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2013 Winner - Jacco van Rheenen

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Tracking the dynamics of cancer stem cells

In early December during the American Society of Hematology (ASH) Annual Meeting, STEM CELLS announces its prestigious Young Investigator Award (YIA) for 2013 to Jacco van Rheenen, Ph.D., of the Hubrecht Institute in The Netherlands.

Each year the $10,000 YIA prize is bestowed on a young scientist whose paper is deemed by a global jury to be of worldwide significance. Dr. van Rheenen’s lab investigates how healthy and tumorigenic tissues are derived from and maintained by cancer stem cells, how tumor cells disseminate from primary tumors and how these disseminated tumor cells grow out in distant organs.

His group develops and utilizes the latest imaging techniques to visualize the adaptive properties of the few cells within the large population of non-metastasizing and differentiated cells that might maintain the heterogeneous tumor and metastasize. 

 

Dr. van Rheenen and his team’s paper, “Brief Report: Intravital Imaging of Cancer Stem Cell Plasticity in Mammary Tumors,” was first published early online in STEM CELLS Express in December 2012. In it, they reported for the first time the existence of cancer stem cells (CSC) in unperturbed mammary tumors and demonstrated CSC plasticity. The data indicates that existing CSCs disappear and new CSCs form during mammary tumor growth, illustrating the cells’ dynamic nature.

Dr. van Rheenen was trained in a variety of imaging techniques during his doctoral work with Dr. Kees Jalink at The Netherlands Cancer Institute. He was among the first to optimize imaging and develop software to quantitatively measure fluorescence resonance energy transfer (FRET) on confocal microscopes.

To broaden his skills, Dr. van Rheenen obtained a postdoctoral fellowship in the United States in the lab of Dr. John Condeelis where he extended his expertise by imaging mammary tumors intravitally, using two-photon microscopy. He also became an expert in intravital FRET imaging.

In 2008 Dr. van Rheenen was appointed a group leader at the Hubrecht Institute, where he uses his imaging techniques to visualize processes in the metastasis of mammary tumor cells in living animals. Today he is senior group leader.

Over the course of his career he has published more than 26 times and earned several prestigious grants, including a 2009 VIDI grant and a research grant from the Dutch Cancer Society. 

STEM CELLS (SC) caught up with Dr. van Rheenen recently to talk about his work and his YIA recognition. We share our conversation here.

SC: What hypothesis were you testing in the paper?
JR: In our paper, we were testing whether every tumor cell in a tumor has exactly the same potential to fuel tumor growth. In healthy tissue, not every cell is equal but there is a hierarchy; you have a very small number of adult stem cells that give rise to new stem cells and to all other more committed cells found in that tissue .

The stem cells are replicative immortal and divide indefinitely, but more differentiated cells can only divide a couple of rounds and are committed to die.

It is important to know whether this hierarchy with cancer stem cells and committed cells is maintained within a tumor. If this hypothesis is true, it means that only a few cells within a tumor are actually causing the tumor to grow. If you want to target a tumor with chemotherapy, that means you need to target specifically these cancer stem cells.

One thing that was only hypothesized, but not really visualized before, is whether committed cells can “de-differentiate” and become a cancer stem cell again. That's really important as well, because if we succeed in killing the cancer stem cells by a therapy, but committed cells reform this dangerous population again, the therapy will fail.

So to summarize, first we were testing the hypothesis that the tumor growth is driven by a small population of cancer stem cells. Second, after confirming the first hypothesis, we were testing whether committed cells can revert back into cancer stem cells and whether this is a natural event happening in tumors.

SC: What was happening that made you wonder about this?
JR: The cancer stem cell hypothesis is based on an assay in which two populations of cells were isolated from a tumor followed by transplantation of the cells into immunodeficient mice: one population expressing stem cell markers, referred to as cancer stem cells, and another population of cells lacking these markers referred to as committed cells.

Interestingly, upon transplantation in mice, only the populations of cells that express stem cell markers are able to grow out into a tumor. Based on this observation, it was hypothesized that the tumor exists of at least two types of cells: cancer stem cells and more committed cells.

However, it is still debated how to interpret this data. First, the capacity to initiate growth of a new tumor may not be the same as the capacity to fuel growth in an intact tumor. Second, isolated cells lose their microenvironment, which is crucial for their functionality and might be important for the maintenance of cancer stem cell properties. Just before our paper came out, two other papers were published in Nature in which the researchers used lineage tracing to study the hierarchy of cancer stem cells and committed cells within unperturbed skin and intestinal tumors. Using promoters thought to be specifically expressed in stem cells, cancer stem cells and their progeny could be genetically labeled and traced over time. Using this innovative approach, the authors could show the existence of a small population of cells with stem cell characteristics that give rise to all shorter-lived committed cells in skin and intestinal tumors.

We were interested in breast tumors. However promoters that drive expression specifically in adult breast stem cells were not available. Moreover, the lineage tracing studies relied on static images and therefore lacked the ability to study tumor cells’ plasticity or to test whether committed cells could revert back to stem cells.

In my lab we have developed a method that allows us to view tumors in mice and visualize the tumor cells’ outgrowth over multiple days and even over multiple weeks. We can do lineage tracing in real time and since we make use of a general promoter that can drive expression of a fluorescent protein in both cancer stem cells and more committed tumor cells we can visualize how committed cells can actually turn into cancer stem cells and the other way around. So in addition to studying hierarchy in tumors, our approach also allows us to study the gain or loss of cancer stem properties, i.e. committed cells that revert to cancer stem cells and the other way around.

SC: So that was the methodological technique that was really important to your study?
JR: Exactly. There are a couple of techniques that were extremely important. First, we make use of intravital imaging using a technique developed in my lab. Through a small imaging window implanted on top of a mouse’s mammary gland, we can visualize individual tumor cells on subcellular resolution in a living mouse. Repetitive microscopy through this imaging window allows us to visualize breast tumors over multiple weeks. So, using this intravital imaging technique we can actually see individual cells, not for a couple of hours, but for a couple of weeks.

We combined this with a Cre-reporter mouse model, termed Confetti, which was originally developed to visualize neuronal circuits. We crossed this mouse with a mouse model in which all cells express a Cre recombinase that can be activated upon injection of Tamoxifen. In the resulting mouse, Tamoxifen injection resulted in activation of Cre which on its turn induces stochastic expression of one of the fluorescent proteins of the Confetti construct leading to the random induction of CFP, GFP, YFP or RFP-expressing cells.

The recombination of the Confetti construct is a genetic change, so it will also be present in the offspring of those cells. Thus, the whole family tree will be coded with the same color.

To study our initial hypotheses, we grew tumors in these mice, implanted our small imaging window on top of the breast tumor and we gave the mice Tamoxifen to induce the random expression of one of the four Confetti colors (CFP, GFP, YFP or RFP). Then, by performing microscopy through the imaging window over several weeks we followed how individual Confetti-expressing tumor cells gave offspring with the same color.

As expected for tumors that contain cancer stem cells, we observed over time the development of small clones of cells with the same color. We could follow the growth of individual clones and were thereby able to actually follow the kinetics of the formation of offspring by cancer stem cells. Based on these experiments, we found that approximately only one out of 10,000 cells was able to fuel tumor growth.

So both the genetic Confetti mouse model and the intravital imaging techniques were key to our research.

SC:  How do you interpret the results and what does that mean for stem cell biology?
JR: Our experiments suggest that only one out of 10,000 cells in a tumor can actually fuel tumor growth. This suggests that tumor cells with stem cell properties produce progeny that are committed to die. This is a really important conclusion from the therapeutic point of view because if treatment would be focused on killing cancer stem cells that fuel tumor growth, we might be able to de-bulk the tumor and treat the patient.

The other very important conclusion that we could draw from our work is that a more committed tumor cell, which is destined to die, can actually revert back to a cancer stem cell state. That means that tumor cells might only temporarily reside in a certain state, i.e. the cancer stem cell or committed cell state, and that tumor cells display extensive dynamic behavior.

SC: What should you test next? What should this information lead to?
JR: One thing we should investigate is the molecular mechanism driving the dynamic behavior of tumor cells. Why does a committed tumor cell become a cancer stem cell and how can cancer stem cells become more committed cells?

This is really important because if you want to design new therapeutic strategies you not only want to kill the cancer stem cells, you also want to avoid that more committed tumor cells regain cancer stem cell properties.

Other very important future experiments should focus on the type of tumor cells that disseminate and grow out at different sites, the process of metastasis. You can imagine that if only one out of 10,000 cells is able to grow and fuel tumor growth, maybe those cells are also the cells that grow metastases. If this is the case, it explains why metastasis formation is such an inefficient process. Maybe many tumor cells are able to go into circulation and spread to a different site — for example, the lungs or the liver — but most cells cannot make it because they don’t have the right stem cell capacity to grow into a metastasis.

On the other hand, what we show is that more committed cells can revert back to becoming cancer stem cells so it may be possible for a more committed cell to leave the primary tumor, travel to the lungs or liver, then revert back into a cancer stem cell state thereby gaining the capacity to grow a metastasis. Our guess is that the plasticity we see in the primary tumor, and the molecular mechanisms needed to have this reversion from a committed cell to a cancer stem cell, may be the same at the distant site during metastasis formation. We are currently making the mouse models to test this hypothesis.

SC: Why did you select STEM CELLS as the journal to publish your paper?
JR: We wanted to share our research with both the stem cell community and the cancer biology community. STEM CELLS is very well known, highly cited and very much read journal by both communities.

SC: Tell us a little about yourself and why you chose to do stem cell research.
JR: I like multidisciplinary work. My undergrad degree was in molecular biology and during my Ph.D. at The Netherlands Cancer Institute in Amsterdam I became interested in cancer. The lab of Kees Jalink where I worked was a biophysics lab, and I gained experience in development of imaging software, computer models, and obviously microscopy. During my Ph.D. I got really interested in the behavior of living cells.

Most molecular and cellular biology techniques provide a static picture of tumor processes. But if you instead can study biological processes in real-time, in living cells or organisms , you can learn so much more. Therefore, during my Ph.D, I started to study molecular processes in real-time using microscopy. and I became very attracted by the idea of visualizing the behavior of individual living cells in living mice.

For my post-doc, I moved to the States to work in the lab of John Condeelis at the Albert Einstein College of Medicine in New York. There I was trained in visualizing individual cells in a living mouse. I realized that an individual cell in in vitro culture conditions behaves totally different from a cell that is present in a living animal. For example, the in vivo microenvironment surrounding a tumor cell is important in determining how that particular cell behaves.

When I moved back to Europe to start my own research group at the Hubrecht Institute, which is an institute for stem cell research, I combined all my knowledge from genetics, microscopy and modeling. I collaborate with people who study biophysics, with medical doctors, with mathematicians and try to incorporate all these disciplines in my research.

I think that’s one of the reasons why my research has so far been a success. In my opinion, combining so many different disciplines really helps to bring my research to the next level.

SC: It sounds like one step of your career led you to the next step and on to the next.
JR: Exactly. I’m currently a senior group leader at the Hubrecht Institute. My group includes four post-docs, four Ph.D. students, four master's students and two technicians. Our main goal is to gain insights into how tumors grow and spread. We investigate how healthy and tumorigenic tissues are derived from and maintained by (cancer) stem cells, how tumor cells disseminate from primary tumors and how these disseminated tumor cells grow out at distant organs.

SC: In addition to your own work, what are some of the more interesting things you think are coming out of the institute right now?
JR: There’s a lot of exciting research going on. For example, the lab of Hans Clevers is located within my institute. He identified Lgr5 as a specific marker for intestinal adult stem cells and he subsequently made mouse models where GFP and Cre are expressed under the control of the Lgr5 promoter which enabled the labeling of and tracing from Lgr5+ stem cells. These models now also enable us to visualize the individual Lgr5+ cells in a living mouse. So we can now combine these mouse models with our intravital imaging techniques, which can really help to make the next step.

I am also very excited by the work from our current director Alexander van Oudenaarden. He's able to sequence the mRNA from just a few cells. We are currently trying to isolate small numbers of cells, for example (cancer) stem cells, and determine the gene expression profile of these unique and dangerous population of cells.

Using these techniques we hope to gain insights into processes determining a cell’s state, i.e. a stem cell state versus a more committed state. When do more committed cells gain stem cell properties, and are we able to visualize this specific process and can we block this reversion?

SC: Well there's a lot going on and that leads to the question, what's next?
JR: There are a couple things. First, we would like to identify the type of tumor cells that is able to metastasize. For example are only cancer stem cells able to metastasize? Can committed cells metastasize? If so, do they need to revert to cancer stem cell state at the metastatic site? If so, what are the molecular mechanisms of this reversion in order to block the formation of metastases?

It has been suggested that for successful chemotherapy responses, the cancer stem cells should be targeted. But our data suggest that committed cells can transfer into cancer stem cells. If we are able to identify the mechanism of this reversion and are able to stop this from happening, we may be able to improve the chemotherapy.

Those are two thoughts we would like to explore more in the coming years.

SC: As far as your own career, what do you see yourself doing down the road?
JR: I would really, really like to continue to work in a multidisciplinary field. So far, that's mainly been biophysics combined with genetics. But in the near future, I definitely want to recruit more medical doctors and students to my lab to make the translation from the basic findings that we have and translate them into the clinic. I think that’s really important.

SC: How do you expect the Young Investigators Award to affect your career?
JR: It's unbelievable. It’s a big honor, and it is great that people recognize my work as being important. This prize does not only inspire me to keep doing this type of research, but it inspires and acknowledges all the people in my lab who worked very hard on this project. That’s especially true for the first author, Anoek Zomer. In addition to Anoek and me, a large multidisciplinary team of people were involved in this research including Anko de Graaff who always makes sure that the microscopes are working and many people who took great care of the mice.

I'm so happy we got this prize, because it's really a team effort.

Click here to read the best papers from our 2012 Young Investigators.