High-resolution mapping of brain tumors reveals possible reason why some patients don’t respond to new drug

The cells that make up cancerous brain tumors are extremely diverse and sometimes create unique three-dimensional shapes. As early as 1932, American neurosurgeon Percival Bailey attempted to label these cells and discovered that they can be divided into several families of cells with similar properties.

More than 90 years later, we still know very little about the identity of the groups of cells that make up different types of brain tumors, how these groups are organized, and how they affect the course of the disease and the outcome of treatment. As a result, the success rate of treatment for most brain cancers is generally low.

Over the past decade, single-cell genetic sequencing technology has made it possible to examine thousands of cells in the same tissue in minute detail at once, to understand which genes they express, and then to categorize them and study the role of each group.

Scientists in the research group of Dr. Itay Tirosh in the Department of Molecular Cell Biology at the Weizmann Institute of Science, in collaboration with the laboratory of Professor Mario L. Suvà at Massachusetts General Hospital, have exploited this technology to reexamine some of the unanswered questions in the field of brain tumors.

The most common type of primary brain tumor is a glioma, which arises from the supporting cells that assist our nerve cells. There are two main types of glioma tumors: those that are generally less aggressive and have a mutation in the gene that codes for an enzyme called IDH, and those that do not have this mutation, which are very aggressive and known in medical terminology as glioblastoma.

Over the past few years, researchers in Tirosh’s lab have used single-cell RNA sequencing to analyze the cellular composition of both tumor types. They have revealed that tumor cells are divided into groups, each expressing a unique genetic program that determines the biological “state” of the cancer cells in that group.

Among other findings, the researchers discovered groups of cells that use their unique genetic programs to mimic normal brain cells.

In a study published in CellResearchers in Tirosh’s lab, led by Dr. Alissa Greenwald, Noam Galili Darnell, and Dr. Rouven Hoefflin, have leveraged technologies that not only allow RNA to be sequenced at the single-cell level, but also allow its expression to be spatially mapped.

This study allowed them, for the first time, to identify the genes uniquely expressed in each of the thousands of areas of a brain tumor. They were thus able to precisely map the organization of glioblastomas and glioma tumors. To carry out this study, they took biopsies from 13 patients with glioblastomas and from six patients with gliomas carrying the IDH mutation.

The researchers’ first discovery was that groups of different cells within a glioma tumor are not evenly distributed throughout the tumor; instead, they are concentrated in various environments within the tumor. These microenvironments are not entirely homogeneous. Cells from other groups are always found in close proximity to other cell types.

In the next step of the study, the researchers checked whether there were groups of tumor cells that usually coexist in close proximity to each other. They found that the cells not only had preferred neighbors, but that these good neighbor pairings were also consistent across different patients.

Some neighboring pairs mimic the natural behavior of brain tissue. For example, cells mimicking oligodendrocyte support cell mother cells have been found in close proximity to endothelial cells, which line the walls of blood vessels. This pairing also occurs in healthy tissue, as endothelial cells release substances vital for the survival and proliferation of oligodendrocyte precursor cells.

Similarly, cells mimicking neural progenitor cells were found in parts of the tumor that had penetrated healthy brain tissue, much as progenitor cells from healthy tissue migrate when tissue is regenerated.

Taking a holistic view to better understand these couplings, the researchers realized that cells created five distinct layers by organizing themselves into separate environments within the tumor. The innermost layer, the core of the tumor, is made up of necrotic cells, which do not receive enough oxygen to survive.

In the layer surrounding the necrotic core, the researchers found cells similar to embryonic connective tissue, as well as other cells, including immune system cells responsible for inflammation. The third layer was mainly composed of blood vessels, endothelial cells that form the walls of blood vessels, and other immune system cells.

The cells in the two outer layers of the tumor do not suffer from a lack of oxygen. This allows groups of tumor cells that mimic healthy brain tissue (neural progenitors and supporting cells) to grow in the fourth layer.

The fifth, outermost layer contains healthy brain tissue, into which the tumor penetrates. These findings on the different layers of a tumor indicate that the driving force behind the layered structure of the tumor is the lack of oxygen, which worsens as the disease progresses and the tumor grows.

Based on these results, the researchers observed a much more chaotic structure in less aggressive tumors – which are also generally smaller – and in areas of the tumor with an abundant oxygen supply.

In most glioma tumors with an IDH mutation, for example, there was usually no necrotic tissue and the tumor structure was disorganized; in the rare cases where there was necrotic tissue, biopsies also showed a relatively well-ordered structure.

“We found that an organized spatial structure is characteristic of the most aggressive tumors,” Tirosh says.

“The lack of oxygen in the environment of tumor cells influences the genetic program they express and therefore affects their state. As the tumor grows, distinct layers form, some of which may be less accessible to drugs and immune system cells, which could make the tumor more resistant.”

The evolution of the status of cancer cells

Researchers in Tirosh’s lab used information they gathered about the cellular makeup of glioma tumors to determine how a promising new drug helped some patients with this type of cancer.

To do this, they used tumor biopsies from three patients who had participated in a clinical trial of the new drug and had responded to treatment, as well as biopsies from six patients who had not received any treatment. To complete the picture, they also used data from biopsies taken from 23 other patients who had taken the drug and 134 patients who had not received treatment.

The research team, led by Dr Avishay Spitzer, found that the drug, which works by inhibiting the mutant IDH enzyme, prompted cells to change the genetic program they expressed. In effect, the treatment encourages cancer stem cells to differentiate into mature cells, compromising their ability to divide rapidly and thereby blocking disease progression.

The researchers postulated that if the drug works by causing cancer cells to differentiate into mature cells, the mutation attacking the gene essential to the differentiation process could explain cases in which the drug does not work.

In biopsies taken from patients who had not received the drug, they identified a certain gene linked to low levels of mature cancer cells. When they silenced this gene in a mouse model of cancer, they found, as expected, that the drug was not effective.

“This indicates that the genetic mutation we identified could be a biological marker to determine in advance which patients will benefit from treatment and which will not,” Tirosh says. These new findings could also help find a treatment that combines IDH inhibitors with another drug that promotes the differentiation process and increases the impact of the treatment on the tumor.

“Our two most recent studies have revealed the forces that shape the character of cancer cells in a tumor, both in their intact environment and in that resulting from therapy that alters the cells’ genetic program,” Tirosh says.

“These results open the way to a new approach to cancer treatment, because once we become familiar with the groups of cells that populate each area of ​​the tumor and know how a cell can switch from one state to another, we may be able to develop new targeted treatments that will modify the course of the disease.

“Understanding that the composition of cells within the tumor and its three-dimensional structure are linked to the level of aggressiveness of the tumor could also lead to new diagnostic methods that do not rely solely on the volume of the tumor and the mutations it contains.”