Directional Cancer Cell Migration Research Articles

LINC00869 Promotes Hepatocellular Carcinoma Metastasis via Protrusion Formation.

LncRNA LINC00869 regulates the activity of CDC42-WASP pathway and positively affects protrusion formation in HCC cells, which expands the current understanding of lncRNA functions as well as gives a better understanding of carcinogenesis.

Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization

Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. Cells depleted of MARK proteins demonstrated that MARK2 promotes phosphorylation of both myosin II and the myosin phosphatase targeting subunit MYPT1 to synergistically drive myosin II contractility and stress fiber formation in cells. Studies with isolated proteins showed that MARK2 directly phosphorylates myosin II regulatory light chain, while its effects on MYPT1 phosphorylation are indirect. Using a mutant lacking the membrane-binding domain, we found that membrane association is required for focal adhesion targeting of MARK2, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of focal adhesions oriented in the direction of migration to mediate directionally persistent cell motility. Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration.

A Novel 3D Model for Visualization and Tracking of Fibroblast-Guided Directional Cancer Cell Migration.

Simple SummaryRecent advances in 3D cell culture have provided new opportunities for investigating interactions between cancer cells and their surrounding stromal cells. The 3D culture platform described herein is both time efficient and economical in the study of direct cell–cell interactions. The unique design of our dumbbell model had allowed us to visualize and monitor the entire recruitment process of cancer cells by fibroblasts under an in vitro condition. Suitable for almost every cell type, our model has the potential for a wider application as it can be adapted for use in drug screening and the study of cellular factors involved in cell–cell attraction.Stromal fibroblasts surrounding cancer cells are a major and important constituent of the tumor microenvironment not least because they contain cancer-associated fibroblasts, a unique fibroblastic cell type that promotes tumorigenicity through extracellular matrix remodeling and secretion of soluble factors that stimulate cell differentiation and invasion. Despite much progress made in understanding the molecular mechanisms that underpin fibroblast–tumor cross-talk, relatively little is known about the way the two cell types interact from a physical contact perspective. In this study, we report a novel three-dimensional dumbbell model that would allow the physical interaction between the fibroblasts and cancer cells to be visualized and monitored by microscopy. To achieve the effect, the fibroblasts and cancer cells in 50% Matrigel suspension were seeded as independent droplets in separation from each other. To allow for cell migration and interaction, a narrow passage of Matrigel causeway was constructed in between the droplets, effectively molding the gel into the shape of a dumbbell. Under time-lapse microscopy, we were able to visualize and image the entire process of fibroblast-guided cancer cell migration event, from initial vessel-like structure formation by the fibroblasts to their subsequent invasion across the causeway, attracting and trapping the cancer cells in the process. Upon prolonged culture, the entire population of fibroblasts eventually infiltrated across the passage and condensed into a spheroid-like cell mass, encapsulating the bulk of the cancer cell population within. Suitable for almost every cell type, our model has the potential for a wider application as it can be adapted for use in drug screening and the study of cellular factors involved in cell–cell attraction.

Abstract 3962: Differential extracellular matrix remodeling induced by VEGF-A and TGF-β activated fibroblasts

Abstract Introduction: Tumor progression is modulated by the presence within the tumor microenvironment of activated fibroblasts called cancer-associated fibroblasts (CAFs). CAFs exhibit an increase in the production of collagen (COL), tenascin C (TNC) and fibronectin (FN), resulting in a remodeling of the extracellular matrix (ECM) within the tumor mass. CAFs-derived ECM guides the cancer cells to migrate directionally increasing metastasis. TGF-β and VEGF-A are highly express in tumors and promote progression, however, their role in ECM remodeling is poorly understood. This study seeks to analyze the ECM protein remodeling mediated by TGF- β o VEGF-A activated fibroblasts. Materials and methods: In vitro and in vivo ECM protein remodeling mediated by TGF- β o VEGF-A activated fibroblasts was analyzed through expression of TNC, FN and COL using immunofluorescence and second harmonic generation (SHG); alignment and thickness of the fibers; Organoleptic invasion and 2D migration assay were conducted to study the effect of the stiffness of collagen gels generated by VEGF-A and TGF-β activated fibroblasts in the migration/invasion of tumor cells. Results and discussion: ECM derived from VEGF-A and TGF-β activated fibroblasts have differential ECM remodeling patterns, including differences in the secretion of TNC, FN, and COL; alignment and thickness of the fibers, and migration/invasion rate. These changes results in a differential stiffness of the ECM, which would alter the directional migration of cancer cells within tumor microenvironment. Citation Format: Muriel A. Nuñez, Javier Cerda-Infante, Francisco Nualart, Gustavo Cerda, Isabel M. Yussef, Viviana P. Montecinos. Differential extracellular matrix remodeling induced by VEGF-A and TGF-β activated fibroblasts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3962.

Phenotypic Heterogeneity and Plasticity of Cancer Cell Migration in a Pancreatic Tumor Three-Dimensional Culture Model

Invasive cancer cell migration is a key feature of metastatic human pancreatic ductal adenocarcinoma (PDAC), yet the underlying mechanisms remain poorly understood. Here, we investigated modes of cancer cell invasion using two pancreatic cancer cell lines with differential epithelial–mesenchymal status, PANC-1 and BxPC-3, under 3D culture conditions. Multicellular tumor spheroids (TSs) were grown in a collagen matrix co-cultured with pancreatic stellate cells (PSCs) using microchannel chips. PANC-1 cells showed individual migration from TSs via invadopodium formation. BxPC-3 cells showed plasticity between collective and individual migration in either mesenchymal mode, with filopodium-like protrusions, or blebby amoeboid mode. These two cell lines showed significantly different patterns of extracellular matrix (ECM) remodeling, with MMP-dependent degradation in a limited area of ECM around invadopodia for PANC-1 cells, or MMP-independent extensive deformation of ECM for BxPC-3 cells. Cancer cell migration out of the collagen channel significantly increased by PSCs and directional cancer cell migration was mediated by fibronectin deposited by PSCs. Our results highlight the phenotypic heterogeneity and plasticity of PDAC cell migration and ECM remodeling under 3D culture conditions. This 3D co-culture model of pancreatic cancer cells and PSCs offers a useful tool for studying cancer cell migration and ECM remodeling to identify and develop potential molecular targets and anti-cancer agents against human PDAC.

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

SummaryThe link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.

Shear stress stimulates integrin β1 trafficking and increases directional migration of cancer cells via promoting deacetylation of microtubules

In egress routes of malignancy, cancer cells are constantly subjected to shear stress imposed by blood/lymph flow. Increasing evidence points toward the regulatory roles of shear stress in tumor cell adhesion and motility. Although it is known that integrin endocytic trafficking governs focal adhesion (FA) turnover and cell migration, the effect and biological consequences of low shear stress (LSS) on integrin trafficking remain unclear. Here, we identified the critical role of integrin β1 trafficking and caveolin-1 (Cav-1) mediated endocytosis in LSS-induced cell directional migration. LSS altered the distribution of integrin β1 in MDA-MB-231 cells and significantly promoted its internalization and recycling, which in turn facilitated FA turnover and directional cell migration. Furthermore, LSS induced cytoskeleton remodeling, which was required for internalization of integrin β1. LSS down-regulated the acetylation level of microtubules (MTs) via activating ROCK/HDAC6 pathway, resulting in elevation of MTs dynamics, Cav-1 motility, and Cav-1-dependent integrin β1 recycling. We also showed that high HDAC6 expression was a ROCK-dependent prognostic factor, which was correlated with poor outcomes in breast cancer patients. Taken together, these results defined a novel mechanism by which LSS enhanced integrin β1 trafficking via actin cytoskeleton remodeling and ROCK/HDAC6 mediated deacetylation of MTs, thereby promoting FAs turnover and directional cell migration.

Anisotropic stiffness gradient-regulated mechanical guidance drives directional migration of cancer cells

Interfacial interactions between cancer cells and surrounding microenvironment involve complex mechanotransduction mechanisms that are directly associated with tumor invasion and metastasis. Matrix remodeling triggers heterogeneity of stiffness in tumor microenvironment and thus generates anisotropic stiffness gradient (ASG). The migration of cancer cells mediated by ASG, however, still remains elusive. Based on a multi-layer polymerization method of microstructured hydrogels with surface topology, we develop an in vitro experimental platform for mechanical interactions of cancer cells with ASG matrix microenvironment. We show that mechanical guidance of mesenchymal cells is essentially modulated by ASG, leading to a spontaneous directional migration along the orientation parallel to the maximum stiffness although there is no stiffness gradient in the direction. The ASG-regulated mechanical guidance presents an alternative way of cancer cell directional migration. Further, our findings indicate that the mechanical guidance occurs only in mesenchymal cancer cells, but not in epithelial cancer cells, implying that cell contractility may contribute to ASG-regulated migration of cells. This work is not only helpful for elucidating the role of matrix remodeling in mediating tumor cell invasion and metastasis, but has potential implications for developing specific cancer treatments. Statement of SignificanceLocal extracellular matrix (ECM) stiffening triggers mechanical heterogeneity in tumor microenvironment, which can exert a crucial impact on interfacial interactions between tumor cells and surrounding ECM. The underlying mechanobiological mechanism that tumor cells are modulated by mechanically heterogeneous ECM, however, still remains mysterious to a great extent. Through our established in vitro platform and analysis, we have demonstrated that anisotropic stiffness gradient (ASG) has the ability to elicit directional migration of cells, essentially depending on local stiffness gradients and the corresponding absolute stiffness values. This study is not only crucial for revealing the role of matrix remodeling in regulating tumor invasion and metastasis, but also offers a valuable guidance for developing anti-tumor therapies from the biomechanical perspective.

Extracellular nanofiber-orchestrated cytoskeletal reorganization and mediated directional migration of cancer cells.

Extracellular matrix anisotropy tunes the organization and movement of surrounding cells. The primary mediators of the extracellular matrix are fiber-based materials. Natural collagen fibers reorganize from curled and isotropic fibers to straightened and anisotropic fibers during tumorigenesis, yet how the cytoskeleton is involved in the directional migration in response to the topography is unknown. To investigate this, we fabricated random, orthogonal, and aligned nanofibers to deconstruct the basic mechanisms for the migration of the human pancreatic cancer cells PANC-1 on different substrates. We found that the extracellular matrix orchestrated actin reorganization by templating the surface topography as an irregular pattern on random fibers, crossover feature on orthogonal fibers, and parallel characteristics on aligned fibers. The intermediate filament as vimentin was upregulated to form a perinuclear shape on orthogonal or aligned surfaces. We also found that the nanofiber topography mediated the directional migration via different mechanisms. The directionality ratio and velocity were statistically analyzed to unveil the pattern of directional migration. Cells on aligned nanofibers yielded a greater velocity. Rac1 and Cdc42 were involved in cell migration through regulating actin polymerization and membrane protrusions. Thus, our findings elucidate that nanofiber alignment orchestrates cytoskeletal reorganization and mediates the directional migration of cancer cells.

Direct interaction between CEP85 and STIL mediates PLK4-driven directed cell migration.

PLK4 has emerged as a prime target for cancer therapeutics, and its overexpression is frequently observed in various types of human cancer. Recent studies have further revealed an unexpected oncogenic activity of PLK4 in regulating cancer cell migration and invasion. However, the molecular basis behind the role of PLK4 in these processes still remains only partly understood. Our previous work has demonstrated that an intact CEP85-STIL binding interface is necessary for robust PLK4 activation and centriole duplication. Here, we show that CEP85 and STIL are also required for directional cancer cell migration. Mutational and functional analyses reveal that the interactions between CEP85, STIL and PLK4 are essential for effective directional cell motility. Mechanistically, we show that PLK4 can drive the recruitment of CEP85 and STIL to the leading edge of cells to promote protrusive activity, and that downregulation of CEP85 and STIL leads to a reduction in ARP2 (also known as ACTR2) phosphorylation and reorganization of the actin cytoskeleton, which in turn impairs cell migration. Collectively, our studies provide molecular insight into the important role of the CEP85-STIL complex in modulating PLK4-driven cancer cell migration.This article has an associated First Person interview with the first author of the paper.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin.

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF-cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α-mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration.

Hypoxic stellate cells of pancreatic cancer stroma regulate extracellular matrix fiber organization and cancer cell motility

Desmoplasia and hypoxia in pancreatic cancer mutually affect each other and create a tumor-supportive microenvironment. Here, we show that microenvironment remodeling by hypoxic pancreatic stellate cells (PSCs) promotes cancer cell motility through alteration of extracellular matrix (ECM) fiber architecture. Three-dimensional (3-D) matrices derived from PSCs under hypoxia exhibited highly organized parallel-patterned matrix fibers compared with 3-D matrices derived from PSCs under normoxia, and promoted cancer cell motility by inducing directional migration of cancer cells due to the parallel fiber architecture. Microarray analysis revealed that procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in PSCs was the gene that potentially regulates ECM fiber architecture under hypoxia. Stromal PLOD2 expression in surgical specimens of pancreatic cancer was confirmed by immunohistochemistry. RNA interference-mediated knockdown of PLOD2 in PSCs blocked parallel fiber architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that hypoxia-induced PLOD2 expression in PSCs creates a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM in pancreatic cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Directional migration of cancer cells induced by a blue light intensity gradient.

We used a spatial light modulator to project an optical micropattern of 473 nm light with a quartic intensity gradient on a single lung cancer cell. We observed that the intracellular amounts of reactive oxygen species (ROS) of the cancer cells were proportional to the intensity of the blue light, and the blue light intensity gradients could drive directional cell migration. This optically induced directional cell migration was inhibited by a ROS scavenger in the culture medium in a dose-dependent manner. In contrast, the ROS levels in fibroblasts were saturated by the blue light at low intensity and therefore the fibroblasts did not exhibit directional migration in the intensity gradient.

Prenylated C17orf37 Induces Filopodia Formation to Promote Cell Migration and Metastasis

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.