The authors thank Donald R. Norwood of Editing Services, Research Medical Library at MD Anderson for editing this article and the multiplex IF and image analysis laboratory in the Department of Translational Molecular Pathology at MD Anderson. This project was supported in part by the Translational Molecular Pathology-Immunoprofiling laboratory (TMP-IL) Moonshots Platform at the Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center and the NCI Cooperative Agreement U24CA224285 (to the MD Anderson Cancer Center CIMAC).

<ol>
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The authors have no conflicts to disclose.

The TME plays an essential role in cancer development, progression, and treatment responses. Additionally, the density of specific tumor-infiltrating lymphocyte subsets in the TME can serve as a prognostic biomarker for certain types of cancer. Remarkably, in addition to the TME&#39;s cellular composition, the spatial characteristics of a tumor can provide an outline for understanding the tumor&#39;s biology and identifying potential prognostic biomarkers12,17.
As numerous immune cell populations are involved in procancer or anticancer responses, a better understanding of these cells and their spatial relationships with each other and with cancer cells will help guide the identification of new immunotherapeutic strategies. Previous studies have stratified the location and spatial distribution of TME cells based on the tissue structure in the intratumoral and peritumoral areas and the invasive margins of the tumor cells18,19. Over the past 15 years, technological advancements have made the phenotypic analysis of individual cells based on their spatial dispersal a novel, influential tool for studying the TME and categorizing potential biomarkers for tumor immunotherapy. Multiplex IF histochemistry can concurrently estimate multiple biological markers20.
Similar to the oligonucleotide-conjugated antibody strategy, four types of protein-based multiplex platforms are used to study the TME: chromogen-, fluorescence-, DNA barcode-, and metal isotope-labeled antibody detection systems. The cost-effective chromogenic IHC platforms enable whole-slide visualization and pathological assessment by using conventional bright-field microscopy. In multiplexed IF and IHC, antibodies conjugated with fluorophores are used. The multiplex IF/IHC platform detects antibodies with high specificity and can quantify targeted antibodies even at the subcellular level6,21. In addition, owing to the nature of chromogens and fluorophores, the use of one antibody panel can capture the expression of up to 10 biomarkers on a single slide. On metal isotope-based platforms, metal-tagged antibodies are used to perform multiplexed imaging with single-cell and spatial resolution, and high sensitivity for individual tissue sections22. Theoretically, these metal-conjugated antibody approaches enable the simultaneous detection of more than 100 biomarkers on a single tissue section. One challenge of the isotope-labeling technique is isobaric interference, which prevents 100% purity of enrichment from being reached23. Moreover, the interference increases as the number of markers increases. DNA-conjugated antibody detection platforms recognize antibodies labeled with unique DNA barcodes. More than 40 biomarkers can be simultaneously captured with high specificity on these platforms6.
Multiplexed imaging is a commercially available DNA barcode-labeled antibody detection platform for applying DNA-conjugated antibodies to a single tissue slide in one step (Figure 8). For the tissue preparation stage, unlike the multiplexed ion beam imaging platform, which requires the use of gold-coated slides obtained from manufacturers, the multiplexed imaging platform requires only regular coverslips or slides coated with 0.1% poly-L-lysine to help the tissue adhere to it and keep tissue intact during the staining and imaging process. The use of tissue sections on coverslips within 4 weeks after sectioning is recommended, as the prolonged storage of unstained slides results in antigenicity reduction. A stained coverslip sample can be maintained in storage buffer at 4 &#176;C for up to 2 weeks without losing its staining signal. No special equipment is required for the storage of the coverslip samples. The multiplexed imaging system has been upgraded to use regular slides instead of coverslips, which enables the staining of larger tissues and easy handling. When using a reduction solution for antibody conjugation (step 3.2.3), the reaction should be limited to no more than 30 min to prevent damage to antibodies. The blocking buffers in step 5.6.6 should be freshly prepared, and the blocking buffers must not be reused.
Compared with chromogen-, fluorescence-, and metal isotope-labeled multiplex antibody detection platforms, the multiplexed imaging technology has certain advantages. For example, more than 60 predesigned antibody panels for multiplexed imaging are commercially available, which helps save time and costs in antibody conjugation and validation, and the number of predesigned antibody panels is growing. These antibodies, which include the carcinoma marker pan-cytokeratin, the melanoma marker SOX10, the vascular marker CD31, the stromal marker SMA, and numerous immune cell markers, are validated and experiment-ready. For antibodies that are not predesigned, the commercially available conjugation kit designed for use with multiplexed imaging is straightforward and user-friendly. Customer-conjugated antibodies are good for 1 year when stored at 4 &#176;C. Additionally, machine warm-up is not required for capturing the images. In this multiplexed imaging technology, the iterative washing, hybridization, and stripping steps in the image acquisition rarely result in decreased marker intensity or degraded tissue morphology5,24,25. Furthermore, composite images are captured in QPTIFF format with a simple three-color fluorescence microscope and can be uploaded and analyzed using third-party digital analysis software. The staining markers can be visualized at single-cell resolution, and cell phenotypes can be characterized via the co-localization of the markers (Figure 6 and Figure 7). The comprehensive analysis of a multiplexed image further reveals the tissue compartments, single-cell marker quantification, and nearest neighbor and proximity data (Figure 8).
A challenge in multiplexed image analysis is cell-type identification. Usually, when more single-object classifiers are applied to an image, more uncommon phenotypes will be annotated. Therefore, using known markers that are not co-expressed in the same classifier and applying only the phenotype-related classifier to the annotation of single cells are recommended. Variations in cell-type annotation will result in substantially different spatial results, such asdifferences in cell spatial distribution and cellular neighborhood analysis26,27.
Multiplexed image analysis has proven to be successful in staining and imaging many sample types, including FFPE tissue, fresh frozen tissue, archived whole slides, and tissue microarrays. Multiplexed images of breast, brain, lung, spleen, kidney, lymph node, and skin tissue sections can be acquired with deep single-cell spatial phenotyping data5,16,25,28.
In the future, more predesigned antibodiesfor multiplexed imaging are expected. Additionally, the development of specific software for multiplexed image analysis is greatly needed. Currently, many commercially available and open-source software programs for Hi-Plex image analysis exist29, but scientists still need help in creating a standard workflow for these analyses30,31. Although the composite images captured using this protocol are compatible with third-party software, this may result in extra costs for the user. Another disadvantage of the multiplexed imaging technology is the signal reduction in nuclear protein detection after iterative washing, hybridization, and stripping with large panels of antibodies. Fortunately, this can be minimized by retrieving the barcoded fluorophores at early cycles when designing the reporter plates. Recently, this platform was upgraded with a new high-speed scanning system, which has dramatically reduced the time to obtain composite images32. Additionally, a new strategy using tyramide-conjugated barcodes has been reported to enhance the oligonucleotide-conjugated antibody barcoding-based imaging. This technology aims to amplify staining signals for which barcode-conjugated antibodies are difficult to obtain33.

The tumor microenvironment (TME) is extremely heterogeneous, consisting of tumor cells, tumor stromal cells, immune cells, noncellular components of the extracellular matrix, and numerous abundant molecules produced and released by tumor, stromal, and immune cells1,2. Accumulating evidence demonstrates that the TME has a pivotal role in reprogramming tumor differentiation, growth, invasion, metastasis, and response to therapies3.
Understanding how different cell types in the TME interact and communicate with each other through signaling networks is essential for improving cancer diagnosis, optimizing immunotherapy, and developing new treatments4. Traditional tissue microscopy techniques, including immunohistochemistry (IHC) and immunofluorescence (IF), have been used for decades to study the cell types, abundance, and communications in tumor samples. Unfortunately, these techniques typically can evaluate only one or two protein markers in a tissue section and cannot reveal the complex spatial and structural relationships among these cells5,8,7.
Over the past two decades, several multiplexed imaging technologies have been established8. These technologies provide much improved views of the composition, function, and location of immune cells within the TME, leading to rapid advancements in the ability to identify and spatially profile complex TMEs at the single-cell level9,10. The spatial and structural relationships of various tumor and immune cells in the TME are now at the forefront of biological and clinical studies using these multiplexed imaging technologies11,12.
The recently developed multiplexed imaging technology using oligonucleotide-conjugated antibody barcoding is an influential single-cell biological research platform based on the detection of oligonucleotide-conjugated antibodies in formalin-fixed, paraffin-embedded (FFPE) samples13,14. Currently, this multiplexed imaging technology allows for the simultaneous imaging of more than 100 markers in a single tissue section15, which has increased the number of cell types that are distinguishable in situ. This enables a level of spatial analysis of tumor and immune cells that is not possible using traditional immunophenotyping approaches16.
Herein, we describe an optimized protocol for conjugating purified antibodies to oligonucleotides and validating this conjugation using the multiplexed imaging platform and a multicycle imaging procedure with FFPE tissue. In addition, we describe the basic image processing and data analysis procedures&#160;used with this technology.

<table><tbody><tr><td>10x AR9 Buffer</td><td>Akoya Biosciences</td><td>AR900250ML</td><td></td></tr><tr><td>10x Buffer</td><td>Akoya Biosciences</td><td>7000001</td><td></td></tr><tr><td>16% paraformaldehyde</td><td>Thermo Fisher Scientific</td><td>28906</td><td></td></tr><tr><td>1X Antibody Diluent/Block</td><td>Akoya Biosciences</td><td>ARD1001EA</td><td></td></tr><tr><td>Antibody Conjugation Kit</td><td>Akoya Biosciences</td><td>7000009</td><td>Contains Filter Blocking Solution, Antibody Reduction Solution 1, Antibody Reduction Solution 2, Conjugation Solution, Purification Solution, Antibody Storage Solution</td></tr><tr><td>Assay Reagent</td><td>Akoya Biosciences</td><td>7000002</td><td></td></tr><tr><td>Diethyl pyrocarbonate</td><td>Sigma-Aldrich</td><td>40718-25ML</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Avantor/VWR</td><td>BDH1115-4LP</td><td></td></tr><tr><td>Ethanol, 200 proof</td><td></td><td></td><td></td></tr><tr><td>G Blocker V2</td><td>Akoya Biosciences</td><td>240199</td><td></td></tr><tr><td>Histoclear</td><td>Thermo Fisher Scientific</td><td>50-329-50</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860-1L-R</td><td></td></tr><tr><td>Milli-Q Integral 10&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>ZRXQ010WW</td><td></td></tr><tr><td>Niknon Fluorescence microscope</td><td>Keyence Corp. of America</td><td>BZ-X810</td><td></td></tr><tr><td>Nuclear Stain</td><td>Akoya Biosciences</td><td>7000003</td><td></td></tr><tr><td>Nuclease-free water</td><td>Thermo Fisher Scientific</td><td>AM9938</td><td></td></tr><tr><td>NuPAGE</td><td>Thermo Fisher Scientific</td><td>NP0008</td><td></td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>14190136</td><td></td></tr><tr><td>PhenoCycler Barcodes/Reporters Combination</td><td>Akoya Biosciences</td><td>5450004 (BX025/RX025)</td><td></td></tr><tr><td></td><td></td><td>5450003 (BX022/RX022)</td><td></td></tr><tr><td></td><td></td><td>5450023 (BX002/RX002)</td><td></td></tr><tr><td></td><td></td><td>&amp;nbsp;5250002 (BX020/RX020)</td><td></td></tr><tr><td></td><td></td><td>2520003 (BX023/RX023)</td><td></td></tr><tr><td></td><td></td><td>5250005 (BX029/RX029)</td><td></td></tr><tr><td></td><td></td><td>5250007 (BX035/RX035)</td><td></td></tr><tr><td></td><td></td><td>5250012 (BX052/RX052)</td><td></td></tr><tr><td></td><td></td><td>5550012 (BX030/RX030)</td><td></td></tr><tr><td></td><td></td><td>5550015 (BX042/RX042)</td><td></td></tr><tr><td></td><td></td><td>5550014 (BX036/RX036)</td><td></td></tr><tr><td>QuPath</td><td>Open-Source</td><td>https://qupath.github.io/</td><td></td></tr><tr><td>SimplyBlue SafeStain</td><td>Thermo Fisher Scientific</td><td>LC6065</td><td></td></tr><tr><td>Staining Kit</td><td>(Akoya Biosciences</td><td>7000008</td><td>Contains Hydration Buffer, N Blocker, J Blocker, S Blocker, Fixative Reagent, Storage Buffer</td></tr></tbody></table>

multiplexed barcoding image analysis for immunoprofiling and spatial mapping characterization in the single-cell analysis of paraffin tissue samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

We employed FFPE tonsil samples to develop a 26 marker immune oncology panel to illustrate the immune status of FFPE tissue using a barcoding image analysis system. Overall, 19 antibodies are currently used in other multiplexed imaging studies in our lab. All of the markers have been tested using FFPE tissue with chromogenic IHC. All the antibodies were conjugated to unique DNA oligonucleotides. When setting up the coverslips using the web-based instrument manager (Figure 4) for this barcoding image analysis technology, it should be noted that the first and last cycles are always &#34;blank&#34; (Figure 2 and Figure 4), which provides the background fluorescence signals to be subtracted from the specific signals from the antibodies. After images in QPTIFF format have been collected with the fluorescence microscope, they can be visualized using several patented automated image analysis software or open-source software programs. Composite images can show all the markers or selected markers for a better view of the signals (Figure 5). Moreover, each antibody can be visually evaluated for nuclear, cytoplasmic, or membranous localization. Immune, tumor, and stromal cells can be easily identified. Subsequently, image analysis can provide information on the signal intensity, dynamic range, and spatial distribution of all the markers (Figure 6). This technique allowed us to analyze all 26 markers at the subcellular level in a single tissue section (Figure 7). By analyzing the markers&#39; co-localization, we could identify the cellular phenotypes, localize the spatial cell position, calculate the distance between cells, and find the distribution of the cells. The crucial impact of this technology is the presentation of a robust 26 marker panel focused on the immune status of the tissue microenvironment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64758/64758fig01.jpg" /> 
Figure 1: Image of custom-conjugated antibody validation using a Bis-Tris protein gel. Lane 1 of the gel shows the protein standard. Lane 2 and Lane 4 show barcode-conjugated antibodies (arrows). Lane 3 and Lane 5 show the heavy and light chain bands from an unconjugated antibody (arrowheads). <a href="https://www.jove.com/files/ftp_upload/64758/64758fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64758/64758fig02.jpg" /> 
Figure 2: Staining plate configuration map for two coverslip samples. Abbreviations: HB = hydration buffer; B = antibody diluent/block; PSFS = post-staining fixative solution; PBS = phosphate-buffered saline; MeOH = methanol; S = storage solution. <a href="https://www.jove.com/files/ftp_upload/64758/64758fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64758/64758fig03.jpg" /> 
Figure 3: Reporter plate configuration. Each conjugated antibody has a barcode that is complementary to a specific reporter. To set up the reporter plate, every conjugated antibody and its corresponding reporter should be listed. Next, each antibody is assigned a cycle number. The performance of two blank cycles (C1 and C18) is used to evaluate the level of autofluorescence in the three fluorescence channels and for post-imaging background subtraction using the image acquisition control software. A software wizard will check the instrument at this stage to ensure all the settings are correct (Figure 4). <a href="https://www.jove.com/files/ftp_upload/64758/64758fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64758/64758fig04.jpg" /> 
Figure 4: Image acquisition using the control software for the experiment setup. (A) Select the Experiment tab in the left-bottom corner of the control software to prepare and start the setup. (B,C) Select New Template to input the experimental settings with a new project and experiment name. (D) Change the start cycle well and the number of cycles to reflect the reporter location in the 96-well reporter plate. (E) Assign the proper fluorescence channels to the four channels designated for the experimental run. <a href="https://www.jove.com/files/ftp_upload/64758/64758fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64758/64758fig05.jpg" /> 
Figure 5: Image visualization using web-based software (QuPath). The viewer window shows 26 markers in the stained tonsil tissue FFPE section. The Brightness &#38; Contrast window shows the markers with the checkmarks. Finally, the viewer window shows the FFPE sample with the selected markers. <a href="https://www.jove.com/files/ftp_upload/64758/64758fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64758/64758fig06.jpg" /> 
Figure 6: Image visualization using web-based software. (A) Tonsil tissue sections were stained for the 26 markers, and the images of the sections in QPTIFF format were visualized using commercial digital slide viewer software or open-source software (QuPath) for annotation and review. (B-F) Six markers were displayed in the same annotation for a better view of the signals. <a href="https://www.jove.com/files/ftp_upload/64758/64758fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64758/64758fig07.jpg" /> 
Figure 7: Views of the 26 individual markers used with web-based software. The marker expression in the tonsil tissue is shown via immunofluorescence staining with an immune oncology panel (top left). Individual markers in two small areas (red rectangles) are shown. The zoomed-in insert shows cells positive for these markers (white arrows). <a href="https://www.jove.com/files/ftp_upload/64758/64758fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64758/64758fig08.jpg" /> 
Figure 8: Summary of the multiplexed image acquisition workflow. FFPE tissue sections were stained using the 26 antibody panel followed by a multicycle reaction. Raw images of the stained sections were computationally processed, and a cell density and spatial analysis was performed using the composite images. <a href="https://www.jove.com/files/ftp_upload/64758/64758fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: IHC validation in tonsil tissue. FFPE tissue sections were stained using an individual antibody. The marker expression in the tonsil tissue is shown at a low magnification, and the zoomed-in insert shows cells positive for the marker (red rectangles). <a href="https://www.jove.com/files/ftp_upload/64758/Suppl Figure.psd" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples at JoVE.c

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

multiplexed-barcoding-image-analysis-for-immunoprofiling-spatial

Research

JoVE Journal

Cancer Research

1.5K Views.  The University of Texas MD Anderson Cancer Center. De grote hoeveelheid biologische informatie die wordt verkregen uit de multiplex barcoding beeldanalyse stelt wetenschappers in staat om de micro-omgeving van de tumor en de speciale verdeling binnen tumorcellen beter te begrijpen. Het belangrijkste voordeel van deze methode is dat het ons veel meer gedetailleerde informatie geeft van hetzelfde weefselmonster met de mogelijkheid om diepere fenotypering van individuele cellen te doen. Deze methode stelde aanpasbare panelen in staat om de micro...

Video: Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

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

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

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

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

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

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 ...

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 ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Developmental Biology

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 ...

Immunology and Infection

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.

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

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

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