Artificial Metastasis: Cancer Cell Migration in Microengineered Environments

Abstract

Cancer metastasis, i.e. the dissemination of cancer cells from the primary tumor to a distant site in the body, is a complex, multistep process that is responsible for more than 90% of cancer –related deaths. A pivotal step in the metastatic cascade is cancer cell migration. During metastatic dissemination, this migration is hindered by several obstacles, such as the interaction with extracellular matrices with varying topologies (e.g. the stroma, the blood vessel endothelium, the vascular system and the tissue at a secondary site), the invasion of surrounding tissues with varying stiffness, and the permeation into or out of blood and lymphatic vessels with shear and compressive forces. The ability of cancer cells to overcome those barriers depends to a great extent on the physical interactions and force transmission between cancer cells and their microenvironment. Migrating cancer cells undergo various biochemical transitions, such as surface receptor expression and cytoskeleton reorganization, which eventually lead to changes in their phenotype and migration modes. Although cell invasion is largely a mechanical process, cancer research has focused mainly on gene regulation and signaling that underlie uncontrolled cell growth. However, the detection and measurement of the mechanical or phenotypic changes that cancer cells go through during metastasis could contribute towards a more integrated understanding of the disease, as well as towards the development of novel diagnostic methods or therapies explicitly targeted at physical biomarkers of cancer cells and their microenvironment. To this end, engineered systems for the study of cancer cell migration represent significant resources for sophisticated investigation of biophysical aspects of metastasis that can provide previously overlooked therapeutic opportunities. In this work, two state-of-the art microfabrication methods were employed for the fabrication of artificial environments that recapitulate key aspects of the cancer microenvironment in a reductionist way. 3D-electrohydrodynamic nanoprinting and laser nanolithography enabled the fast and on-demand production of complex microenvironments with topographical features of well controlled shape and size, that were used for live cell migration experiments of very high spatial and temporal resolution. The first part of this study, using nanoprinted pores that impede cell migration, is presented in Chapter 2 of this dissertation. The analysis of individual cell interactions with nanoprinted pores indicated a temporal correlation between cell division and pore penetration. Soon after cell division, the dynamic remodeling of actin cytoskeleton and of its interaction with the nucleus, as well as the modulation of cell adhesion to the substrate enable cells to efficiently squeeze their mass into extremely small pores. Differences in DNA condensation between cancer cells of different origin and/or malignancy may further contribute to the complexity of this picture by either increasing or decreasing nuclear compliance and size. Our results indicate that the outcome of chromatin decondensation are both cell cycle and cell type dependent. In this frame, the decondensation of DNA may impinge on cell migration, proliferation, and deformability by interfering with the cell cytoskeleton. Depending on the cell type these perturbations may contribute to increased or decreased pore penetration efficiency. In summary, the first part of our study supports a model in which a population of malignant cancer cells dividing more often or featuring prolonged permanence in permissive cell cycle phases may penetrate interstitial tissues faster and with higher efficiency, thus establishing a functional link between proliferation and the colonization of distant tissues. A pivotal role in regulating both cell proliferation and migration is played by cell adhesions to ECM, which are established upon integrin activation. Integrin contacts are partially lost during cell division and gradually reassembled by daughter cells upon abscission. We showed that cancer cells feature a higher number of large adhesions to the substrate in the G1 and S phase, it was therefore logical to speculate that in these specific phases of the cell cycle, the transmission of cellular forces to the substrate is sufficient to actuate the necessary deformation and squeezing of a stiff nucleus. We set to investigate this by employing a reference-free, continuum traction force method, recently developed in our laboratory, that allows the generation of spatially-resolved, overlapping maps of protein activity and traction forces (Chapter 3). Analysis of non invasive as well as metastatic cancer cells stably expressing a cell cycle marker demonstrated a cell cycle phase-dependent force variation. Detected forces were invariably higher in the G1 and early S phases as compared to the ensuing late S/G2, and locally co-localized with high levels of paxillin phosphorylation. The biochemical inhibition of focal adhesion kinase (FAK) reduced paxillin phosphorylation and significantly diminished the force transmitted to the substrate. These data demonstrate a reproducible modulation of force transmission during the cell cycle progression of cancer cells. Importantly, the emergence of G1 phase as the point where traction forces peak is of clinical relevance in regards to widely used chemotherapeutic drugs that arrest cells in exactly this phase. Invasion of dense tissues by cancer cells is defined largely by the interplay between the resistance imposed by interstitial pores and cell deformability. Metastatic cancer finds optimal paths of minimal resistance through an adaptive “path-finding” process which leads to successful cancer cell dissemination. The physical limits of nuclear deformation define the minimal cross section of pores that can be successfully penetrated. However, this single biophysical parameter does not describe completely the architectural anisotropy of tissues with pores of similar area but variable aspect ratio. In the last part of this dissertation (Chapter 4), we fabricated by means of laser nanolithography an artificial environment for the migration of cancer cells that contained constrictions of varying shape. We were able to highlight the pore shape as a major and independent determinant of the efficiency of cancer penetration. In complex architectures that contain pores small enough to require large deformations from invading cells, we demonstrated that low aspect ratio openings facilitated cancer migration. Moreover, we investigated the characteristics of the explorative behavior of metastatic cells, which allows them to select and preferentially migrate through paths of least resistance. In summary, through our artificial platforms for the study of cancer cell migration, the cell cycle (during which cell size, deformability and force actuation fluctuate) and the pore shape (which defines paths of least resistance) emerged as new important determinants of cancer cell migration efficiency. Our approach aims to serve as a paradigm of how microengineered models can contribute to the elucidation of complex biological processes, such as metastasis, by deciphering their dynamic and synergistic elements.