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Immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay.

ABSTRACT: Monitoring functional competence of immune cell populations in clinical routine represents a major challenge. We developed a whole-blood assay to monitor functional competence of peripheral innate immune cells including NK cells, dendritic and monocyte cell subsets through their ability to produce specific cytokines after short-term stimulation, detected through intra-cytoplasmic staining and multi-parametric flow-cytometry. A PMA/ionomycin T cell activation assay complemented this analysis. Comparing cohorts of healthy women and breast cancer (BC) patients at different stages, we identified significant functional alteration of circulating immune cells during BC progression prior to initiation of treatment. Of upmost importance, as early as the localized primary tumor (PT) stage, we observed functional alterations in several innate immune populations and T cells i.e. (i) reduced TNF? production by BDCA-1⁺ DC and non-classical monocytes in response to Type-I IFN, (ii) a strong drop in IFN? production by NK cells in response to either Type-I IFN or TLR7/8 ligand, and (iii) a coordinated impairment of cytokine (IL-2, IFN?, IL-21) production by T cell subpopulations. Overall, these alterations are further accentuated according to the stage of the disease in first-line metastatic patients. Finally, whereas we did not detect functional modification of DC subsets in response to TLR7/8 ligand, we highlighted increased IL-12p40 production by monocytes specifically at first relapse (FR). Our results reinforce the importance of monitoring both innate and adaptive immunity to better evaluate dysfunctions in cancer patients and suggest that our whole-blood assay will be useful to monitor response to treatment, particularly for immunotherapeutic strategies.

SUBMITTER: Verronese E

PROVIDER: S-EPMC4839376 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay.

Verronèse E E Delgado A A Valladeau-Guilemond J J Garin G G Guillemaut S S Tredan O O Ray-Coquard I I Bachelot T T N'Kodia A A Bardin-Dit-Courageot C C Rigal C C Pérol D D Caux C C Ménétrier-Caux C C

Oncoimmunology 20151110 3

Monitoring functional competence of immune cell populations in clinical routine represents a major challenge. We developed a whole-blood assay to monitor functional competence of peripheral innate immune cells including NK cells, dendritic and monocyte cell subsets through their ability to produce specific cytokines after short-term stimulation, detected through intra-cytoplasmic staining and multi-parametric flow-cytometry. A PMA/ionomycin T cell activation assay complemented this analysis. Com ...[more]

PMID: 27141361

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Project description:Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer's software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay's ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.

Dataset Information

Immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay.

Publications

Immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay.

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