Protocol for investigating tertiary lymphoid structures in human and murine fixed tissue sections using Opal™-TSA multiplex immunohistochemistry

Quigley, Luke; Pang, Lokman; Tavancheh, Elnaz; Ernst, Matthias; Behren, Andreas; Huynh, Jennifer; Duarte, Jessica Da Gama

doi:10.1016/j.xpro.2022.101961

Cited by 5 publications

(3 citation statements)

References 11 publications

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“…Nonetheless, we cannot ascertain whether they were TLS as we did not include the necessary markers for identification in our panel. 27 …”

Section: Resultsmentioning

confidence: 99%

Immune cell topography of head and neck cancer

Muijlwijk,

Nijenhuis,

Ganzevles

et al. 2024

J Immunother Cancer

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BackgroundApproximately 50% of head and neck squamous cell carcinomas (HNSCC) recur after treatment with curative intent. Immune checkpoint inhibitors are treatment options for recurrent/metastatic HNSCC; however, less than 20% of patients respond. To increase this response rate, it is fundamental to increase our understanding of the spatial tumor immune microenvironment (TIME).MethodsIn total, 53 HNSCC specimens were included. Using a seven-color multiplex immunohistochemistry panel we identified tumor cells, CD163+macrophages, B cells, CD8+T cells, CD4+T helper cells and regulatory T cells (Tregs) in treatment-naive surgical resection specimens (n=29) and biopsies (n=18). To further characterize tumor-infiltrating CD8+T cells, we stained surgical resection specimens (n=12) with a five-color tumor-resident panel including CD103, Ki67, CD8 and pan-cytokeratin. Secretome analysis was performed on matched tumor suspensions (n=11) to measure protein levels.ResultsBased on CD8+T cell infiltrates, we identified four different immunotypes: fully infiltrated, stroma-restricted, immune-excluded, and immune-desert. We found higher cytokine levels in fully infiltrated tumors compared with other immunotypes. While the highest immune infiltrates were observed in the invasive margin for all immune cells, CD163+macrophages and Tregs had the highest tendency to infiltrate the tumor center. Within the tumor center, especially B cells stayed at the tumor stroma, whereas CD163+macrophages, followed by T cells, were more often localized within tumor fields. Also, B cells were found further away from other cells and often formed aggregates while T cells and CD163+macrophages tended to be more closely located to each other. Across resection specimens from various anatomical sites within the head and neck, oral cavity tumors exhibited the highest densities of Tregs. Moreover, the distance from B cells and T cells to tumor cells was shortest in oral cavity squamous cell carcinoma (OCSCC), suggesting more interaction between lymphocytes and tumor cells. Also, the fraction of T cells within 10 µm of CD163+macrophages was lowest in OCSCC, indicating fewer myeloid/T-cell suppressive interactions in OCSCC.ConclusionsWe comprehensively described the TIME of HNSCC using a unique data set of resection specimens. We discovered that the composition, as well as the relative localization of immune cells in the TIME, differed in distinct anatomical sites of the head and neck.

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“…Nonetheless, we cannot ascertain whether they were TLS as we did not include the necessary markers for identification in our panel. 27 …”

Section: Resultsmentioning

confidence: 99%

Immune cell topography of head and neck cancer

Muijlwijk,

Nijenhuis,

Ganzevles

et al. 2024

J Immunother Cancer

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“… 31 These concepts cannot be proven without quantitative spatial analysis. Previous studies using light microscopy 32 and multispectral immunofluorescence imaging 17 rely on cell morphology and minimizing spectral overlap of traditional immunofluorescence microscopy 33 to characterize lesion cells. These methods are less scalable than the sequential imaging method we use because they (1) are limited by the recognition of morphology or the imaging spectrum of the microscope, and (2) rely on visual inspection instead of computational algorithms to perform quantitative analyses.…”

Section: Discussionmentioning

confidence: 99%

Multiplex Imaging for Cell Phenotyping of Early Human Atherosclerosis

Elishaev,

Li,

Zhou

et al. 2024

JAHA

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Background Previous studies using animal models and cultured cells suggest that vascular smooth muscle cells (SMCs) and inflammatory cytokines are important players in atherogenesis. Validating these findings in human disease is critical to designing therapeutics that target these components. Multiplex imaging is a powerful tool for characterizing cell phenotypes and microenvironments using biobanked human tissue sections. However, this technology has not been applied to human atherosclerotic lesions and needs to first be customized and validated. Methods and Results For validation, we created an 8‐plex imaging panel to distinguish foam cells from SMC and leukocyte origins on tissue sections of early human atherosclerotic lesions (n=9). The spatial distribution and characteristics of these foam cells were further analyzed to test the association between SMC phenotypes and inflammation. Consistent with previous reports using human lesions, multiplex imaging showed that foam cells of SMC origin outnumbered those of leukocyte origin and were enriched in the deep intima, where the lipids accumulate in early atherogenesis. This new technology also found that apoptosis or the expression of pro‐inflammatory cytokines were not more associated with foam cells than with nonfoam cells in early human lesions. More CD68 + SMCs were present among SMCs that highly expressed interleukin‐1β. Highly inflamed SMCs showed a trend of increased apoptosis, whereas leukocytes expressing similar levels of cytokines were enriched in regions of extracellular matrix remodeling. Conclusions The multiplex imaging method can be applied to biobanked human tissue sections to enable proof‐of‐concept studies and validate theories based on animal models and cultured cells.

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“…However, the steps and mechanisms through which TLS formation is initiated remain largely unclear, limiting our ability to enhance TLS-inducing therapeutics. To date, most studies of TLS in human cancer have relied on immunohistochemical stainings of sequential tissue sections, or multiplex immunostainings including less than ten parameters [25][26][27] . These studies have been important in elucidating that TLS exhibit different maturation stages which have an impact on patients' prognosis, with TLS that resemble secondary follicles being associated with the most positive outcomes 4,5 .…”

Section: Discussionmentioning

confidence: 99%

Cellular and molecular events organizing the assembly of tertiary lymphoid structures in glioblastoma

Vaccaro,

Yang,

van de Walle

et al. 2024

Preprint

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Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates associated with improved prognosis in numerous tumors. To date, their prognostic value in central nervous system cancers and the events underlying their formation remain unclear. Here, we find that TLS correlate with improved survival in glioblastoma patients. Furthermore, combining spatial transcriptomics of human tissues with longitudinal studies in murine glioma, we establish that the assembly of T cell-rich clusters is a prerequisite for the development of canonical TLS, and show that CD4 T cells play a central role in this process. Indeed, we provide evidence that IL7R+CCR7+Th1 lymphocytes with lymphoid tissue-inducing potential are recruited to TLS nucleation sites, where they can initiate TLS assembly by expressing lymphotoxin β. Our work defines TLS as a potential prognostic factor in glioblastoma and identifies cellular and molecular mechanisms of TLS formation. These findings have broader implications for the development of TLS-inducing therapies for cancer.Graphical abstract

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Protocol for investigating tertiary lymphoid structures in human and murine fixed tissue sections using Opal™-TSA multiplex immunohistochemistry

Cited by 5 publications

References 11 publications

Immune cell topography of head and neck cancer

Immune cell topography of head and neck cancer

Multiplex Imaging for Cell Phenotyping of Early Human Atherosclerosis

Cellular and molecular events organizing the assembly of tertiary lymphoid structures in glioblastoma

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