreadsheet file. In this representative experiment, on of four random images were analyzed from a series of formalin-fixed paraffin-embedded pretreatment tumor samples obtained from primary oropharyngeal tumors, diagnosed in the Leiden University Medical Center between 1970 to 2011, that were prepared as just demonstrated.In this experimental sample, some autofluorescent cells were observed, as distinguished by their complete yellow appearance, while negative control slides did not demonstrate any specific staining above background tissue autofluorescence. All of the tumor samples were determined to be infiltrated by varying numbers of fox p3 positive, CD3 positive T cells with only a small T cell subset expressing IL-17. Samples from patients with a high number of total infiltrating CD3 positive T cells demonstrate a trend toward a correlation with an improved disease-free survival compared to patient samples with low T cell members.Further, in samples with a low number of IL-17 positive cells, a high number of total infiltrating T cells is also correlated with an improved disease-free survival, suggesting that the effective tumor infiltrating T cells in oropharyngeal squamous cell carcinoma maybe related to the low number of IL-17 positive cells present. Once mastered, these sample preparation steps can be completed in one and a half days for a maximum of 25 slides per staining session. It's very important to de-paraffanize the sample, otherwise you'll get a lot of background.After its development, this technique paved the way for researchers in the fields of pathology and immunology. For exploring the microenvironment for the localisation of various immune cell subpopulations. After watching this video, you should have a good understanding of how to visualize various proteins and cell types in a single archival tissue section.Don't forget that working with silo is very dangerous, you should always work in a flow cabinet.

four-color-fluorescence-immunohistochemistry-t-cell-subpopulations

Research

JoVE Journal

Cancer Research

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

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 ...

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

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.

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 ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.