The emerging roles of the cytoskeleton in intestinal epithelium homeostasis.

The intestinal epithelium must absorb many nutrients and water while forming a barrier that is impermeable to pathogens present in the external environment. Concurrently to fulfill this dual role, the intestinal epithelium is challenged by a rapid renewal of cells and forces resulting from digestion. Hence, intestinal homeostasis requires precise control of tissue integrity, tissue renewal, cell polarity, and force generation/transmission. In this review, we highlight the contribution of the cell cytoskeleton- actin, microtubules, and intermediate filaments- to intestinal epithelium homeostasis. With a focus on enterocytes, we first discuss the role of these networks in the formation and maintenance of cell-cell and cell-matrix junctions. Then, we cover their role in intracellular trafficking related to the apicobasal polarity of enterocytes. Finally, we report on the cytoskeletal changes that occur during tissue renewal. In conclusion, the importance of the cytoskeleton in maintaining intestinal homeostasis is emerging, and we think this field will keep evolving.

Invadopodia enable cooperative invasion and metastasis of breast cancer cells.

Invasive and non-invasive cancer cells can invade together during cooperative invasion. However, the events leading to it, role of the epithelial-mesenchymal transition and the consequences this may have on metastasis are unknown. In this study, we demonstrate that the isogenic 4T1 and 67NR breast cancer cells sort from each other in 3D spheroids, followed by cooperative invasion. By time-lapse microscopy, we show that the invasive 4T1 cells move more persistently compared to non-invasive 67NR, sorting and accumulating at the spheroid-matrix interface, a process dependent on cell-matrix adhesions and independent from E-cadherin cell-cell adhesions. Elimination of invadopodia in 4T1 cells blocks invasion, demonstrating that invadopodia requirement is limited to leader cells. Importantly, we demonstrate that cells with and without invadopodia can also engage in cooperative metastasis in preclinical mouse models. Altogether, our results suggest that a small number of cells with invadopodia can drive the metastasis of heterogeneous cell clusters.

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users' needs.

Frontiers in Intravital Multiphoton Microscopy of Cancer.

Background: Cancer is a highly complex disease which involves the co-operation of tumor cells with multiple types of host cells and the extracellular matrix. Cancer studies which rely solely on static measurements of individual cell types are insufficient to dissect this complexity. In the last two decades, intravital microscopy has established itself as a powerful technique that can significantly improve our understanding of cancer by revealing the dynamic interactions governing cancer initiation, progression and treatment effects, in living animals. This review focuses on intravital multiphoton microscopy (IV-MPM) applications in mouse models of cancer. Recent Findings: IV-MPM studies have already enabled a deeper understanding of the complex events occurring in cancer, at the molecular, cellular and tissue levels. Multiple cells types, present in different tissues, influence cancer cell behavior via activation of distinct signaling pathways. As a result, the boundaries in the field of IV-MPM are continuously being pushed to provide an integrated comprehension of cancer. We propose that optics, informatics and cancer (cell) biology are co-evolving as a new field. We have identified four emerging themes in this new field. First, new microscopy systems and image processing algorithms are enabling the simultaneous identification of multiple interactions between the tumor cells and the components of the tumor microenvironment. Second, techniques from molecular biology are being exploited to visualize subcellular structures and protein activities within individual cells of interest, and relate those to phenotypic decisions, opening the door for "in vivo cell biology". Third, combining IV-MPM with additional imaging modalities, or omics studies, holds promise for linking the cell phenotype to its genotype, metabolic state or tissue location. Finally, the clinical use of IV-MPM for analyzing efficacy of anti-cancer treatments is steadily growing, suggesting a future role of IV-MPM for personalized medicine. Conclusion: IV-MPM has revolutionized visualization of tumor-microenvironment interactions in real time. Moving forward, incorporation of novel optics, automated image processing, and omics technologies, in the study of cancer biology, will not only advance our understanding of the underlying complexities but will also leverage the unique aspects of IV-MPM for clinical use.

Invadopodia-mediated ECM degradation is enhanced in the G1 phase of the cell cycle.

The process of tumor cell invasion and metastasis includes assembly of invadopodia, protrusions capable of degrading the extracellular matrix (ECM). The effect of cell cycle progression on invadopodia has not been elucidated. In this study, by using invadopodia and cell cycle fluorescent markers, we show in 2D and 3D cultures, as well as in vivo, that breast carcinoma cells assemble invadopodia and invade into the surrounding ECM preferentially during the G1 phase. The expression (MT1-MMP, also known as MMP14, and cortactin) and localization (Tks5; also known as SH3PXD2A) of invadopodia components are elevated in G1 phase, and cells synchronized in G1 phase exhibit significantly higher ECM degradation compared to the cells synchronized in S phase. The cyclin-dependent kinase inhibitor (CKI) p27kip1 (also known as CDKN1B) localizes to the sites of invadopodia assembly. Overexpression and stable knockdown of p27kip1 lead to contrasting effects on invadopodia turnover and ECM degradation. Taken together, these findings suggest that expression of invadopodia components, as well as invadopodia function, are linked to cell cycle progression, and that invadopodia are controlled by cell cycle regulators. Our results caution that this coordination between invasion and cell cycle must be considered when designing effective chemotherapies.

Intravital Imaging of Tumor Cell Motility in the Tumor Microenvironment Context.

Cancer cell motility and invasion are key features of metastatic tumors. Both are highly linked to tumor microenvironmental parameters, such as collagen architecture or macrophage density. However, due to the genetic, epigenetic and microenvironmental heterogeneities, only a small portion of tumor cells in the primary tumor are motile and furthermore, only a small portion of those will metastasize. This creates a challenge in predicting metastatic fate of single cells based on the phenotype they exhibit in the primary tumor. To overcome this challenge, tumor cell subpopulations need to be monitored at several timescales, mapping their phenotype in primary tumor as well as their potential homing to the secondary tumor site. Additionally, to address the spatial heterogeneity of the tumor microenvironment and how it relates to tumor cell phenotypes, large numbers of images need to be obtained from the same tumor. Finally, as the microenvironment complexity results in nonlinear relationships between tumor cell phenotype and its surroundings, advanced statistical models are required to interpret the imaging data. Toward improving our understanding of the relationship between cancer cell motility, the tumor microenvironment context and successful metastasis, we have developed several intravital approaches for continuous and longitudinal imaging, as well as data classification via support vector machine (SVM) algorithm. We also describe methods that extend the capabilities of intravital imaging by postsacrificial microscopy of the lung as well as correlative immunofluorescence in the primary tumor.

EP4 receptor promotes invadopodia and invasion in human breast cancer.

The production of Prostaglandin E2 (PGE2) is elevated in human breast cancer cells. The abnormal expression of COX-2, which is involved in the synthesis of PGE2, was recently reported as a critical determinant for invasiveness of human breast cancer cells. Autocrine and paracrine PGE2-mediated stimulation of the PGE2 receptor EP4 transduces multiple signaling pathways leading to diverse patho-physiological effects, including tumor cell invasion and metastasis. It is known that PGE2-induced EP4 activation can transactivate the intracellular signaling pathway of the epidermal growth factor receptor (EGFR). In malignant cancer cells, EGFR pathway activation promotes invadopodia protrusions, which further leads to degradation of the surrounding extracellular matrix (ECM). Despite the known influence of EP4 on the EGFR signaling pathway, the effect of EP4 stimulation on invadopodia formation in human breast cancer was never tested directly. Here we demonstrate the involvement of EP4 in invasion and its effect on invadopodia in breast cancer MDA-MB-231 cells using 2D invadopodia and 3D invasion in vitro assays as well as intravital microscopy. The results show that stimulation with the selective EP4 agonist CAY10598 or PGE2 promotes invadopodia-mediated degradation of the ECM, as well as the invasion of breast cancer cells in in vitro models. The effect on matrix degradation can be abrogated via direct inhibition of EP4 signaling as well as via inhibition of EGFR tyrosine kinase, indicating that EP4-mediated effects on invadopodia-driven degradation are EGFR dependent. Finally, using xenograft mouse models, we show that short-term systemic treatment with CAY10598 results in a >9-fold increase in the number of invadopodia. These findings highlight the importance of further investigation on the role of EP4-EGFR crosstalk in invadopodia formation.

SIRT1 Overexpression Maintains Cell Phenotype and Function of Endothelial Cells Derived from Induced Pluripotent Stem Cells.

Endothelial cells (ECs) that are differentiated from induced pluripotent stem cells (iPSCs) can be used in establishing disease models for personalized drug discovery or developing patient-specific vascularized tissues or organoids. However, a number of technical challenges are often associated with iPSC-ECs in culture, including instability of the endothelial phenotype and limited cell proliferative capacity over time. Early senescence is believed to be the primary mechanism underlying these limitations. Sirtuin1 (SIRT1) is an NAD(+)-dependent deacetylase involved in the regulation of cell senescence, redox state, and inflammatory status. We hypothesize that overexpression of the SIRT1 gene in iPSC-ECs will maintain EC phenotype, function, and proliferative capacity by overcoming early cell senescence. SIRT1 gene was packaged into a lentiviral vector (LV-SIRT1) and transduced into iPSC-ECs at passage 4. Beginning with passage 5, iPSC-ECs exhibited a fibroblast-like morphology, whereas iPSC-ECs overexpressing SIRT1 maintained EC cobblestone morphology. SIRT1 overexpressing iPSC-ECs also exhibited a higher percentage of canonical markers of endothelia (LV-SIRT1 61.8% CD31(+) vs. LV-empty 31.7% CD31(+), P < 0.001; LV-SIRT1 46.3% CD144(+) vs. LV-empty 20.5% CD144(+), P < 0.02), with a higher nitric oxide synthesis, lower ß-galactosidase production indicating decreased senescence (3.4% for LV-SIRT1 vs. 38.6% for LV-empty, P < 0.001), enhanced angiogenesis, increased deacetylation activity, and higher proliferation rate. SIRT1 overexpressing iPSC-ECs continued to proliferate through passage 9 with high purity of EC-like characteristics, while iPSC-ECs without SIRT1 overexpression became senescent after passage 5. Taken together, SIRT1 overexpression in iPSC-ECs maintains EC phenotype, improves EC function, and extends cell lifespan, overcoming critical hurdles associated with the use of iPSC-ECs in translational research.

Enabling non-invasive assessment of an engineered endothelium on ePTFE vascular grafts without increasing oxidative stress.

Magnetic resonance imaging (MRI) in combination with contrast enhancement is a potentially powerful tool to non-invasively monitor cell distribution in tissue engineering and regenerative medicine. The most commonly used contrast agent for cell labeling is super paramagnetic iron oxide nanoparticles (SPIONs). However, uptake of SPIONs triggers the production of reactive oxygen species (ROS) in cells often leading to a pro-inflammatory phenotype. The objective of this study was to develop a labeling system to non-invasively visualize an engineered endothelium in vascular grafts without creating excessive oxidative stress. Specifically, we investigated: (1) chitosan-coated SPIONs (CSPIONs) as an antioxidant contrast agent for contrast enhancement, and (2) poly(1,8-octamethylene citrate) (POC) as an antioxidant interface to support cell adhesion and function of labeled cells on the vascular graft. While SPION-labeled endothelial cells (ECs) experienced elevated ROS formation and altered cell morphology, CSPION-labeled ECs cultured on POC-coated surfaces mitigated SPION-induced ROS formation and maintained EC morphology, phenotype, viability and functions. A monolayer of labeled ECs exhibited sufficient contrast with T2-weighed MR imaging. CSPION labeling of endothelial cells in combination with coating the graft wall with POC allows non-invasive monitoring of an engineered endothelium on ePTFE grafts without increasing oxidative stress.