eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. In Situ Immunofluorescence Staining of tumor-infiltrating lymphocytes (TILs)
<ol>
	<li>Procedural guidelines
	<ol>
		<li>Perform all experimental steps at room temperature.</li>
		<li>For all steps, perform the dilutions with Tris Buffer Saline (TBS; see Table of Materials).</li>
		<li>Perform the wash in TBS for all the steps except after the primary antibody incubation. For the latter, use Tris Buffer Saline Tween20 (TBST, see Table of Materials). For buffer composition and reconstitution, see Table 1.</li>
		<li>Use a humidity chamber for antibody incubations and a staining jar for washing.</li>
		<li>Do not let the section dry out during the procedure.</li>
	</ol>
	</li>
	<li>Pretreatment
	<ol>
		<li>Thaw the slide containing the tissue sample and carefully dry the slide around the specimen with a paper towel. Delimitate the reaction area containing the tissue with a hydrophobic barrier pen (see Table of Materials). Dry for 2 min.</li>
		<li>Fix the samples in 100% acetone for 5 min, dry for 2 min, and wash with TBS for 10 min.</li>
	</ol>
	</li>
	<li>Saturation and blockade
	<ol>
		<li>Pretreat the slides for 10 min with 3 drops of avidin 0.1%, tap and/or flick the slide to distribute the avidin and remove air bubbles, and then treat for 10 min with 3 drops of biotin 0.01% (see Table of Materials), tap, and/or flick. Wash with TBS.</li>
		<li>Perform an Fc receptors blockade. Apply 100 &#181;L of a 5% volume/volume of normal serum diluted in TBS. Use serum from the same host species as the labeled secondary or tertiary antibody (see Table of Materials); donkey serum was used here. Incubate for 30 min.</li>
		<li>During the incubation, briefly spin the anti-CD8, program cell death protein 1 (PD-1), and T-cell immunoglobulin and mucin-containing protein-3 (Tim-3) antibodies (Table of Materials). Prepare the mix of primary antibodies in TBS as described in Table 2.</li>
	</ol>
	</li>
	<li>Immuno-staining for CD8, PD-1, and Tim-3
	<ol>
		<li>Prepare the primary antibody mixture. Refer to Table of Materials and Table 2 for the antibodies used (anti-CD8, PD-1, Tim-3, and their corresponding secondary antibodies) and their concentrations.</li>
		<li>Tap and/or flick the remaining donkey serum (added in step 1.3.2). Incubate the slides with 100 &#181;L of the non-labeled primary antibodies mix for 1 h in a humidified chamber.</li>
		<li>During the incubation, briefly spin the Cyanine 5 anti-rabbit, Alexa Fluor 488-anti-goat, and biotinylated anti-mouse antibodies and prepare the mix of secondary antibodies as described in Table 2.</li>
		<li>Wash the slides in TBST for 5 min. Dry the slides.</li>
		<li>Incubate the slides with 100 &#181;L of the secondary antibodies for 30 min in a humidified chamber.</li>
		<li>Prepare the mixture of tertiary antibodies containing Cyanine 3-streptavidin, as described in Table 2.</li>
		<li>Wash the slides in TBS for 10 min. Dry the slides.</li>
		<li>Incubate the slides with 100 &#181;L of the tertiary antibody mixture for 30 min.</li>
		<li>Wash the slides in TBS for 10 min. Dry the slides.</li>
	</ol>
	</li>
	<li>Cell mounting
	<ol>
		<li>Mount the slides in a 4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)-containing mounting medium (1.5 &#181;g/mL DAPI) with a coverslip compatible for fluorescence microscopy (see Table of Materials). 
		NOTE: As a negative control for each experiment, one slide with isotype-matched antibodies was used at the same concentration as the corresponding antibodies. For this staining, we used mouse IgG1, rabbit IgG, and goat IgG-negative control antibodies (see Table of Materials). As a positive control, we used human hyperplasic tonsil, which is known to be positive for the tested markers, for each experiment. A typical staining is described in the results (Figure 1). Mono-staining of CD8, PD-1, and Tim-3 should be performed individually (one staining per slide) with the same pre-treatment and without DAPI. A slide in the same experimental conditions without any antibody and only a DAPI mounting medium should also be performed. A slide should be imaged using identical experimental conditions without any antibody and without DAPI.</li>
	</ol>
	</li>
</ol>
2. Fluorescence Analysis and Automated Cell Count
<ol>
	<li>Image acquisition with the automated microscope
	<ol>
		<li>Create a protocol.
		<ol>
			<li>Turn on the automated microscope software and the fluorescence illuminator.</li>
			<li>In the menu bar, click on &#34;File,&#34; &#34;Create protocol,&#34; select &#34;DAPI,&#34; &#34;FITC,&#34; and &#34;TRITC.&#34;</li>
			<li>Click &#34;Next,&#34; &#34;Tissue section,&#34; and click &#34;Next,&#34; &#34;Protocol name.&#34; Choose a name, for example, &#34;CD8-PD-1-Tim-3,&#34; click &#34;Next&#34; and &#34;Save.&#34;</li>
			<li>Manually place a slide on the stage.</li>
			<li>In the menu bar, click &#34;setup&#34; and &#34;settings.&#34; Click &#34;Set home Z&#34; in the new window.</li>
			<li>Adjust exposure time with a positive control slide, i.e., a CD8, PD-1, and Tim-3 triple-stained with a DAPI sample.</li>
			<li>In the control bar, click &#34;set exposure.&#34;</li>
			<li>Adjust the exposure time for each filter until a sufficient but not saturating signal is obtained.</li>
			<li>For monochrome imaging, perform the following:</li>
			<li>Select acquisition, and under &#39;autofocus,&#39; choose the DAPI filter.</li>
			<li>In the &#34;scan area limit,&#34; move the objective upper to the upper left corner of the slide. Click &#34;Mark&#34;. Do the same for the lower right corner. 
			NOTE: This step delimitates the area of low-power imaging (LP imaging) at 4X; be sure to place the mark well to surround all the samples of the series.</li>
			<li>For high power (HP) imaging, perform the following:</li>
			<li>Under &#39;Autofocus,&#39; choose &#34;DAPI.&#34;</li>
			<li>Under the &#39;Acquisition&#39; band, choose &#34;DAPI,&#34; &#34;FITC,&#34; &#34;Cy3&#34;, and &#34;Cy5&#34;. Click &#34;OK&#34;. Click &#34;File&#34; and &#34;Save protocol&#34; on the menu bar.</li>
		</ol>
		</li>
		<li>Scan the slides.
		<ol>
			<li>Load the protocol by clicking &#34;File&#34; and &#34;Load protocol&#34; from the menu bar. Click &#34;Start&#34;.</li>
			<li>Enter &#39;Lab ID&#39; (folder location for image storage). Enter Slide ID to identify the slide. Click &#34;Next&#34;.</li>
			<li>Click &#34;Monochrome Imaging&#34; (to scan the whole slide under bright field light and acquire sequential images using a 4x objective). 
			NOTE: This produces a grayscale overview of the slide.</li>
			<li>Click &#34;Find Specimen&#34;. Select the area of tissue to be scanned at low resolution (4x): hold the &#39;Ctrl&#39; key and click on a field using the cursor to select or deselect fields (Figure 2A).</li>
			<li>Click &#34;LP Imaging&#34; (4x imaging) for fluorescent Red Blue Green image acquisition of each field.</li>
			<li>For HP field selection, hold down the &#39;Ctrl&#39; key and click on a field using the cursor to select or deselect fields corresponding to the tissue area that will be scanned at high resolution (20x). Select 5 fields (Figure 2B).</li>
			<li>Click &#34;HP Imaging&#34; (20x imaging) for a multispectral image acquisition of each field (Figure 2C).</li>
			<li>Click &#34;Data Storage&#34; to store images in the folder selected at the beginning in LabID. 
			NOTE: The process is summarized and illustrated in Figure 2. For each step (tissue detection, field selection), an algorithm can be incremented and trained by the user for a whole-automated scan process.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
Table 1: Buffer composition of TBS and TBST. TBS is used for antibody mixes and dilutions. For washes, all steps are performed with TBS except for the washes following primary antibodies, where TBST is recommended.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>TBS</td>
			<td>TBST</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>0.05 M</td>
			<td>0.05 M</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>0.15 M</td>
			<td>0.15 M</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>-</td>
			<td>0.05%</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.6</td>
			<td>7.6</td>
		</tr>
		<tr>
			<td>preparation</td>
			<td colspan="2">1 tablet in 500 mL H2O</td>
		</tr>
	</tbody>
</table>
Table 2: Detailed antibodies mix composition for CD8-PD-1-Tim-3 immunofluorescence staining. For each primary, secondary, and tertiary antibody mixes, the dilution to perform in the corresponding volume of TBS is given for a total volume of 1 mL. For one slide, the total reaction volume is 100 &#181;L for a tissue area of 2 cm2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">The volume of primary antibody</td>
			<td>Final V= 1mL</td>
		</tr>
		<tr>
			<td>Rabbit anti-CD8</td>
			<td>Mouse anti-PD-1</td>
			<td>Goat anti-Tim-3</td>
			<td>TBS</td>
		</tr>
		<tr>
			<td>1.6 &#181;L</td>
			<td>2 &#181;L</td>
			<td>30 &#181;L</td>
			<td>966.4 &#181;L</td>
		</tr>
		<tr>
			<td colspan="3">The volume of secondary antibody</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyan 5 anti-rabbit</td>
			<td>Biotinylated anti-mouse</td>
			<td>Alexa Fluor 488 anti-goat</td>
			<td>TBS</td>
		</tr>
		<tr>
			<td>3.3 &#181;L</td>
			<td>2 &#181;L</td>
			<td>2.5 &#181;L</td>
			<td>992.2</td>
		</tr>
		<tr>
			<td colspan="3">The volume of tertiary antibody</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Cy3 labeled streptavidin</td>
			<td></td>
			<td>TBS</td>
		</tr>
		<tr>
			<td></td>
			<td>3.3 &#181;L</td>
			<td></td>
			<td>996.7</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vectra 3 Automated Quantitative Pathology Imaging</td>
			<td>Perkin Elmer</td>
			<td>CLS142338</td>
			<td></td>
		</tr>
		<tr>
			<td>inForm cell analysis 2.1.</td>
			<td>Perkin Elmer</td>
			<td>CLS135781</td>
			<td></td>
		</tr>
		<tr>
			<td>R software</td>
			<td>https://www.r-project.org</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dakopen delimiting pen</td>
			<td>Dako</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Buffer Saline TBS Tablets</td>
			<td>Takara, Bio Inc.</td>
			<td>TAKT9141Z</td>
			<td>pH7.6 100 tablet</td>
		</tr>
		<tr>
			<td>Tris Buffer Saline Tween 20 TBS(+Tween20)</td>
			<td>Takara, Bio Inc.</td>
			<td>TAKT9142Z</td>
			<td>pH 7.6 100 tablets</td>
		</tr>
		<tr>
			<td>Biotin blocking system</td>
			<td>Dako</td>
			<td>X0590</td>
			<td>Avidin 0.1% and Biotin 0.01%</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-001</td>
			<td>5% vol./vol. concentration</td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-aldrich</td>
			<td>F6057</td>
			<td>1.5 &#181;g/mL concentration</td>
		</tr>
		<tr>
			<td>Knittel glass coverslip</td>
			<td>Knittel Gl&#228;ser,</td>
			<td>100039</td>
			<td>24x60 mm 100 cover slips</td>
		</tr>
		<tr>
			<td>Rabbit anti-CD8 Clone P17-V</td>
			<td>novus</td>
			<td>NBP1-79055</td>
			<td>use at 4&#181;g/mL</td>
		</tr>
		<tr>
			<td>Mouse anti-PD-1 Clone NAT</td>
			<td>abcam</td>
			<td>ab52587</td>
			<td>use at 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-Tim-3</td>
			<td>R&#38;D</td>
			<td>AF2365</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Rabbit anti-PD-L1 Clone SP142</td>
			<td>Roche</td>
			<td>7309457001</td>
			<td>use at 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Mouse AF647 labeled pan- Keratin Clone C11</td>
			<td>Cell Signalling</td>
			<td>4528</td>
			<td>use at 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-human gal9</td>
			<td>R&#38;D</td>
			<td>AF2045</td>
			<td>use at 0.3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cyan 5 conjugated donkey anti-rabbit</td>
			<td>Jackson Immunoresearch</td>
			<td>711-175-152</td>
			<td>use at 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Biotinylated F(ab&#39;2) donkey anti-mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>715-066-150</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor488 conjugated donkey anti-goat IgG</td>
			<td>abcam</td>
			<td>ab150133</td>
			<td>use at 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cy3 labeled streptavidin</td>
			<td>Amersham</td>
			<td>PA43001</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Negative control mouse IgG1</td>
			<td>Dako</td>
			<td>X0931</td>
			<td>use at 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IgG from goat serum</td>
			<td>Sigma-aldrich</td>
			<td>I5256</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IgG from rabbit serum</td>
			<td>Sigma-aldrich</td>
			<td>I5006</td>
			<td>use at 4&#181;g/mL</td>
		</tr>
	</tbody>
</table>

multiplexed immunofluorescence imaging to analyze immunotherapy-resistant t cell subpopulations

Source: Granier, C., et al. <a href="https://app.jove.com/v/56606">Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells.</a> J. Vis. Exp. (2018).
This video demonstrates an in-situ fluorescence multispectral imaging technique to analyze different subpopulations of infiltrating T cells in tumor tissue. A renal cell carcinoma tissue cryosection is treated with antibodies to label CD8+ T cells that express specific inhibitory receptors and exhibit an immunotherapy-resistant phenotype. The labeled section is visualized using a multispectral microscope to identify the distinct CD8+ T cell subpopulations.

<img alt="Figure 1" src="/files/ftp_upload/22139/22139_Figure1.jpg" /> 
Figure 1: CD8-PD-1-Tim-3 immunostaining on a tonsil paraffin-embedded tissue. Paraffin-embedded tissue sections instead of cryopreserved sections were selected for triple immunostaining. After dewaxing and rehydration, the same protocol of staining previously developed for cryopreserved sections was applied. CD8+ T cells are located outside the germinal center, and non-CD8+ T cells expressing PD-...

Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. In Situ Immunofluorescence ...

Take a renal cell carcinoma tissue cryosection exhibiting infiltrating CD8+ T cells in its tumor microenvironment.
Treat it with acetone to fix and permeabilize the tissue. Wash to remove excess acetone.
Add avidin to saturate endogenous tissue biotin. Next, add biotin to block immobilized avidin-binding sites, minimizing non-specific interactions.
Apply serum containing immunoglobulins to block immune cell Fc receptors, preventing non-specific antibody binding.
Add a primary antibody cocktail targeting CD8, a cytotoxic T cell marker, and PD-1 and Tim-3, inhibitory receptors expressed by CD8+ T cell subpopulations resistant to immunotherapy.
Introduce distinct fluorophore-labeled secondary antibodies targeting anti-CD8 and anti-Tim-3 antibodies.
Add biotinylated secondary antibodies targeting anti-PD-1 antibodies and a different fluorophore-labeled streptavidin binding to biotin.
Apply a dye-containing mounting medium to stain cell nuclei and place a coverslip.
Acquire multispectral fluorescence images to identify the tumor-infiltrating CD8+ T cells and the immunotherapy-resistant subpopulations expressing PD-1, Tim-3, or both.

multiplexed-immunofluorescence-imaging-to-analyze-immunotherapy

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

84 vistas. Fuente: Granier, C., et al. Análisis de inmunofluorescencia multiplexada y cuantificación de células T intratumorales PD-1+ Tim-3+ CD8+. J. Vis. Exp. (2018). Este video muestra una técnica de imagen multiespectral de fluorescencia in situ para analizar diferentes subpoblaciones de células T infiltrantes en el tejido tumoral. La criosección de tejido de un carcinoma de células renales se trata con anticuerpos para marcar las células T CD8+ que expresan receptores inhibidores específicos...

Video: Imágenes de inmunofluorescencia multiplexadas para analizar subpoblaciones de células T resistentes a la inmunoterapia - Concepto

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

La implantación ortotópica de células cancerosas derivadas de pacientes en ratones recapitula el cáncer colorrectal avanzado

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Investigación

JoVE Journal

Investigación sobre el cáncer

Inmunofluorescencia múltiple y análisis de imágenes espaciales del microambiente tumoral

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Autor destacado: Inmunofluorescencia multiplex combinada con análisis de imágenes espaciales para la evaluación clínica y biológica del microambiente tumoral  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

Inmunofluorescencia múltiple combinada con análisis de imágenes espaciales para la evaluación clínica y biológica del microambiente tumoral

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Un método rápido para imágenes de fluorescencia multiespectral de secciones de tejido congelado

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

Transcripción

Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations