Changes in the Characteristics of AC133+ Stem Cells in Hypoxic U251 Glioblastoma Cell Microenvironment
Angiogenesis, the formation of blood vessels, is a critical factor in brain cancer growth. Anti-angiogenic drugs block oxygen supply to the tumor by preventing blood vessel formation. Previous in vivo studies suggested a link between anti-angiogenic treatment and increased tumor growth post-remission. Our study attempts to examine the role of AC133+ stem cells in the glioblastoma microenvironment to understand tumor response mechanisms, in vitro. We proposed that glioblastoma cells in an hypoxic environment would release angiogenic factors, signaling to AC133+ cells, resulting in tumor growth. In this study, AC133+ cells were exposed to hypoxic U251 glioblastoma environment and they responded by elongating, indicating possible blood vessel formation. Flow cytometry was performed to determine the markers expressed and the cell types that the AC133+ cells differentiated into post-elongation. The results indicate that the stem cells differentiated into blood vessels under hypoxic condition.
Brain Cancer is the deadliest cancer in the US, killing over 14000 people every year. ((National Cancer Institute, 2010)). About 85% to 90% of all primary central nervous system (CNS) tumors are brain tumors ((Mehta, 2011)). Glioblastoma, also known as Glioblastoma Multiforme (GBM), is the most common form of primary brain cancer in adults. Approximately 10,000 new cases of GBM (affecting 3.2 out of every 100,000 people) are diagnosed in the United States each year occurring more commonly in older male individuals ((Novocure, 2014)). There are very few conclusive observations that have been made either about the environmental or occupational causes of primary CNS tumors ((Mehta, 2011)). For example, exposure to vinyl chloride may cause the development of glioma. The infection of the virus Epstein-Barr has been linked to cause primary CNS lymphoma. The risks for primary CNS lymphoma are greatly increased for patients with AIDS (acquired immunodeficiency syndrome) and transplant patients ((Mehta, 2011, Schabet, 1999)).
Cancer is caused by DNA mutations of a cell, which causes the cell to divide uncontrollably (American Cancer Society, 2014). GBM tumors grow from the astrocytes-star-shaped cells that make up the tissue supporting the brain's nerve cells. Usually, GBM tumors occur within the two larger lobes (cerebral hemispheres) of the brain more often than the spinal cord and don’t spread to other parts of the body ((Novocure, 2014)).
GBM cells grow rapidly and in large numbers. As a tumor mass grows, it leads to pressure on the brain, causing symptoms such as headaches, nausea and vomiting, and drowsiness. It can also lead to a variety of symptoms such as seizures, weakness on one side of the body, difficulty with memory or speech, or changes in vision. The symptoms depend on the location of the tumor in the brain and the part of the body that the brain controls ((Novocure, 2014)).
GBM is one of the most aggressive forms of brain cancer and currently there are no effective treatments for it. As the cells within the tumor are very resistant to most types of treatments, doctors rather focus on using various treatments in order to slow tumor growth. It is also difficult to surgically remove these tumors, as they tend to have tentacle-like structures ((Novocure, 2014)).
GBM is a fast-growing type of central nervous system tumor that forms from glial (supportive) tissue of the brain and spinal cord and has cells that look very different from normal cells. ((National Cancer Institute, 2014)). However, like all cells, brain tumor cells also need blood vessels to grow. Angiogenesis is the physiological process of formation of new blood vessels from pre-existing blood vessels. Brain cancer drugs such as angiogenesis inhibitors aim to prevent the growth of new blood vessels ((Paez-Ribes, 2013)). Although the effectiveness of these drugs is not established, these inhibitors are still used in the treatment of cancer with the intent to extend the life of the patient for a couple of weeks or months. Anti-angiogenic drugs have been developed based on the fact that tumor cells need blood vessels to grow and blood vessels bring oxygen to the cells. Therefore, limiting the blood vessel’s growth will prevent and eventually stop cancerous tumor growth. Thus, the goal of the anti-angiogenic drug is to create a hypoxic (lack of oxygen) environment at the site of the tumor.
Recent literature disputed the efficacy of the anti-angiogenic drugs in treating cancer (Hayden, 2009). When cancerous cells form small solids at the early stage of cancer, they are supplied with nutrients from nearby blood vessels. In order to grow, these solid cancers need their own blood vessels, which they create by angiogenesis promoters such as vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGF). FGFs and VEGFs impart their effects via specific binding to cell
surface-expressed receptors equipped with tyrosine kinase activity. Activation of the receptor kinase activity allows coupling to downstream signal transduction pathways. These pathways regulate proliferation, migration and differentiation of endothelial cells ((Cross and Claesson-Welsh, 2001)). In theory, anti-angiogenic drugs are expected to interrupt that process and thus have been hailed as opening a new era in cancer treatment. However, a number of in vivo studies suggest that such drugs may in certain situations actually accelerate the growth and spread of cancer ((Paez–Ribes, 2013)). Figure 1 illustrates adaptive-evasive responses by tumors to anti-angiogenic therapies ((Paez-Ribes, 2013)).
Figure 1: Schematic summary of adaptive responses to anti-angiogenic therapy that elicit “Evasive Resistance”. Figure adapted from Paez-Ribes, 2013
Paez-Ribes investigated the impact of anti-angiogenic drugs on angiogenic inhibitor based cancer therapy. These investigators observed increased invasiveness of cancer after the treatment and identified strong hypoxic condition in the cancer cell. This research takes a closer look at the physio-chemical processes near hypoxic cancer cells and the interaction of stem cells with cancer cell environment. According to the hypothesis, glioblastoma cells in hypoxic environment release angiogenic factors and these factors signal to the stem cells for help. In response, the stem cells rush to the hypoxic environment, change their phenotypical expression, and transform themselves into blood vessels and help cancer grow. The results will provide new insight into the cancer growth as a result of anti-angiogenic drugs treatment.
Materials & Methods
In this research, U251 cells are modeled as a specific type of brain cancer cells, also known as Glioblastoma Multiforme (GBM). AC133+ stem cells from a human placenta were used in the experiment due to their pluripotency. Pluripotency is important in this experiment because it allowed all possible cells to form from the stem cell, as opposed to totipotent and multipotent cells, which have limited differentiation. The experiment was conducted in the following steps:
Preparation of U251 cells
We grew U251 cells in six different T-75 flasks in an incubator (20% O2) via serum-based growth media. After the cells had grown to 5 million cells per flask, the cells were collected and separated from the growth media using a centrifuge. 10 ml of RPMI (Roswell Park Memorial Institute) media was as introduced to all the flasks in order to keep the cells alive but prevent any further cell growth aka a suspension media.
Subject U251 cells to hypoxic condition
Three flasks were placed in hypoxic conditions (2% oxygen, 37 degrees Celsius), for 24 hours, 48 hours, and 72 hours. The other three flasks were used as the control and were subjected normoxic condition (21% O2, 37 degrees Celsius) for 24, 48, and 72 hours. The environmental condition is expected to cause U251 cells to release angiogenic factors in the RPMI. The supernatant (extracellular fluid) was then separated from the U251 cells using a centrifuge. The supernatant contains all the angiogenic factors released by the cancer cells during hypoxic and normoxic conditions. U251 cells were separated in order to avoid any secondary interaction with stem cells.
Introduction of stem cells into supernatant and incubation
Stem cells were then introduced to the flasks containing supernatants procured from the U251 in six different conditions. Three wells for each of the simulated environmental conditions were prepared, in order to ensure that the results were accurate. The flasks containing stem cells and the U251 supernatant were then incubated for 7 days at 37° Celsius, 1 atmosphere, and 21% O2. The objective
was to allow sufficient time for the stem cells to interact with the chemicals released by U251. After the incubation period of 7 days had ended, pictures were taken of the stem cells in order to observe the physical changes in the cells. The stem cells placed in 72-hour supernatant were used to determine the phenotypical expression by flowcytometry of different markers after incubation in different conditioned media. Flow cytometry measures the amount (percentage) of cells of a sample that display a marker. We performed flow cytometry in order to determine if the stem cells had transformed into cells found in blood vessels. For example, if 70 percent of the stem cells display several markers indicating the presence of hematopoietic cells, then we will know that the majority of the stem cells have indeed differentiated into cells found in blood vessels. All the experimental steps are illustrated in the following flowchart.
SC = Stem Cells
St = Supernatant
H = Hypoxic
N = Normoxic
In this experiment, a normoxic environment is used as a control. Hypoxic and normoxic conditions were imposed for two different durations (24 hours and 48 hours) in order to investigate the impact of time on the stem cell behavior. The incubation period of seven days was used to obtain stable data. Moreover different markers were analyzed to determine the phenotypical expression of stem cells by flow cytometry following 72 hours exposure to tumor microenvironments in an in vitro set up.
Figures 2-5: Stem cell images in various U251 supernatants
The stem cells subjected to 24 hour and 48 hours of normoxic condition are generally circular in shape and do not show any major changes. The apparent non-uniformity of the cells within the sample is the natural variation of the cells within the supernatant media.
The stark difference in the shape of the stem cells subjected to hypoxic condition for 24 hours and 48 hours respectively, compared to the same under normoxic conditions, is very evident. The progression of cell elongation and the higher number of elongated cells per sample, in the case of 48 hours, indicate that the longer the stem cells are subjected to hypoxic condition, the greater is the shape change in stem cells. The shape change of the stem cells is a clear indication that these cells are undergoing significant changes. As shown, the shape change is not uniform. Some of the stem cells changed from circular cells to elongated cells and the shape of the elongation varies within the sample.
While figures 2-5 demonstrate that the AC133+ stem cells in hypoxic U251 glioblastoma cell microenvironment is undergoing changes (demonstrated by elongated shape), the cell type cannot be
confirmed from these results. Therefore, we have conducted flow cytometry analysis to determine the changes in phenotypes and discover if the stem cells differentiated into blood vessels. AC133+ stem cells were exposed to supernatant collected from different conditions and stained with specific antibodies.
Figure 6: Shows the markers displayed by the AC133+ Stem Cells (Flow Cytometry)
Flow cytometry shows the expression of different markers on the surface of stem cells in control and hypoxic media (Figure 6). Figure 6 shows the changes in phenotypes of AC133+ cell following exposure to hypoxic condition compared to control condition. Comparing these two sets of data, it is evident that the phenotypes of AC133+ changed significantly in the hypoxic condition. The decrease of the cell markers CD45 (Hematopoietic cell precursor marker), CD31 (cell adhesion), CD144 (cell adhesion) and the significant increase of CD105 (angiogenesis), CD195 (Monocytes, T cells) and CD150 are clearly visible in hypoxic condition. The most remarkable change is that CD105 doubled in the hypoxic condition compared to the control; CD150 is a marker that indicates the presence of hematopoietic stem cells (Kiel, 2005). This indicates that the chemicals released by the U251 caused the amount of hematopoietic stem cells (HSCs) present to double. HSCs are different from AC133+ stem cells in that HSCs can only differentiate into cells in the blood, while AC133+ stem cells can transform into any cell. Therefore, figure 6 clearly demonstrates that many AC133+ cells are turning into blood vessels under a simulated hypoxic condition.
The increases and decreases in the other cell markers mentioned previously are quite notable.
The decrease in CD45 indicates that the AC133+ stem cells are changing from precursors to HSCs into the HSC cells themselves. The decrease in CD31 and CD144 indicate a decrease in endothelial cells, which may indicate that the cells that were formerly endothelial transformed into another type of cell. The increase in CD195 is a definite indicator that blood cells such as monocytes and T cells are formed from the AC133+ cells, while the increase in CD105 is an indicator that angiogenesis is increasing.
The physical changes in the AC133+ stem cells lead to the conclusion that the U251 cells, when under hypoxic or nutrient deprived conditions, cause stem cells to differentiate and assist in angiogenesis. This change may play a major factor in the failure of anti-angiogenic drugs to treat cancer, and ultimately result in the death of the patient.
Discussions and Conclusion
The objective of this study was to investigate the interaction of AC133+ stem cells with hypoxic U251 glioblastoma cell microenvironment. The microenvironment was modeled as the supernatant separated from the U251 cells subjected to hypoxic condition. The supernatant contained all the angiogenic factors released by the U251 glioblastoma cells. AC133+ stem cells were added to the supernatant to model its interaction with U251 cells under hypoxic condition. According to our hypothesis, the stem cells differentiation into blood vessels would only occur when U251 cells are in a hypoxic condition. This research contributes in understanding the fundamental process of interaction between glioblastoma cells and AC133+ stem cells. The hypothesis provides a plausible explanation about the poor efficacy of anti-angiogenic drugs for brain cancer therapy.
Dr. Ali Syed Arbab, Senior Scientist and Director of Molecular and Cellular Research Laboratory at Henry Ford Hospital, was my mentor for the research project. He provided all materials for my research work and helped me with data analysis. He also trained me in the basic cellular protocols and staining protocols of stem cells and U251 glioblastoma cells. Safa Khan and Thomas Ittoop, summer undergraduate volunteers at the laboratory, performed Flow Cytometry for all cells.
Featured image credit: 3Fx Medical Animation & Visual Media