Organ on Chip Technology to Model Cancer Growth and Metastasis

Organ on chip (OOC) has emerged as a major technological breakthrough and distinct model system revolutionizing biomedical research and drug discovery by recapitulating the crucial structural and functional complexity of human organs in vitro. OOC are rapidly emerging as powerful tools for oncology research. Indeed, Cancer on chip (COC) can ideally reproduce certain key aspects of the tumor microenvironment (TME), such as biochemical gradients and niche factors, dynamic cell–cell and cell–matrix interactions, and complex tissue structures composed of tumor and stromal cells. Here, we review the state of the art in COC models with a focus on the microphysiological systems that host multicellular 3D tissue engineering models and can help elucidate the complex biology of TME and cancer growth and progression. Finally, some examples of microengineered tumor models integrated with multi-organ microdevices to study disease progression in different tissues will be presented.

Impact of Electrospun Piezoelectric Core–Shell PVDFhfp/PDMS Mesh on Tenogenic and Inflammatory Gene Expression in Human Adipose-Derived Stem Cells: Comparison of Static Cultivation with Uniaxial Cyclic Tensile Stretching

Specific microenvironments can trigger stem cell tenogenic differentiation, such as specific substrates or dynamic cell cultivation. Electrospun meshes composed by core–shell fibers (random or aligned; PDMS core; piezoelectric PVDFhfp shell) were fabricated by coaxial electrospinning. Elastic modulus and residual strain were assessed. Human ASCs were seeded on such scaffolds either under static conditions for 1 week or with subsequent 10% dynamic stretching for 10,800 cycles (1 Hz, 3 h), assessing load elongation curves in a Bose® bioreactor system. Gene expression for tenogenic expression, extracellular matrix, remodeling, pro-fibrotic and inflammatory marker genes were assessed (PCR). For cell-seeded meshes, the E modulus increased from 14 ± 3.8 MPa to 31 ± 17 MPa within 3 h, which was not observed for cell-free meshes. Random fibers resulted in higher tenogenic commitment than aligned fibers. Dynamic cultivation significantly enhanced pro-inflammatory markers. Compared to ASCs in culture flasks, ASCs on random meshes under static cultivation showed a significant upregulation of Mohawk, Tenascin-C and Tenomodulin. The tenogenic commitment expressed by human ASCs in contact with random PVDFhfp/PDMS paves the way for using this novel highly elastic material as an implant to be wrapped around a lacerated tendon, envisioned as a functional anti-adhesion membrane.

Unsupervised discovery of dynamic cell phenotypic states from transmitted light movies

Identification of cell phenotypic states within heterogeneous populations, along with elucidation of their switching dynamics, is a central challenge in modern biology. Conventional single-cell analysis methods typically provide only indirect, static phenotypic readouts. Transmitted light images, on the other hand, provide direct morphological readouts and can be acquired over time to provide a rich data source for dynamic cell phenotypic state identification. Here, we describe an end-to-end deep learning platform, UPSIDE (Unsupervised Phenotypic State IDEntification), for discovering cell states and their dynamics from transmitted light movies. UPSIDE uses the variational auto-encoder architecture to learn latent cell representations, which are then clustered for state identification, decoded for feature interpretation, and linked across movie frames for transition rate inference. Using UPSIDE, we identified distinct blood cell types in a heterogeneous dataset. We then analyzed movies of patient-derived acute myeloid leukemia cells, from which we identified stem-cell associated morphological states as well as the transition rates to and from these states. UPSIDE opens up the use of transmitted light movies for systematic exploration of cell state heterogeneity and dynamics in biology and medicine.

Bmp Signal Gradient Modulates Convergent Cell Movement via Xarhgef3.2 during Gastrulation of Xenopus Embryos

Gastrulation is a critical step in the establishment of a basic body plan during development. Convergence and extension (CE) cell movements organize germ layers during gastrulation. Noncanonical Wnt signaling has been known as major signaling that regulates CE cell movement by activating Rho and Rac. In addition, Bmp molecules are expressed in the ventral side of a developing embryo, and the ventral mesoderm region undergoes minimal CE cell movement while the dorsal mesoderm undergoes dynamic cell movements. This suggests that Bmp signal gradient may affect CE cell movement. To investigate whether Bmp signaling negatively regulates CE cell movements, we performed microarray-based screening and found that the transcription of Xenopus Arhgef3.2 (Rho guanine nucleotide exchange factor) was negatively regulated by Bmp signaling. We also showed that overexpression or knockdown of Xarhgef3.2 caused gastrulation defects. Interestingly, Xarhgef3.2 controlled gastrulation cell movements through interacting with Disheveled (Dsh2) and Dsh2-associated activator of morphogenesis 1 (Daam1). Our results suggest that Bmp gradient affects gastrulation cell movement (CE) via negative regulation of Xarhgef3.2 expression.

Dynamic cell cycle-dependent phosphorylation modulates CENP-L-CENP-N centromere recruitment

The kinetochore is a macromolecular structure that is required to ensure proper chromosome segregation during each cell division. The kinetochore is assembled upon a platform of the 16-subunit Constitutive Centromere Associated Network (CCAN), which is present at centromeres throughout the cell cycle. The nature and regulation of CCAN assembly, interactions, and dynamics required to facilitate changing centromere properties and requirements remain to be fully elucidated. The CENP-LN CCAN sub-complex displays a unique cell cycle-dependent localization behavior, peaking in S phase. Here, we demonstrate that phosphorylation of CENP-L and CENP-N controls CENP-LN complex formation and localization in a cell cycle-dependent manner. Mimicking constitutive phosphorylation of either CENP-L or CENP-N or simultaneously preventing phosphorylation of both proteins prevents CENP-LN localization and disrupts chromosome segregation. Together, our work suggests that cycles of phosphorylation and dephosphorylation are critical for CENP-LN complex recruitment and dynamics at centromeres to enable cell cycle-dependent CCAN reorganization.

The Adaptor Protein Lurap1 Is Required for Cell Cohesion during Epiboly Movement in Zebrafish

Cell adhesion and polarized cellular behaviors play critical roles in a wide variety of morphogenetic events. In the zebrafish embryo, epiboly represents an important process of epithelial morphogenesis that involves differential cell adhesion and dynamic cell shape changes for coordinated movements of different cell populations, but the underlying mechanism remains poorly understood. The adaptor protein Lurap1 functions to link myotonic dystrophy kinase-related Rac/Cdc42-binding kinase with MYO18A for actomyosin retrograde flow in cell migration. We previously reported that it interacts with Dishevelled in convergence and extension movements during gastrulation. Here, we show that it regulates blastoderm cell adhesion and radial cell intercalation during epiboly. In zebrafish mutant embryos with loss of both maternal and zygotic Lurap1 function, deep cell multilayer of the blastoderm exhibit delayed epiboly with respect to the superficial layer. Time-lapse imaging reveals that these deep cells undergo unstable intercalation, which impedes their expansion over the yolk cell. Cell sorting and adhesion assays indicate reduced cellular cohesion of the blastoderm. These defects are correlated with disrupted cytoskeletal organization in the cortex of blastoderm cells. Thus, the present results extend our previous works by demonstrating that Lurap1 is required for cell adhesion and cell behavior changes to coordinate cell movements during epithelial morphogenesis. They provide insights for a further understanding of the regulation of cytoskeletal organization during gastrulation cell movements.

Dynamic Cell Imaging: application to the diatom Phaeodactylum tricornutum under environmental stresses

The dynamic movement of cell organelles is an important and poorly understood component of cellular organisation and metabolism. In this work we present a non-invasive non-destructive method (Dynamic Cell Imaging, DCI) based on light scattering and interferometry to monitor dynamic events within photosynthetic cells using the diatom Phaeodactylum tricornutum as a model system. For this monitoring we acquire few seconds movies of the signals that are related to the motion of dynamic structures within the cell (denoted scatterers), followed by a statistical analysis of each pixel time series. Illuminating P.tricornutum with LEDs of different wavelengths associated to short pulsed or continuous-wave modes of illumination revealed that dynamic movements depend on chloroplast activity, in agreement with the reduction in the number of pixels with dynamic behaviour after addition of photosystemII inhibitors. We studied P. tricornutum under two environmentally relevant stresses, iron and phosphate deficiency. The major dynamic sites were located within lipid droplets and chloroplast envelope membranes. By comparing standard deviation and cumulative sum analysis of the time series, we showed that within the droplets two types of scatterer movement could be observed: random motions (Brownian type) but also anomalous movements corresponding to a drift which may relate to molecular fluxes within a cell. The method appears valuable for studying the effects of various environments on a large variety of microalgae in the laboratory as well as in natural aquatic environments.