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Quantitation of calreticulin exposure associated with immunogenic cell death
Peng Liu1, Liwei Zhao1, Oliver Kepp1
1Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France; Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Institut National de la Santé et de la Recherche Médicale (INSERM), UMR1138, Equipe labellisée Ligue Nationale Contre le Cancer, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie, Paris, France.
Abstract:
Tumor cells treated by immunogenic cell death (ICD) inducers emit danger associated molecular patterns (DAMP), including but not limited to calreticulin (CALR), which translocates from the ER lumen to the surface of the cellular membrane where it serves as de novo uptake signal for antigen presenting cells of the immune system. CALR is exposed at an early stage of ICD and dictates tumor antigen transfer and therefore the immunogenicity of cancer cell death. Here, we provide a bi-color flow cytometry protocol for the quantification of ICD-associated CALR cell surface exposure in fixed samples. As compared to the detection of surface exposed CALR by confocal microscopy, the present flow cytometry-based analysis is cost-efficient and does not require sophisticated equipment. Moreover, the staining panel can be extended to a multicolor analysis for the parallel assessment of additional parameters.
Insights
Immunogenic cell death (ICD) inducers expose calreticulin (CALR) on tumor cells, signaling immune cells. This study presents a cost-effective flow cytometry method to quantify CALR exposure, enhancing cancer immunotherapy research.
Area of Science:
- Immunology
- Cell Biology
- Cancer Research
Background:
- Immunogenic cell death (ICD) is a form of cell death that elicits an immune response.
- Danger associated molecular patterns (DAMPs), such as calreticulin (CALR), are released by dying tumor cells.
- Surface exposure of CALR acts as a signal for antigen-presenting cells, promoting tumor antigen uptake and immune response.
Purpose of the Study:
- To develop and validate a bi-color flow cytometry protocol for quantifying cell surface calreticulin (CALR) exposure.
- To provide a cost-efficient and accessible method for assessing ICD-associated CALR exposure in fixed samples.
- To establish a foundation for multicolor flow cytometry panels to assess additional ICD parameters.
Main Methods:
- Utilized a bi-color flow cytometry protocol.
- Quantified cell surface exposure of calreticulin (CALR) on fixed tumor cells.
- Compared flow cytometry method to confocal microscopy for CALR detection.
Main Results:
- The developed flow cytometry protocol accurately quantifies ICD-associated CALR cell surface exposure.
- Flow cytometry offers a cost-efficient alternative to confocal microscopy for CALR detection.
- The protocol is adaptable for multicolor analysis, allowing assessment of multiple parameters simultaneously.
Conclusions:
- Cell surface calreticulin (CALR) exposure is a key early event in immunogenic cell death (ICD).
- Flow cytometry provides a practical and scalable method for quantifying CALR exposure in cancer research.
- This protocol facilitates the study of ICD and the development of novel cancer immunotherapies.
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