Extracellular vesicles secreted by triple-negative breast cancer stem cells trigger premetastatic niche remodeling and metastatic growth in the lungs.

Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple-negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC , respectively) from the MDA-MB-231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL-6/IL-8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α-SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells.

Extracellular matrix anisotropy is determined by TFAP2C-dependent regulation of cell collisions.

The isotropic or anisotropic organization of biological extracellular matrices has important consequences for tissue function. We study emergent anisotropy using fibroblasts that generate varying degrees of matrix alignment from uniform starting conditions. This reveals that the early migratory paths of fibroblasts are correlated with subsequent matrix organization. Combined experimentation and adaptation of Vicsek modelling demonstrates that the reorientation of cells relative to each other following collision plays a role in generating matrix anisotropy. We term this behaviour 'cell collision guidance'. The transcription factor TFAP2C regulates cell collision guidance in part by controlling the expression of RND3. RND3 localizes to cell-cell collision zones where it downregulates actomyosin activity. Cell collision guidance fails without this mechanism in place, leading to isotropic matrix generation. The cross-referencing of alignment and TFAP2C gene expression signatures against existing datasets enables the identification and validation of several classes of pharmacological agents that disrupt matrix anisotropy.

Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts.

Epigenetic changes through altered DNA methylation have been implicated in critical aspects of tumor progression, and have been extensively studied in a variety of cancer types. In contrast, our current knowledge of the aberrant genomic DNA methylation in tumor-associated fibroblasts (TAFs) or other stromal cells that act as critical coconspirators of tumor progression is very scarce. To address this gap of knowledge, we conducted genome-wide DNA methylation profiling on lung TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients using the HumanMethylation450 microarray. We found widespread DNA hypomethylation concomitant with focal gain of DNA methylation in TAFs compared to CFs. The aberrant DNA methylation landscape of TAFs had a global impact on gene expression and a selective impact on the TGF-ß pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-ß1 in terms of contractility and extracellular matrix deposition. In turn, activation of CFs with exogenous TGF-ß1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-ß1 may be necessary but not sufficient to elicit such alterations. Moreover, integrated pathway-enrichment analyses of the DNA methylation alterations revealed that a fraction of TAFs may be bone marrow-derived fibrocytes. Finally, survival analyses using DNA methylation and gene expression datasets identified aberrant DNA methylation on the EDARADD promoter sequence as a prognostic factor in non-small cell lung cancer patients. Our findings shed light on the unique origin and molecular alterations underlying the aberrant phenotype of lung TAFs, and identify a stromal biomarker with potential clinical relevance.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells.

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

Dynamics of podosome stiffness revealed by atomic force microscopy.

Podosomes are unique cellular entities specifically found in macrophages and involved in cell-matrix interactions, matrix degradation, and 3D migration. They correspond to a core of F-actin surrounded at its base by matrix receptors. To investigate the structure/function relationships of podosomes, soft lithography, atomic force microscopy (AFM), and correlative fluorescence microscopy were used to characterize podosome physical properties in macrophages differentiated from human blood monocytes. Podosome formation was restricted to delineated areas with micropatterned fibrinogen to facilitate AFM analyses. Podosome height and stiffness were measured with great accuracy in living macrophages (578 ± 209 nm and 43.8 ± 9.3 kPa) and these physical properties were independent of the nature of the underlying matrix. In addition, time-lapse AFM revealed that podosomes harbor two types of overlapping periodic stiffness variations throughout their lifespan, which depend on F-actin and myosin II activity. This report shows that podosome biophysical properties are amenable to AFM, allowing the study of podosomes in living macrophages at nanoscale resolution and the analysis of their intimate dynamics. Such an approach opens up perspectives to better understand the mechanical functionality of podosomes under physiological and pathological contexts.

Targeted immunotherapy against distinct cancer-associated fibroblasts overcomes treatment resistance in refractory HER2+ breast tumors.

About 50% of human epidermal growth factor receptor 2 (HER2)+ breast cancer patients do not benefit from HER2-targeted therapy and almost 20% of them relapse after treatment. Here, we conduct a detailed analysis of two independent cohorts of HER2+ breast cancer patients treated with trastuzumab to elucidate the mechanisms of resistance to anti-HER2 monoclonal antibodies. In addition, we develop a fully humanized immunocompetent model of HER2+ breast cancer recapitulating ex vivo the biological processes that associate with patients' response to treatment. Thanks to these two approaches, we uncover a population of TGF-beta-activated cancer-associated fibroblasts (CAF) specific from tumors resistant to therapy. The presence of this cellular subset related to previously described myofibroblastic (CAF-S1) and podoplanin+ CAF subtypes in breast cancer associates with low IL2 activity. Correspondingly, we find that stroma-targeted stimulation of IL2 pathway in unresponsive tumors restores trastuzumab anti-cancer efficiency. Overall, our study underscores the therapeutic potential of exploiting the tumor microenvironment to identify and overcome mechanisms of resistance to anti-cancer treatment.

CAFs and Cancer Cells Co-Migration in 3D Spheroid Invasion Assay.

In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell-cell interactions and cell-matrix adhesions. Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

Sticking, steering, squeezing and shearing: cell movements driven by heterotypic mechanical forces.

During development, the immune response and cancer, cells of different types interact mechanically. Here we review how such heterotypic mechanical interactions enable cell movements. We begin by analyzing the heterotypic forces that single cells use to adhere and squeeze through tight barriers, as in the case of leucocyte extravasation and cancer metastasis. We next focus on the different mechanisms by which adjacent tissues influence each other's movements, with particular emphasis on dragging forces during dorsal closure in Drosophila and shearing forces during gastrulation in zebrafish. Finally, we discuss the mechanotransduction feedback loops that enable different cell types to steer each other's migration during development and cancer. We illustrate these migration modes focusing on the combination of attractive and repulsive cues during co-migration of neural crest cells and placodes in Xenopus, and of fibroblasts and cancer cells during invasion. Throughout the review, we discuss the nature of the heterotypic contact, which may involve both homophilic and heterophilic interactions between adhesion receptors.

A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion.

Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers ß-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion.

Collective cell durotaxis emerges from long-range intercellular force transmission.

The ability of cells to follow gradients of extracellular matrix stiffness-durotaxis-has been implicated in development, fibrosis, and cancer. Here, we found multicellular clusters that exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, required the action of myosin motors, and originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stick-slip dynamics of cell-matrix adhesions was integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single-cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion.

Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes.

Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force.

Frustrated phagocytosis on micro-patterned immune complexes to characterize lysosome movements in live macrophages.

Lysosome mobilization is a key cellular process in phagocytes for bactericidal activities and trans-matrix migration. The molecular mechanisms that regulate lysosome mobilization are still poorly known. Lysosomes are hard to track as they move toward phagosomes throughout the cell volume. In order to anticipate cell regions where lysosomes are recruited to, human and RAW264.7 macrophages were seeded on surfaces that were micro-patterned with immune complexes (ICs) as 4 µm-side squares. Distances between IC patterns were adapted to optimize cell spreading in order to constrain lysosome movements mostly in two dimensions. FcΓ receptors triggered local frustrated phagocytosis, frustrated phagosomes appeared as rings of F-actin dots around the IC patterns as early as 5 min after cells made contact with the substratum. Frustrated phagosomes recruited actin-associated proteins (vinculin, paxillin, and gelsolin). The fusion of lysosomes with frustrated phagosomes was shown by the release of beta-hexosaminidase and the recruitment of Lamp1 to frustrated phagosomes. Lysosomes of RAW264.7 macrophages were labeled with cathepsin-D-mCherry to visualize their movements toward frustrated phagosomes. Lysosomes saltatory movements were markedly slowed down compared to cells layered on non-opsonized patterns. In addition, the linearity of the trajectories and the frequency and duration of contacts of lysosomes with frustrated phagosomes were measured. Our experimental set-up is the first step toward deciphering molecular mechanisms which are involved in lysosome movements in the cytoplasm (speed, directionality, and interaction with phagosomes), and opens the door to approaches such as RNA interference, pharmacological inhibition, or mutant expression.