Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Cl&#233;mence Granier; Emeline Vinatier; Elia Colin; Marion Mandavit; Charles Dariane; Virginie Verkarre; Lucie Biard; Rami El Zein; Corinne Lesaffre; Isabelle Galy-Fauroux; H&#233;l&#232;ne Roussel; El&#233;onore De Guillebon; Charlotte Blanc; Antonin Saldmann; C&#233;cile Badoual; Alain Gey; &Eacute;ric Tartour

doi:10.3791/56606

Multiplexado imunofluorescência análise e quantificação de Intratumoral PD-1+ +

JoVE Journal
Cancer Research

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JoVE Journal Cancer Research

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1⁺ Tim-3⁺ CD8⁺ T Cells

Multiplexado imunofluorescência análise e quantificação de Intratumoral PD-1+ + Tim-3 CD8+ T células

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09:32 min

February 8, 2018

DOI: 10.3791/56606-v

Clémence Granier¹, Emeline Vinatier^2,3,4, Elia Colin^2,3, Marion Mandavit^2,3, Charles Dariane^2,3,5, Virginie Verkarre⁶, Lucie Biard⁷, Rami El Zein², Corinne Lesaffre², Isabelle Galy-Fauroux², Hélène Roussel^2,3,6, Eléonore De Guillebon⁸, Charlotte Blanc^2,3, Antonin Saldmann⁴, Cécile Badoual^2,6, Alain Gey*⁴, Éric Tartour*^2,4

¹PARCC-INSERM U970,Université Paris Descartes, ²INSERM U970,Université Paris Descartes, ³Equipe Labellisée Ligue Contre le Cancer, ⁴Department of Immunology,Hopital Européen Georges Pompidou, ⁵Department of Medical Urology,Hopital Européen Georges Pompidou, ⁶Department of Pathology,Hopital Européen Georges Pompidou, ⁷Université Paris Diderot Paris 7, ⁸Department of medical oncology,Hopital Européen Georges Pompidou

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Overview

This study presents an innovative multiplexed immunofluorescence analysis method for automatic counting of tumor immune cells based on their phenotype. The technique is designed to analyze the tumor microenvironment and identify prognostic biomarkers for immunotherapy response.

Key Study Components

Area of Science

Immunology
Oncology
Cell Biology

Background

The tumor microenvironment plays a crucial role in cancer progression and treatment response.
CD8 + T cells are important for immune response in tumors.
Existing methods for cell counting can be time-consuming and require extensive data processing.
This study aims to streamline the analysis of immune cell phenotypes.

Purpose of Study

To develop a reliable method for automatic counting of immune cells in tumor samples.
To facilitate large cohort analyses in cancer research.
To improve the understanding of immune interactions within the tumor microenvironment.

Methods Used

Multiplexed immunofluorescence for cell phenotype analysis.
In situ fluorescence multispectral imaging for automatic counting.
Preparation of tissue sections with hydrophobic barriers for imaging.
Data processing techniques to analyze cell populations.

Main Results

The method allows for efficient counting of CD8 + T cell subpopulations.
Results indicate potential prognostic markers derived from the tumor microenvironment.
The technique is reproducible and suitable for large-scale studies.
Improved understanding of immune cell dynamics in tumors.

Conclusions

This innovative method enhances the analysis of tumor immune cells.
It provides a framework for identifying biomarkers for immunotherapy.
The approach can be adapted for various research applications in oncology.

Frequently Asked Questions

What is the significance of CD8 + T cells in tumors?

CD8 + T cells are crucial for mediating anti-tumor immunity and can influence treatment outcomes.

How does this method improve upon traditional cell counting?

It automates the counting process, reducing time and potential human error in data analysis.

Can this technique be used for other types of immune cells?

Yes, the method can be adapted to analyze various immune cell types within the tumor microenvironment.

What challenges do researchers face when using this method?

New users may find the phenotyping procedure lengthy and data processing complex.

Is this method suitable for clinical applications?

While primarily for research, it has potential clinical applications in identifying biomarkers for immunotherapy.

Estudar o microambiente do tumor pode identificar biomarcadores prognósticos ou preditivos de resposta clínica à imunoterapia. Apresentado aqui, é um método inovador baseado em situ fluorescência multiespectral de imagens para analisar e contar automaticamente várias subpopulações de CD8⁺T células. Esta técnica reprodutível e confiável é apropriada para análises de grandes coorte.

O objetivo geral desta análise de imunofluorescência multiplexada é contar automaticamente as células com base em seu fenótipo. Este método permite a multifluorescência e a contagem automática de células para analisar o fenótipo e a função das células imunes tumorais dentro de um microambiente tumoral. Essa tecnologia facilitou a geração de dois tipos de prognóstico composto para previsto por seus marcadores derivados diretamente do microambiente tumoral.

Geralmente, os indivíduos novos neste método podem ter dificuldades porque o procedimento de fenotipagem é muito longo e os dados brutos resultantes requerem processamento adicional. Tivemos a ideia para este método pela primeira vez quando tentamos caracterizar o fenótipo e as interações imunológicas das células annhodge miranty. Após o descongelamento, use uma toalha de papel para secar cuidadosamente as lâminas ao redor das seções de tecido e, em seguida, circule os tecidos com uma caneta barreira hidrofóbica.

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Imunologia questão 132 Multiplex em situ imagens multiespectrais técnica de imunofluorescência imagem digital carcinoma de células renais Tim-3 o PD-1 microambiente do tumor inibidores de ponto de verificação análise de célula única em situ