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Research objectives

Cancer biophysics

Introduction:
     Complication of metastasis, the process in which cells detach from the primary tumor to form new distant tumor sites, is the primary reason why patients die from cancer. In the van Rheenen lab we try to investigate the cellular mechanisms underlying cancer-related death using high resolution microscopy (1).

Our research focuses on four areas:
1) The cellular mechanisms of tissue development and homeostasis, tumor initiation, and tumor progression.
2) The cellular mechanisms of migration and metastasis of cancer.
3) The role of microvesicles in tumor heterogeneity and tumor progression.
4) The molecular and cellular mechanisms of chemotherapy resistance and side effects.

High resolution intravital microscopy
    Our group develops and utilizes state-of-the-art imaging techniques to visualize the adaptive properties of the few cells (e.g. stem and/or migratory cells) within the large population of non-metastasizing and differentiated cells that may maintain the heterogeneous tumor and metastasize. We combine the latest genetic tumor models with intravital imaging (the visualization of single cells in living mice) (Movie1). For this, we have developed techniques (1 ,2, 3, 4, 5) to trace individual tumor cells within the primary tumor and at distant organs in a living mouse for several weeks at subcellular resolution.

Intestinal crypt homeostasis revealed at single stem cell level by in vivo live imaging:
    Adult stem cells (SCs) are long-lived, able to self-renew and differentiate into specialized cells to drive tissue homeostasis and tissue repair, and in addition are considered to be crucial for the initiation of tumors. Deletion of the tumor suppressor gene adenomatous polyposis coli (APC) (initiating alteration in >80% of all human colorectal tumors) in Lgr5+ intestinal SCs leads to adenoma formation while deletion of this gene in differentiated cells does not (6). However, not every SC that loses APC will initiate a tumor. To better understand these stem cell dynamics and the effects of potential tumor-initiating mutations, the van Rheenen lab has developed a novel approach for continuous intravital imaging of intestinal SCs to study how the progeny of dividing SCs compete for niche space (7). We found that intestinal SCs in the upper part of the stem cell niche (termed ‘border cells’) can be passively displaced out of the niche, after the division of proximate SCs (7). Nevertheless, since SCs at the border of the niche are still able to displace cells at the center of the niche, they are also able, though at a lower frequency, to completely outcompete the other SCs. This data shows that all SCs compete for niche space and that adenoma formation depends on the outcome of competition between healthy SCs and SCs that acquired a genetic alteration.

Intravital imaging of cancer stem cell plasticity in mammary tumors:
     It is widely debated whether all tumor cells within a tumor have the same potential to propagate and maintain tumor growth, or whether there is a hierarchical organization. Lineage tracing experiments have recently shown the existence of small populations of cells, referred to as cancer stem cells (CSCs), that maintain and provide growth of squamous skin tumors and intestinal adenomas (8, 9). Using intravital lineage tracing experiments, the van Rheenen lab showed that mammary adenomas and carcinomas also contain CSCs (10). However, in contrast to the aforementioned lineage tracing techniques which provide static images and lack the ability to study whether stem cell properties can be obtained or lost, the use of intravital lineage tracing in their study demonstrated that stemness is a state rather than an intrinsic property of these cancer cells (10).

Intravital imaging of dissemination and metastasis formation:
    Complications caused by metastasis are the major cause of cancer-related death. Metastasis is a multistep process in which only a minority of cells within a tumor acquire traits and are surrounded by microenvironments that enable them to disseminate and form distant metastases. Using intravital microscopy, the van Rheenen lab dissected various critical steps of metastasis, and monitored the dynamic processes that are involved in the rare but crucial individual cells that metastasize. For example, they showed that tumor cells surrounding major blood vessels are motile, intravasate and get transported to the lungs, in contrast to tumor cells surrounding microvessels, which only display intratumoral motility (2). Moreover, we showed that the presence of T cells in the microenvironment supports the motile behavior of mammary tumors (4). Furthermore, we filmed for the first time the formation of metastases from individual cells that arrived in the liver (3). We showed that single extravasated tumor cells proliferate to form “pre-micrometastases”, in which cells are motile. Genetic and chemical suppression of this motility reduces the metastatic load by 50%, suggesting that tumor cell migration is important for the formation of liver metastases (3).

In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior:
    Tumor are heterogeneous due to genetic variations and diverging microenvironment surrounding tumor cells. A growing number of studies suggest that extracellular vesicles (EVs) may also be an important microenvironmental factor that could potentially affect tumor heterogeneity. Many different cell types have been shown to transfer biomolecules including proteins, lipids and nucleic acids through the release and uptake of EVs. The van Rheenen lab has combined high resolution intravital imaging with a Cre recombinase-based method to study EV exchange between tumor cells in living mice (11). In our system, a DsRed-to-GFP color-switch is induced specifically in reporter-expressing tumor cells (reporter+) that take up EVs released from tumor cells expressing the Cre recombinase (Cre) (Cre+ cells). We demonstrated, in living mice, that malignant tumor cells, through local and systemic transfer of EVs, can phenocopy their migratory behavior and metastatic capacity to less malignant cells(11). Our study illustrates that tumor heterogeneity contains an additional layer of complexity with tumor cells sharing biomolecules through local and systemic transfer of EVs, which profoundly affects cell behavior.

Final conclusions:
    The unique intravital imaging techniques developed in the van Rheenen lab enabled this group to gain detailed information on the cellular behavior at single-cell level during both tissue homeostasis and tumor initiation and progression. This unique way of studying these processes has led to major breakthroughs in the fundamental understanding of cancer.

References:
1. Ellenbroek SI, van Rheenen J. (2014) Nature Reviews  Cancer, 14(6): 406-18

2. Kedrin D, Gligorijevic B, Wyckoff J, Verkusha VV, Condeelis J, Segall JE, van Rheenen J. (2008). Nature Methods, (12):1019-21

3. Ritsma, L., Steller, E.J.A., Beerling, E., Loomans, C.J.M., Zomer, A., Gerlach, C., Vrisekoop, N., Seinstra, D., van Gurp, L., Schäfer, R., Raats, D.A., de Graaff, A. Schumacher, T.N., de Koning, E.J.P. Borel Rinkes, I.H., Kranenburg, O. van Rheenen, J. (2012) Sci Transl Med 4, 158ra145.

4. Ritsma L, Vrisekoop N, van Rheenen (2013) In vivo imaging and histochemistry are combined in the cryosection labelling and intravital microscopy technique. Nat Commun., 4:2366

5. Ritsma L, Steller EJA, Ellenbroek SIJ, Kranenburg O, Borel Rinkes IHM, van Rheenen (2013) Surgical implantation of the Abdominal Imaging Window for intravital imaging. Nat Protoc. 2013 Mar;8(3):583-94. doi: 10.1038/nprot.2013.026. Epub 2013 Feb 21

6. Barker, N., Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M, Danenberg E, Clarke AR, Sansom OJ, Clevers H. 2009. Nature.

7. Ritsma L, Ellenbroek S, Zomer A, Snippert H, de Sauvage F, Simons B, Clevers H, van Rheenen J, (2014), Nature, 507(7492):362-365

8. Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C, Nature 2012;488:527–530.

9. Schepers AG, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, Clevers H., Science 2012 337:730–735.

10. Zomer A, Ellenbroek SIJ, Ritsma L, Beerling E, Vrisekoop N, van Rheenen J (2013), Stem Cells, Mar;31(3):602-6. doi: 10.1002/stem.1296 .

11. Zomer A, Maynard C, Verweij FJ, Kamermans A, Schäfer R, Beerling E, Schiffelers RM, de Wit E, Berenguer J, Ellenbroek SIJ, Wurdinger T, Pegtel DM, van Rheenen J, (2015), In Vivo Imaging Reveals Extracellular Vesicle-Mediated Phenocopying of Metastatic Behavior. Cell, 161(5):1046–1057


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vanRheenen IVM

Movie 1: We visualize the motility of single invasive lobular carcinoma cells (P53-/-;E-cad-/-) in a living mouse. Time series of IVM images of tumor cells (green) and type I collagen (purple) were taken with a two-photon microscope. The movie is a maximum projection of a 30um-thick Z-stack at 50-80um deep into the tumor. Scale bar represents 25 um.

 

moving colorectal tumor cells in a liver pre-micrometastasis
Movie 2: Single colorectal tumor cells that arrive in the liver grow into a pre-micrometastasis where cells are motile and predominantly adjacent to liver tissue. Pre-micrometastasis grow into micrometastasis where tumor cells are non-motile. In this movie, the movement of tumor cells in pre-micrometastasis is visible. This movie is taken through the Abdominal Imaging Window (Ritsma et al, Sci Trans Med, 2012). Scale bar is 25 um.

 

 

 

 

 

 

 

 

 

 

image description

Figure 2: intravital imaging of metastasis. Top panel: Through a mammary gland imaging window, we image tumors for multiple days. By photomarking a population of tumor cells within a tumor (here we photomarked a square, which is depicted as green), we can track the cells for multiple days (here 6 (depicted as blue) and 24 (depicted as red) hours later), and observe migration, invasion and intravasation. Lower left panel: Tumors in which the tumor cells express GFP were imaged with intravital imaging. The blood vessels were labelled with fluorescent dextrans and are here depicted in red. Lower right panel, a tumor cell that enter the blood. The periphery of the blood vessel is shown in red.