Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Surace, Michael; Dacosta, Karma; Huntley, Anna; Zhao, Wei‐Guang; Bagnall, Christopher; Brown, C. M.; Wang, Tao; Roman, Kristin; Cann, Jennifer A.; Lewis, Arthur B.; Steele, Keith; Rebelatto, Marlon C.; Parra, Edwin R.; Hoyt, Clifford; Rodriguez‐Canales, Jaime

doi:10.3791/58390-v

Cited by 8 publications

(3 citation statements)

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“…The mIF proved to be an invaluable tool for tumor tissue immune profiling [8][9][10]. In this study, we established an mIF panel specially applied in mice tumors and explored the characteristics of TAICs in mice lung adenocarcinoma using the Opal workflow according to the rising research needs of mice tumors.…”

Section: Discussionmentioning

confidence: 99%

Immune profiling of mouse lung adenocarcinoma paraffin tissues using multiplex immunofluorescence panel: a pilot study

Zhai,

Tamegnon,

Jiang

et al. 2024

Lab Anim Res

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Background Immune profiling has become an important tool for identifying predictive, prognostic and response biomarkers for immune checkpoint inhibitors from tumor microenvironment (TME). We aimed to build a multiplex immunofluorescence (mIF) panel to apply to formalin-fixed and paraffin-embedded tissues in mice tumors and to explore the programmed cell death protein 1/ programmed cell death 1 ligand 1 (PD-1/PD-L1) axis. Results An automated eight-color mIF panel was evaluated to study the TME using seven antibodies, including cytokeratin 19, CD3e, CD8a, CD4, PD-1, PD-L1, F4-80 and DAPI, then was applied in six mice lung adenocarcinoma samples. Cell phenotypes were quantified by software to explore the co-localization and spatial distribution between immune cells within the TME. This mice panel was successfully optimized and applied to a small cohort of mice lung adenocarcinoma cases. Image analysis showed a sparse degree of immune cell expression pattern in this cohort. From the spatial analysis we found that T cells and macrophages expressing PD-L1 were close to the malignant cells and other immune cells. Conclusions Comprehensive immune profiling using mIF in translational studies improves our ability to correlate the PD-1/PD-L1 axis and spatial distribution of lymphocytes and macrophages in mouse lung cancer cells to provide new cues for immunotherapy, that can be translated to human tumors for cancer intervention.

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Section: Discussionmentioning

confidence: 99%

Immune profiling of mouse lung adenocarcinoma paraffin tissues using multiplex immunofluorescence panel: a pilot study

Zhai,

Tamegnon,

Jiang

et al. 2024

Lab Anim Res

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“…We developed a seven-color TSA-based mIF panel using Opal fluorophores with simultaneous staining on a single paraffin tissue section [38, 40‒43], evaluating single conventional IHC and IF stains. This approach uses TSA reagents, significantly increasing sensitivity while maintaining specificity and resolution, allowing detection in low-expression targets [38, 40‒43]. However, it is essential to select the areas that can represent the whole tumor section [44].…”

Section: Discussionmentioning

confidence: 99%

Exploring the Expression of Adenosine Pathway-Related Markers CD73 and CD39 in Colorectal and Pancreatic Carcinomas Characterized by Multiplex Immunofluorescence: A Pilot Study

Lima,

Tamegnon,

Rodriguez

et al. 2023

Pathobiology

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Introduction: Generating high levels of immunosuppressive adenosine in the tumor microenvironment contributes to cancer immune evasion. CD39 and CD73 hydrolyze adenosine triphosphate into adenosine; thus, efforts have been made to target this pathway for cancer immunotherapy. Our objective was optimizing a multiplex immunofluorescence (mIF) panel to explore the role of CD39 and CD73 within the tumor microenvironment. Materials and methods: In three-time points, a small cohort (n=8 ) of colorectal and pancreatic adenocarcinomas were automated staining using an mIF panel against CK, CD3, CD8, CD20, CD39, CD73 and CD68 to compare them with individual markers immunohistochemistry (IHC) for internal panel validation. Densities of immune cells and distances from different tumor-associated immune cells to tumor cells were exploratory assessment and compared with clinicopathologic variables and outcomes. Results: Comparing the three-time points and individual IHC staining results, we demonstrated high reproducibility of the mIF panel. CD39 and CD73 expression was low in malignant cells; the exploratory analysis showed higher densities of CD39 expression by various cells, predominantly stromal cells, followed by T cells, macrophages, and B cells. No expression of CD73 by B cells or macrophages was detected. Distance analysis revealed proximity of cytotoxic T cells, macrophages, and T cells expressing CD39 to malignant cells, suggesting a close regulatory signal driven by this adenosine marker. Conclusions: We optimized an mIF panel for detection of markers in the adenosine pathway, an emerging clinically relevant pathway. The densities and spatial distribution demonstrated that this pathway may modulate aspects of the tumor immune microenvironment.

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“…Multiplex IHC was performed as described previously 29 . The antibody panel was listed in Supporting Table S3 .…”

Section: Methodsmentioning

confidence: 99%

Genipin-activating PPARγ impedes CCR2-mediated macrophage infiltration into postoperative liver to suppress recurrence of hepatocellular carcinoma

Wu,

Chan,

et al. 2023

Int. J. Biol. Sci.

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A high postoperative tumour recurrence rate has significantly rendered a poorer prognosis in hepatocellular carcinoma (HCC) patients. The aim of this study is to identify a natural compound genipin as a potential and effective candidate to suppress the postoperative recurrence of HCC. Clinical analysis revealed that infiltration of macrophage into the adjacent tissue but not HCC predicted patients' poor prognosis on recurrence-free survival. Genipin intervention suppressed the Ly6C+CD11b+F4/80+ pro-inflammatory macrophage infiltration in the postoperative liver of mice. Adoptive transfer of pro-inflammatory monocytic cells completely abolished the inhibitory effect of genipin on HCC recurrence. Transcriptomic analysis on FACs-sorted macrophages from the postoperative livers of mice revealed that PPARγ signalling was involved in the regulatory effect of genipin. Genipin is directly bound to PPARγ, causing PPARγ-induced p65 degradation, which in turn suppressed the transcriptional activation of CCR2 signalling. PPARγ antagonist GW9662 abrogated the effects of genipin on CCR2-medaited macrophage infiltration as well as HCC recurrence. Cytokine array analysis identified that genipin intervention potently suppressed the secretion of CCL2 further partially contributed to the pro-inflammatory macrophage infiltration into the postoperative liver. Multiplex immunostaining on tissue array of human HCC revealed that PPARγ expression was inversely associated with CCL2 and the macrophage infiltration in the adjacent liver of HCC patients. Our works provide scientific evidence for the therapeutic potential of genipin as a PPARγ agonist in preventing postoperative recurrence of HCC.

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Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Cited by 8 publications

References 0 publications

Immune profiling of mouse lung adenocarcinoma paraffin tissues using multiplex immunofluorescence panel: a pilot study

Immune profiling of mouse lung adenocarcinoma paraffin tissues using multiplex immunofluorescence panel: a pilot study

Exploring the Expression of Adenosine Pathway-Related Markers CD73 and CD39 in Colorectal and Pancreatic Carcinomas Characterized by Multiplex Immunofluorescence: A Pilot Study

Genipin-activating PPARγ impedes CCR2-mediated macrophage infiltration into postoperative liver to suppress recurrence of hepatocellular carcinoma

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