Name: spatial profiling of protein and rna expression in tissue: an approach to fine-tune virtual microdissection
Uploaded: 2022-07-06T12:30:00
Description: Watch this Scientific Journal Video about Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection at JoVE.com

The authors acknowledge Thomas Wu for processing NGS files. We thank James Ziai for the results discussions and manuscript review and Meredith Triplet and Rachel Taylor for internal manuscript revision.

All human tissues were acquired from commercial biobanks or accredited tissue banks under warranty that appropriate Institutional Review Board approval and informed consent were obtained.
NOTE: The protocol is performed using the Discovery Ultra and the GeoMx Digital Spatial Profiler. See the Table of Materials for details about reagents, equipment, and software used in this protocol.
1. Automated visualization protocol for spatial proteomics ...

<ol>
	<li>Nerurkar, S. N., 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=Transcriptional+spatial+profiling+of+cancer+tissues+in+the+era+of+immunotherapy:+The+potential+and+promise.">Transcriptional spatial profiling of cancer tissues in the era of immunotherapy: The potential and promise.</a> Cancers. 12 (9), 2572 (2020).</li><li>Decalf, J., Albert, M. L., Ziai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+pathology:+a+user's+review+of+a+highly+multiplexed+method+for+in+situ+analysis+of+protein+and+RNA+expression+in+tissue.">New tools for pathology: a user's review of a highly multiplexed method for in situ analysis of protein and RNA expression in tissue.</a> Journal of Pathology. 247 (5), 650-661 (2019).</li><li>McGinnis, L. M., Ibarra-Lopez, V., Rost, S., Ziai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+research+applications+of+multiplexed+immunohistochemistry+and+in+situ+hybridization.">Clinical and research applications of multiplexed immunohistochemistry and in situ hybridization.</a> Journal of Pathology. 254 (4), 405-417 (2021).</li><li>. NanoString. GeoMx Digital Spatial Profiler Available from: <a target="_blank" href="https://nanostring.com/products/geomx-digital-spatial-profiler/geomx-dsp-overview">https://nanostring.com/products/geomx-digital-spatial-profiler/geomx-dsp-overview</a> (2022)</li><li>. NanoString. GeoMx Digital Spatial Profiler Available from: <a target="_blank" href="https://nanostring.com/wp-content/uploads/BR_MK0981_GeoMx_Brochure_r19_FINAL_Single_WEB.pdf">https://nanostring.com/wp-content/uploads/BR_MK0981_GeoMx_Brochure_r19_FINAL_Single_WEB.pdf</a> (2022)</li><li>McCart Reed, A. E., 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=Digital+spatial+profiling+application+in+breast+cancer:+a+user's+perspective.">Digital spatial profiling application in breast cancer: a user's perspective.</a> Virchows Arch: An International Journal of Pathology. 477 (6), 885-890 (2020).</li><li>McNamara, K. L., 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=Spatial+proteomic+characterization+of+HER2-positive+breast+tumors+through+neoadjuvant+therapy+predicts+response.">Spatial proteomic characterization of HER2-positive breast tumors through neoadjuvant therapy predicts response.</a> Nature Cancer. 2 (4), 400-413 (2021).</li><li>Kim, S. W., Roh, J., Park, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+for+pathologists:+Protocols,+pitfalls,+and+tips.">Immunohistochemistry for pathologists: Protocols, pitfalls, and tips.</a> Journal of Pathology and Translational Medicine. 50 (6), 411-418 (2016).</li><li>Mu&#241;oz, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecularly+targeted+photothermal+ablation+improves+tumor+specificity+and+immune+modulation+in+a+rat+model+of+hepatocellular+carcinoma.">Molecularly targeted photothermal ablation improves tumor specificity and immune modulation in a rat model of hepatocellular carcinoma.</a> Communications Biology. 3 (1), 783 (2020).</li><li>Coleman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronidase+impairs+neutrophil+function+and+promotes+Group+B+Streptococcus+invasion+and+preterm+labor+in+nonhuman+primates.">Hyaluronidase impairs neutrophil function and promotes Group B Streptococcus invasion and preterm labor in nonhuman primates.</a> mBio. 12 (1), 03115-03120 (2021).</li><li>Gupta, S., Zugazagoitia, J., Martinez-Morilla, S., Fuhrman, K., Rimm, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+quantitative+assessment+of+PD-L1+using+digital+spatial+profiling.">Digital quantitative assessment of PD-L1 using digital spatial profiling.</a> Laboratory Investigation; A Journal of Technical Methods and Pathology. 100 (10), 1311-1317 (2020).</li><li>Carter, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+spatially+defined+immune+(micro)landscapes+of+early-stage+PD-L1-positive+triple-negative+breast+cancer.">Characteristics and spatially defined immune (micro)landscapes of early-stage PD-L1-positive triple-negative breast cancer.</a> Clinical Cancer Research. 27 (20), 5628-5637 (2021).</li><li>Busse, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprofiling+in+neuroendocrine+neoplasms+unveil+immunosuppressive+microenvironment.">Immunoprofiling in neuroendocrine neoplasms unveil immunosuppressive microenvironment.</a> Cancers. 12 (11), 3448 (2020).</li><li>Kulasinghe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+lung+SARS-CoV-2+and+influenza+virus+infection+dissects+virus-specific+host+responses+and+gene+signatures.">Profiling of lung SARS-CoV-2 and influenza virus infection dissects virus-specific host responses and gene signatures.</a> European Respiratory Journal. 59 (1), (2021).</li><li>Li, X., Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+bulk,+single-cell+to+spatial+RNA+sequencing.">From bulk, single-cell to spatial RNA sequencing.</a> International Journal of Oral Science. 13 (36), (2021).</li><li>Merritt, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+digital+spatial+profiling+of+proteins+and+RNA+in+fixed+tissue.">Multiplex digital spatial profiling of proteins and RNA in fixed tissue.</a> Nature Biotechnology. 38 (5), 586-599 (2020).</li><li>Van, T. M., Blank, C. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+perspective+on+GeoMxTM+digital+spatial+profiling.">A user's perspective on GeoMxTM digital spatial profiling.</a> Immuno-Oncology Technology. 1, 11-18 (2019).</li><li>Introduction to GeoMx Normalization: Protein. White Paper. Nanostring Available from: <a target="_blank" href="https://nanostring.com/wp-content/uploads/MK2593_GeoMx_Normalization-Protein.pdf">https://nanostring.com/wp-content/uploads/MK2593_GeoMx_Normalization-Protein.pdf</a> (2020)</li><li>Bergholtz, H., 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=Best+practices+for+spatial+profiling+for+breast+cancer+research+with+the+geomx+spatial+profiler.">Best practices for spatial profiling for breast cancer research with the geomx spatial profiler.</a> Cancers. 13 (17), 4456 (2021).</li><li>Hwang, W. L., 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=Single-nucleus+and+spatial+transcriptomics+of+archival+pancreatic+cancer+reveals+multi-compartment+reprogramming+after+neoadjuvant+treatment.">Single-nucleus and spatial transcriptomics of archival pancreatic cancer reveals multi-compartment reprogramming after neoadjuvant treatment.</a> BioRxiv. , (2020).</li><li>Jerby-Arnon, L., 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=Opposing+immune+and+genetic+mechanisms+shape+oncogenic+programs+in+synovial+sarcoma.">Opposing immune and genetic mechanisms shape oncogenic programs in synovial sarcoma.</a> Nature Medicine. 27 (2), 289-300 (2021).</li></ol>

To date, directly conjugated fluorescent antibodies in a manual protocol are most commonly used as visualization panels for spatial proteomics or spatial transcriptomics assays9,10. However, the use of directly conjugated fluorescent antibodies can be challenging for less abundant markers, limiting the selection of suitable antibodies. This protocol shows that labeling of visualization markers can be automated on an automated staining platform using TSA technolog...

Advances in multiplexing techniques continue to provide better characterization tools for targets present in tumors. The tumor microenvironment (TME) is a complex system of tumor cells, infiltrating immune cells, and stroma, where spatial information is critical to better understand and interpret mechanisms of interaction between biomarkers of interest1. With emerging techniques such as the GeoMx Digital Spatial Profiler (DSP) and 10x Visium, multiple targets can be detected and quantified simultaneously within their spatial context. The use of immunofluorescence protocols that facilitate tissue visualization can further improve the spatial profiling capabilities of these technologies.
The Spatial Omics technology we focused on for this method development consists of spatial proteomics and transcriptomics assays where oligonucleotides are attached to antibodies or RNA probes via a UV-sensitive photocleavable linker. Histological slides are labeled with these oligo-conjugated antibodies or probes and then imaged on the spatial profiling platform. Next, ROIs of different sizes and shapes are selected for illumination, and the photocleaved oligonucleotides are aspirated and collected in a 96-well plate. The photocleaved oligonucleotides are prepared to be quantified with either the Nanostring nCounter system or Next Generation Sequencing (NGS)2,3 (Figure 1)4,5.
Cell distributions vary within tissues, and the ability to characterize specific locations of cells using selected markers and different ROI sizes is of great importance to fully understand the tissue environment and identify specific features. In the Spatial Omics technology mentioned here, the standard visualization protocol uses directly conjugated antibodies and is a manual protocol. The standard markers to distinguish between tumor and stroma are panCytokeratin (panCK) and CD456,7, but additional markers are necessary to target specific cell populations of interest. Furthermore, the use of directly conjugated fluorescent antibodies lacks amplification, which limits antibody selection to abundant markers. Additionally, manual assays are subject to more variability than automated workflows8. Therefore, it is desirable to have a customizable, automated, and amplified visualization protocol for ROI selection.
Here, the study demonstrates that, for spatial proteomics assays, TSA technology can be used for visualization protocols on an automated platform resulting in a more targeted and standardized assay. In addition, TSA based assays enable the use of low-expressing markers, increasing the range of targets that can be selected for visualization. A 3-plex assay for panCK, FAP, and Antibody X was developed using an automated platform where panCK and FAP were used to differentiate between tumor and stroma, respectively. Antibody X is a stromal protein frequently encountered in tumors, but its biology and impact on anti-tumor immunity are not fully understood. Characterizing the immune contexture in areas rich in Antibody X can elucidate its role in anti-tumor immunity and therapeutic response, as well as its potential as a drug target.
While customized automated TSA visualization panels proved to be successful for spatial proteomics assays, the application of these assays for spatial transcriptomics assays could not be confirmed. This is most likely due to the reagents and the protocol used for the automated visualization protocols, which seem to compromise RNA integrity. Recognizing that an automated labeling protocol for visualization markers can be used for spatial proteomics assays but not for spatial transcriptomics assays provides important guidance on Spatial Omics technology assay designs.
Additionally, the study demonstrates that the spatial transcriptomics assay can be used to profile targets in regions as small as 50 &#181;m in diameter, or approximately 15 cells. Two different-sized ROIs were selected to test the ability of the assay to also detect transcripts in small ROIs. For each region of interest, oligos corresponding to 1,800 mRNA targets were collected and made into libraries according to the spatial profiling platform protocol. Libraries were individually indexed, subsequently pooled, and sequenced. This allowed the evaluation of both pooling efficiency and the capability of identifying specific cell populations in small ROIs.
This paper shows that for spatial proteomics assays, an automated protocol to guide ROI selection on specific markers of interest can be used to selectively target the interrogation of relevant tissue areas and characterize the spatial environment of the tissue. Furthermore, we demonstrate that smaller ROIs can be used for spatial transcriptomic assays to detect and characterize specific cell populations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Tris buffered saline (TBS)</td>
			<td>Cell Signaling Technologies</td>
			<td>12498S</td>
			<td>Diluted to 1x TBS in DEPC treated water</td>
		</tr>
		<tr>
			<td>Antibody X (not disclosed)</td>
			<td></td>
			<td></td>
			<td>antibody blinded due to confidentiality</td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>ThermoFisher</td>
			<td>AM9922</td>
			<td>Another can be used</td>
		</tr>
		<tr>
			<td>DISCOVERY Cell Conditioning ( CC1)</td>
			<td>Ventana</td>
			<td>950-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DISCOVERY Cy5 Kit</td>
			<td>Ventana</td>
			<td>760-238</td>
			<td>Referred as Cy5</td>
		</tr>
		<tr>
			<td>DISCOVERY FAM Kit</td>
			<td>Ventana</td>
			<td>760-243</td>
			<td>Referred as FAM</td>
		</tr>
		<tr>
			<td>DISCOVERY Goat Ig Block</td>
			<td>Ventana</td>
			<td>760-6008</td>
			<td>Referred as Gt Ig Block</td>
		</tr>
		<tr>
			<td>DISCOVERY OmniMap anti-Ms HRP</td>
			<td>Ventana</td>
			<td>760-4310</td>
			<td>Referred as OMap anti-Ms HRP</td>
		</tr>
		<tr>
			<td>DISCOVERY OmniMap anti-Rb HRP</td>
			<td>Ventana</td>
			<td>760-4311</td>
			<td>Referred as OMap anti-Rb HRP</td>
		</tr>
		<tr>
			<td>DISCOVERY Rhodamine 6G Kit</td>
			<td>Ventana</td>
			<td>760-244</td>
			<td>Referred as Rhodamine 6G</td>
		</tr>
		<tr>
			<td>DISCOVERY ULTRA Automated Slide Preparation System</td>
			<td>Ventana</td>
			<td>05 987 750 001 / N750-DISU-FS</td>
			<td>Referred as autostainer on the manuscript</td>
		</tr>
		<tr>
			<td>FAP [EPR20021] Antibody</td>
			<td>Abcam</td>
			<td>Ab207178</td>
			<td></td>
		</tr>
		<tr>
			<td>GeoMx Digital Spatial Profiler</td>
			<td>NanoString</td>
			<td>GMX-DSP-1Y</td>
			<td>Referred as spatial profiling platform on the manuscript</td>
		</tr>
		<tr>
			<td>Humidity chamber</td>
			<td>Simport</td>
			<td>M920-2</td>
			<td>Another can be used</td>
		</tr>
		<tr>
			<td>Pan-Cytokeratin [AE1/AE3] Antibody</td>
			<td>Abcam</td>
			<td>Ab27988</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Python</td>
			<td>Python</td>
			<td></td>
			<td>Statistical analysis</td>
		</tr>
		<tr>
			<td>Reaction Buffer (10x)</td>
			<td>Ventana</td>
			<td>950-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical analysis software</td>
			<td>GraphPad</td>
			<td>Prism 7</td>
			<td>Statistical analysis</td>
		</tr>
		<tr>
			<td>SYTO 64</td>
			<td>ThermoFisher</td>
			<td>S11346</td>
			<td></td>
		</tr>
		<tr>
			<td>ULTRA Cell Conditioning (ULTRA CC2)</td>
			<td>Ventana</td>
			<td>950-223</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventana Antibody Diluent with Casein</td>
			<td>Ventana</td>
			<td>760-219</td>
			<td>Referred as specified diluent on the manuscript</td>
		</tr>
		<tr>
			<td>Ventana Primary antibody dispenser</td>
			<td>Ventana</td>
			<td></td>
			<td>Catalog number depends on dispenser number</td>
		</tr>
	</tbody>
</table>

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.

Automated visualization protocol to guide ROI selection 
In this paper, we present the use of an automated, custom TSA-based IF protocol to visualize the tissue and select specific ROIs. Visualization panel development using melanoma and human normal skin as control tissues consisted of epitope stability testing, fine-tuning of marker intensities, and bleedthrough assessment through leave one out controls. To test if epitope stability of the antibodies is affected by repetitive elution steps, FAP an...

Watch this Scientific Journal Video about Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection at JoVE.com

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

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 ...

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