Seeking New Treatment Methods by Analyzing the Fate of Single Cancer Stem Cells

Highly Malignant Stem Cells of the Uppermost Stage

There is a hierarchy of cancer cells, with some that have difficulty in dying, and other cells that die easily. The top of this hierarchy is occupied by cancer stem cells. The “Cancer Stem Cell Hypothesis,” which states that cancer emerges and progresses from these cancer stem cells, has been proposed, and has been verified, to some extent.

Stem cells have two characteristics: a self-replicative potential, which allows them to produce identical stem cells through division, and pluripotency, which allows them to differentiate into various kinds of cell. Differentiated cancer cells are thought to have a greater tendency to die eventually, and are expected to be less malignant. In contrast, because cancer stem cells repeatedly undergo asymmetric cell division, they continually produce both stem cells and non-stem cells while proliferating tremendously, making them an extremely malignant cell type.

The existence of such cancer stem cells is a big problem in the treatment of cancer. Cancer cells differentiated from stem cells can be considered as being only mildly malignant of themselves; however, mutations can cause the drugs used up to that point to become ineffective. Finding a drug that is effective for a given kind of cancer cell does not imply that treatment is completed. Moreover, cancer stem cells have considerable tumorigenicity such that they exhibit strong tumor-forming potential when implanted onto the backs of immune-deficient mice. Proliferation occurs rapidly even from single cells in vitro, and it is possible this also reflects metastases in vivo.

Mr. Ishii said that, “Given these characteristics, if one can kill the cancer stem cells, this is expected to have some therapeutic efficacy, and treatments that target them are being developed; however, in practice, it is not so easy.”

Initially, Fucci-expressing cells were produced to investigate the fate of cancer stem cells. Fucci-expressing cells exhibit a red nucleus in the G0/G1 phase and a green nucleus in the G2/M phase. A single, podoplanin-positive, Fucci-expressing cancer stem cell was seeded and its fate observed over time for a week (168 hours). 

25 hr following inoculation: single tumor cell (green) found.
After 38 hr: the tumor cell divided into two (red).
After 76 hr: tumor cells further divided into four. Of these, one is in the G2/M phase.
After 168 hr: tumor cells have proliferated to become 10 cells. Of these, two are in the S/G2/M phase.

Following Single Cells Using Time lapse

To achieve high-precision cancer therapy, it is firstly important to fully capture the image of the living cancer cell. Rather than perceiving cancer as a vague mass of cancer cells, it is especially necessary to perceive the fate of each individual cancer stem cell. It is important to remember that the narrative of cancer stem cells dividing to produce numerous non-stem cancer cells that die, is only a model; there had been no investigations that had actually observed this process by eye.

Therefore, an experimental setup was established using the IncuCyte® system. This system is installed in an incubator to observe cells over time and quantify them, completely automatically. Using time-lapse photography, it is possible to image the life cycle of cells and store the analysis data.

Podoplanin and Fucci Cells Are the Key to Experimentation

Podoplanin molecules are feasible markers for discerning cancer stem cells. In 2008, Mr. Ishii et al. reported that squamous cancer cells that expressed extremely high levels of podoplanin had the characteristics of cancer stem cells (Ref: Biochem Biophys Res Commun. 2008 Aug 15; 373 (1): 36−41).

It has been suggested that podoplanin may play an important role in the progression of cancer. In squamous cancers, podoplanin is expressed more focally in cells in the undifferentiated region at the basal layer. Additionally, research by the Cancer Institute of the Japanese Foundation for Cancer Research has shown that metastases are promoted by growth factors released by the platelets through interactions between podoplanin and platelets. Meanwhile, in the cytoplasm, it activates malignant transformation-related Rho proteins.

On actually seeding single podoplanin-positive or podoplanin-negative cells, the colony-forming potential was clearly greater for the podoplanin-positive cells. This may reflect that, for example, just a single cancer cell circulating in the blood could cause a metastasis.

Although the formation of colonies from single cells has been confirmed, the kinds of process through which larger colonies are actually formed are completely unknown.

“I thought it important to look at what was occurring during the processes and investigate the molecular mechanisms involved in tumor formation,” said Mr. Ishii. Therefore, he formed a research team centered on Mr. Tomoyuki Miyashita, a Ph.D. student of the Graduate School of Frontier Sciences, the University of Tokyo, to investigate the molecular mechanisms involved in tumor formation by following the fate of single cancer stem cells.

It should be possible to obtain a deeper picture of cancer stem cells by observing how colonies form over time from the division of a single, podoplanin-positive cancer stem cell, as well as by observing whether podoplanin negative cells really all die, or whether they survive.

Another technological revolution that enabled the experiment is fluorescent, ubiquitination-based cell cycle indicator (Fucci), a fluorescent probe for observing the progress of the cell cycle developed by Atsushi Miyawaki of the Riken institute. Fucci-expressing cancer cells turn red at the G0/G1 phase, and green at the later S/G2/M phase, enabling the real-time visualization of the state of cancer cell division. By using IncuCyte®, two-color fluorescence imaging becomes possible.

Single podoplanin-positive cells (≒ cancer stem cells) and single negative cells (≒non-stem cancer cells) were each seeded into wells, with an IncuCyte® used to image them using time-lapse photography (every hour) for a week.

Surviving Cells Form Large Colonies

After a week, rather than all of the cancer cells being red or green, there was a mixture of both colors. Moreover, in the seven days that proliferation occurred, while some cells turned green and entered cell death, others divided again and turned red, so a fairly dramatic process was occurring over the week. It was impossible to understand such intermediate processes just using the traditional method of counting the final number of cells.

The experiment was conducted five times, and a genealogical tree of the fates of all 253 non-stem cancer cells and 272 cancer stem cells was produced and further analyzed.

As a result, it was found that the cell death was better avoided through the division process for the cancer stem cells, compared to the non-stem cancer cells. In other words, the mechanism of cell death evasion is likely involved in the formation of larger colonies by cancer stem cells. While it was expected that the cell cycle was changing, it was found from the actual observations that there were no such changes.

Subsequently, podoplanin-knockdown cells were produced for a similar experiment. The results of this experiment showed a significant increase in the proportion of cells that died. This shows that podoplanin is a functional molecule for evading cell death and supporting cancer stem cells.

Additionally, using Rho-associated coiled-coil forming kinase (ROCK) inhibitors, the formation of colonies by podoplanin-positive cells was significantly reduced. This is considered likely to be important in overcoming cancer stem cells.

Some of these results were published in Scientific Reports in January 2017.

Considering the Microenvironment in the Next Steps

Mr. Ishii developed an interest in the fact that cells have various shapes from his training in pathology during his time in the medical department, and has leaped into the world of pathology with its focus on morphology. Although this field provides less opportunities to see patients directly, if one can accurately attribute diagnoses to received specimens and link this to new treatment methods, this will have a considerable knock-on effect.

The finding from this time-lapse analysis of single cells—that podoplanin is one part of the molecular mechanism that augments colony-forming potential and survival in squamous cancer cells—is quite a huge step. This is not a simple narrative of a single cancer stem cell regularly proliferating, rather it was found that there are some cells that divide and undergo cell death, and others that exhibit cessation of the cell cycle.

When considering an in vivo perspective, the complexities increase further, making cancer treatment an extremely difficult issue. Because cancer cells exist and grow amongst a convolution of fibroblasts, blood vessels, and immune cells, it is necessary to consider this microenvironment.

“It has become clear that there are considerable differences in the picture of live cancer stem cells and non-stem cancer cells, so next, I would like to investigate the influence of other cells being added.”

Having again verified the stubbornness of cancer cells, the merits of such research seem even greater than before. Work continues day and night towards the day when cancer is eliminated from the world and “cancer research centers” are no longer needed.