Central Nervous System Brain Tumors: A New Odyssey

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The Greek (Trojan) Horse standing in front of the citadel of Troy. Illustration: Ismaïl Hermelo

By: Ismaïl Hermelo

The extent of what we do not know predetermines the drawn assumptions, a priori. These assumptions can help us to move forward the frontiers of our understanding in virtually any given research project, including those related to human health and disease. Within human biology, the central nervous system (CNS) represents a bastion of the unknown, tantamount to embarking into a new odyssey.

Over 30 centuries have passed since the Greek author Homer wrote the Odyssey. The epic poem relates how the Greek hero Odysseus (in Latin Ulysses) returns home, Ithaca, after the Trojan war. Perhaps the Trojan war is the most famous episode of this journey. The Trojan horse was an effigy built by the Greeks as a means to persuade the Trojans into thinking as if they had given up and fled the battle. Beguiled by hubris, the Trojans considered the wooden statue a battle trophy, and decided to exhibit it in their citadel. Not suspecting at all that inside the statue, Odysseus was waiting among an elite force. The rest is legend.

The New Troy

Let’s imagine our CNS as the citadel of Troy. In turn, the blood brain barrier (BBB), could be the citadel’s wall, with the main function of keeping intruders away. Current available systemic treatments aiming at the CNS are able to pass through BBB, but 70% of the concentration that circulates in peripheral blood is left out.

The BBB is also responsible for regulating cellular trafficking. Nonetheless, several cells, as the ones coming from systemic circulation, can still access the CNS. Interestingly not only in health, but also in brain tumor disease, the cells secrete a myriad of chemokines and cytokines, co-creating an inflammatory milieu which contributes to the entrance of immune cells from peripheral blood.

The Greek Horse Infiltration

Notice that despite the fact that the horse statue was made by the Greeks, it is commonly referred to as the Trojan horse. Hence, referencing the actual builders, the immune cells accessing the CNS micro-environment could be the Greek Horse of this proposed new odyssey.

The original tumor lesion continues to invade the CNS due to the highly infiltrative nature of diffuse gliomas, while at the same time, the inflow of immune cells from the blood stream to the CNS continues. As part of this complex process, the immune cells altogether with the neoplastic cells generate the tumor-immune micro-environment, or TiME. However, a fundamental question is brought up:

“Exactly which immune cell types do comprise the TiME?”  

This question can be addressed in many ways. In fact, regarding the CNS immunology, there are many “unknowns” even if considering healthy conditions. If the question is addressed in the context of tumors, the necessity of following unbiased approaches becomes greater. Therefore, we decided to follow a non-biased analysis. Consequently, computational unsupervised methods were used. But before explaining them, let’s walk through the experimental side.

One of the most used methods over the last half a century is flow cytometry. A powerful method, based on the light scattering properties of cells, and the fluorescent signals emitted by fluorochrome-bearing antibodies. In order to study the cells, it is needed to suspend them individually in buffer solution to pass them through the laser beam. Thus, to obtain a cell suspension, solid tissue dissociation into single cells is required. This provides cell population characteristics of the cells that compose a tissue.

Starting with cell suspension, the immune cellular fraction was sorted using microbead particles bearing CD45 antibody, the leukocyte common antigen. Hence the flow cytometry analysis was focused on immune cells from human brain tumor biopsies. CD45 negative fraction contained most of the tumor and other stromal cell. SOC: Standard of care, including maximal safe resection. Illustration: Ismail Hermelo

The Nykter’s and Rautajoki’s Labs research regarding brain tumors include projects that focus on tumors originating from CNS supportive cells, collectively grouped under the term glia. These include astrocytes and oligodendrocytes, to name some. Tumors with this origin are referred to as diffuse gliomas, and they are the most common CNS malignant tumors in adults. Recent molecular characterizations, stepping away from histopathological features, have provided accurate biomarkers that better diagnose tumor entities.

As an example, a relevant biomarker is the mutation status of the isocitrate dehydrogenase 1 (or 2) gene, or IDH1/2, an enzyme of the Krebs cycle. An IDH1/2 mutant tumor can indicate a survival rate of five years in about 80% of diagnosed cases. Since transformed cells undergo such a process inside the CNS already, candidates that might fit to the Greek Horse concept may be surprising.

How Many Are There Engaged?

There are several immune cell types in peripheral blood, including comprehensive categories like myeloid cells. To cite a few, the monocytes (precursors of macrophages), the granulocytes (being the most abundant population neutrophils), and the lymphoid cells: helper T cells (CD4 T cells) and cytotoxic T cells (CD8 T cells).

Interestingly, it has been reported that myeloid cells are accumulated within the tumor lesion in diffuse gliomas. This accumulation can add up to 80% of the entire tumor mass. However, there are other myeloid cells already present, known as the microglia, the resident macrophage-like cells of the CNS.

General overview of the development of main hematopoietic stem cell-derived immune cells. In the case of microglia (CNS resident macrophage-like cells), are derived from early hematopoietic progenitors in the fetal yolk sac, which migrate to the developing CNS during early fetal development. Illustration: Ismaïl Hermelo

The Myrmidons or Achilles, Are Many..

The elite Thessalian soldiers commanded by Achilles were known as the Myrmidons. Under the guidance of the son of the Nereid Thetis, the Myrmidons executed formidably the art of the war, accounting for many battle victories, even in the most difficult circumstances. For this reason, the Myrmidons’ deployment was reserved for when most needed.

Therefore, to a certain degree, a parallelism can be drawn between the Myrmidons and a subset of leukocytes, termed as cytotoxic T-cells. However, while they can reach the tumor lesion easily, our Myrmidons are not engaged in tumor clearance activities. And precisely this becomes a known diffuse glioma’s hallmark: the suppressive TiME.

Continuing with the analogy, another couple of subtypes of Myrmidons could be considered. In one hand, the infiltrating monocyte-derived macrophages, which undergo a TiME conditioning that confers them tumor promoting functions. In the other, another infiltrating myeloid-lineage are the neutrophils, which undergo an alternative activation due to the suppressive cytokines present in the tumor milieu.

Analogies, albeit limited, serve the purpose of illustrating how immune cells entering the CNS to clear and stop the neoplastic expansion, change their main duties and become tumor promoting elements. Still in line with the Homer’s Odyssey analogy, it is like if the elite squat switched sides and become allies to the Trojans.

Every single dot on the graphs shows a single cell. The graphs visualize t-distributed stochastic neighbor embedding (t-sne) a statistical method for visualizing high-dimensional data. In this case every staining parameter is used as a separate dimension. In both graphs, CD45 sorted positive cells from all tumor samples are combined and displayed together. On the left graph, we can observe how the CD45 signal varies across all cells. On the right, after running the clustering algorithm PhenoGraph, t-sne shows groups of cells (clusters) that have a similar staining profile for one or more parameters. Analyzing next e.g. what proteins are co-expressed in each cluster, proposes a cell phenotype. Devoid of subjectiveness, this pipeline allows phenotype discovery. Computational analysis is performed by PhD candidate Tuomo Virtanen. Image: Ismaïl Hermelo

However, biological systems are way more complex. There is scientific evidence to asseverate that several myeloid populations seem to facilitate equally tumor progression by suppressing immune cells. Collectively, these suppressive cells of myeloid origin, are grouped under the term myeloid-derived suppressor cells. Continuing with the analogy, it would imply that the Myrmidon squad was comprised by several Achilles, as each component has equal relevance when performing their deeds. 

To conclude in regard to this new Odyssey, our approach aims to unravel which previously unknown Myrmidons (immune cells) comprise the squad entering the citadel of Troy (CNS), and to layout possible threads to follow by which understanding why they are switching sides.

The research project is supervised by Prof. Matti Nykter and Adj.Prof. Kirsi Rautajoki from Tampere University in collaboration with the Adj.Prof. Joonas Haapasalo MD from Tampere University Hospital.

Ismaïl Hermelo MSc. PhD Candidate from the Computational Biology Group, and Cancer Regulation and Immunology Group at Tampere University. Image credit: Sonja Mäntylä

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