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Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.

ABSTRACT: Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.

SUBMITTER: Mori H

PROVIDER: S-EPMC7960613 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Every day, more evidence is revealed regarding the importance of the relationship between the response to cancer immunotherapy and the cancer immune microenvironment. It is well established that a profound characterization of the immune microenvironment is needed to identify prognostic and predictive immune biomarkers. To this end, we find phenotyping cells by multiplex immunofluorescence (mIF) a powerful and useful tool to identify cell types in biopsy specimens. Here, we describe the use of mIF tyramide signal amplification for labeling up to eight markers on a single slide of formalin-fixed, paraffin-embedded tumor tissue to phenotype immune cells in tumor tissues. Different panels show different markers, and the different panels can be used to characterize immune cells and relevant checkpoint proteins. The panel design depends on the research hypothesis, the cell population of interest, or the treatment under investigation. To phenotype the cells, image analysis software is used to identify individual marker expression or specific co-expression markers, which can differentiate already selected phenotypes. The individual-markers approach identifies a broad number of cell phenotypes, including rare cells, which may be helpful in a tumor microenvironment study. To accurately interpret results, it is important to recognize which receptors are expressed on different cell types and their typical location (i.e., nuclear, membrane, and/or cytoplasm). Furthermore, the amplification system of mIF may allow us to see weak marker signals, such as programmed cell death ligand 1, more easily than they are seen with single-marker immunohistochemistry (IHC) labeling. Finally, mIF technologies are promising resources for discovery of novel cancer immunotherapies and related biomarkers. In contrast with conventional IHC, which permits only the labeling of one single marker per tissue sample, mIF can detect multiple markers from a single tissue sample, and at the same time, deliver extensive information about the cell phenotypes composition and their spatial localization. In this matter, the phenotyping process is critical and must be done accurately by a highly trained personal with knowledge of immune cell protein expression and tumor pathology.

Project description:BackgroundGastroesophageal adenocarcinomas (GEAs) are heterogeneous cancers where immune checkpoint inhibitors have robust efficacy in heavily inflamed microsatellite instability (MSI) or Epstein-Barr virus (EBV)-positive subtypes. Immune checkpoint inhibitor responses are markedly lower in diffuse/genome stable (GS) and chromosomal instable (CIN) GEAs. In contrast to EBV and MSI subtypes, the tumor microenvironment of CIN and GS GEAs have not been fully characterized to date, which limits our ability to improve immunotherapeutic strategies.Patients and methodsHere we aimed to identify tumor-immune cell association across GEA subclasses using data from The Cancer Genome Atlas (N = 453 GEAs) and archival GEA resection specimen (N = 71). The Cancer Genome Atlas RNAseq data were used for computational inferences of immune cell subsets, which were correlated to tumor characteristics within and between subtypes. Archival tissues were used for more spatial immune characterization spanning immunohistochemistry and mRNA expression analyses.ResultsOur results confirmed substantial heterogeneity in the tumor microenvironment between distinct subtypes. While MSI-high and EBV+ GEAs harbored most intense T cell infiltrates, the GS group showed enrichment of CD4+ T cells, macrophages and B cells and, in ∼50% of cases, evidence for tertiary lymphoid structures. In contrast, CIN cancers possessed CD8+ T cells predominantly at the invasive margin while tumor-associated macrophages showed tumor infiltrating capacity. Relatively T cell-rich 'hot' CIN GEAs were often from Western patients, while immunological 'cold' CIN GEAs showed enrichment of MYC and cell cycle pathways, including amplification of CCNE1.ConclusionsThese results reveal the diversity of immune phenotypes of GEA. Half of GS gastric cancers have tertiary lymphoid structures and are therefore promising candidates for immunotherapy. The majority of CIN GEAs, however, exhibit T cell exclusion and infiltrating macrophages. Associations of immune-poor CIN GEAs with MYC activity and CCNE1 amplification may enable new studies to determine precise mechanisms of immune evasion, ultimately inspiring new therapeutic modalities.

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Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.

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