Name: automated multiplex immunofluorescence panel for immuno-oncology studies on formalin-fixed carcinoma tissue specimens
Uploaded: 2019-01-21T13:00:00
Description: Watch this Scientific Journal Video about Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens at JoVE.com

Editorial support was provided by Deborah Shuman of MedImmune.

NOTE: The protocol presented here describes how to perform immunoprofiling of an mIF panel by using TSA for six antibodies (CD68, ki67, PD-L1, PD-1, CD8, and AE1/AE3) on an automated stainer (see Table of Materials). The protocol also describes how to perform the drop controls for a quality control of a new mIF panel (see Supplemental Materials). In this protocol, staining is performed with eight unstained FFPE slides from human tonsil (positive control) and eight unstai...

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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=Quantitative+and+pathologist-read+comparison+of+the+heterogeneity+of+programmed+death-ligand+1+(PD-L1)+expression+in+non-small+cell+lung+cancer.">Quantitative and pathologist-read comparison of the heterogeneity of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer.</a> Modern Pathology. 30 (3), 340-349 (2017).</li><li>Dixon, A. R., 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=Recent+developments+in+multiplexing+techniques+for+immunohistochemistry.">Recent developments in multiplexing techniques for immunohistochemistry.</a> Expert Review of Molecular Diagnostics. 15 (9), 1171-1186 (2015).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+a+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</li><li>Feng, Z., 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=Multiparametric+immune+profiling+in+HPV-+oral+squamous+cell+cancer.">Multiparametric immune profiling in HPV- oral squamous cell cancer.</a> JCI Insight. 2 (14), (2017).</li><li>Feng, Z., 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=Multispectral+imaging+of+formalin-fixed+tissue+predicts+ability+to+generate+tumor-infiltrating+lymphocytes+from+melanoma.">Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.</a> Journal for ImmunoTherapy of Cancer. 3, 47 (2015).</li><li>Granier, C., 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=Multiplexed+immunofluorescence+analysis+and+quantification+of+intratumoral+PD-1++Tim-3++CD8++T+cells.">Multiplexed immunofluorescence analysis and quantification of intratumoral PD-1+ Tim-3+ CD8+ T cells.</a> Journal of Visualized Experiments. (132), e56606 (2018).</li><li>Parra, E. R., 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=Validation+of+multiplex+immunofluorescence+panels+using+multispectral+microscopy+for+immune-profiling+of+formalin-fixed+and+paraffin-embedded+human+tumor+tissues.">Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.</a> Scientific Reports. 7 (1), 13380 (2017).</li><li>Ribas, A., Wolchok, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunotherapy+using+checkpoint+blockade.">Cancer immunotherapy using checkpoint blockade.</a> Science. 359 (6382), 1350-1355 (2018).</li><li>Gorris, M. A. 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The ongoing cancer immunotherapy revolution is opening novel and promising therapeutic options for cancer patients13. Advances in the field of immuno-oncology will require increased knowledge of the inflammatory tumor microenvironment, not only to understand the biology of the immunological mechanisms involved in carcinogenesis but also to find predictive biomarkers for new immunotherapy-based treatments1,2. Due to the complex biology of c...

Immunoprofiling analysis of FFPE tumor tissue samples has become an essential component of immuno-oncology studies, particularly for the discovery and validation of novel predictive biomarkers for cancer immunotherapy in the context of clinical trials1,2. Chromogenic IHC, using chemical chromogens such as diaminobenzidine, remains the standard technique in diagnostic pathology for the immunolabeling of biopsy tissue3. Standard IHC can also be used for cancer tissue immunoprofiling, including the quantitation of subpopulations of tumor-associated lymphocytes and the assessment of expression levels of immune checkpoints such as programmed cell death ligand 1 (PD-L1)4,5. Standard IHC is limited, however, in that only one antigen can be labeled per tissue section. Because immunoprofiling studies typically require the analysis of the combined expression of several markers, the use of standard IHC would require the staining of multiple tissue sections, each stained with a single marker, and would, therefore, be substantially limited for the analysis of small tissue samples such as core needle biopsies. Standard IHC methods are also limited for the assessment of markers that are coexpressed by diverse cell populations, as is common with immune checkpoint markers such as PD-L1, which is expressed by both tumor-associated macrophages and cancer cells. This limitation has been reported in, for instance, the use of standard monoplex IHC by pathologists for the quantitative analysis of an IHC marker expressed by diverse cell types6. The development of multiplex chromogenic IHC techniques employing diverse colored chromogens on the same tissue section represents an advancement over the standard IHC monoplex method,7 although they remain limited by the immunolabeling of just a few markers and also present an important technical challenge for the proper evaluation of markers expressed in the same subcellular compartments of the same cell populations.
The aforementioned caveats regarding tissue availability from clinical samples, as well as the limitations of multiplex chromogenic IHC techniques, have given rise to the need to develop improved multiplex methods for immuno-oncology studies based on fluorescent labeling combined with imaging systems that can effectively separate the signals of multiple fluorophores from the same slide. One such technique is based on tyramide signal amplification (TSA) combined with multispectral microscopy imaging for efficient color separation8. A commercially available TSA-based kit employs fluorophores optimized for multispectral imaging8 (see Table of Materials). A critical advantage of this system is its compatibility with the same unlabeled primary antibodies that have already been validated and optimized for standard chromogenic IHC9,10,11. This allows not only faster optimization but also flexibility in the optimization and panel modifications incorporating new targets. Furthermore, the multiplex immunofluorescence (mIF) TSA method can be optimized for commercially available automated IHC stainer systems, allowing for a straightforward transfer from monoplex chromogenic IHC to mIF.
Here we present a protocol for an mIF panel for immuno-oncology studies that is based on automated mIF TSA staining and uses a multispectral scanner for imaging. This protocol can be adapted and modified by any laboratory user with access to the described instrumentation and reagents. The protocol includes a panel of six primary antibodies for the immunoprofiling of carcinomas: PD-L1, PD-1, CD68 (as a pan-macrophage marker), CD8 (T-cytotoxic cells), Ki-67, and AE1/AE3 (pan-cytokeratin, used as an epithelial marker for the identification of carcinoma cells). A recent study describes the optimization of a manual TSA mIF protocol by using chromogenic IHC as a standard reference to validate the multiplex staining12. The updated method presented here has been developed by using a commercially available, seven-color TSA kit optimized in an automated stainer, drastically shortening the staining time from 3&#8211;5 days to 14 h, while also improving the consistency of the staining. In addition to the detailed main protocol presented here, a Supplemental Materials section includes the &#34;drop-control&#34; method, an additional quality control process to evaluate a new mIF panel, as well as technical notes for the optimization, troubleshooting, and development of new multiplex panels to help the laboratory user to set up and optimize the mIF TSA method for customized mIF panels.

<table border="0" cellpadding="0" cellspacing="0" style="width:813px;" width="814">
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		<col />
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		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">&#34;InForm 2.4.2&#34; Software for Spectral Unmixing and Image Analysis</td>
			<td style="width:123px;">PerkinElmer</td>
			<td style="width:119px;">CLS151066</td>
			<td style="width:299px;">Called &#34;spectral unmixing software&#34; in text</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">&#34;Phenochart 1.0.9&#34; QPTIFF Software for Selection of MSI and Overall Slide Scan Viewing</td>
			<td style="width:123px;">PerkinElmer</td>
			<td style="width:119px;">CLS151067</td>
			<td>Called &#34;QPTIFF software&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#1.5 Coverslips</td>
			<td>Sigma Aldrich</td>
			<td>2975246</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 Proof Ethanol</td>
			<td>Koptec</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20x Tris-Buffered Saline</td>
			<td>VWR</td>
			<td>J640-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Antibody Diluent</td>
			<td>DAKO</td>
			<td>S2203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD68 Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M087601-2</td>
			<td>Clone PG-M1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD8 Rabbit Monoclonal</td>
			<td>Ventana</td>
			<td>M5392</td>
			<td>Clone SP239</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CK Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M351501-2</td>
			<td>Clone AE1/AE3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-ki67 Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M724001-2</td>
			<td>Clone MIB-1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PD-1 Rabbit Monoclonal</td>
			<td>Cell Signaling</td>
			<td>#86163</td>
			<td>Clone D4W2J</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PD-L1 Rabbit Monoclonal</td>
			<td>Ventana</td>
			<td>790-4905</td>
			<td>Clone SP263</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Dewax Solution</td>
			<td>Leica</td>
			<td>AR9222</td>
			<td>Called &#34;dewax solution&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Epitope Retrieval Solution 1</td>
			<td>Leica</td>
			<td>AR9961</td>
			<td>Called &#34;ER1&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Epitope Retrieval Solution 2</td>
			<td>Leica</td>
			<td>AR9640</td>
			<td>Called &#34;ER2&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Open Containers, 30 mL</td>
			<td>Leica</td>
			<td>OP309700</td>
			<td>Called &#34;30 mL open containers&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Open Containers, 7 mL</td>
			<td>Leica</td>
			<td>OP79193</td>
			<td>Called &#34;7 mL open containers&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bond Polymer Refine Detection</td>
			<td>Leica</td>
			<td>DS9800</td>
			<td>Called &#34;chromogenic detection kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Research Detection Kit</td>
			<td>Leica</td>
			<td>DS9455</td>
			<td>Called &#34;research detection kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Titration Kit</td>
			<td>Leica</td>
			<td>OPT9049</td>
			<td>Called &#34;titration kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Universal Covertile Novocastra</td>
			<td>Leica</td>
			<td>S21.2001</td>
			<td>Called &#34;covertiles&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Wash Solution 10X Concentrate</td>
			<td>Leica</td>
			<td>AR9590</td>
			<td>Called &#34;10x wash solution&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BondRX Autostainer</td>
			<td>Leica</td>
			<td style="width:119px;"></td>
			<td>Called &#34;automated stainer&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BondRX Software Version 5.2.1.204</td>
			<td>Leica</td>
			<td style="width:119px;"></td>
			<td>Called &#34;automated stainer software&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Opal 7-Color Automation IHC Kit</td>
			<td style="width:123px;">PerkinElmer</td>
			<td>NEL801001KT</td>
			<td style="width:299px;">Called &#34;multispectral staining kit&#34; in text</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase Block</td>
			<td>Leica</td>
			<td>RE7101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond Antifade Mountant</td>
			<td>Thermo</td>
			<td>P36965</td>
			<td>Called &#34;slide mountant&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Starfrost Slides</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">15-183-51</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">Vectra Polaris Multispectral Microscope with &#34;Vectra 3.0.5&#34; Software for Multispectral Microscope Control</td>
			<td style="width:123px;">PerkinElmer</td>
			<td>CLS143455</td>
			<td>Called &#34;microscope control software&#34; in text</td>
		</tr>
	</tbody>
</table>

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.

The protocol described here will provide results like those shown in Figure 2. Start with an evaluation of the staining in the tonsil control, beginning with the surface squamous cell epithelium. The histology of the tonsil sample can be reviewed with a pathologist, using the H&#38;E slide as a reference. If chromogenic IHC sections are performed with the same markers on the same tissue block, then these can be used to confirm the density and distribution of ...

Watch this Scientific Journal Video about Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens at JoVE.com

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

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 ...

This protocol is significant because the development optimization and transfer of multiplex immunofluorescence protocols is complex and challenging. For many labs the toe hold of an optimized protocol is invaluable. Multi-spectral immunofluorescence allows us to assess both the location and the identity of multiple cell types in and around the tumor.This is a powerful combination of information in immune oncology studies. The most compelling use for multiplex immunofluorescence is immune oncology. But the ability to survey multiple immune cell types in setu can be leveraged across all auto-immune and inflammatory therapies.Knowing the location and phenotype of immuno cells in and around solidual cells is both prognostic and predictable responses to tumor specific immunotherapy. The optimization and validation of the protocol are key steps that should not be rushed. New developers should familiarize themselves with the new kinds of artifacts associated with multi-spectral imaging.To locate the base multi-plex protocol preload it on the automated stainer. Filter for all'instead of preferred'on the protocol screen. Before making changes save the original protocol and click the protocols tab, and right click Opal 6 Multiplex to select copy.Change the name to 7 Multiplex Protocol 1'in the new window and select the tab associated with the correct device. Match the protocol in supplemental table S1'and click add reagent to add a ten minute paroxidies inhibition step, selecting the peroxidase inhibitor as the reagent. Confirm that the blocking steps utilize a protein kinase inhibitor blocking buffer, that each primary anti-body refers to the appropriate reagent, that the secondary anti-body steps utilize a horseradish peroxidase polymer and that the spectral dapi provided with the multi-spectral automation kit is utilized.Make sure that the protocol contains the peroxidase inhibitor, followed by six sequential staining runs each containing a protein blocking step, a primary anti-body, a secondary anti-body, a Tyramide floor, and then antigen retrieval to remove the anti-bodies. To add the drop controls, copy the 7 Multiplex Protocol 1'name, and change it to 7 Multiplex Protocol 1 CD68 Drop. Change the CD68 anti-body reagent to Mouse IgG and save the protocol.Then, create six more new protocols, one for each drop control, and one for the complete iso-type control in the same manner. Make a drop control protocol for each target in the same way as I've shown you here. To prepare a research detection kit, fill the 30 milliliter open container marked for 1X tris-buffered saline with 1X tris-buffered saline, and place the container in the position farthest from the handle in the Multiplex Research Kit 1.Label two 30 milliliter open containers muli-spectral block'and multi-spectral secondary, and fill the mutli-spectral block container with 30 milliliters of blocking buffer antibody diluent from the Multi-Spectral Staining Kit. Fill the multi-spectral secondary container with 30 milliliters of anti-Mouse anti-rabid secondary antibody polymer from the Multi-spectral Staining Kit and add 600 microliters of antibody diluent to each of ten 7 milliliter open containers. Next, add the concentrated primary antibody to the appropriate tubes in the volumes indicated in the table, and mix by gentle pipetting.Add 3 milliliters of double distilled water, plus 12 drops of spectral dapi into one 7 milliliter open container, and 3 milliliters of peroxidase block into a second 7 milliliter open container. Re-suspend each liathalized floor with 75 microliters of dimethyl sulfoxide and mix by pipetting. Then, store the Tyramide signal amplification floor stocks at 4 degrees Celsius until ready.While preparing the individual TSA floor dilutions. Register all reagents on the auto-stainer, then place each container into a reagent rack and load all of the reagents onto the automated stainer. The stainer will detect the presence and confirm the volume of each reagent.Once all of the reagents have been loaded, activate the protocol to run overnight. At the end of the staining procedure, mount the samples with cover slips and load the samples into the Multi-Spectral Imaging Platform Scanner for scanning. When all of the samples have been scanned, the images will be formatted as qptiff files.Open an appropriate qptiff analysis software program, and click load slide'to open a five filter whole slide scan of the slide of interest. Log in and use the stamp tool to select five regions for multi-spectral imaging. Under Select for:select Acquisition'under Size in Fields:select 1x1 and under Field resolution'select 0.5 micrometers 20x.Based on this cytokeratin staining select several regions with both cytokeratin positive tumor tissue, and cytokeratin negative stromal tissue to be imaged from the overall scan. When all of the multi spectral images have been selected, switch to the Vectra software and change the task from scan'to multi-spectral imaging'for each slide. Then, click scan to perform the multi-spectral imaging collection.When the multi-spectral image acquisition is complete, use the spectral un-mixing software to open files within the multi-spectral image folder. Under image format'select multi-spectral'Under sample format'select fluorescence'And under Spectral Library Source'select inForm'To select the floors, select and load all six floors, and dapi from the sample library slides, and click prepare all. The spectral un-mixing software will perform spectral unmixing on all of the open images using the provided spectral profiles.Identify and annotate the auto-fluorescent spectral profile. Be sure to check the auto-fluorescent feature to insure that it is completely captured in the auto-fluorescent channel after un-mixing and to test different samplings until it is removed acceptably. Then assess the staining pattern against the chromogenic single stains of the same target in sequential slides of the same tissue with a pathologist.To assess the fluorescence intensity of each channel, use the counts tool to survey the open images. Then, return to the staining protocol, and adjust the TSA concentration to address any normalized counts that exceed or fail to reach the acceptable range. In this representative analysis the tonsil tissue demonstrated a clearly defined cytokeratin staining within the tonsil surface, squamous epithelium without background in the lymphoid tissue with cytokeratin and PDL-1 staining apparent in the reticulated epithelium of the crypts.The follicular germinal centers were easily recognizable and rich in Ki-67 positive lymphocytes. The macrophages present within the germinal centers were identified by their CD-68 expression with some macrophages also observed outside of the germinal center. CD-8 stained lymphocytes with a T-cell distribution and PD-1 expression strongly stained small lymphocytes clustered at the periphery of the germinal center, as well as scattered throughout the interfollicular region.After the expected staining pattern was confirmed in the tonsil tissue control, the same staining could then be evaluated in a lung cancer sample. Isotype negative controls and drop controls should also be evaluated;not only for the detection of background and auto-fluorescence, but also for umbrella effects and spectral bleed. Drop controls are important for evaluating potential artifacts in the staining due to the multi-plex immunofluorescence method during the optimization of a new multi-plex panel.Each drop control should show the same staining pattern as the full multi-plex panel, except for the single primary antibody which should be removed on each specific control. Immuno profiling with multi-spectral immunofluorescence allows for the stratification of tumors and regions by immune criteria which can then be investigated by massively multiplex genomic, transcriptomic, and proteomic studies. I've used multi-plex immunofluorescence to investigate mechanisms of action in oncology and auto-immune disorders which allows for the identification and characterization of immune and pathogenic cells in a single section in setu.General precautions should be taken when working with anti-bodies against human proteins, Tyramide and dapi, but no reagent or material in this protocol is known to be particularly hazardous.

automated-multiplex-immunofluorescence-panel-for-immuno-oncology

Research

JoVE Journal

Cancer Research

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 ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

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 ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

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.

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

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.

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

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 ...

Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture

To begin the tissue dissociation, collect one to two cubic centimeters of hepatocellular carcinoma or HTC tissue from untreated patients. Then place the tissue in the tissue preservation solution and maintain it at four degrees Celsius until processing. Now, preheat the ultra-low attachment surface cell culture plates for one hour in an incubator set at 37 degrees Celsius.Thaw the basement membrane extract overnight at four degrees Celsius. Using surgical scissors, cut the tumor tissue into small pieces on a Petri dish inside a laminar flow hood. Transfer these pieces into a 15-milliliter conical tube and add five to 10 milliliters of basal medium.With a barotropic pipette, wash away as much blood as possible. Allow the mixture to settle for one to two minutes before removing some of the supernatant, including any blood cells and floating fat clots. Next, centrifuge the mixture at 300 G for five minutes at room temperature.Then carefully extract the supernatant and add five milliliters of pre-warmed digestion solution to the trimmed tissue. Place the tube at 37 degrees Celsius in a rotor for optimum digestion. After 30 minutes of initial digestion, use a microscope to look for small cell clusters.To stop the digestion, add five milliliters of cold basal medium to the tube. Then use a 100-micrometer cell filter to sieve into a new 50-milliliter tube. Fill up the tube with cold basal medium to reach a total volume of 50 milliliters.Next, centrifuge the cells at two G for 10 minutes at eight degrees Celsius. Once complete, carefully remove the supernatant liquid. Re-suspend the pellet in 50 milliliters of cold basal medium to obtain the cell pellet for organoid plating.

Preparation of Slides for Multiplex Cyclic Immunohistochemistry of Cancer Tissue Sections

Multiplex Cyclic Fluorescent Immunohistochemical Staining of Cancer Tissue Sections

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.