Spatial heterogeneity in tumor adhesion qualifies collective cell invasion.

Collective cell invasion (CCI), a canon of most invasive solid tumors, is an emergent property of the interactions between cancer cells and their surrounding extracellular matrix (ECM). However, tumor populations invariably consist of cells expressing variable levels of adhesive proteins that mediate such interactions, disallowing an intuitive understanding of how tumor invasiveness at a multicellular scale is influenced by spatial heterogeneity of cell-cell and cell-ECM adhesion. Here, we have used a Cellular Potts model-based multiscale computational framework that is constructed on the histopathological principles of glandular cancers. In earlier efforts on homogenous cancer cell populations, this framework revealed the relative ranges of interactions, including cell-cell and cell-ECM adhesion that drove collective, dispersed, and mixed multimodal invasion. Here, we constitute a tumor core of two separate cell subsets showing distinct intra- and inter-subset cell-cell or cell-ECM adhesion strengths. These two subsets of cells are arranged to varying extents of spatial intermingling, which we call the heterogeneity index (HI). We observe that low and high inter-subset cell adhesion favors invasion of high-HI and low-HI intermingled populations with distinct intra-subset cell-cell adhesion strengths, respectively. In addition, for explored values of cell-ECM adhesion strengths, populations with high HI values collectively invade better than those with lower HI values. We then asked how spatial invasion is regulated by progressively intermingled cellular subsets that are epithelial, i.e., showed high cell-cell but poor cell-ECM adhesion, and mesenchymal, i.e., with reversed adhesion strengths to the former. Here too, inter-subset adhesion plays an important role in contextualizing the proportionate relationship between HI and invasion. An exception to this relationship is seen for cases of heterogeneous cell-ECM adhesion where sub-maximal HI patterns with higher outer localization of cells with stronger ECM adhesion collectively invade better than their relatively higher-HI counterparts. Our simulations also reveal how adhesion heterogeneity qualifies collective invasion, when either cell-cell or cell-ECM adhesion type is varied but results in an invasive dispersion when both adhesion types are simultaneously altered.

An Endosomal Acid-Regulatory Feedback System Rewires Cytosolic cAMP Metabolism and Drives Tumor Progression.

Although suppressed cAMP levels have been linked to cancer for nearly five decades, the molecular basis remains uncertain. Here, we identify endosomal pH as a novel regulator of cytosolic cAMP homeostasis and a promoter of transformed phenotypic traits in colorectal cancer. Combining experiments and computational analysis, we show that the Na+/H+ exchanger NHE9 contributes to proton leak and causes luminal alkalinization, which induces resting [Ca2+], and in consequence, represses cAMP levels, creating a feedback loop that echoes nutrient deprivation or hypoxia. Higher NHE9 expression in cancer epithelia is associated with a hybrid epithelial-mesenchymal (E/M) state, poor prognosis, tumor budding, and invasive growth in vitro and in vivo. These findings point to NHE9-mediated cAMP suppression as a pseudostarvation-induced invasion state and potential therapeutic vulnerability in colorectal cancer. Our observations lay the groundwork for future research into the complexities of endosome-driven metabolic reprogramming and phenotype switching and the biology of cancer progression. IMPLICATIONS: Endosomal pH regulator NHE9 actively controls cytosolic Ca2+ levels to downregulate the adenylate cyclase-cAMP system, enabling colorectal cancer cells to acquire hybrid E/M characteristics and promoting metastatic progression.

The senescent mesothelial matrix accentuates colonization by ovarian cancer cells.

Ovarian cancer is amongst the most morbid of gynecological malignancies due to its diagnosis at an advanced stage, a transcoelomic mode of metastasis, and rapid transition to chemotherapeutic resistance. Like all other malignancies, the progression of ovarian cancer may be interpreted as an emergent outcome of the conflict between metastasizing cancer cells and the natural defense mounted by microenvironmental barriers to such migration. Here, we asked whether senescence in coelom-lining mesothelia, brought about by drug exposure, affects their interaction with disseminated ovarian cancer cells. We observed that cancer cells adhered faster on senescent human and murine mesothelial monolayers than on non-senescent controls. Time-lapse epifluorescence microscopy showed that mesothelial cells were cleared by a host of cancer cells that surrounded the former, even under sub-confluent conditions. A multiscale computational model predicted that such colocalized mesothelial clearance under sub-confluence requires greater adhesion between cancer cells and senescent mesothelia. Consistent with the prediction, we observed that senescent mesothelia expressed an extracellular matrix with higher levels of fibronectin, laminins and hyaluronan than non-senescent controls. On senescent matrix, cancer cells adhered more efficiently, spread better, and moved faster and persistently, aiding the spread of cancer. Inhibition assays using RGD cyclopeptides suggested the adhesion was predominantly contributed by fibronectin and laminin. These findings led us to propose that the senescence-associated matrisomal phenotype of peritoneal barriers enhances the colonization of invading ovarian cancer cells contributing to the metastatic burden associated with the disease.

Targeting metabolic fluxes reverts metastatic transitions in ovarian cancer.

The formation of spheroids during epithelial ovarian cancer progression is correlated with peritoneal metastasis, disease recurrence, and poor prognosis. Although metastasis has been demonstrated to be driven by metabolic changes in transformed cells, mechanistic associations between metabolism and phenotypic transitions remain ill-explored. We performed quantitative proteomics to identify protein signatures associated with three distinct phenotypic morphologies (2D monolayers and two geometrically distinct three-dimensional spheroidal states) of the high-grade serous ovarian cancer line OVCAR-3. We obtained disease-driving phenotype-specific metabolic reaction modules and elucidated gene knockout strategies to reduce metabolic alterations that could drive phenotypic transitions. Exploring the DrugBank database, we identified and evaluated drugs that could impair such transitions and, hence, cancer progression. Finally, we experimentally validated our predictions by confirming the ability of one of our predicted drugs, the neuraminidase inhibitor oseltamivir, to inhibit spheroidogenesis in three ovarian cancer cell lines without any cytotoxic effects on untransformed stromal mesothelia.

Tunable encapsulation of sessile droplets with solid and liquid shells.

Droplet encapsulations using liquid or solid shells are of significant interest in microreactors, drug delivery, crystallization, and cell growth applications. Despite progress in droplet-related technologies, tuning micron-scale shell thickness over a large range of droplet sizes is still a major challenge. In this work, we report capillary force assisted cloaking using hydrophobic colloidal particles and liquid-infused surfaces. The technique produces uniform solid and liquid shell encapsulations over a broad range (5-200 µm shell thickness for droplet volume spanning over four orders of magnitude). Tunable liquid encapsulation is shown to reduce the evaporation rate of droplets by up to 200 times with a wide tunability in lifetime (1.5 h to 12 days). Further, we propose using the technique for single crystals and cell/spheroid culture platforms. Stimuli-responsive solid shells show hermetic encapsulation with tunable strength and dissolution time. Moreover, scalability, and versatility of the technique is demonstrated for on-chip applications.

Jammed microgel growth medium prepared by flash-solidification of agarose for 3D cell culture and 3D bioprinting.

Although cells cultured in three-dimensional (3D) platforms are proven to be beneficial for studying cellular behavior in settings similar to their physiological state, due to the ease, convenience, and accessibility, traditional 2D culturing approaches are widely adopted. Jammed microgels are a promising class of biomaterials extensively suited for 3D cell culture, tissue bioengineering, and 3D bioprinting. However, existing protocols for fabricating such microgels either involve complex synthesis steps, long preparation times, or polyelectrolyte hydrogel formulations that sequester ionic elements from the cell growth media. Hence, there is an unmet need for a broadly biocompatible, high-throughput, and easily accessible manufacturing process. We address these demands by introducing a rapid, high-throughput, and remarkably straightforward method to synthesize jammed microgels composed of flash-solidified agarose granules directly prepared in a culture medium of choice. Our jammed growth media are optically transparent, porous, yield stress materials with tunable stiffness and self-healing properties, which makes them ideal for 3D cell culture as well as 3D bioprinting. The charge-neutral and inert nature of agarose make them suitable for culturing various cell types and species, the specific growth media for which do not alter the chemistry of the manufacturing process. Unlike several existing 3D platforms, these microgels are readily compatible with standard techniques such as absorbance-based growth assays, antibiotic selection, RNA extraction, and live cell encapsulation. In effect, we present a versatile, highly accessible, inexpensive, and easily adoptable biomaterial for 3D cell culture and 3D bioprinting. We envision their widespread application not just in routine laboratory settings but also in designing multicellular tissue mimics and dynamic co-culture models of physiological niches.

How safe are magnetic nanomotors: From cells to animals.

Helical magnetic nanomotors can be actuated using an external magnetic field and have potential applications in drug delivery, colloidal manipulation, and bio-microrheology. Recently, they have been maneuvered in biological environments such as vitreous humour, dentinal tubules, peritoneal fluid, stromal matrix, and blood, which are promising developments for clinical applications. However, their biocompatibility and biodistribution are vital parameters that must be assessed before further use. An extensive quantitative evaluation has been performed for these parameters for the first time through in vitro and in vivo experiments. Investigations of cell death, proliferation, and DNA damage ascertain that the motors are non-toxic. Also, an unbiased transcriptomic analysis affirms that the motors are not genotoxic till 20 motors/ cell. Toxicity studies in mice reveal that the motors show no signs of toxicity up to a dose of 55 mg/ kg body weight. Further, the biodistribution studies show that they remain in the blood circulation after injection and at later stages possibly adhere to the walls of the blood vessel because of adsorption. However, perfusion with physiological saline decreases this adsorption/adhesion. Overall, we demonstrate the biocompatibility of nanomotors in live cellular and organismal systems, and a systemic biodistribution analysis reveals organ-specific retention of motors.

Extracellular matrix as a driver for intratumoral heterogeneity.

The architecture of an organ is built through interactions between its native cells and its connective tissue consisting of stromal cells and the extracellular matrix (ECM). Upon transformation through tumorigenesis, such interactions are disrupted and replaced by a new set of intercommunications between malignantly transformed parenchyma, an altered stromal cell population, and a remodeled ECM. In this perspective, we propose that the intratumoral heterogeneity of cancer cell phenotypes is an emergent property of such reciprocal intercommunications, both biochemical and mechanical-physical, which engender and amplify the diversity of cell behavioral traits. An attempt to assimilate such findings within a framework of phenotypic plasticity furthers our understanding of cancer progression.

Galectin-9 Signaling Drives Breast Cancer Invasion through Extracellular Matrix.

Aberrations in glycan and lectin expression and function represent one of the earliest hallmarks of cancer. Among galectins, a conserved family of ß-galactoside-binding lectins, the role of Galectin-9 in immune-tumor interactions is well-established, although its effect on cancer cell behavior remains unclear. In this study, we assayed for, and observed, an association between Galectin-9 expression and invasiveness of breast cancer cells in vitro and in vivo. Genetic perturbation and pharmacological inhibition using novel cognate inhibitors confirmed a positive correlation between Galectin-9 levels and the adhesion of invasive cancer cells to─and their invasion through─constituted organomimetic extracellular matrix microenvironments. Signaling experiments and unbiased quantitative proteomics revealed Galectin-9 induction of Focal Adhesion Kinase activity and S100A4 expression, respectively. FAK inhibition decreased S100A4 mRNA levels. Our results provide crucial insights into how elevated Galectin-9 expression potentiates the invasiveness of breast cancer cells during early steps of invasion.

Spatial waves and temporal oscillations in vertebrate limb development.

The mesenchymal tissue of the developing vertebrate limb bud is an excitable medium that sustains both spatial and temporal periodic phenomena. The first of these is the outcome of general Turing-type reaction-diffusion dynamics that generate spatial standing waves of cell condensations. These condensations are transformed into the nodules and rods of the cartilaginous, and eventually (in most species) the bony, endoskeleton. In the second, temporal periodicity results from intracellular regulatory dynamics that generate oscillations in the expression of one or more gene whose products modulate the spatial patterning system. Here we review experimental evidence from the chicken embryo, interpreted by a set of mathematical and computational models, that the spatial wave-forming system is based on two glycan-binding proteins, galectin-1A and galectin-8 in interaction with each other and the cells that produce them, and that the temporal oscillation occurs in the expression of the transcriptional coregulator Hes1. The multicellular synchronization of the Hes1 oscillation across the limb bud serves to coordinate the biochemical states of the mesenchymal cells globally, thereby refining and sharpening the spatial pattern. Significantly, the wave-forming reaction-diffusion-based mechanism itself, unlike most Turing-type systems, does not contain an oscillatory core, and may have evolved to this condition as it came to incorporate the cell-matrix adhesion module that enabled its pattern-forming capability.

Extracellular matrix mediates moruloid-blastuloid morphodynamics in malignant ovarian spheroids.

Ovarian cancer metastasizes into peritoneum through dissemination of transformed epithelia as multicellular spheroids. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues: lumen surrounded by smoothly contoured and adhered epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid "moruloid" clusters that permit intercellular movement, cell penetration, and interspheroidal coalescence. Moruloid clusters subsequently mature into "blastuloid" spheroids with smooth contours, a temporally dynamic lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow cell penetration. Ultrastructural examination reveals a basement membrane-like extracellular matrix coat on the surface of blastuloid, but not moruloid, spheroids. Quantitative proteomics reveals down-regulation in ECM protein Fibronectin-1 associated with the moruloid-blastuloid transition; immunocytochemistry also confirms the relocalization of basement membrane ECM proteins: collagen IV and laminin to the surface of blastuloid spheroids. Fibronectin depletion accelerates, and enzymatic basement membrane debridement impairs, lumen formation, respectively. The regulation by ECM dynamics of the morphogenesis of cancer spheroids potentially influences the progression of the disease.

Theragnostic nanomotors: Successes and upcoming challenges.

The idea of "fantastic voyagers" carrying out medical tasks within the human body has existed as part of popular culture for many decades. The concept revolved around a miniaturized robot that can travel inside the human body and perform complicated functions such as surgery, navigation of otherwise inaccessible biological environments, and delivery of therapeutics. Since the last decade, significant developments have occurred in this arena that are yet to enter mainstream biomedical practises. Here, we define the challenges to make this fiction into reality. We begin by chalking the journey from pills, nanoparticles, and then to micro-nanomotors. The review describes the principles, physicochemical contexts, and advantages that micro-nanomotors provide. The article then describes micro-nanomotors' obstacles such as maneuverability, in vivo imaging, toxicity, and biodistribution. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

An interplay of resource availability, population size and mutation rate potentiates the evolution of metabolic signaling.

BACKGROUND: Asexually reproducing populations of single cells evolve through mutation, natural selection, and genetic drift. Environmental conditions in which the evolution takes place define the emergent fitness landscapes. In this work, we used Avida-a digital evolution framework-to uncover a hitherto unexplored interaction between mutation rates, population size, and the relative abundance of metabolizable resources, and its effect on evolutionary outcomes in small populations of digital organisms. RESULTS: Over each simulation, the population evolved to one of several states, each associated with a single dominant phenotype with its associated fitness and genotype. For a low mutation rate, acquisition of fitness by organisms was accompanied with, and dependent on, an increase in rate of genomic replication. At an increased mutation rate, phenotypes with high fitness values were similarly achieved through enhanced genome replication rates. In addition, we also observed the frequent emergence of suboptimal fitness phenotype, wherein neighboring organisms signaled to each other information relevant to performing metabolic tasks. This metabolic signaling was vital to fitness acquisition and was correlated with greater genotypic and phenotypic heterogeneity in the population. The frequency of appearance of signaling populations increased with population size and with resource abundance. CONCLUSIONS: Our results reveal a minimal set of environment-genotype interactions that lead to the emergence of metabolic signaling within evolving populations.

Vortex chip incorporating an orthogonal turn for size-based isolation of circulating cells.

Size-based label-free separation of rare cells such as CTCs is attractive due to its wider applicability, simpler sample preparation, faster turnaround and better efficiency. Amongst such methods, vortex-trapping based techniques offer high throughput but operate at high flow velocities where the resulting hydrodynamic shear stress is likely to damage cells and compromise their viability for subsequent assays. We present here an orthogonal vortex chip which can carry out size-differentiated trapping at significantly lower (38% of previously reported) velocities. Composed of entry-exit channels that couple orthogonally to a trapping chamber, fluid flow in such configuration results in formation of a vortex which selectively traps larger particles above a critical velocity while smaller particles get ejected with the flow. We call this phenomenon the turn-effect. Critical velocities and optimal architectures for trapping of cells and particles of different sizes are characterized. We explain how shear-gradient lift, centrifugal and Dean flow drag forces contribute to the turn-effect by pushing particles into specific vortex orbits in a size- and velocity-dependent fashion. Selective trapping of human breast cancer cells mixed with whole blood at low concentration is demonstrated. The device shows promising results for gentle isolation of rare cells from blood.

A biphasic response of polymerized Type 1 collagen architectures to dermatan sulfate.

Collagen I, the most abundant extracellular matrix (ECM) protein in vertebrate tissues provides mechanical durability to tissue microenvironments and regulates cell function. Its fibrillogenesis in biological milieu is predominantly regulated by dermatan sulfate proteoglycans, proteins conjugated with iduronic acid-containing dermatan sulfate (DS) glycosaminoglycans (GAG). Although DS is known to regulate tissue function through its modulation of Coll I architecture, a precise understanding of the latter remains elusive. We investigated this problem by visualizing the fibrillar pattern of fixed Coll I gels polymerized in the presence of varying concentrations of DS using second harmonic generation microscopy. Measuring mean second harmonic generation signal (which estimates the ordering of the fibrils), and surface occupancy (which estimates the space occupied by fibrils) supported by confocal reflectance microscopy, our observations indicated that the effect on fibril pattern of DS is contextual upon the latter's concentrations: Lower levels of DS resulted in sparse disorganized fibrils; higher levels restore organization, with fibrils occupying greater space. An appropriate change in elasticity as a result of DS levels was also observed through atomic force microscopy. Examination of dye-based GAG staining and scanning electron microscopy suggested distinct constitutions of Coll I gels when polymerized with higher and lower levels of DS. We observed that adhesion of the invasive ovarian cancer cells SKOV3 decreased for lower DS levels but was partially restored at higher DS levels. Our study shows how the Coll I gel pattern-tuning of DS is of relevance for understanding its biomaterial applications and possibly, pathophysiological functions.

Heterogeneity in 2,6-Linked Sialic Acids Potentiates Invasion of Breast Cancer Epithelia.

Heterogeneity in phenotypes of malignantly transformed cells and aberrant glycan expression on their surface are two prominent hallmarks of cancers that have hitherto not been linked to each other. In this paper, we identify differential levels of a specific glycan linkage: α2,6-linked sialic acids within breast cancer cells in vivo and in culture. Upon sorting out two populations with moderate, and relatively higher, cell surface α2,6-linked sialic acid levels from the triple-negative breast cancer cell line MDA-MB-231, both populations (denoted as medium and high 2,6-Sial cells, respectively) stably retained their levels in early passages. Upon continuous culturing, medium 2,6-Sial cells recapitulated the heterogeneity of the unsorted line whereas high 2,6-Sial cells showed no such tendency. Compared with high 2,6-Sial cells, the medium 2,6-Sial counterparts showed greater adhesion to reconstituted extracellular matrices (ECMs) and invaded faster as single cells. The level of α2,6-linked sialic acids in the two sublines was found to be consistent with the expression of a specific glycosyl transferase, ST6GAL1. Stably knocking down ST6GAL1 in the high 2,6-Sial cells enhanced their invasiveness. When cultured together, medium 2,6-Sial cells differentially migrated to the edge of growing tumoroid-like cocultures, whereas high 2,6-Sial cells formed the central bulk. Multiscale simulations in a Cellular Potts model-based computational environment calibrated to our experimental findings suggest that differential levels of cell-ECM adhesion, likely regulated by α2,6-linked sialic acids, facilitate niches of highly invasive cells to efficiently migrate centrifugally as the invasive front of a malignant breast tumor.

Nanomotors Sense Local Physicochemical Heterogeneities in Tumor Microenvironments*.

The invasion of cancer is brought about by continuous interaction of malignant cells with their surrounding tissue microenvironment. Investigating the remodeling of local extracellular matrix (ECM) by invading cells can thus provide fundamental insights into the dynamics of cancer progression. In this paper, we use an active untethered nanomechanical tool, realized as magnetically driven nanomotors, to locally probe a 3D tissue culture environment. We observed that nanomotors preferentially adhere to the cancer-proximal ECM and magnitude of the adhesive force increased with cell lines of higher metastatic ability. We experimentally confirmed that sialic acid linkage specific to cancer-secreted ECM makes it differently charged, which causes this adhesion. In an assay consisting of both cancerous and non-cancerous epithelia, that mimics the in vivo histopathological milieu of a malignant breast tumor, we find that nanomotors preferentially decorate the region around the cancer cells.

Author Correction: Mutually exclusive locales for N-linked glycans and disorder in human glycoproteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.