Abstract LB-352: Identification of myeloid-derived suppressor cells in solid tumors by multiplex IHC
Authors: BURRER Renaud , ARIBO Océane , BENCHAABEN Assil , TLIBA Maroua , LAZZARO Domenico , COTON Jean-Philippe 
Published: August 2018

In healthy individuals, immature myeloid cells undergo differentiation into Macrophages, Dendritic Cells or Neutrophils. In individuals with several types of cancers, Tumor-Derived Factors promote a pathological differentiation of these immature cells into Myeloid-Derived Suppressor Cells (MDSCs) that phenotypically resemble either Macrophages (M-MDSCs) or Neutrophils (PMN-MDSCs). Rather than supporting antitumoral immunity like their regular counterparts, these abnormal, highly immunosuppressive cells, impair antitumoral immunity and promote tumor development. In addition to MDSCs, the tumor micro-environment is also infiltrated by Tumor-Associated Macrophages (TAMs), which can be roughly divided in two main subgroups, M1- and M2-polarized Macrophages, that exhibit anti- or pro-tumor activity, respectively.

Over the last years, MDSCs have been recognized as valuable prognostic and/or predictive biomarkers, and there is a raising need for methods to phenotype these cells in clinical trials. Myeloid cell subpopulations are all closely related, and as a consequence, the phenotyping of each subset is complex, due to the high number of markers needed to discriminate them from the others. Recent advances have been made to define standard protocols to discriminate M- and PMN-MDSCs from TAMs and physiological Macrophages or Neutrophils combining flow cytometry and T-cell suppression assays. There are differences in the composition of subpopulations at the systemic level and those that infiltrate the tumor micro-environment, and a method for accurate phenotyping of MDSCs on tissue sections by ImmunoHistoChemistry (IHC) still need to be defined, but has been hampered by the limited number of antibodies that could be associated on a single slide.

Using multiplex IHC and multispectral imaging, we have designed an approach to phenotype each myeloid cell population in the tumor micro-environment, in different solid tumor types. Antibodies to cell surface markers, cytoplasmic proteins contributing to the immune-regulatory activity and transcription factors, have first been validated as simplex IHC markers, tested as parts of multiplex panels, and evaluated in various combinations, to define a phenotyping strategy. A new Image Analysis Software has been designed to assess the co-expression of each of the markers for individual cells even for stains that do not strictly colocalize at the pixel level (e.g. nuclear and membrane proteins). Using a successive gating strategy, cells displaying MDSCs and M1/M2-polarized macrophages phenotypes have been numbered in sections from various tumor types. Our method is compatible with standard automated IHC platforms and an automated multispectral scanner, and provides a solution for the analysis of MDSCs in FFPE samples in clinical trials.

Citation Format: Renaud BURRER, Océane ARIBO, Assil BENCHAABEN, Maroua TLIBA, Domenico LAZZARO, Jean-Philippe COTON. Identification of myeloid-derived suppressor cells in solid tumors by multiplex IHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-352.