Editorial support was provided by Deborah Shuman of MedImmune.

<ol>
	<li>Bethmann, D., Feng, Z., Fox, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprofiling+as+a+predictor+of+patient's+response+to+cancer+therapy-promises+and+challenges.">Immunoprofiling as a predictor of patient's response to cancer therapy-promises and challenges.</a> Current Opinion in Immunology. 45, 60-72 (2017).</li><li>Taube, J. 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=Implications+of+the+tumor+immune+microenvironment+for+staging+and+therapeutics.">Implications of the tumor immune microenvironment for staging and therapeutics.</a> Modern Pathology. 31 (2), 214-234 (2018).</li><li>Idikio, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+in+diagnostic+surgical+pathology:+contributions+of+protein+life-cycle,+use+of+evidence-based+methods+and+data+normalization+on+interpretation+of+immunohistochemical+stains.">Immunohistochemistry in diagnostic surgical pathology: contributions of protein life-cycle, use of evidence-based methods and data normalization on interpretation of immunohistochemical stains.</a> International Journal of Clinical and Experimental Pathology. 3 (2), 169-176 (2009).</li><li>Rebelatto, M. 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=Development+of+a+programmed+cell+death+ligand-1+immunohistochemical+assay+validated+for+analysis+of+non-small+cell+lung+cancer+and+head+and+neck+squamous+cell+carcinoma.">Development of a programmed cell death ligand-1 immunohistochemical assay validated for analysis of non-small cell lung cancer and head and neck squamous cell carcinoma.</a> Diagnostic Pathology. 11 (1), 95 (2016).</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=Immunohistochemical+and+image+analysis-based+study+shows+that+several+immune+checkpoints+are+co-expressed+in+non-small+cell+lung+carcinoma+tumors.">Immunohistochemical and image analysis-based study shows that several immune checkpoints are co-expressed in non-small cell lung carcinoma tumors.</a> Journal of Thoracic Oncology. 13 (6), 779-791 (2018).</li><li>Rehman, J. 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. J., 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=Eight-color+multiplex+immunohistochemistry+for+simultaneous+detection+of+multiple+immune+checkpoint+molecules+within+the+tumor+microenvironment.">Eight-color multiplex immunohistochemistry for simultaneous detection of multiple immune checkpoint molecules within the tumor microenvironment.</a> Journal of Immunology. 200 (1), 347-354 (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=Effect+of+neoadjuvant+chemotherapy+on+the+immune+microenvironment+in+non-small+cell+lung+carcinomas+as+determined+by+multiplex+immunofluorescence+and+image+analysis+approaches.">Effect of neoadjuvant chemotherapy on the immune microenvironment in non-small cell lung carcinomas as determined by multiplex immunofluorescence and image analysis approaches.</a> Journal for ImmunoTherapy of Cancer. 6 (1), 48 (2018).</li><li>Rimm, D., Schalper, K., Pusztai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unvalidated+antibodies+and+misleading+results.">Unvalidated antibodies and misleading results.</a> Breast Cancer Research and Treatment. 147 (2), 457-458 (2014).</li><li>Baskin, D. G., Hewitt, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+state+of+the+science+of+immunohistochemistry:+the+Histochemical+Society's+standards+of+practice.">Improving the state of the science of immunohistochemistry: the Histochemical Society's standards of practice.</a> Journal of Histochemistry &amp; Cytochemistry. 62 (10), 691-692 (2014).</li><li>Freedman, L. P., 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=The+need+for+improved+education+and+training+in+research+antibody+usage+and+validation+practices.">The need for improved education and training in research antibody usage and validation practices.</a> Biotechniques. 61 (1), 16-18 (2016).</li><li>Hewitt, S. 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This work was supported by MedImmune, the global biologics R&amp;D arm of AstraZeneca. C.W., K.R., and C.C.H. are employees of Perkin Elmer, which produces the reagents (TSA kit) and multispectral scanners that were used in this work. M.S., K.D., A.H., W.Z., C. Bagnall, C. Brown, J.C., A.L., K.S., M.R., and J.R.C. are employees of MedImmune with stock ownership and/or stock interests or options in AstraZeneca.

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

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

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Cancer Research

20.5K Views.  MedImmune. Dit protocol is belangrijk omdat de optimalisatie en overdracht van multiplex immunofluorescentie protocollen complex en uitdagend is. Voor veel laboratoria is het vasthouden van een geoptimaliseerd protocol van onschatbare waarde. Multispectrale immunofluorescentie stelt ons in staat om zowel de locatie als de identiteit van meerdere celtypen in en rond de tumor te beoordelen.Dit is een krachtige combinatie van informatie in immuunoncologiestudies. Het meest dwingende gebruik voor mul...

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

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

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 ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of ...

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 ...

Mapping the Tumor Immune Landscape: An In-Depth Multiplex IHC Analysis Protocol

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...

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 ...

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Immunology and Infection

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

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

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