Mechanical compression regulates tumor spheroid invasion into a 3D collagen matrix.

Uncontrolled growth of tumor cells in confined spaces leads to the accumulation of compressive stress within the tumor. Although the effects of tension within 3D extracellular matrices (ECMs) on tumor growth and invasion are well established, the role of compression in tumor mechanics and invasion is largely unexplored. In this study, we modified a Transwell assay such that it provides constant compressive loads to spheroids embedded within a collagen matrix. We used microscopic imaging to follow the single cell dynamics of the cells within the spheroids, as well as invasion into the 3D ECMs. Our experimental results showed that malignant breast tumor (MDA-MB-231) and non-tumorigenic epithelial (MCF10A) spheroids responded differently to a constant compression. Cells within the malignant spheroids became more motile within the spheroids and invaded more into the ECM under compression; whereas cells within non-tumorigenic MCF10A spheroids became less motile within the spheroids and did not display apparent detachment from the spheroids under compression. These findings suggest that compression may play differential roles in healthy and pathogenic epithelial tissues and highlight the importance of tumor mechanics and invasion.

Functional Contribution and Clinical Implication of Cancer-Associated Fibroblasts in Glioblastoma.

PURPOSE: The abundance and biological contribution of cancer-associated fibroblasts (CAF) in glioblastoma (GBM) are poorly understood. Here, we aim to uncover its molecular signature, cellular roles, and potential tumorigenesis implications. EXPERIMENTAL DESIGN: We first applied single-cell RNA sequencing (RNA-seq) and bioinformatics analysis to identify and characterize stromal cells with CAF transcriptomic features in human GBM tumors. Then, we performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant GBM cells. RESULTS: We found that CAF abundance was low but significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling using three large cohorts containing bulk RNA-seq data and clinical information. Proteomic analysis of a GBM-derived CAF line and its secretome revealed fibronectin (FN1) as a critical candidate factor mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF-conditioned media and recombinant FN1 could facilitate the migration and invasion of GBM cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of GBMs. Interestingly, cell lines resembling the proneural state responded to the CAF signaling better for the migratory and invasive phenotypes. CONCLUSIONS: Overall, this study characterized the molecular features and functional impacts of CAFs in GBM, alluding to novel cell interactions mediated by CAFs in the GBM microenvironment.

CD44 and ß1-integrin are both engaged in cell traction force generation in hyaluronic acid-rich extracellular matrices.

Mechanical properties of the extracellular matrices (ECMs) critically regulate a number of important cell function including growth, differentiation and migration. Type I collagen and glycosaminoglycans (GAGs) are two primary components of ECMs that contribute to tissue mechanics with the collagen fiber network sustaining tension and GAGs withstanding compression. Collagen stiffness as well as its architecture are known to be important role players in cell-ECM mechanical interactions, however, much less is known about how GAGs within ECMs regulate cell force generation and invasion. Inspired by a recent theoretical work from the Shenoy lab that GAGs play important roles in cell - ECM interactions, we hereby present experimental studies on the role of hyaluronic acid (HA, an unsulfated GAG) in single tumor cell traction force generation within HA collagen cogels using a recently developed 3D cell traction force microscopy. Our work revealed that CD44, a cell surface adhesion receptor to HA, was engaged in cell traction force generation in conjunction with ß1-integrin. Furthermore, we found that HA significantly modified the architecture and mechanics of the collagen fiber network, decreased tumor cells' propensity to remodel the collagen network, decreased traction force generation and transmission distance, and attenuated tumor invasion in agreement with theoretical predictions. Our findings highlighted the significance of CD44 and HA engagement in cell-ECM mechanical interactions, providing new insights on the mechanical model of cellular force transmission.

Mechanical compression regulates tumor spheroid invasion into a 3D collagen matrix.

Uncontrolled growth of tumor cells in confined spaces leads to the accumulation of compressive stress within the tumor. Although the effects of tension within 3D extracellular matrices on tumor growth and invasion are well established, the role of compression in tumor mechanics and invasion is largely unexplored. In this study, we modified a Transwell assay such that it provides constant compressive loads to spheroids embedded within a collagen matrix. We used microscopic imaging to follow the single cell dynamics of the cells within the spheroids, as well as invasion into the 3D extracellular matrices (EMCs). Our experimental results showed that malignant breast tumor (MDA-MB-231) and non-tumorigenic epithelial (MCF10A) spheroids responded differently to a constant compression. Cells within the malignant spheroids became more motile within the spheroids and invaded more into the ECM under compression; whereas cells within non-tumorigenic MCF10A spheroids became less motile within the spheroids and did not display apparent detachment from the spheroids under compression. These findings suggest that compression may play differential roles in healthy and pathogenic epithelial tissues and highlights the importance of tumor mechanics and invasion.

Tumor spheroid invasion in epidermal growth factor gradients revealed by a 3D microfluidic device.

Epidermal growth factor (EGF), a potent cytokine, is known to promote tumor invasion bothin vivoandin vitro. Previously, we observed that single breast tumor cells (MDA-MB-231 cell line) embedded within a 3D collagen matrix displayed enhanced motility but no discernible chemotaxis in the presence of linear EGF gradients using a microfluidic platform. Inspired by a recent theoretical development that clustered mammalian cells respond differently to chemical gradients than single cells, we studied tumor spheroid invasion within a 3D extracellular matrix (ECM) in the presence of EGF gradients. We found that EGF gradients promoted tumor cell detachment from the spheroid core, and the position of the tumor spheroid core showed a mild chemotactic response towards the EGF gradients. For those tumor cells detached from the spheroids, they showed an enhanced motility response in contrast to previous experimental results using single cells embedded within an ECM. No discernible chemotactic response towards the EGF gradients was found for the cells outside the spheroid core. This work demonstrates that a cluster of tumor cells responds differently than single tumor cells towards EGF gradients and highlights the importance of a tumor spheroid platform for tumor invasion studies.

Spatial and temporal dynamics of RhoA activities of single breast tumor cells in a 3D environment revealed by a machine learning-assisted FRET technique.

One of the hallmarks of cancer cells is their exceptional ability to migrate within the extracellular matrix (ECM) for gaining access to the circulatory system, a critical step of cancer metastasis. RhoA, a small GTPase, is known to be a key molecular switch that toggles between actomyosin contractility and lamellipodial protrusion during cell migration. Current understanding of RhoA activity in cell migration has been largely derived from studies of cells plated on a two-dimensional (2D) substrate using a FRET biosensor. There has been increasing evidence that cells behave differently in a more physiologically relevant three-dimensional (3D) environment. However, studies of RhoA activities in 3D have been hindered by low signal-to-noise ratio in fluorescence imaging. In this paper, we present a a machine learning-assisted FRET technique to follow the spatiotemporal dynamics of RhoA activities of single breast tumor cells (MDA-MB-231) migrating in a 3D as well as a 2D environment. We found that RhoA activity is more polarized along the long axis of the cell for single cells migrating on 2D fibronectin-coated glass versus those embedded in 3D collagen matrices. In particular, RhoA activities of cells in 2D exhibit a distinct front-to-back and back-to-front movement during migration in contrast to those in 3D. Finally, regardless of dimensionality, RhoA polarization is found to be moderately correlated with cell shape.

Live tumor imaging shows macrophage induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.

Microglial-stimulation of glioma invasion involves the EGFR ligand amphiregulin.

High grade glioma is one of the deadliest human cancers with a median survival rate of only one year following diagnosis. The highly motile and invasive nature of high grade glioma makes it difficult to completely remove surgically. Therefore, increasing our knowledge of the mechanisms glioma cells use to invade normal brain is of critical importance in designing novel therapies. It was previously shown by our laboratory that tumor-associated microglia (TAMs) stimulate glioma cell invasion and this process is dependent on CSF-1R signaling. In this study, we seek to identify pro-invasive factors that are upregulated in microglia in a CSF-1R-dependent manner. We assayed cDNA and protein from microglia treated with conditioned media from the murine glioma cell line GL261, and discovered that several EGFR ligands including amphiregulin (AREG) are strongly upregulated. This upregulation is blocked by addition of a pharmacological CSF-1R inhibitor. Using RNA interference, we show that AREG-depleted microglia are less effective at promoting invasion of GL261 cells into Matrigel-coated invasion chambers. In addition, an AREG blocking antibody strongly attenuates the ability of THP-1 macrophages to activate human glioma cell line U87 invasion. Furthermore, we have identified a signaling pathway which involves CSF-1 signaling through ERK to upregulate AREG expression in microglia. Interfering with ERK using pharmacological inhibitors prevents AREG upregulation in microglia and microglia-stimulated GL261 invasion. These data highlight AREG as a key factor in produced by tumor associated microglia in promoting glioma invasion.

TC10 regulates breast cancer invasion and metastasis by controlling membrane type-1 matrix metalloproteinase at invadopodia.

During breast cancer metastasis, cancer cell invasion is driven by actin-rich protrusions called invadopodia, which mediate the extracellular matrix degradation required for the success of the invasive cascade. In this study, we demonstrate that TC10, a member of a Cdc42 subfamily of p21 small GTPases, regulates the membrane type 1 matrix metalloproteinase (MT1-MMP)-driven extracellular matrix degradation at invadopodia. We show that TC10 is required for the plasma membrane surface exposure of MT1-MMP at these structures. By utilizing our Förster resonance energy transfer (FRET) biosensor, we demonstrate the p190RhoGAP-dependent regulation of spatiotemporal TC10 activity at invadopodia. We identified a pathway that regulates invadopodia-associated TC10 activity and function through the activation of p190RhoGAP and the downstream interacting effector Exo70. Our findings reveal the role of a previously unknown regulator of vesicular fusion at invadopodia, TC10 GTPase, in breast cancer invasion and metastasis.

Palbociclib Renders Human Papilloma Virus-Negative Head and Neck Squamous Cell Carcinoma Vulnerable to the Senolytic Agent Navitoclax.

We demonstrate that inhibition of cyclin-dependent kinases 4/6 (CDK4/6) leads to senescence in human papillomavirus (HPV)-negative (-) head and neck squamous cell carcinoma (HNSCC), but not in HPV-positive (+) HNSCC. The BCL-2 family inhibitor, navitoclax, has been shown to eliminate senescent cells effectively. We evaluated the efficacy of combining palbociclib and navitoclax in HPV- HNSCC. Three HPV- HNSCC cell lines (CAL27, HN31, and PCI15B) and three HPV+ HNSCC cell lines (UPCI-SCC-090, UPCI-SCC-154, and UM-SCC-47) were treated with palbociclib. Treatment drove reduced expression of phosphorylated Rb (p-Rb) and phenotypic evidence of senescence in all HPV- cell lines, whereas HPV+ cell lines did not display a consistent response by Rb or p-Rb and did not exhibit morphologic changes of senescence in response to palbociclib. In addition, treatment of HPV- cells with palbociclib increased both ß-galactosidase protein expression and BCL-xL protein expression compared with untreated controls in HPV- cells. Co-expression of ß-galactosidase and BCL-xL occurred consistently, indicating elevated BCL-xL expression in senescent cells. Combining palbociclib with navitoclax led to decreased HPV- HNSCC cell survival and led to increased apoptosis levels in HPV- cell lines compared with each agent given alone. IMPLICATIONS: This work exploits a key genomic hallmark of HPV- HNSCC (CDKN2A disruption) using palbociclib to induce BCL-xL-dependent senescence, which subsequently causes the cancer cells to be vulnerable to the senolytic agent, navitoclax.

The architecture of co-culture spheroids regulates tumor invasion within a 3D extracellular matrix.

Tumor invasion, the process by which tumor cells break away from their primary tumor and gain access to vascular systems, is an important step in cancer metastasis. Most current 3D tumor invasion assays consisted of single tumor cells embedded within an extracellular matrix (ECM). These assays taught us much of what we know today on how key biophysical (e.g. ECM stiffness) and biochemical (e.g. cytokine gradients) parameters within the tumor microenvironment guided and regulated tumor invasion. One limitation of the single tumor cell invasion assay was that it did not account for cell-cell adhesion within the tumor. In this article, we developed a micrometer scale 3D co-culture spheroid invasion assay that was compatible with microscopic imaging. Micrometer scale co-culture spheroids (1:1 ratio of metastatic breast cancer MDA-MB-231 and non-tumorigenic epithelial MCF-10A cells) were made using an array of microwells, and then were embedded within a collagen matrix in a microfluidic platform. Real time imaging of tumor spheroid invasion revealed that the spatial distribution of the two cell types within the tumor spheroid critically regulated tumor invasion. This work linked tumor architecture with tumor invasion and highlighted the importance of the biophysical cues within the bulk of the tumor in tumor invasion.

Tumor spheroids under perfusion within a 3D microfluidic platform reveal critical roles of cell-cell adhesion in tumor invasion.

Tumor invasion within the interstitial space is critically regulated by the force balance between cell-extracellular matrix (ECM) and cell-cell interactions. Interstitial flows (IFs) are present in both healthy and diseased tissues. However, the roles of IFs in modulating cell force balance and subsequently tumor invasion are understudied. In this article, we develop a microfluidic model in which tumor spheroids are embedded within 3D collagen matrices with well-defined IFs. Using co-cultured tumor spheroids (1:1 mixture of metastatic and non-tumorigenic epithelial cells), we show that IFs downregulate the cell-cell adhesion molecule E-cadherin on non-tumorigenic cells and promote tumor invasion. Our microfluidic model advances current tumor invasion assays towards a more physiologically realistic model using tumor spheroids instead of single cells under perfusion. We identify a novel mechanism by which IFs can promote tumor invasion through an influence on cell-cell adhesion within the tumor and highlight the importance of biophysical parameters in regulating tumor invasion.

Microscopic Methods for Analysis of Macrophage-Induced Tunneling Nanotubes.

Macrophages are known to play multiple roles in the breast cancer microenvironment including the promotion of tumor cell invasion that is dependent on soluble factors or through direct contact. Macrophages can also enhance the production of Tunneling Nanotubes (TNTs) in tumor cells which can be mimicked using macrophage-conditioned medium. TNTs are long thin F-actin structures that connect two or more cells together that have been found in many different cell types including macrophages and tumor cells and have been implicated in enhancing tumor cells functions, such as invasion. Here we describe basic procedures used to stimulate tumor cell TNT formation through macrophage-conditioned medium along with methods for quantifying TNTs.

Glycation of collagen matrices promotes breast tumor cell invasion.

Cancer metastasis is a physical process in which tumor cells break away from the primary tumor, enter, and then exit the blood or lymph vessels, and establish secondary tumors in distant organs. Current clinical studies report a higher risk of cancer metastasis for diabetics than non-diabetics. However, due to complex overlapping risk factors between diabetes and cancer, the mechanism underlying this correlation is largely unknown. Elevated lifetime blood sugar levels in diabetics are known to increase glycation of collagen, causing stiffening of the ECM and connective tissue. In this study, we explored the roles of glycation of 3D collagen matrices in tumor cell invasion and migration. Using time-lapse images, we quantitatively compared the motility behavior of malignant breast tumor cells (MDA-MB-231) and co-culture spheroids (1:1 ratio of MDA-MB-231 cells with normal epithelial MCF-10A cells) embedded in glycated and non-glycated collagen matrices of various concentrations. Experimental results demonstrated that glycation increased tumor invasion within collagen matrices. More specifically, the average speed of MDA-MB-231 cells was higher in glycated collagen gels than in non-glycated collagen gels for all three gel concentrations tested. Cell spreading characterized by its diffusion coefficient or the effective spheroid radii at various time points was significantly greater in glycated collagen than in non-glycated collagen at a concentration of 3.5 mg/mL. This enhancement was moderate and less evident at lower collagen concentrations of 1.0 and 2.0 mg/mL. These results suggest a possible biomechanical link that relates to the high blood sugar level in diabetic patients and the cancer metastatic outcome.

Macrophages enhance 3D invasion in a breast cancer cell line by induction of tumor cell tunneling nanotubes.

Background: Metastasis is the cause of most cancer-related deaths. It is known that breast cancer cells in proximity to macrophages become more invasive in an Epidermal Growth Factor (EGF) dependent manner. Tunneling nanotubes (TNTs) are thin, F-actin containing, cellular protrusions that mediate intercellular communication and have been identified in many tumors. The mechanism of TNT formation varies between different cell types. M-Sec (TNFAIP2) has been demonstrated to be involved in TNT formation in some cell types including macrophages. Yet, the requirement of M-Sec in tumor cell TNT formation in response to macrophages has not been explored. Aim: The aim of this study was to determine whether EGF was required for macrophage induced tumor cell TNTs in an M-Sec dependent manner and what possible roles tumor cell TNTs play in tumor cell migration and invasion. Methods and Results: Macrophage Conditioned Media (CM) was used to induce an increase in TNTs in a number of breast cancer cell lines as measured by live cell microscopy. Tumor cell TNT formation by CM was dependent on the presence of EGF which was sufficient to induce TNT formation. CM treatment enhanced the level of M-Sec identified using western blot analysis. Reduction of endogenous M-Sec levels via shRNA in MTLn3 mammary adenocarcinoma cells inhibited the formation of TNTs. The role of tumor cell TNTs in cell behavior was tested using in vitro transwell and 3D invasion assays. No effect on chemotaxis was detected but 3D invasion was reduced following the knockdown of M-Sec in tumor cell TNTs. Conclusions: Our results show that EGF was necessary and sufficient for tumor cell TNT formation which was dependent on cellular M-Sec levels. While tumor cell TNTs may not play a role in individual cell behaviors like chemotaxis, they may be important in more complex tumor cell behaviors such as 3D invasion.

Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth.

BACKGROUND: Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. METHODS: We crossed AREG-null (AREG-/-) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG-/- PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. RESULTS: Intriguingly, PyMT-induced lesions progress more rapidly in AREG-/- mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG-/- mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG-/- PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. CONCLUSIONS: Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer.

A novel neuregulin - jagged1 paracrine loop in breast cancer transendothelial migration.

BACKGROUND: The interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Invasion and intravasation, two critical steps in the metastatic process, are influenced by these interactions. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages involving colony stimulating factor 1 (CSF-1) produced by tumor cells and epidermal growth factor (EGF) production by macrophages. In this paper, we identify a novel paracrine loop between tumor cells and macrophages involving neuregulin (NRG1) and notch signaling. METHODS: The aim of this study was to determine the role of NRG1, a ligand of the ErbB3 receptor, in macrophage stimulation of tumor cell transendothelial migration and intravasation. We used fluorescence-activated cell sorting (FACS) and western blot to determine ErbB3 and NRG1 expression, respectively. An in vitro transendothelial migration (iTEM) assay was used to examine the effects of short hairpin (sh)RNA targeting NRG1 in tumor cells and clustered regularly interspaced short palindromic repeats (CRISPR) knockout of jagged 1 (JAG1) in macrophages. Orthotopic xenograft injections in mice were used to confirm results in vivo. RESULTS: In our system, macrophages were the primary cells showing expression of ErbB3, and a blocking antibody against ErbB3 resulted in a significant decrease in macrophage-induced transendothelial migration of breast cancer cells. Stimulation of macrophages with NRG1 upregulated mRNA and protein expression of JAG1, a ligand of the Notch receptor, and JAG1 production by macrophages was important for transendothelial migration of tumor cells. CONCLUSIONS: This study demonstrates that stimulation of macrophages by tumor cell NRG1 can enhance transendothelial migration and intravasation. We also demonstrate that this effect is due to induction of macrophage JAG1, an important ligand of the Notch signaling pathway.

An Akt3 Splice Variant Lacking the Serine 472 Phosphorylation Site Promotes Apoptosis and Suppresses Mammary Tumorigenesis.

The Akt pathway is a well-known promoter of tumor malignancy. Akt3 is expressed as two alternatively spliced variants, one of which lacks the key regulatory serine 472 phosphorylation site. Whereas the function of full-length Akt3 isoform (Akt3/+S472) is well-characterized, that of Akt3/-S472 isoform remains unknown. Despite being expressed at a substantially lower level than Akt3/+S472 in triple-negative breast cancer cells, specific ablation of Akt3/-S472 enhanced, whereas overexpression, suppressed mammary tumor growth, consistent with a significant association with patient survival duration relative to Akt3/+S472. These effects were due to striking induction of apoptosis, which was mediated by Bim upregulation, leading to conformational activation of Bax and caspase-3 processing. Bim accumulation was caused by marked endocytosis of EGF receptors with concomitant ERK attenuation, which stabilizes BIM. These findings demonstrate an unexpected function of an endogenously expressed Akt isoform in promoting, as opposed to suppressing, apoptosis, underscoring that Akt isoforms may exert dissonant functions in malignancy.Significance: These results illuminate an unexpected function for an endogenously expressed Akt isoform in promoting apoptosis, underscoring the likelihood that different Akt isoforms exert distinct functions in human cancer. Cancer Res; 78(1); 103-14. ©2017 AACR.

Rac3 regulates breast cancer invasion and metastasis by controlling adhesion and matrix degradation.

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and ßPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.