Invadopodia are actin‐rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they have been visualized as localized and dot‐like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein–Barr virus (EBV)‐associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live‐cell imaging and molecular assays revealed the interaction of macrophage‐released TNFα and EBV‐encoded latent membrane protein 1 (LMP1) in co‐activating the EGFR/Src/ERK/cortactin and Cdc42/N‐WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot‐like digestion patterns on a 2D gelatin to a dendrite‐like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot‐like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα‐rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Vascular smooth muscle cells (VSMCs) play a central role in the onset and progression of atherosclerosis. In pre-atherosclerotic lesions, VSMCs switch from a contractile to a synthetic phenotype and subsequently remodel the microenvironment, leading to further disease progression. Ageing and associated mechanical changes of the extracellular matrix as well as hypertension are major risk of atherosclerosis. Consequently, we sought here to systematically study the impact of mechanical stimulations on VSMC phenotypic switching, by modulating both stiffness and hydrodynamic pressure. Thereby we find that hemodynamic pressure and matrix stiffness individually affect the VSMC phenotype. However, only the combination of hypertensive pressure and matrix compliance, and as such mechanical stimuli that are prevalent during atherosclerosis, lead to a full phenotypic switch including the formation of matrix degrading podosomes. We further analyse the molecular mechanism in stiffness and pressure sensing and identify a regulation through different, but overlapping pathways, culminating in the regulation of the actin cytoskeleton through cofilin. Altogether, our data shows how different pathological mechanical signals combined, but through distinct pathways accelerate a phenotypic switch that will ultimately contribute to atherosclerotic disease progression. ### Competing Interest Statement The authors have declared no competing interest.

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.

Invadosomes are invasive protrusions generated by cells which can secrete matrix metalloproteinases for focal digestion of extracellular matrix. They also aid invasive cancer cells in their transmigration through vascular endothelium. However, how the physical and chemical cues in a three-dimensional (3D) system signal the spatial localization of invadosomes remains largely unknown. Here we study the topographic guidance of invadosome formation in invasive nasopharyngeal cells under the stimulation of an inflammatory cytokine, TGF-β1, using engineered gratings with different width and depth. We first report that TGF-β1 can act as an external signal to upregulate the formation of invadosomes with a random distribution on a plane 2D surface. When the cells were seeded on parallel 3D gratings of 5 µm width and 1 µm depth, most of the invadosomes aligned to the edges of the gratings, indicating a topographic cue to the control of invadosome localization. While the number of invadosomes per cell were not upregulated when the cells were seeded on 3D topography, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges when the cells move at a lower speed and are guided along narrow gratings. The invadosomes forming at 3D edges also have a longer half-life than those forming on a plane surface. These data suggest that there are integrated biochemical and 3D geometric cues underlying the spatial regulation of invasive structures so as to elicit efficient invasion or metastasis of cells. STATEMENT OF SIGNIFICANCE: Nasopharyngeal cells were integrated with the biological cues and matrix topography to govern the activity and spatial distribution of invadosomes. The biochemical induction of invadosome formation by TGF-β1 in nasopharyngeal cells was observed. When the cells were seeded on parallel 3D gratings, most of the invadosomes aligned to the edges of the gratings due to topographical induced invadosome localization. While the number of invadosomes per cell were not upregulated, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges with a higher stability when the cells are guided along narrow gratings. The integrated biochemical and 3D geometric cues could elicit efficient invasion or metastasis of cells.

Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors. First, we describe the process of podosome disassembly induced by activation of myosin-IIA filament assembly. Next, we find that podosome integrity and dynamics depends upon membrane tension and can be experimentally perturbed by osmotic swelling and deoxycholate treatment. We have also found that podosomes can be disrupted in a reversible manner by single or cyclic radial stretching of the substratum. We show that disruption of podosomes induced by osmotic swelling is independent of myosin-II filaments. The inhibition of the membrane sculpting protein, dynamin-II, but not clathrin, resulted in activation of myosin-IIA filament formation and disruption of podosomes. The effect of dynamin-II inhibition on podosomes was, however, independent of myosin-II filaments. Moreover, formation of organized arrays of podosomes in response to microtopographic cues (the ridges with triangular profile) was not accompanied by reorganization of myosin-II filaments. Thus, mechanical elements such as myosin-II filaments and factors affecting membrane tension/sculpting independently modulate podosome formation and dynamics, underlying a versatile response of these adhesion structures to intracellular and extracellular cues. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes. Myosin IIA filaments are indispensable effectors in the microtubule-driven regulation of integrin-mediated adhesions. Myosin IIA filament assembly depends on Rho activation by the RhoGEF GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.

p21-Activated kinase 4 (PAK4), a serine/threonine kinase, is purported to localize to podosomes: transient adhesive structures that degrade the extracellular matrix to facilitate rapid myeloid cell migration. We find that treatment of transforming growth factor β (TGF-β)-differentiated monocytic (THP-1) cells with a PAK4-targeted inhibitor significantly reduces podosome formation and induces the formation of focal adhesions. This switch in adhesions confers a diminution of matrix degradation and reduced cell migration. Furthermore, reduced PAK4 expression causes a significant reduction in podosome number that cannot be rescued by kinase-dead PAK4, supporting a kinase-dependent role. Concomitant with PAK4 depletion, phosphorylation of Akt is perturbed, whereas a specific phospho-Akt signal is detected within the podosomes. Using superresolution analysis, we find that PAK4 specifically localizes in the podosome ring, nearer to the actin core than other ring proteins. We propose PAK4 kinase activity intersects with the Akt pathway at the podosome ring:core interface to drive regulation of macrophage podosome turnover.

High-density analysis methods for localization microscopy increase acquisition speed but produce artifacts. We demonstrate that these artifacts can be eliminated by the combination of Haar wavelet kernel (HAWK) analysis with standard single-frame fitting. We tested the performance of this method on synthetic, fixed-cell, and live-cell data, and found that HAWK preprocessing yielded reconstructions that reflected the structure of the sample, thus enabling high-speed, artifact-free super-resolution imaging of live cells.

Cancer metastasis is a major cause of mortality, accounting for approximately 90% of cancer related deaths. Emerging evidences suggest that tumour microenvironment plays an indispensable role in cancer metastasis. Recruitment of innate and adaptive immune cells to the tumour results in chronic inflammation which favours tumour growth and progression. Particularly, tumour associated macrophages (TAMs) are found to promote the initiation, growth, and metastasis in several types of cancers. Nasopharyngeal carcinoma (NPC) is one of the highly invasive and metastatic cancers and is closely associated with EBV infection. We postulate that TAMs may increase the invasiveness of NPC by enhancing the formation of invadopodia, which are membranous actin-rich protrusions with digestive ability of extra-cellular matrix. In this study, monocyte THP-1 cells were polarized to M2-macrophages, and co-cultured with nasopharyngeal cells in a contained chamber-insert. The stimulated nasopharyngeal cells then formed massive numbers of invadopodia and digested larger area of gelatin compared with the unstimulated counterparts. A cytokine antibody array was used to identify the upregulated cytokines in the co-culture medium of the M2-macrohphages and the NPC cells. Tumour necrosis factor alpha (TNFα) was revealed to be the responsible cytokine to induce the formation of invadopodia and digestion of gelatin. Besides, western blot analysis identified that several actin-associated proteins such as Src, Erk, and cortactin were involved in the TNFα-mediated invadopodia formation. We also found that the expression of latent membrane protein 1 (LMP1), an EBV protein that has similar structure to TNF receptor was elevated in NPC cells after co-culturing with the M2-polarized macrophages. Through transient transfection and stable expression of LMP1 in the NPC cells, LMP1 could also promote the formation of invadopodia through cdc42 activation. Taken together, these data indicate macrophages can promote the invasiveness of EBV-infected NPC cells by enhancing their ability in forming invadopodia through an activated TNFα signaling axis and LMP1 upregulation. Citation Format: Wilson Wing Chung Tang, Chan Ping You, Gareth E. Jones, George Sai-Wah Tsao, Anna Chi Man Tsang. M2-polarized macrophages increase invasiveness of EBV-associated nasopharyngeal carcinoma by inducing invadopodia formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1108.

AbstractMotivationClustering analysis is a key technique for quantitatively characterizing structures in localization microscopy images. To build up accurate information about biological structures, it is critical that the quantification is both accurate (close to the ground truth) and precise (has small scatter and is reproducible).ResultsHere, we describe how the Rényi divergence can be used for cluster radius measurements in localization microscopy data. We demonstrate that the Rényi divergence can operate with high levels of background and provides results which are more accurate than Ripley’s functions, Voronoi tesselation or DBSCAN.Availability and implementationThe data supporting this research and the software described are accessible at the following site: https://dx.doi.org/10.18742/RDM01-316. Correspondence and requests for materials should be addressed to the corresponding author.Supplementary informationSupplementary data are available at Bioinformatics online.

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesions, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin-IIA filaments. Myosin-IIA filaments, augmenting the focal adhesions and disrupting the podosomes, are indispensable effectors in the microtubule-dependent regulation of integrin-mediated adhesions. Myosin-IIA filament assembly depends on Rho activation by the RhoGEF, GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capturing by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the myosin-IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR–ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR–ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott–Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR – ABL1 and is particularly low in blast crisis. Enforced expression of BCR – ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR–ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR–ABL1-induced tumorigenesis in vitro and in vivo . Taken together, our data reveal a novel molecular mechanism that operates in BCR–ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).

Podosomes represent a special class of integrin-mediated cell-matrix adhesions formed by migrating and matrix degrading cells. Here, we demonstrated that in macrophage-like THP1 cells and in fibroblasts stimulated to produce podosomes, down-regulation of ARF1 by siRNA, and by pharmacological inhibitors led to striking podosome elimination. Treatments that induced podosome formation increased the level of GTP-bound ARF1. Furthermore, siRNA knockdown of the ARF1-GEF ARNO also resulted in dramatic podosome inhibition. ARNO was found to co-localize with the adhesive rings of podosomes while ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells that resemble sarcomere-like organization typically observed in fibroblasts, whilst the suppression of myosin-IIA rescued podosome formation despite ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors; actin-rich puncta that coincided with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. We conclude that ARNO-ARF1 regulates formation of podosomes by inhibition of Rho/myosin II and promotion of actin core assembly.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)–bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

Podosomes are adhesive structures formed on the plasma membrane abutting the extracellular matrix of macrophages, osteoclasts, and dendritic cells. They consist of an f-actin core and a ring structure composed of integrins and integrin-associated proteins. The podosome ring plays a major role in adhesion to the underlying extracellular matrix, but its detailed structure is poorly understood. Recently, it has become possible to study the nano-scale structure of podosome rings using localization microscopy. Unlike traditional microscopy images, localization microscopy images are reconstructed using discrete points, meaning that standard image analysis methods cannot be applied. Here, we present a pipeline for podosome identification, protein position calculation, and creating a podosome ring model for use with localization microscopy data.