Developing an enhanced 7-color multiplex IHC protocol to dissect immune infiltration in human cancers
The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among ind...
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description | The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa. |
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This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0247238</identifier><identifier>PMID: 33596250</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Alcohol ; Animal species ; Antibodies ; Antigens ; Automation ; Biological markers ; Biology and Life Sciences ; Biomarkers ; Breast cancer ; Buffers ; Cancer ; Cancer immunotherapy ; Citric acid ; Data analysis ; Editing ; Fluorescence ; Head & neck cancer ; Head and neck cancer ; Heat ; Heat treatment ; Immune checkpoint ; Immunohistochemistry ; Immunotherapy ; Kidney cancer ; Lung cancer ; Lung diseases ; Medical prognosis ; Medicine and Health Sciences ; Melanoma ; Metastases ; Methodology ; Methods ; Microwaves ; Non-small cell lung carcinoma ; Oncology, Experimental ; Paraffin ; Paraffins ; Polymers ; Protocol ; Protocol (computers) ; Renal cell carcinoma ; Research and Analysis Methods ; Retrieval ; Reviews ; Small cell lung carcinoma ; Squamous cell carcinoma ; Staining ; Stains & staining ; Tissues</subject><ispartof>PloS one, 2021-02, Vol.16 (2), p.e0247238-e0247238</ispartof><rights>COPYRIGHT 2021 Public Library of Science</rights><rights>2021 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2021 Sun et al 2021 Sun et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c758t-cf0b07c0ad17319276b25d5811ea8de2790cdb0c373c28daf49bbcb8c7f163173</citedby><cites>FETCH-LOGICAL-c758t-cf0b07c0ad17319276b25d5811ea8de2790cdb0c373c28daf49bbcb8c7f163173</cites><orcidid>0000-0002-2572-1731</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888634/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888634/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33596250$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Schmitt, Nicole</contributor><creatorcontrib>Sun, Zhaoyu</creatorcontrib><creatorcontrib>Nyberg, Richard</creatorcontrib><creatorcontrib>Wu, Yaping</creatorcontrib><creatorcontrib>Bernard, Brady</creatorcontrib><creatorcontrib>Redmond, William L</creatorcontrib><title>Developing an enhanced 7-color multiplex IHC protocol to dissect immune infiltration in human cancers</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa.</description><subject>Alcohol</subject><subject>Animal species</subject><subject>Antibodies</subject><subject>Antigens</subject><subject>Automation</subject><subject>Biological markers</subject><subject>Biology and Life Sciences</subject><subject>Biomarkers</subject><subject>Breast cancer</subject><subject>Buffers</subject><subject>Cancer</subject><subject>Cancer immunotherapy</subject><subject>Citric acid</subject><subject>Data analysis</subject><subject>Editing</subject><subject>Fluorescence</subject><subject>Head & neck cancer</subject><subject>Head and neck cancer</subject><subject>Heat</subject><subject>Heat treatment</subject><subject>Immune checkpoint</subject><subject>Immunohistochemistry</subject><subject>Immunotherapy</subject><subject>Kidney cancer</subject><subject>Lung cancer</subject><subject>Lung diseases</subject><subject>Medical prognosis</subject><subject>Medicine and Health Sciences</subject><subject>Melanoma</subject><subject>Metastases</subject><subject>Methodology</subject><subject>Methods</subject><subject>Microwaves</subject><subject>Non-small cell lung carcinoma</subject><subject>Oncology, Experimental</subject><subject>Paraffin</subject><subject>Paraffins</subject><subject>Polymers</subject><subject>Protocol</subject><subject>Protocol (computers)</subject><subject>Renal cell carcinoma</subject><subject>Research and Analysis Methods</subject><subject>Retrieval</subject><subject>Reviews</subject><subject>Small cell lung carcinoma</subject><subject>Squamous cell carcinoma</subject><subject>Staining</subject><subject>Stains & 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enhanced 7-color multiplex IHC protocol to dissect immune infiltration in human cancers</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2021-02-17</date><risdate>2021</risdate><volume>16</volume><issue>2</issue><spage>e0247238</spage><epage>e0247238</epage><pages>e0247238-e0247238</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>33596250</pmid><doi>10.1371/journal.pone.0247238</doi><tpages>e0247238</tpages><orcidid>https://orcid.org/0000-0002-2572-1731</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Alcohol Animal species Antibodies Antigens Automation Biological markers Biology and Life Sciences Biomarkers Breast cancer Buffers Cancer Cancer immunotherapy Citric acid Data analysis Editing Fluorescence Head & neck cancer Head and neck cancer Heat Heat treatment Immune checkpoint Immunohistochemistry Immunotherapy Kidney cancer Lung cancer Lung diseases Medical prognosis Medicine and Health Sciences Melanoma Metastases Methodology Methods Microwaves Non-small cell lung carcinoma Oncology, Experimental Paraffin Paraffins Polymers Protocol Protocol (computers) Renal cell carcinoma Research and Analysis Methods Retrieval Reviews Small cell lung carcinoma Squamous cell carcinoma Staining Stains & staining Tissues |
title | Developing an enhanced 7-color multiplex IHC protocol to dissect immune infiltration in human cancers |
url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-02T21%3A08%3A28IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Developing%20an%20enhanced%207-color%20multiplex%20IHC%20protocol%20to%20dissect%20immune%20infiltration%20in%20human%20cancers&rft.jtitle=PloS%20one&rft.au=Sun,%20Zhaoyu&rft.date=2021-02-17&rft.volume=16&rft.issue=2&rft.spage=e0247238&rft.epage=e0247238&rft.pages=e0247238-e0247238&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0247238&rft_dat=%3Cgale_plos_%3EA652074678%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2490579541&rft_id=info:pmid/33596250&rft_galeid=A652074678&rft_doaj_id=oai_doaj_org_article_d49f2a80270540e99660d7eb5a55ff9b&rfr_iscdi=true |