Tumor Invasion

The migration of cancer cells away from a primary tumor, a process known as tumor invasion is the first step in metastasis. Despite the importance of this process in cancer progression, surprisingly little is known about how cancer cells escape the tumor and migrate through the surrounding tissue. Histological studies have shown that invading cancer cells can have a wide variety of morphologies (Reviewed in Friedl et al, 2012). It has recently been shown, using in vitro systems that mimic the cancer environement, that the properties of the environemnt surrounding a tumor may affect cancer cell migration (Haeger et al, 2012). For my postdoctoral work in the lab of Dr. Danijela Matic Vignjevic at the Institute Curie in Paris, France, I am working toward understanding the process of tumor invasion. As a model system, I primarily use confined cellular spheroids, which have previously been used in the Vignjevic group in collaboration with others at the Institute Curie. Confined cellular spheroids are aggregates of cancer cells that are grown inside of a spherical capsule of alginate, an elastic polymer (Figure 1; Alessandri et al, 2013). Cell spheroids comprised of invasive cancer cells have been shown to expand over time, and the cells eventually burst the alginate shell and migrate away from the aggregate (Movie 1).

Figure 1. Cellular spheroid generation. Left, spheroids are generated using a microfluidics device in which a cell solution (CS) is flown in parallel with an intermediate solution (IF) an an alginate solution (AL). Drops are formed, causing the alginate to form a sphere around the other solutions. The alginate is cross-linked when the drop is submersed in a calcium bath. Right, growth of mouse CT26 colon carcinoma cells in a spheriod over a period of 10 days. (Modified from Alessandri et al, 2013.)

Movie 1. Growth and migration of a CT26 cell spheroid. After the cell spheroid grows and bursts the alginate cell, the cells migrate away from the cell aggregate. Scale bar: 100 μm. (From Alessandri et al, 2013.)

The Actin Cortex

The actin cortex is a thin network of F-actin, myosin and associated proteins that underlies the plasma membrane (PM). The cortex provides stability to the cell surface and is the primary determinant of cell surface mechanics in most animal cells. As a result, the the cortex allows cells to resist external forces and change shape.

In my previous work in the lab of Professor Ewa K Paluch, I developed a novel method to measure the thickness of the cortical network in live cells. This method was inspired by Single-molecule High Resolution Colocalization (SHREC), which relies on the fact that although sub-resolution geometry cannot be resolved, point-like objects can be localized with up to nanometer precision, even with a light microscope. To measure cortex thickness, we labeled the PM and cortex with chromatically different fluorophores (Figure 2A,B; Clark et al, 2013). Using a theoretical description of cortex geometry, we could use the information about the relative localization of the cortex and PM to determine cortex thickness (Figure 2C; Clark et al, 2013). We performed a number of controls to verify this method, including using different fluorescent probes for actin, changing the colors of the probes and analyzing computer-generated images of cells.

Figure 2. Cortex thickness measurements. A. Schematic representation of method to measure cortex thickness. Cortical actin and the plasma membrane are labelled with chromatically different fluorophores, and distance between the fluorescence peaks, Δ is measured and related to cortex thickness, h. B. A HeLa cell expressing GFP-Actin and mCherry-CAAX, a marker for the plasma membrane. Bottom, a straightened image of the cell border. Scale bars: 10 μm. C. A simple theoretical description of cortex geometry showing the actin model, a convolution of the actin model (to mimic the imaging process) and the position of the membrane. (Modified from Clark et al, 2013.)

To test if this method could be used to used in live cells undergoing shape change, we measured cortex thickness in blebs. Blebs are membrane protrusions that can form from a local weak point in the cortex. Because intracellular pressure is higher than extracellular pressure, cytosol flows from the cell body through the weak point in the cortex, blowing up the plasma membrane like a balloon. The bleb continues to expand until a new actin cortex reforms under the bleb membrane. The contractile actin cortex in the bleb first slows bleb growth and eventually leads to retraction of the bleb back into the cell body. Blebs have been shown to play a role in cytokinesis (Movie 2) and are also used in some forms of cell migration (Movie 3).

Movie 2. Blebs in cytokinesis. A HeLa cell blebbing at the polar regions during cytokinesis. Such polar blebbing has been hypothesized to stabilize the position of the cleavage furrow and ensure successful cytokinesis. (From Sedzinski and Biro et al, 2011.)

Movie 3. Blebs in migration. A non-adherent Walker rat 256 carcinosarcoma cell in confinement can move via polarized bleb formation at the leading edge. Scale bar: 10 μm. (From Bergert et al, 2012.)

Because blebs initially lack an actin cortex, this provided an ideal system to test our method to measure cortex thickness. By measuring thickness in blebs, we would initially expect a low thickness, when the cortex is just starting to reform, and thickness would then increase over time as the cortex regrew. We induced blebs by locally ablating the cortex with a pulsed UV laser, which induced a bleb at the site of ablation (Movie 4; Clark et al, 2013). By following cortex thickness over time in blebs, we found that the bleb cortex was indeed initially thin and grew thicker over time, eventually returning to the pre-ablation thickness (Clark et al, 2013).

Movie 4. Inducing blebs by laser ablation. A HeLa cell expressing GFP-Actin and mCherry-CAAX. At t=0, the cortex is ablated with a pulsed laser to induce a bleb. Scale bar: 5 μm. (From Clark et al, 2013.)

The Paluch lab has continued this investigation of cortical architecture and is currently exploring how cortex thickness is regulated and what are the effects of cortex thickness on cell mechanics.

Wound Healing & Cytokinesis (The Cortex, Part 2)

In my previous work in the Bement Lab at the University of Wisconsin-Madison, I also studied the actin cortex, but in a very different context. The Bement lab uses the African Clawed Frog, Xenopus laevis, (Figure X) as a model system to study how single cells repair wounds and how cells divide.

Cells in the body can be subject to a number of stresses that may induce physical damage to the cell. For example, when you exercise, muscle cells are stretched and strained, which can lead to small tears in the cells; if this tearing in extensive enough, it can also be quite painful! For many cell types in the body, when a cell experiences damage, it would be inefficient or even impossible to simply get rid of the damaged cell and generate a new replacement. Muscle cells, for example, can be up to several cm in length and can fuse together to increase the length of a single cell. Single neurons can be over a meter long in humans (depending on a person's height) and do not have the capacity to regenerate in adults. Thus, when muscle cells and neurons experience physical damage, intrinsic processes must be in place to repair this damage and prevent cell death.

One system that the Bement lab uses to study wound healing is the Xenopus oocyte, which are approximately 1 mm in diameter (clearly visible by the naked eye) and are very robust wound healers. These oocytes can be poked with glass needles or shot with pulsed lasers and continue to survive happily after they have repaired the damage. Oocytes, like most other animal cells, have an actin cortex underlying the plasma membrane. When cells are wounded, not only is the plasma membrane torn, but the cortex is disrupted as well. In order to repair the damage to the cortex, actin and myosin accumulate in a contratile ring around the wound and the ring contracts over several minutes to seal the hole in the cortex. Movie 5 (below) from Craig Mandato, a former postdoc in the Bement lab, shows a the accumulation of actin into a contractile ring following wounding of a Xeonpus oocyte with a pulsed laser.

Movie 5. Wound healing in a Xenopus laevis oocyte. A Xenopus oocyte injected with fluorescent phalloidin (a probe for F-actin) is wounded using a pulsed laser. F-Actin accumulates into a contractile ring which constricts, closing the hole in the cortex. (From Mandato and Bement, 2001.)

One of my projects in the Bement lab was to peform a screen of small molecule inhibitors to find new chemicals that could prevent actomyosin contraction during single-cell wound healing and cytokinesis. I performed a manual screen of 1990 small molecules by incubating Xenopus oocytes in the chemicals, stabbing them with a glass needle and scoring their survival and efficacy in wound healing. In parallel, collaborators Jenny Sider and George von Dassow screened the same set of chemicals for inhibition of cytokinesis in Sand Dollar (Dendraster excentricus) embryos. We followed up on hits from the parallel screens and discovered two potent inhibitors of wound healing and cytokinesis, Sphinctostatin-1 and -2 (Clark et al, 2012).

In the Bement lab, I also studied how cells heal wounds in the context of a multicellular tissue. For this study, we wounded cells in early Xenopus embryos using a pulsed laser. Surprisingly, we found that if cells were wounded in the proximity of neighboring cells, the neighboring cells would also upregulate contractile actomyosin and help the cell to heal its wound (Movie 6).

Movie 6. Wound healing in an early Xenopus embryo. A single cell in a Xenopus embryo expressing EGFP-rGBD (a probe for active RhoA) and mRFP-Utrophin (a probe for F-actin) is wounded. A RhoA is activated and F-actin accumulates in a contractile ring, neighboring cells also upregulate RhoA and F-Actin, which contributes to healing the wounded cell (From Clark et al, 2009.)

The Bement lab continues to study both wound healing and cytokinesis and also focuses on cross-talk between actin filaments and microtubules. Be sure to check out Bill and George's websites for great movies of these processes!

References

Alessandri, K., Sarangi, B.R., Gurchenkov, V. V., Sinha, B., Kießling, T.R., Fetler, L., Rico, F., Scheuring, S., Lamaze, C., Simon, A., Geraldo, S., Vignjevic, D., Doméjean, H., Rolland, L., Funfak, A., Bibette, J., Bremond, N. and Nassoy, P. (2013) Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro. Proc Natl Acad Sci USA, 110(37):14843-14848.

Bergert, M., Chandradoss, S.D., Desai, R.A. and Paluch, E. (2012) Cell mechanics control rapid transitions between blebs and lamellipodia during migration. Proc Natl Acad Sci USA, 109(36):14434-14439.

Clark, A.G., Miller, A.L., Vaughan, E., Yu, H.-Y.E., Penkert, R. and Bement, W.M. (2009) Integration of Single and Multicellular Wound Responses. Current Biology. 19:1389-1395.

Clark A.G., Sider J.R., Verbrugghe K., Fenteany G., von Dassow G. and Bement, W.M. (2012) Identification of Small Molecule Inhibitors of Cytokinesis and Single Cell Wound Repair. Cytoskeleton. 69(11):1010-1020.

Clark A.G., Dierkes K. and Paluch, E.K. (2013) Monitoring Actin Cortex Thickness in Live Cells. Biophysical Journal. 105(3):570-580.

Friedl, P., Locker, J., Sahai, E. and Segall, J.E. (2012) Classifying collective cancer cell invasion. Nat Cell Biol, 14(8):777-783.

Haeger, A., Krause, M., Wolf, K. and Friedl, P. (2014) Cell jamming: Collective invasion of mesenchymal tumor cells imposed by tissue confinement. BBA-Gen Subjects, 1840(8):2386-2395.

Mandato, C.A. and Bement W.M (2001) Contraction and polymerization cooperate to assemble and close actomyosin rings around Xenopus oocyte wounds. J Cell Biol, 154(4):785-798.

Sedzinski, J., Biro, M., Oswald, A., Tinevez, J.-Y., Salbreux G., and Paluch. E. (2011) Polar actomyosin contractility destabilizes the position of the cytokinetic furrow. Nature, 476(7361):462-466.