Talin2 binds to non-muscle myosin IIa and regulates cell attachment and fibronectin secretion.

Talin2 is localized to large focal adhesions and is indispensable for traction force generation, invadopodium formation, cell invasion as well as metastasis. Talin2 has a higher affinity toward ß-integrin tails than talin1. Moreover, disruption of the talin2-ß-integrin interaction inhibits traction force generation, invadopodium formation and cell invasion, indicating that a strong talin2-ß-integrin interaction is required for talin2 to fulfill these functions. Nevertheless, the role of talin2 in mediation of these processes remains unknown. Here we show that talin2 binds to the N-terminus of non-muscle myosin IIA (NMIIA) through its F3 subdomain. Moreover, talin2 co-localizes with NMIIA at cell edges as well as at some cytoplasmic spots. Talin2 also co-localizes with cortactin, an invadopodium marker. Furthermore, overexpression of NMIIA promoted the talin2 head binding to the ß1-integrin tail, whereas knockdown of NMIIA reduced fibronectin and matrix metalloproteinase secretion as well as inhibited cell attachment on fibronectin-coated substrates. These results suggest that talin2 binds to NMIIA to control the secretion of extracellular matrix proteins and this interaction modulates cell adhesion.

The Oncolytic Avian Reovirus p17 Protein Inhibits Invadopodia Formation in Murine Melanoma Cancer Cells by Suppressing the FAK/Src Pathway and the Formation of theTKs5/NCK1 Complex.

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

Unveiling epithelial plasticity regulation in lung cancer: Exploring the cross-talk among Tks4 scaffold protein partners.

The epithelial-to-mesenchymal transition (EMT) represents a hallmark event in the evolution of lung cancer. This work aims to study a recently described EMT-regulating protein, Tks4, and to explore its potential as a prognostic biomarker in non-small cell lung cancer. In this study, we used CRISPR/Cas9 method to knockout (KO) Tks4 to study its functional roles in invadopodia formation, migration, and regulation of EMT marker expressions and we identified Tks4-interacting proteins. Tks4-KO A549 cells exhibited an EMT-like phenotype characterized by elongated morphology and increased expression of EMT markers. Furthermore, analyses of a large-scale lung cancer database and a patient-derived tissue array data revealed that the Tks4 mRNA level was decreased in more aggressive lung cancer stages. To understand the regulatory role of Tks4 in lung cancer, we performed a Tks4-interactome analysis via Tks4 immunoprecipitation-mass spectrometry on five different cell lines and identified CAPZA1 as a novel Tks4 partner protein. Thus, we propose that the absence of Tks4 leads to disruption of a connectome of multiple proteins and that the resulting undocking and likely mislocalization of signaling molecules impairs actin cytoskeleton rearrangement and activates EMT-like cell fate switches, both of which likely influence disease severity.

Fibronectin induces a transition from amoeboid to a fan morphology and modifies migration in Entamoeba histolytica.

Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.

Cancer cell extravasation requires iplectin-mediated delivery of MT1-MMP at invadopodia.

BACKGROUND: Invadopodia facilitate cancer cell extravasation, but the molecular mechanism whereby invadopodia-specific proteases such as MT1-MMP are called to invadopodia is unclear. METHODS: Mass spectrometry and immunoprecipitation were used to identify interactors of MT1-MMP in metastatic breast cancer cells. After identification, siRNA and small molecule inhibitors were used to assess the effect these interactors had on cellular invasiveness. The chicken embryo chorioallantoic membrane (CAM) model was used to assess extravasation and invadopodia formation in vivo. RESULTS: In metastatic breast cancer cells, MT1-MMP was found to associate with plectin, a cytolinker and scaffolding protein. Complex formation between plectin and MT1-MMP launches invadopodia formation, a subtype we termed iplectin (i = invadopodial). iPlectin delivers MT1-MMP to invadopodia and is indispensable for regulating cell surface levels of the enzyme. Genetic depletion of plectin with siRNA reduced invadopodia formation and cell invasion in vitro. In vivo extravasation efficiency assays and intravital imaging revealed iplectin to be a key contributor to invadopodia ultrastructure and essential for extravasation. Pharmacologic inhibition of plectin using the small molecule Plecstatin-1 (PST-1) abrogated MT1-MMP delivery to invadopodia and extravasation efficiency. CONCLUSIONS: Anti-metastasis therapeutic approaches that target invadopodia are possible by disrupting interactions between MT1-MMP and iplectin. CLINICAL TRIAL REGISTRATION NUMBER: NCT04608357.

Hypoxia Promotes Invadosome Formation by Lung Fibroblasts.

Lung parenchymal hypoxia has emerged as a cardinal feature of idiopathic pulmonary fibrosis (IPF). Hypoxia promotes cancer cell invasion and metastasis through signaling that is dependent upon the lysophosphatidic acid (LPA) receptor, LPA1 (LPAR1). Abundant data indicate that LPA1-dependent signaling also enhances lung fibrogenesis in IPF. We recently reported that fibroblasts isolated from the lungs of individuals with IPF have an increased capacity to form subcellular matrix-degradative structures known as invadosomes, an event that correlates with the degree of lung fibrosis. We therefore hypothesized that hypoxia promotes invadosome formation in lung fibroblasts through LPA1-dependent signaling. Here, it is demonstrated that invadosome formation by fibroblasts from the lungs of individuals with advanced IPF is inhibited by both the tyrosine receptor kinase inhibitor nintedanib and inhibition of LPA1. In addition, exposure of normal human lung fibroblasts to either hypoxia or LPA increased their ability to form invadosomes. Mechanistically, the hypoxia-induced invadosome formation by lung fibroblasts was found to involve LPA1 and PDGFR-Akt signaling. We concluded that hypoxia increases the formation of invadosomes in lung fibroblasts through the LPA1 and PDGFR-Akt signaling axis, which represents a potential target for suppressing lung fibrosis.

In Vitro Cell Culture Model for Osteoclast Activation during Estrogen Withdrawal.

Estrogen (17ß-estradiol) deficiency post-menopause alters bone homeostasis whereby bone resorption by osteoclasts exceeds bone formation by osteoblasts, leading to osteoporosis in females. We established an in vitro model to examine the consequences of estrogen withdrawal (E2-WD) on osteoclasts derived from the mouse macrophage RAW 264.7 cell line and utilized it to investigate the mechanism behind the enhanced osteoclast activity post-menopause. We found that a greater population of osteoclasts that underwent E2-WD contained a podosome belt necessary for osteoclasts to adhere and resorb bone and possessed elevated resorptive activity compared to osteoclasts exposed to estrogen (E2) continuously. Our results show that compared to osteoclasts that received E2 continuously, those that underwent E2-WD had a faster rate of microtubule (MT) growth, reduced RhoA activation, and shorter podosome lifespan. Thus, altered podosome and MT dynamics induced by the withdrawal of estrogen supports podosome belt assembly/stability in osteoclasts, which may explain their enhanced bone resorption activity.

The ß-arrestin1/endothelin axis bolsters ovarian fibroblast-dependent invadosome activity and cancer cell metastatic potential.

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of ß-arrestin1 (ß-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might "educate" human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/ß-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin ß1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or ß-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/ß-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.

Tumor Microenvironment Modulates Invadopodia Activity of Non-Selected and Acid-Selected Pancreatic Cancer Cells and Its Sensitivity to Gemcitabine and C18-Gemcitabine.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug. METHODS: For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pHe 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine. RESULTS: We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. CONCLUSIONS: We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.

MTM1-mediated production of phosphatidylinositol 5-phosphate fuels the formation of podosome-like protrusions regulating myoblast fusion.

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.

PAK6 acts downstream of IQGAP3 to promote contractility in triple negative breast cancer cells.

Breast cancer is a heterogeneous disease that remains the most common malignancy among women worldwide. During genomic analysis of breast tumours, mRNA levels of IQGAP3 were found to be upregulated in triple negative tumours. IQGAP3 was subsequently found to be expressed across a panel of triple negative breast cancer (TNBC) cell lines. Depleting expression levels of IQGAP3 delivered elongated cells, disrupted cell migration, and inhibited the ability of cells to form specialised invasive adhesion structures, termed invadopodia. The morphological changes induced by IQGAP3 depletion were found to be dependent on RhoA. Indeed, reduced expression of IQGAP3 disrupted RhoA activity and actomyosin contractility. Interestingly, IQGAP3 was also found to interact with p-21 activated kinase 6 (PAK6); a protein already associated with the regulation of cell morphology. Moreover, PAK6 depletion phenocopied IQGAP3 depletion in these cells. Whereas PAK6 overexpression rescued the IQGAP3 depletion phenotype. Our work points to an important PAK6-IQGAP3-RhoA pathway that drives the cellular contractility of breast cancer cells promoting both cell migration and adhesive invasion of these cells. As this phenotype is relevant to the process of metastasis and re-seeding of metastasis, the pharmacological targeting of PAK6 could lead to clinical benefit in TNBC patients.

MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

Understanding Actin Remodeling in Neuronal Cells Through Podosomes.

Cytoskeletal dysregulation forms an important aspect of many neurodegenerative diseases such as Alzheimer's disease. Cytoskeletal functions require the dynamic activity of the cytoskeletal proteins-actin, tubulin, and the associated proteins. One of such important phenomena is that of actin remodeling, which helps the cell to migrate, navigate, and interact with extracellular materials. Podosomes are complex actin-rich cytoskeletal structures, abundant in proteins that interact and degrade the extracellular matrix, enabling cells to displace and migrate. The formation of podosomes requires extensive actin networks and remodeling. Here we present a novel immunofluorescence-based approach to study actin remodeling in neurons through the medium of podosomes.

Isoprenylcysteine carboxyl methyltransferase (ICMT) promotes invadopodia formation and metastasis in cancer cells.

Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis in vivo and that its expression is repressed by the p53 tumor suppressor. Based on evidence suggesting that some ICMT substrates affect invasive traits, we wondered if this enzyme may promote metastasis. In this work, we found that ICMT overexpression enhanced lung metastasis in vivo. Accordingly, ICMT overexpression also promoted cellular functions associated with aggressive phenotypes such as migration and invasion in vitro. Considering that some ICMT substrates are involved in the regulation of actin cytoskeleton, we hypothesized that actin-rich structures, associated with invasion and metastasis, may be affected. Our findings revealed that ICMT enhanced the formation of invadopodia. Additionally, by analyzing cancer patient databases, we found that ICMT is overexpressed in several tumor types. Furthermore, the concurrent expression of ICMT and CTTN, which encodes a crucial component of invadopodia, showed a significant correlation with clinical outcome. In summary, our work identifies ICMT overexpression as a relevant alteration in human cancer that promotes the development of metastatic tumors.

A Model for Membrane Degradation Using a Gelatin Invadopodia Assay.

One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work.

The stiffness and collagen control differentiation of osteoclasts with an altered expression of c-Src in podosome.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

A MAP1B-cortactin-Tks5 axis regulates TNBC invasion and tumorigenesis.

The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.

A synergistic regulation works in matrix stiffness-driven invadopodia formation in HCC.

Growing evidence has suggested that increased matrix stiffness can significantly strengthen the malignant characteristics of hepatocellular carcinoma (HCC) cells. However, whether and how increased matrix stiffness regulates the formation of invadopodia in HCC cells remain largely unknown. In the study, we developed different experimental systems in vitro and in vivo to explore the effects of matrix stiffness on the formation of invadopodia and its relevant molecular mechanism. Our results demonstrated that increased matrix stiffness remarkably augmented the migration and invasion abilities of HCC cells, upregulated the expressions of invadopodia-associated genes and enhanced the number of invadopodia. Two regulatory pathways contribute to matrix stiffness-driven invadopodia formation together in HCC cells, including direct triggering invadopodia formation through activating integrin ß1 or Piezo1/ FAK/Src/Arg/cortactin pathway, and indirect stimulating invadopodia formation through improving EGF production to activate EGFR/Src/Arg/cortactin pathway. Src was identified as the common hub molecule of two synergistic regulatory pathways. Simultaneously, activation of integrin ß1/RhoA/ROCK1/MLC2 and Piezo1/Ca2+/MLCK/MLC2 pathways mediate matrix stiffness-reinforced cell migration. This study uncovers a new mechanism by which mechanosensory pathway and biochemical signal pathway synergistically regulate the formation of invadopodia in HCC cells.

POTEE promotes breast cancer cell malignancy by inducing invadopodia formation through the activation of SUMOylated Rac1.

The small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) has been implicated in cancer progression and in the poor prognosis of various types of tumors. Rac1 SUMOylation occurs during epithelial-mesenchymal transition (EMT), and it is required for tumor cell migration and invasion. Here we identify POTEE (POTE Ankyrin domain family member E) as a novel Rac1-SUMO1 effector involved in breast cancer malignancy that controls invadopodium formation through the activation of Rac1-SUMO1. POTEE activates Rac1 in the invadopodium by recruiting TRIO-GEF (triple functional domain protein), and it induces tumor cell proliferation and metastasis in vitro and in vivo. We found that the co-localization of POTEE with Rac1 is correlated with more aggressive breast cancer subtypes. Given its role in tumor dissemination, the leading cause of cancer-related deaths, POTEE could represent a potential therapeutic target for these types of cancer.

Fibroblast activation protein drives tumor metastasis via a protease-independent role in invadopodia stabilization.

During metastasis, tumor cells invade through the basement membrane and intravasate into blood vessels and then extravasate into distant organs to establish metastases. Here, we report a critical role of a transmembrane serine protease fibroblast activation protein (FAP) in tumor metastasis. Expression of FAP and TWIST1, a metastasis driver, is significantly correlated in several types of human carcinomas, and FAP is required for TWIST1-induced breast cancer metastasis to the lung. Mechanistically, FAP is localized at invadopodia and required for invadopodia-mediated extracellular matrix degradation independent of its proteolytic activity. Live cell imaging shows that association of invadopodia precursors with FAP at the cell membrane promotes the stabilization and growth of invadopodia precursors into mature invadopodia. Together, our study identified FAP as a functional target of TWIST1 in driving tumor metastasis via promoting invadopodia-mediated matrix degradation and uncovered a proteolytic activity-independent role of FAP in stabilizing invadopodia precursors for maturation.