A Toolkit for Profiling the Immune Landscape of Pediatric Central Nervous System Malignancies

Abstract

The prognosis of pediatric central nervous system (CNS) malignancies remains dismal due to limited treatment options, resulting in high mortality rates and long-term morbidities. Immunotherapies, including checkpoint inhibition, cancer vaccines, engineered T cell therapies, and oncolytic viruses, have promising results in some hematological and solid malignancies, and are being investigated in clinical trials for various high-grade CNS malignancies. However, the role of the tumor immune microenvironment (TIME) in CNS malignancies is mostly unknown for pediatric cases. In order to successfully implement immunotherapies and to eventually predict which patients would benefit from such treatments, in-depth characterization of the TIME at diagnosis and throughout treatment is essential. In this review, we provide an overview of techniques for immune profiling of CNS malignancies, and detail how they can be utilized for different tissue types and studies. These techniques include immunohistochemistry and flow cytometry for quantifying and phenotyping the infiltrating immune cells, bulk and single-cell transcriptomics for describing the implicated immunological pathways, as well as functional assays. Finally, we aim to describe the potential benefits of evaluating other compartments of the immune system implicated by cancer therapies, such as cerebrospinal fluid and blood, and how such liquid biopsies are informative when designing immune monitoring studies. Understanding and uniformly evaluating the TIME and immune landscape of pediatric CNS malignancies will be essential to eventually integrate immunotherapy into clinical practice.

Keywords: central nervous system malignancy; flow cytometry; immune monitoring; immunohistochemistry; immunotherapy; transcriptomics; tumor immune microenvironment.

Figure 1

Multi-dimensional immunophenotyping of TIME from tumor tissue using IHC, flow cytometry, bulk- and scRNA-seq, and functional assays.

Figure 2

(A) Cell-type markers for identification of immunological, tumor, and stromal cells in CNS malignancies. (B) Immunohistochemistry detects leukocytes in tumor tissue, and can be used to stratify patient tumor tissue (as demonstrated in Murata et al.). *Cell surface expression may vary between tumor cells and indications. (C) Example of flow cytometry gating strategy. More elaborate flow panels can be used for in-depth phenotyping of immune cells, providing information on specific cell subsets and status (activation/anergic/suppressive).

Figure 3

Bulk and single-cell transcriptomics reveals differentially expressed genes and pathways between tumor sub-types or cell sub-populations, respectively.

Figure 4

Characterization of immune cells from longitudinal liquid biopsies (as shown, blood and CSF) using scRNA-seq and flow cytometry.

Figure 5

Integrating methodologies allows for multi-dimensional characterization of TIME and will enable personalized immunotherapeutic strategies.