A new pipeline for pathophysiological analysis of the mammary gland based on organoid transplantation and organ clearing.

Recent developments in techniques for tissue clearing and size reduction have enabled optical imaging of whole organs and the study of rare tumorigenic events in vivo The adult mammary gland provides a unique model for investigating physiological or pathological processes such as morphogenesis or epithelial cell dissemination. Here, we establish a new pipeline to study rare cellular events occurring in the mammary gland, by combining orthotopic transplantation of mammary organoids with the uDISCO organ size reduction and clearing method. This strategy allows us to analyze the behavior of individually labeled cells in regenerated mammary gland. As a proof of concept, we analyzed the localization of rare epithelial cells overexpressing atypical protein kinase C iota (also known as PRKCI, referred to here as aPKCι) with an N-terminal eGFP fusion (GFP-aPKCι+) in the normal mammary gland. Using this analytical pipeline, we were able to visualize epithelial aPKCι+ cells escaping from the normal mammary epithelium and disseminating into the surrounding stroma. This technical resource should benefit mammary development and tumor progression studies.

Cancer cells in the tumor core exhibit spatially coordinated migration patterns.

In the early stages of metastasis, cancer cells exit the primary tumor and enter the vasculature. Although most studies have focused on the tumor invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we imaged tumor explants from spontaneously forming tumors in mice in real time using long-term two-photon microscopy. Cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local 'currents' and large-scale tissue dynamics. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Use of pharmacological inhibitors indicates that migration patterns in tumors are actively driven by the actin cytoskeleton. Under these conditions, we also observed a relationship between migration speed and correlation length, suggesting that cells in tumors are near a jamming transition. Our study provides new insight into the dynamics of cancer cells in the tumor core, opening new avenues of research in understanding the migratory properties of cancer cells and later metastasis.This article has an associated First Person interview with the first author of the paper.

IGF1R undergoes active and directed centripetal transport on filopodia upon receptor activation.

Filopodia are thin, actin-based membrane protrusions with roles in sensing external mechanical and chemical cues, such as growth factor gradients in tissues. It was proposed that the chemical sensing role of filopodia is achieved through clearance of activated signaling receptors from filopodia. Type I insulin-like growth factor receptor (IGF1R) is a key regulator of normal development and growth, as well as tumor development and progression. Its biological roles depend on its activation upon IGF1 binding at the cell membrane. IGF1R behavior at the cell membrane and in particular in filopodia, has not been established. We found that IGF1 activation led to a gradual reduction in IGF1R puncta in filopodia, and that this clearance depended on actin, non-muscle myosin II, and IGF1R kinase activity. Using single particle tracking of filopodial IGF1R, we established that ligand-free IGF1R undergoes non-directional unidimensional diffusion along the filopodium. Moreover, after initial diffusion, the ligand-bound IGF1R is actively transported along the filopodium towards the filopodium base, and consequently cleared from the filopodium. Our results show that IGF1R can move directionally on the plasma membrane protrusions, supporting a sensory role for filopodia in interpreting local IGF1 gradients.

IGF-1 drives chromogranin A secretion via activation of Arf1 in human neuroendocrine tumour cells.

Hypersecretion is the major symptom of functional neuroendocrine tumours. The mechanisms that contribute to this excessive secretion of hormones are still elusive. A key event in secretion is the exit of secretory products from the Golgi apparatus. ADP-ribosylation factor (Arf) GTPases are known to control vesicle budding and trafficking, and have a leading function in the regulation of formation of secretory granula at the Golgi. Here, we show that Arf1 is the predominant Arf protein family member expressed in the neuroendocrine pancreatic tumour cell lines BON and QGP-1. In BON cells Arf1 colocalizes with Golgi markers as well as chromogranin A, and shows significant basal activity. The inhibition of Arf1 activity or expression significantly impaired secretion of chromogranin A. Furthermore, we show that the insulin-like growth factor 1 (IGF-1), a major regulator of growth and secretion in BON cells, induces Arf1 activity. We found that activation of Arf1 upon IGF-1 receptor stimulation is mediated by MEK/ERK signalling pathway in BON and QGP-1 cells. Moreover, the activity of Arf1 in BON cells is mediated by autocrinely secreted IGF-1, and concomitantly, autocrine IGF1 secretion is maintained by Arf1 activity. In summary, our data indicate an important regulatory role for Arf1 at the Golgi in hypersecretion in neuroendocrine cancer cells.

The phosphatase of regenerating liver 3 (PRL-3) promotes cell migration through Arf-activity-dependent stimulation of integrin α5 recycling.

The formation of metastasis is one of the most critical problems in oncology. The phosphatase of regenerating liver 3 (PRL-3) is a new target in colorectal cancer, mediating metastatic behavior through a promigratory function. However, detailed explanations for this effect have remained elusive. Here we show that PRL-3 interacts with the ADP-ribosylation factor 1 (Arf1). PRL-3 colocalizes with Arf1 in an endosomal compartment and associates with transmembrane proteins such as the transferrin receptor and α5 integrins. PRL-3 interacts with Arf1 through a distinct motif and regulates activation of Arf1. PRL-3-mediated migration depends on expression and activation of Arf1 and is sensitive to treatment with Brefeldin A. We also demonstrate that PRL-3 modulates recycling of α5 integrins and that its phosphatase activity as well as Arf activation and compartmentalization with Arf1 are required for this effect. In summary our data identify a new function for PRL-3 and show that Arf1 is a new PRL-3-dependent mediator of enhanced migration of cancer cells through enhanced recycling of matrix receptors.

Correction: Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions.

Correction for 'Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions' by Marine Verhulsel et al., Lab Chip, 2021, 21, 365-377, https://doi.org/10.1039/d0lc00672f.

Targeted Transgenic Mice Using CRISPR /Cas9 Technology.

CRISPR /Cas9 is a powerful technology that has transformed gene editing of mammalian genomes, being faster and more cost-effective than standard gene targeting techniques. In this chapter, we provide a step-by-step protocol to obtain Knock-Out (KO ) or Knock-In (KI ) mouse models using CRISPR /Cas9 technology. Detailed instructions for the design of single guide RNAs (sgRNA ) for KO approaches and single-strand oligonucleotide (ssODN ) matrix for generation of KI animals are included. We also describe two independent CRISPR /Cas9 delivery methods to produce gene-edited animals starting from zygote-stage embryos, based either on cytoplasmic injection or electroporation.

Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions.

Organoids are widely used as a model system to study gut pathophysiology; however, they fail to fully reproduce the complex, multi-component structure of the intestinal wall. We present here a new gut on chip model that allows the co-culture of primary epithelial and stromal cells. The device has the topography and dimensions of the mouse gut and is based on a 3D collagen I scaffold. The scaffold is coated with a thin layer of laminin to mimic the basement membrane. To maintain the scaffold structure while preserving its cytocompatibility, the collagen scaffold was rigidified by threose-based post-polymerization treatment. This treatment being cytocompatible enabled the incorporation of primary intestinal fibroblasts inside the scaffold, reproducing the gut stromal compartment. We observed that mouse organoids, when deposited into crypts, opened up and epithelialized the scaffold, generating a polarized epithelial monolayer. Proper segregation of dividing and differentiated cells along the crypt-villus axis was achieved under these conditions. Finally, we show that the application of fluid shear stress allows the long-term culture of this intestinal epithelium. Our device represents a new biomimetic tool that captures key features of the gut complexity and could be used to study gut pathophysiology.

Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration.

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium.

Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system.

BACKGROUND: Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions. RESULTS: We have quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to mis-calculation of migration rates of up to 410%. In order to provide for objective measurements of cell migration rates, we have employed multi-target tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high throughput screening of video sequences of unstained living cells. CONCLUSION: We demonstrate that our automatic multi target tracking system identifies cell objects, follows individual cells and computes migration rates with high precision, clearly outperforming manual procedures.

Tumor biology and cancer therapy - an evolving relationship.

The aim of palliative chemotherapy is to increase survival whilst maintaining maximum quality of life for the individual concerned. Although we are still continuing to explore the optimum use of traditional chemotherapy agents, the introduction of targeted therapies has significantly broadened the therapeutic options. Interestingly, the results from current trials put the underlying biological concept often into a new, less favorable perspective. Recent data suggested that altered pathways underlie cancer, and not just altered genes. Thus, an effective therapeutic agent will sometimes have to target downstream parts of a signaling pathway or physiological effects rather than individual genes. In addition, over the past few years increasing evidence has suggested that solid tumors represent a very heterogeneous group of cells with different susceptibility to cancer therapy. Thus, since therapeutic concepts and pathophysiological understanding are continuously evolving a combination of current concepts in tumor therapy and tumor biology is needed. This review aims to present current problems of cancer therapy by highlighting exemplary results from recent clinical trials with colorectal and pancreatic cancer patients and to discuss the current understanding of the underlying reasons.

Protein kinase D2 regulates chromogranin A secretion in human BON neuroendocrine tumour cells.

Chromogranin A is a member of the granin family of acidic secretory glycoproteins that is found in secretory granules of many endocrine cells including neuroendocrine tumour cells. This hormone serves as a model system for autonomous hormone secretion by the so called functional neuroendocrine tumours of the gastrointestinal tract. The precise regulation of chromogranin secretion at the level of the Golgi apparatus is a subject of intense research. The protein kinase D (PKD) family of serine threonine kinases has so far been implicated in the regulation of constitutive secretion in epithelial cells. Here we examined whether PKD2 expression and activity could also play a role in the release of secretory granules from the trans Golgi network (TGN) in neuroendocrine tumour cells and hence be a target to block autonomous secretion by these tumours. Our data show that expression and catalytic activity of PKD2 are required for the release of chromogranin A containing secretory vesicles. Inhibition of PKD2 activity or siRNA knockdown of PKD2 resulted in a marked perinuclear retention of chromogranin A immunofluorescence in the trans Golgi network and led to a marked reduction in basal as well as phorbol ester stimulated secretion of chromogranin A into the supernatant of cells. Thus, PKD2 controls the release of secretory granules in neuroendocrine tumour cells at the level of the Golgi apparatus and could hence serve as a novel target to block hormone secretion in functional neuroendocrine tumours.

Active cell migration is critical for steady-state epithelial turnover in the gut.

Steady-state turnover is a hallmark of epithelial tissues throughout adult life. Intestinal epithelial turnover is marked by continuous cell migration, which is assumed to be driven by mitotic pressure from the crypts. However, the balance of forces in renewal remains ill-defined. Combining biophysical modeling and quantitative three-dimensional tissue imaging with genetic and physical manipulations, we revealed the existence of an actin-related protein 2/3 complex-dependent active migratory force, which explains quantitatively the profiles of cell speed, density, and tissue tension along the villi. Cells migrate collectively with minimal rearrangements while displaying dual-apicobasal and front-back-polarity characterized by actin-rich basal protrusions oriented in the direction of migration. We propose that active migration is a critical component of gut epithelial turnover.

Focal adhesion kinase mediates defects in the force-dependent reinforcement of initial integrin-cytoskeleton linkages in metastatic colon cancer cell lines.

Micro-environmental clues, including the biophysical interpretation of the extracellular matrix, are critical to proliferation, apoptosis and migration. Here, we show that metastatic human colon cancer cell lines display altered matrix interaction. Interaction of colon cancer cells with collagen I depends on integrins (mainly alpha(1)/beta(1)) but metastatic cells display delayed spreading and reduced extension of lamellipodia. In addition, cells show defective strengthening of integrin-cytoskeleton linkages upon mechanical stimulation, as determined by laser trapping experiments and binding of large beads to the cell surface. However, adhesion to pliable surfaces is ameliorated in metastatic variants. These changes are caused by constitutive activation of focal adhesion kinase (FAK) and can be modulated by changing expression and/or activity of FAK via RNA-interference or expression of inhibitory constructs, respectively. In addition, consistent with defective strengthening of integrin-cytoskeleton linkages, metastatic cell lines show reduced random motility. Taken together these data suggest that constitutive activation of FAK causes defects in spreading, reinforcement of integrin-cytoskeleton linkages and migration and at the same time could ameliorate the adhesion of metastatic cells to suboptimal surfaces.

Cell Migration in Tissues: Explant Culture and Live Imaging.

Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.

Phosphorylation and turnover of paxillin in focal contacts is controlled by force and defines the dynamic state of the adhesion site.

Micro-environmental clues are critical to cell behavior. One of the key elements of migration is the generation and response to forces. Up to now there is no definitive concept on how the generation and responses to cellular forces influence cell behavior. Here, we show that phosphorylation of paxillin is a crucial event in the response to exogenous forces. Application of force induced growth of adhesion sites and this phenomenon was accompanied by a downregulation of Src family kinase activity, which in turn led to a decrease in the phosphorylation of paxillin at the tyrosine residues Y31 and Y118. The force-dependent growth of adhesion sites is mediated by a decrease in the turnover-rate of paxillin in focal contacts. This turnover critically depended on the phosphorylation state of paxillin at Y31/118. Paxillin is an important regulator in the control of the aggregate state of the whole adhesion site since the turnover of other adhesion site proteins such as vinculin is influenced by the phosphorylation state of paxillin as well. Taken together these data suggest that SFK dependent phosphorylation of paxillin is a crucial event in the regulation of adhesion site function in response to force.

Plug-and-play pairing via defined divalent streptavidins.

Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4Å resolution and trans-divalent streptavidin to 1.6Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.

Hydroxy-terminated conjugated polymer nanoparticles have near-unity bright fraction and reveal cholesterol-dependence of IGF1R nanodomains.

Fluorescent nanoparticles have enabled many discoveries regarding how molecular machines function. Quantum dots have been the dominant class of fluorescent nanoparticles but suffer from blinking and from a substantial dark fraction--particles where the fluorescence is never seen--complicating any analysis of biological function. Nanoparticles composed of conjugated fluorescent polymers (Pdots) have recently been shown to have high brightness and no blinking. Here we develop a robust and efficient means to measure the dark fraction of Pdots, conjugating Atto dyes to the nanoparticles and testing fluorescence colocalization of dye and Pdot puncta. This established that the Pdots we generated had minimal dark fraction: ∼3%. The application of nanoparticles in biological environments is highly sensitive to surface functionalization. For Pdots we found that passivation with uncharged hydroxy-terminated polyethylene glycol caused a dramatic reduction in nonspecific cell binding and aggregation compared to a charged coating. Using carbonyl di-imidazole the hydroxy-Pdots were functionalized efficiently with streptavidin for high stability targeting, allowing specific labeling of mammalian cells. Type I insulin-like growth factor receptor (IGF1R) regulates cell survival and development, with roles in aging, heart disease, and cancer. We used hydroxy-Pdots to track the dynamics of IGF1R on a breast cancer cell-line, determining the diffusion characteristics and showing cholesterol-containing membrane nanodomains were important for receptor mobility at the plasma membrane. The near-unity bright fraction and low nonspecific binding of hydroxy-Pdots, combined with Pdot photostability and lack of blinking, provides many advantages for investigations at the single molecule level.

Role of the second cysteine-rich domain and Pro275 in protein kinase D2 interaction with ADP-ribosylation factor 1, trans-Golgi network recruitment, and protein transport.

Protein kinase D (PKD) isoenzymes regulate the formation of transport carriers from the trans-Golgi network (TGN) that are en route to the plasma membrane. The PKD C1a domain is required for the localization of PKDs at the TGN. However, the precise mechanism of how PKDs are recruited to the TGN is still elusive. Here, we report that ADP-ribosylation factor (ARF1), a small GTPase of the Ras superfamily and a key regulator of secretory traffic, specifically interacts with PKD isoenzymes. ARF1, but not ARF6, binds directly to the second cysteine-rich domain (C1b) of PKD2, and precisely to Pro275 within this domain. Pro275 in PKD2 is not only crucial for the PKD2-ARF1 interaction but also for PKD2 recruitment to and PKD2 function at the TGN, namely, protein transport to the plasma membrane. Our data suggest a novel model in which ARF1 recruits PKD2 to the TGN by binding to Pro275 in its C1b domain followed by anchoring of PKD2 in the TGN membranes via binding of its C1a domain to diacylglycerol. Both processes are critical for PKD2-mediated protein transport.