One‐dimensional patterning of cells in silicone wells via compression‐induced fracture

We have adapted our existing compression‐induced fracture technology to cell culture studies by generating linear patterns on a complex cell culture well structure rather than on simple solid constructs. We present a simple method to create one‐dimensional (1D), submicron, and linear patterns of extracellular matrix on a multilayer silicone material. We identified critical design parameters necessary to optimize compression‐induced fracture patterning on the wells, and applied stresses using compression Hoffman clamps. Finite‐element analyses show that the incorporation of the well improves stress homogeneity (stress variation = 25%), and, thus, crack uniformity over the patterned region. Notably, a shallow well with a thick base (vs. deeper wells with thinner bases) reduces out‐of‐plane deflections by greater than a sixth in the cell culture region, improving clarity for optical imaging. The comparison of cellular and nuclear shape indices of a neuroblast line cultured on patterned 1D lines and unpatterned 2D surfaces reveals significant differences in cellular morphology, which could impact many cellular functions. Because 1D cell cultures recapitulate many important phenotypical traits of 3D cell cultures, our culture system offers a simple means to further study the relationship between 1D and 3D cell culture environments, without demanding expensive engineering techniques and expertise. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1361–1369, 2014.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106690/1/jbma34814.pd

Microfabricated Physical Spatial Gradients for Investigating Cell Migration and Invasion Dynamics

We devise a novel assay that introduces micro-architectures into highly confining microchannels to probe the decision making processes of migrating cells. The conditions are meant to mimic the tight spaces in the physiological environment that cancer cells encounter during metastasis within the matrix dense stroma and during intravasation and extravasation through the vascular wall. In this study we use the assay to investigate the relative probabilities of a cell 1) permeating and 2) repolarizing (turning around) when it migrates into a spatially confining region. We observe the existence of both states even within a single cell line, indicating phenotypic heterogeneity in cell migration invasiveness and persistence. We also show that varying the spatial gradient of the taper can induce behavioral changes in cells, and different cell types respond differently to spatial changes. Particularly, for bovine aortic endothelial cells (BAECs), higher spatial gradients induce more cells to permeate (60%) than lower gradients (12%). Furthermore, highly metastatic breast cancer cells (MDA-MB-231) demonstrate a more invasive and permeative nature (87%) than non-metastatic breast epithelial cells (MCF-10A) (25%). We examine the migration dynamics of cells in the tapered region and derive characteristic constants that quantify this transition process. Our data indicate that cell response to physical spatial gradients is both cell-type specific and heterogeneous within a cell population, analogous to the behaviors reported to occur during tumor progression. Incorporation of micro-architectures in confined channels enables the probing of migration behaviors specific to defined geometries that mimic in vivo microenvironments

Engineered Models of Metastasis with Application to Study Cancer Biomechanics

Three-dimensional complex biomechanical interactions occur from the initial steps of tumor formation to the later phases of cancer metastasis. Conventional monolayer cultures cannot recapitulate the complex microenvironment and chemical and mechanical cues that tumor cells experience during their metastatic journey, nor the complexity of their interactions with other, noncancerous cells. As alternative approaches, various engineered models have been developed to recapitulate specific features of each step of metastasis with tunable microenvironments to test a variety of mechanistic hypotheses. Here the main recent advances in the technologies that provide deeper insight into the process of cancer dissemination are discussed, with an emphasis on three-dimensional and mechanical factors as well as interactions between multiple cell types

Integrated Micro/Nanoengineered Functional Biomaterials for Cell Mechanics and Mechanobiology: A Materials Perspective

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106681/1/adma201304431.pd

The Role Of Force Generation In Metastatic Cancer Progression

Metastasis, or the process by which cancer cells escape a primary tumor and travel through the body to form secondary tumors, is believed to be responsible for over 90% of the 7.9 million annual cancer-related fatalities reported worldwide. To migrate from the original tumor, cancer cells must navigate an extremely dense and heterogeneous stromal environment to arrive at a blood or lymph vessel, which they can then penetrate to enter the circulatory or lymphatic system. Each of these steps requires cells to pull on its matrix using contractile, or traction, forces. However, the precise relationship of force generation to metastatic cell structure and function remains largely unknown. Herein, I demonstrate that metastatic cells exert increased contractile forces which facilitate the invasion of the extracellular microenvironment (ECM). Using traction force microscopy, I show that human metastatic breast, prostate, and lung cancer cell lines exhibit increased traction stresses compared to non-metastatic counterparts on physiologically-relevant substrates. Additionally, I find that the increased collagen density and matrix stiffness previously shown to be a hallmark of the tumor microenvironment promote increased traction forces through cell spread area-dependent and independent mechanisms. Finally, I develop a novel 3D model for one mode of metastatic migration in which secondary cancer cells follow microtracks that are formed by leading tumor cells secreting proteases and cleaving ECM fibers. iii By using physiologically relevant 3D collagen channels to study cancer cell migration, I specifically assessed the role of force in protease-independent migration, and, surprisingly, found that contractile force was dispensable for this form of protease independent migration. Instead, my results point to focal adhesion, actin filaments, and microtubules being key mediators of protease-independent migration within patterned collagen microtracks. Ultimately, these studies help to define the role that cellular force generation plays in metastatic invasion, and also yield insight into the biophysical mechanisms that tumor cells use to migrate. These insights could potentially lead to a targeted therapeutic approach to combating those mechanisms to delay or prevent metastasis and its subsequent fatal damage. i

Cellular traction stresses increase with increasing metastatic potential.

Cancer cells exist in a mechanically and chemically heterogeneous microenvironment which undergoes dynamic changes throughout neoplastic progression. During metastasis, cells from a primary tumor acquire characteristics that enable them to escape from the primary tumor and migrate through the heterogeneous stromal environment to establish secondary tumors. Despite being linked to poor prognosis, there are no direct clinical tests available to diagnose the likelihood of metastasis. Moreover, the physical mechanisms employed by metastatic cancer cells to migrate are poorly understood. Because metastasis of most solid tumors requires cells to exert force to reorganize and navigate through dense stroma, we investigated differences in cellular force generation between metastatic and non-metastatic cells. Using traction force microscopy, we found that in human metastatic breast, prostate and lung cancer cell lines, traction stresses were significantly increased compared to non-metastatic counterparts. This trend was recapitulated in the isogenic MCF10AT series of breast cancer cells. Our data also indicate that increased matrix stiffness and collagen density promote increased traction forces, and that metastatic cells generate higher forces than non-metastatic cells across all matrix properties studied. Additionally, we found that cell spreading for these cell lines has a direct relationship with collagen density, but a biphasic relationship with substrate stiffness, indicating that cell area alone does not dictate the magnitude of traction stress generation. Together, these data suggest that cellular contractile force may play an important role in metastasis, and that the physical properties of the stromal environment may regulate cellular force generation. These findings are critical for understanding the physical mechanisms of metastasis and the role of the extracellular microenvironment in metastatic progression

Metastatic cancer cells exert greater forces than non-metastatic cells.

<p>(A) Representative traction maps (<i>left</i>), corresponding phase images (<i>middle</i>), and overall net traction force (|<i>F</i>|, <i>right</i>) of non-metastatic mammary epithelial (MCF10A) and highly metastatic (MDAMB231) cancer cells. (B) Representative traction maps (<i>left</i>), corresponding phase images (<i>middle</i>), and |<i>F</i>| (<i>right</i>) of non-metastatic primary prostate epithelial cells (PrEC) and highly metastatic prostate cancer cells (PC3). (C) Representative traction maps (<i>left</i>), corresponding phase images (<i>middle</i>), and |<i>F</i>| (<i>right</i>) of non-metastatic bronchial epithelial cells (BEAS2B) and metastatic lung adenocarcinoma cells (A549). All cells are on polyacrylamide substrates with Young's Modulus (E) = 5 kPa and type I collagen concentration of 0.1 mg/mL. Scale bar = 50 µm. Mean+SEM; *** indicates p<0.001.</p

Metastatic derivative in a series of isogenic cell lines exerts greater forces.

<p>(A) Phase images of parental untransformed cells (MCF10A), transformed premalignant (MCF10AT1) and highly metastatic (MCF10CA1a) derivatives. (B) Net traction forces increase with increasing metastatic potential, with the highest forces exerted by the metastatic MCF10CA1a cells. (C) Average traction stress (|<i>F</i>|/A) increases with increasing metastatic potential. Mean ± SEM; ** indicates p<0.01; *** indicates p<0.001.</p