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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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+updated+landscape+of+tumor+microenvironment+and+drug+repurposing.">The updated landscape of tumor microenvironment and drug repurposing.</a> Signal Transduction and Targeted Therapy. 5 (1), 166 (2020).</li><li>Waldman, A. D., Fritz, J. M., Lenardo, M. 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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=Overview+of+multiplex+immunohistochemistry/immunofluorescence+techniques+in+the+era+of+cancer+immunotherapy.">Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy.</a> Cancer Communications. 40 (4), 135-153 (2020).</li><li>Radtke, 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=IBEX:+A+versatile+multiplex+optical+imaging+approach+for+deep+phenotyping+and+spatial+analysis+of+cells+in+complex+tissues.">IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (52), 33455-33465 (2020).</li><li>Allam, M., Cai, S., Coskun, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+bioimaging+of+single-cell+spatial+profiles+for+precision+cancer+diagnostics+and+therapeutics.">Multiplex bioimaging of single-cell spatial profiles for precision cancer diagnostics and therapeutics.</a> NPJ Precision Oncology. 4, 11 (2020).</li><li>Eng, 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=A+framework+for+multiplex+imaging+optimization+and+reproducible+analysis.">A framework for multiplex imaging optimization and reproducible analysis.</a> Communications Biology. 5 (1), 438 (2022).</li><li>Taube, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td>Millipore&#160;</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>&#160;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

1.4K Views.  The University of Texas MD Anderson Cancer Center. 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 analysis technique.

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

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

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. ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...