Colin Paul

Physical confinement alters tumor cell adhesion and migration phenotypes

Cell migration on planar surfaces is driven by cycles of actin protrusion, integrin‐mediated adhesion, and myosin‐mediated contraction; however, this mechanism may not accurately describe movement in 3‐dimensional (3D) space. By subjecting cells to restrictive 3D environments, we demonstrate that physical confinement constitutes a biophysical stimulus that alters cell morphology and suppresses mesenchymal motility in human breast carcinoma (MDA‐MB‐231). Dorsoventral polarity, stress fibers, and focal adhesions are markedly attenuated by confinement. Inhibitors of myosin, Rho/ROCK, or β1‐integrins do not impair migration through 3‐μm‐wide channels (confinement), even though these treatments repress motility in 50‐μm‐wide channels (unconfined migration) by ≥50%. Strikingly, confined migration persists even when F‐actin is disrupted, but depends largely on microtubule (MT) dynamics. Interfering with MT polymerization/depolymerization causes confined cells to undergo frequent directional changes, thereby reducing the average net displacement by ≥80% relative to vehicle controls. Live‐cell EB1‐GFP imaging reveals that confinement redirects MT polymerization toward the leading edge, where MTs continuously impact during advancement of the cell front. These results demonstrate that physical confinement can induce cytoskeletal alterations that reduce the dependence of migrating cells on adhesion‐contraction force coupling. This mechanism may explain why integrins can exhibit reduced or altered function during migration in 3D environments.—Balzer, E. M., Tong, Z., Paul, C. D., Hung, W.‐C., Stroka, K. M., Boggs, A. E., Martin, S. S., Konstantopoulos, K. Physical confinement alters tumor cell adhesion and migration phenotypes. FASEB J. 26, 4045–4056 (2012). www.fasebj.org

Distinct signaling mechanisms regulate migration in unconfined versus confined spaces

α4β1 integrin promotes migration of fibroblast-like cells in confined environment by enhancing myosin IIA via Rac1 inhibition, whereas unconfined migration requires Rac1 and myosin IIB.

Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

Mesothelin (MSLN) and cancer antigen125/mucin 16 (CA125/MUC16) are potential biomarkers for pancreatic cancer (PC) that are co-overexpressed at the invading edges of PC tissues, and their expression correlates with poor survival rates. However, the role of MSLN-MUC16 molecular interaction in PC cell motility and invasion has yet to be elucidated. Using sophisticated bioengineering and molecular biology tools, we report that the binding of MSLN to MUC16 markedly enhances PC cell motility and invasion via the selective induction of matrix metalloproteinase (MMP)-7. MSLN-mediated MMP-7 upregulation in MUC16-expressing PC cells occurs via a p38 MAPK-dependent pathway. Depletion of MMP-7 or inhibition of p38 activity abolishes MSLN-mediated PC motility and invasion. These findings provide a novel perspective on the enhanced invasive potential associated with MSLN and MUC16 co-overexpression, and the mechanism underlying MMP-7 activation in PC invasion and metastasis.

Cancer cell motility: lessons from migration in confined spaces

Time-lapse, deep-tissue imaging made possible by advances in intravital microscopy has demonstrated the importance of tumour cell migration through confining tracks in vivo. These tracks may either be endogenous features of tissues or be created by tumour or tumour-associated cells. Importantly, migration mechanisms through confining microenvironments are not predicted by 2D migration assays. Engineered in vitro models have been used to delineate the mechanisms of cell motility through confining spaces encountered in vivo. Understanding cancer cell locomotion through physiologically relevant confining tracks could be useful in developing therapeutic strategies to combat metastasis.

Fluid shear promotes chondrosarcoma cell invasion by activating matrix metalloproteinase 12 via IGF-2 and VEGF signaling pathways

Interstitial fluid flow in and around the tumor tissue is a physiologically relevant mechanical signal that regulates intracellular signaling pathways throughout the tumor. Yet, the effects of interstitial flow and associated fluid shear stress on the tumor cell function have been largely overlooked. Using in vitro bioengineering models in conjunction with molecular cell biology tools, we found that fluid shear (2 dyn/cm2) markedly upregulates matrix metalloproteinase 12 (MMP-12) expression and its activity in human chondrosarcoma cells. MMP-12 expression is induced in human chondrocytes during malignant transformation. However, the signaling pathway regulating MMP-12 expression and its potential role in human chondrosarcoma cell invasion and metastasis have yet to be delineated. We discovered that fluid shear stress induces the synthesis of insulin growth factor-2 (IGF-2) and vascular endothelial growth factor (VEGF) B and D, which in turn transactivate MMP-12 via PI3-K, p38 and JNK signaling pathways. IGF-2-, VEGF-B- or VEGF-D-stimulated chondrosarcoma cells display markedly higher migratory and invasive potentials in vitro, which are blocked by inhibiting MMP-12, PI3-K, p38 or JNK activity. Moreover, recombinant human MMP-12 or MMP-12 overexpression can potentiate chondrosarcoma cell invasion in vitro and the lung colonization in vivo. By reconstructing and delineating the signaling pathway regulating MMP-12 activation, potential therapeutic strategies that interfere with chondrosarcoma cell invasion may be identified.

Probing cell traction forces in confined microenvironments

Cells migrate in vivo within three-dimensional (3D) extracellular matrices. Cells also migrate through 3D longitudinal channels formed between the connective tissue and the basement membrane of muscle, nerve, and epithelium. Although traction forces have been measured during 2D cell migration, no assay has been developed to probe forces during migration through confined microenvironments. We thus fabricated a novel microfluidic device consisting of deflectable PDMS microposts incorporated within microchannels of varying cross-sectional areas. Using NIH-3T3 fibroblasts and human osteosarcoma (HOS) cells as models, we found that the average traction forces per post decreased upon increasing confinement. Inhibition of myosin-II function by blebbistatin in HOS cells decreased traction forces in unconfined (wide) channels but failed to alter them in confined spaces. Myosin activation by calyculin A also failed to affect traction forces in confining channels but increased them in wide channels. These observations underlie the importance of the physical microenvironment in the regulation of cell migration and cellular traction forces.

Engineered Models of Confined Cell Migration

Cells in the body are physically confined by neighboring cells, tissues, and the extracellular matrix. Although physical confinement modulates intracellular signaling and the underlying mechanisms of cell migration, it is difficult to study in vivo. Furthermore, traditional two-dimensional cell migration assays do not recapitulate the complex topographies found in the body. Therefore, a number of experimental in vitro models that confine and impose forces on cells in well-defined microenvironments have been engineered. We describe the design and use of microfluidic microchannel devices, grooved substrates, micropatterned lines, vertical confinement devices, patterned hydrogels, and micropipette aspiration assays for studying cell responses to confinement. Use of these devices has enabled the delineation of changes in cytoskeletal reorganization, cell-substrate adhesions, intracellular signaling, nuclear shape, and gene expression that result from physical confinement. These assays and the physiologically relevant signaling pathways that have been elucidated are beginning to have a translational and clinical impact.

Interplay of the physical microenvironment, contact guidance, and intracellular signaling in cell decision making.

The peritumoral physical microenvironment consists of complex topographies that influence cell migration. Cell decision making, upon encountering anisotropic, physiologically relevant physical cues, has yet to be elucidated. By integrating microfabrication with cell and molecular biology techniques, we provide a quantitative and mechanistic analysis of cell decision making in a variety of well‐defined physical microenvironments. We used MDA‐MB‐231 breast carcinoma and HT1080 fibrosarcoma as cell models. Cell decision making after lateral confinement in 2‐dimensional microcontact printed lines is governed by branch width at bifurcations. Cells confined in narrow feeder microchannels prefer to enter wider branches at bifurcations. In contrast, in feeder channels that are wider than the cell body, cells elongate along one side wall of the channel and are guided by contact with the wall to the contiguous branch channel independent of its width. Knockdown of β1‐integrins or inhibition of cellular contractility suppresses contact guidance. Concurrent, but not individual, knockdown of nonmuscle myosin iso‐forms IIA and IIB also decreases contact guidance, which suggests the existence of a compensatory mechanism between myosin IIA and myosin IIB. Conversely, knockdown or inhibition of cell division control protein 42 homolog promotes contact guidance‐mediated decision making. Taken together, the dimensionality, length scales of the physical microenvironment, and intrinsic cell signaling regulate cell decision making at intersections.—Paul, C. D., Shea, D. J., Mahoney, M. R., Chai, A., Laney, V., Hung, W.‐C., Konstantopoulos, K. Interplay of the physical microenvironment, contact guidance, and intracellular signaling in cell decision making. FASEB J. 30, 2161–2170 (2016). www.fasebj.org

Giant obscurins regulate the PI3K cascade in breast epithelial cells via direct binding to the PI3K/p85 regulatory subunit

Obscurins are a family of giant cytoskeletal proteins, originally identified in striated muscles where they have structural and regulatory roles. We recently showed that obscurins are abundantly expressed in normal breast epithelial cells where they play tumor and metastasis suppressing roles, but are nearly lost from advanced stage breast cancer biopsies. Consistent with this, loss of giant obscurins from breast epithelial cells results in enhanced survival and growth, epithelial to mesenchymal transition (EMT), and increased cell migration and invasion in vitro and in vivo. In the current study, we demonstrate that loss of giant obscurins from breast epithelial cells is associated with significantly increased phosphorylation and subsequent activation of the PI3K signaling cascade, including activation of AKT, a key regulator of tumorigenesis and metastasis. Pharmacological and molecular inhibition of the PI3K pathway in obscurin-depleted breast epithelial cells results in reversal of EMT, (re)formation of cell-cell junctions, diminished mammosphere formation, and decreased cell migration and invasion. Co-immunoprecipitation, pull-down, and surface plasmon resonance assays revealed that obscurins are in a complex with the PI3K/p85 regulatory subunit, and that their association is direct and mediated by the obscurin-PH domain and the PI3K/p85-SH3 domain with a KD of ∼50 nM. We therefore postulate that giant obscurins act upstream of the PI3K cascade in normal breast epithelial cells, regulating its activation through binding to the PI3K/p85 regulatory subunit.