“Identification of a Cancer Stem Cell in Human Brain Tumors” (2003), by Sheila Singh, Ian Clarke, Mizuhiko Terasaki, Victoria Bonn, Cynthia Hawkins, Jeremy Squire, Peter Dirks

By: Akaash Surendra

Published: 2025-08-11

In 2003, Sheila Singh and colleagues published “Identification of a Cancer Stem Cell in Human Brain Tumors” in the journal Cancer Research. The study examines a small population of cells within brain cancers that have abilities similar to those of neural stem cells and can maintain the growth of a brain tumor. The researchers identified the brain cancer stem cells by looking at the expression of proteins CD133 and nestin, which are usually present in neural stem cells. In addition, they characterized brain cancer stem cells as having the ability to quickly proliferate and self-renew, form tumor spheres, as well as differentiate in a manner that resembles characteristics of the original brain tumor from which those cells originated. “Identification of a Cancer Stem Cell in Human Brain Tumors” was one of the first studies to identify cancer stem cells in brain cancer and laid the framework for future research investigating the role of brain cancer stem cells in response to treatment, as well as the recurrence of a tumor after treatment.

Background and Context

At the time of publication, the researchers who published "Identification of a Cancer Stem Cell in Human Brain Tumors" were all affiliated with different departments at the University of Toronto in Toronto, Ontario. As of 2025, the primary author, Sheila Singh, is a neurosurgeon and professor in the department of surgery and biochemistry at McMaster University in Hamilton, Ontario. Her work involves using principles of developmental biology to better understand brain tumors. In addition, the senior author, Peter Dirks, is the chief of neurosurgery at the University of Toronto as of 2025. Dirks investigates how stem cells, development, and brain tumor growth are all linked together.

Throughout "Identification of a Cancer Stem Cell in Human Brain Tumors," the authors discuss brain tumors and the stem cells associated with them. Brain tumors are abnormal growths of cells that form into cancerous tissue in or around the brain. Brain tumors are benign or malignant. Benign, or non-cancerous, brain tumors grow in their location of origin but do not invade nearby tissues or spread throughout the body. However, malignant brain tumors are made up of cells that can grow uncontrollably and invade nearby tissues and other parts of the body. Stem cells are cells in the body that can develop into more specialized cells through a process known as differentiation. Stem cells can also produce more of the same type of stem cell, called self-renewal. Those two traits of self-renewal and differentiation distinguish stem cells from other cell types, which cannot self-renew and differentiate. Neural stem cells are specific stem cells that can give rise to more specialized cells in the central nervous system, which includes the brain and the spinal cord.

Additionally, the cancer stem cell, or CSC, hypothesis states that a small number of stem cells within a tumor are likely responsible for initiating a tumor and maintaining its growth. The theory refers to the idea that even if treatments like chemotherapy and radiation kills a majority of cancer cells, if a few cancer stem cells survive, those stem cells can initiate and grow a recurrent tumor. As a result, cancer researchers focus on further characterizing cancer stem cells in different cancer types to reveal more about how a specific cancer type stays in the body. Other researchers had already identified cancer stem cells in leukemia, multiple myeloma, and breast cancer by 2003, but researchers had not yet identified cancer stem cells in brain cancer.

Article Roadmap

“Identification of a Cancer Stem Cell in Human Brain Tumors” consists of four sections, titled “Introduction,” “Materials and Methods,” “Results,” and “Discussion.” In “Introduction,” Singh and colleagues point to the lack of understanding of the behavior of brain tumors and assert that other researchers have not identified cancer stem cells in brain cancer. “Materials and Methods” has eight subsections that each describe a laboratory technique that the researchers used in the experiment to identify and characterize brain cancer stem cells. In “Results,” the researchers demonstrate that they have identified brain cancer stem cells through the expression of proteins CD133 and nestin, as well as characteristics like self-renewal and differentiation. In “Discussion,” Singh and colleagues summarize their findings and state that a better understanding of brain tumor stem cells can lead to new treatment options.

In “Introduction,” Singh and colleagues discuss how difficult it is to understand how brain tumors behave, because tumors that share similar appearances and cellular characteristics may still behave very differently and can vary in how they respond to treatments. The authors attribute part of the lack of understanding of how brain tumors maintain themselves in the body to the fact that there is not currently an experimental test to identify which specific cells in a brain tumor are responsible for perpetuating the growth of that tumor. They state that determining a method to identify those cells will improve understanding of the origins of a brain tumor and how that tumor develops.

Additionally, in the “Introduction,” Singh and colleagues draw on previous studies performed in other cancers, such as leukemia. They state that in leukemia, other researchers have demonstrated that there is a small proportion of blood cancer stem cells, or leukemic stem cells, that have the unique ability to not only self-renew to produce more leukemic stem cells but also differentiate into more specialized cancer cells that can resemble normal blood cells such as white blood cells and red blood cells but do not contribute to normal body functions due to their cancerous abnormalities.

Next, in the “Materials and Methods,” Singh and colleagues describe various laboratory assays and techniques that they used throughout their experiments. The authors explain that they obtained their brain tumor samples from consenting patients at the Hospital for Sick Children in Toronto, and those samples also differed in brain cancer type and location. They then added enzymes, which are proteins in the body that facilitate biological reactions, to the brain tumors to break down proteins that hold together the cells in a tumor. That allowed the researchers to separate the brain tumor tissue into single brain cancer cells. Singh and colleagues then kept those brain cancer cells in a solution medium that contained growth factors and nutrients that encouraged those cells to grow. Singh and colleagues measured the number of cells and the number of stem cells in the sample tumors periodically over seven days.

Two methods that the researchers used to study those brain cancer cells and identify the brain cancer stem cells were immunocytochemistry, or ICC, and immunohistochemistry, or IHC. ICC is a laboratory technique that uses antibodies, which can bind to proteins in a cell, to visualize proteins of interest. Singh and colleagues used ICC to both identify brain cancer stem cells and analyze their behavior as they differentiate into more specialized brain cancer cells. They also used ICC to look at whether brain cancer cells expressed certain proteins of interest. In particular, they looked at proteins CD133 and nestin, which are common in neural stem cells. Singh and colleagues also added a dye that binds to a cell’s nucleus, which is a membrane-bound organelle that contains genetic material. Because each of those brain cancer cells has a nucleus, researchers utilized that dye to quantify how many cells expressed their proteins of interest. Singh and colleagues also performed a similar technique to ICC called IHC. The main difference between ICC and IHC is that researchers usually use ICC to analyze cells that they derived from a tissue and then grew in a culture, whereas they usually use IHC to analyze a tissue sample as a whole. Singh and colleagues used IHC to understand whether proteins of interest were present in both the original tumor sample and the brain cancer cells that the researchers were growing.

Also, in “Materials and Methods,” other techniques that Singh and colleagues describe are magnetic cell sorting and flow cytometry. Magnetic cell sorting is a technique in which magnetic particles attach to cells of interest in order to isolate those cells from a broader sample using a magnetic field. Flow cytometry is a technique that uses laser beams to assess the properties of cells and identify cells of interest. Singh and colleagues used both of those techniques to isolate brain cancer cells that expressed their proteins of interest, particularly CD133, from a broader population of brain cancer cells. Researchers in the study also used another laboratory technique called Spectral Karyotype, or SKY, analysis to visualize a cell’s chromosomes, which are thread-like structures in a cell’s nucleus that contain genetic information. Singh and colleagues used SKY to analyze abnormalities in the chromosomes of brain cancer cells.

Next, in “Results,” Singh and colleagues identify a potential marker for brain cancer stem cells, which they used to isolate those cells to then test their ability to carry out cancer stem cell functions like rapid proliferation, tumor sphere formation, and self-renewal. They identified the protein CD133 as that marker. Researchers found that brain cancer cells with the CD133 protein had the ability to self-renew, proliferate, and form tumor spheres. They termed brain cancer cells with the CD133 protein, brain tumor stem cells, or BTSCs. The researchers stated that they now had a marker that they could use to isolate those BTSCs and further characterize their abilities.

Additionally, in “Results,” the researchers further characterized those BTSCs first by looking at the ability for those cells to rapidly proliferate, form spheres and multiply, and express proteins CD133 and nestin, all of which are abilities of stem cells. In brain cancer cells derived from different brain tumor types from multiple patient samples that had varying appearances and cellular characteristics, the researchers observed that, in all of the samples, there was a consistent small population of cells within each sample that formed tumor spheres. In addition, Singh and colleagues analyzed those tumor spheres for proteins of interest and found that the cells in those spheres expressed CD133 and nestin, which are both protein markers of stem cells. The researchers also found that those cells did not express markers of more differentiated cell types, which they stated reaffirms that those cells that formed the tumor spheres were likely stem cells.

To further understand the characteristics of those cells within the tumor spheres, the researchers discuss in “Results” how they performed additional experiments to test proliferation and self-renewal capabilities. A small proportion of normal brain stem cells can self-renew, a process by which stem cells divide to create more stem cells. Singh and colleagues found that brain cancer cells within those tumor spheres had an increased ability to self-renew, especially in brain cancer cells that came from more aggressive types of brain cancer, such as medulloblastoma. The researchers write that the ability to self-renew might be a factor explaining why some types of brain cancer are more aggressive than others.

In addition, in “Results,” Singh and colleagues report that they investigated how those brain cancer cells with stem cell-like properties differentiate into more specialized cell types. After assessing multiple types of brain cancers such as medulloblastomas and pilocytic astrocytomas, Singh and colleagues found that after differentiation, the brain cancer cells with stem cell properties began to express the same proteins as in the original tumor sample, suggesting that the small population of brain cancer stem cells are key for tumor development and reestablishment.

Lastly, in “Results,” the researchers confirmed that those brain tumor stem cells that they identified were not actually normal stem cells found in the brain. They found that those cells did not have the same differentiation patterns as a normal neural stem cell. Additionally, they looked at the chromosomes of those tumor cells and compared them to the chromosomes of normal neural stem cells and saw abnormal chromosomes that differed from those of normal neural stem cells.

In “Discussion,” Singh and colleagues claim that across various types of brain tumors, they have identified a population of brain cancer stem cells. They again affirm that those brain cancer stem cells can form tumor spheres, rapidly proliferate and self-renew, and differentiate in a way that represents the characteristics of the original brain tumor that they came from. The researchers also state that their findings suggest that brain tumors might originate from normal stem cells found in the brain. Additionally, because brain tumors are very heterogeneous and composed of many different types of cells that are both differentiated or undifferentiated, they state that there may be a hierarchy of cells within a brain tumor, and brain cancer stem cells may be at the top of the hierarchy because of their ability to maintain growth of a brain tumor. Similarly, other researchers report that the cancer stem cells maintain the growth of leukemia in the body. Singh and colleagues also assert that other researchers have identified cancer stem cells in multiple myeloma and breast cancer. Singh and colleagues assert that further studying the brain cancer stem cells can provide insight into how brain tumors spread throughout the body, especially in the central nervous system. In addition, they claim that further analysis of those cells could lead to new treatment strategies that target the brain tumor stem cells that are responsible for maintaining a tumor’s growth.

Impact

According to Google Scholar, researchers have cited “Identification of a Cancer Stem Cell in Human Brain Tumors” almost 7,000 times as of 2025. Studies that have cited “Identification of a Cancer Stem Cell in Human Brain Tumors” have investigated the role of brain cancer stem cells and their contribution to treatment resistance. In one study in 2006, for example, researchers at Duke University Medical Center in Durham, North Carolina, investigated the role of brain cancer stem cells in resistance to radiation. They demonstrated that in an aggressive brain cancer known as glioblastoma, brain cancer stem cells contributed to resistance to radiation and suggested that those glioma stem cells may be one of the reasons why the tumor came back after radiation. Other researchers also cited “Identification of a Cancer Stem Cell in Human Brain Tumors” in the discoveries of cancer stem cells in other cancers as well. For example, in 2007, researchers from various medical and cancer centers identified cancer stem cells in pancreatic cancer and cited “Identification of a Cancer Stem Cell in Human Brain Tumors” to highlight background information in the field and show one of the origins of the idea to look for cancer stem cells in pancreatic cancer.

“Identification of a Cancer Stem Cell in Human Brain Tumors” describes the origins of brain cancer and how brain tumors can grow and maintain themselves. Because of the role that brain cancer stem cells have in tumor growth and recurrence, they have become a target for treatment. A 2024 study from researchers at the University of Health Sciences in Istanbul, Turkey, asserts that finding ways to directly target brain cancer stem cells may improve outcomes for brain cancer patients. Singh’s and her colleagues’ work in “Identification of a Cancer Stem Cell in Human Brain Tumors” created opportunities for future investigation of the role of brain cancer stem cells, including the contribution of those cells to treatment resistance as well as recurrence.