Biology subjects

Bhat, R.

Publications and source records attributed to Bhat, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Dicarbonyl stress debilitates mesothelial defense against metastasizing ovarian cancer

Chronic metabolic disorders and aging result in accumulation of active dicarbonyls that glycate biomolecules rendering them dysfunctional. Although metabolic aberrations are known to be epidemiologically associated with faster cancer progression, cell biological determinants of such associations remain elusive. The formation of micro-metastases in epithelial ovarian cancer involves its colonization of visceral peritonea through clearance of mesothelia that line the coelom. In this study, we observe that cocultures of immortalized human coelomic MeT-5A mesothelia with human ovarian cancer cells OVCAR-3 and SK-OV-3 show greater infiltration by the latter when exposed to increasing concentrations of the dicarbonyl methylglyoxal (MG). Treatment with increasing concentrations of MG caused death and senescence within human and murine serosal mesothelia. Cells showed higher levels of advanced glycation end products, dysregulated occludens junction protein ZO-1, and disrupted localization of cortical filamentous actin and its regulator ezrin, indicating poor inter-cell adhesion. Time lapse imaging also showed impaired migration for MG-treated single mesothelia and for their collective monolayers. Agent-based computer modeling of coculture dynamics predicted that a combined effect of confluence and migration allows inter-adherent mesothelia to contain the spread of colonizing cancer cells, which was confirmed through coculture time lapses of cancer colonization within higher and lower mesothelial densities. We found ovarian cancer cells showed higher levels of glyoxalase-1 (GLO-1) enzyme, which catabolizes MG, suggesting how they escaped its cytotoxic effects. Consistent with this, treatment of OVCAR-3 with MG concurrently with pharmacological inhibition of GLO-1 showed greater cell death. Our results suggest dicarbonyl stress helps colonizing cancer cells overcome the resistance of natural homoeostatic barriers and its inhibition may, in supplementation with chemotherapy, stem metastasis.

cancer biology↗

bioRxiv2024

The ER Thioredoxin-Related Transmembrane Protein TMX2 Controls Redox-Mediated Tethering of ER-Mitochondria Contacts (ERMCS)

Thioredoxin-related transmembrane proteins (TMX) of the endoplasmic reticulum (ER) have emerged as key regulators of ER membrane properties. Within the ER lumen, TMX proteins and other ER redox enzymes determine oxidative conditions, which control the formation of ER-mitochondria membrane contacts (ERMCS) and determine their function. ERMCS exhibit cytoplasmic redox nanodomains, derived from ER and mitochondrial reactive oxygen species (ROS), whose mechanistic regulation is uncharacterized. Our research has identified the ER protein TMX2, which uses its unique cytosolic thioredoxin domain to prevent cytosolic sulfenylation of mitochondrial outer membrane proteins such as TOM70 through a functional interaction with peroxiredoxin-1 (PRDX1). By doing so, TMX2 interferes with the TOM70 ERMCS tethering function and reduces mitochondrial Ca2+ flux and metabolism. Recently, TMX2 mutations have been identified to cause a neurodevelopmental disorder with microcephaly, cortical malformations, and spasticity (NEDMCMS). Using TMX2-mutated NEDMCMS patient cells, we demonstrate that compromising TMX2 through mutation reproduces mitochondrial defects. In a fly in vivo model, TMX2 knockdown manifests predominantly in glial cells. Our results therefore provide important mechanistic insight into NEDMCMS and mechanistically link TMX2-mediated control of ERMCS to brain development and function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/589228v2_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@809587org.highwire.dtl.DTLVardef@1d8ef0corg.highwire.dtl.DTLVardef@135fdc6org.highwire.dtl.DTLVardef@1a7f7b6_HPS_FORMAT_FIGEXP M_FIG C_FIG The transmembrane thioredoxin-related TMX2 prevents TOM70 sulfenylation at ERMCS, thus maintaining normal mitochondria metabolism in wild-type cells. TMX2 knockout leads to TOM70 sulfenylation and tight ERMCS formation. This then increases ROS production, unbalances mitochondrial lipids, and relatively shifts OXPHOS electron supply to complex II.

cell biology↗

bioRxiv2023

Spatial heterogeneity in tumor adhesion qualifies collective cell migration

Collective cell migration, 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 migrations. Here, we constitute a tumor core of two separate cell subsets showing distinct intra-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). Our simulations show that for a given two intra-subset cell-cell adhesions for two subsets of cells, low and high inter-subset cell adhesion favors migration of high HI and low HI intermingled populations, respectively. In addition, for the most explored values of cell-ECM adhesion strengths, populations with high HI values collectively migrate better than those with lower HI values. We then asked how spatial migration is regulated by progressively intermingled cellular subsets that were 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 migration. We also observe an exception to this relationship for cases of heterogeneous cell-ECM adhesion where sub-maximal HI patterns with higher outer localization of cells with stronger ECM adhesion collectively migrate better than their relatively higher HI counterparts. Our simulations also reveal how adhesion heterogeneity qualifies migrative dynamics through collective cellular unjamming, when either cell-cell or -ECM adhesion type is varied but incorporates dispersion when both adhesion types are simultaneously altered.

systems biology↗

bioRxiv2023

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 non-senescent controls. Time-lapse epifluorescent 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 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 and their clearance contributing to the metastatic burden associated with the disease.

cancer biology↗

bioRxiv2023

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

1. Introduction ABSTRACT 1. Introduction 2. Materials and methods 3. Results and discussion 4. Conclusion Author contributions Supplementary Information References Cells grown as monolayers on conventional two-dimensional (2D) culture plates are significantly different from their in vivo counterparts in many different ways [1]. In standard flat plate cultures, not only do they exhibit altered morphologies and dynamics [2-4], their gene expression and signal transduction profiles are dramatically altered [5]. Furthermore, cells in vivo are exposed to more complex mechanical cues and chemical fields due to their three-dimensional (3D) packing and access to systemic circulation [ ...

bioengineering↗

bioRxiv2022

T-cell cellular stress and reticulocyte signatures, but not loss of naive T lymphocytes, characterize severe COVID-19 in older adults

In children and younger adults up to 39 years of age, SARS-CoV-2 usually elicits mild symptoms that resemble the common cold. Disease severity increases with age starting at 30 and reaches astounding mortality rates that are ~330 fold higher in persons above 85 years of age compared to those 18-39 years old. To understand age-specific immune pathobiology of COVID-19 we have analyzed soluble mediators, cellular phenotypes, and transcriptome from over 80 COVID-19 patients of varying ages and disease severity, carefully controlling for age as a variable. We found that reticulocyte numbers and peripheral blood transcriptional signatures robustly correlated with disease severity. By contrast, decreased numbers and proportion of naive T-cells, reported previously as a COVID-19 severity risk factor, were found to be general features of aging and not of COVID-19 severity, as they readily occurred in older participants experiencing only mild or no disease at all. Single-cell transcriptional signatures across age and severity groups showed that severe but not moderate/mild COVID-19 causes cell stress response in different T-cell populations, and some of that stress was unique to old severe participants, suggesting that in severe disease of older adults, these defenders of the organism may be disabled from performing immune protection. These findings shed new light on interactions between age and disease severity in COVID-19.

immunology↗

bioRxiv2022

Kinetic control in amyloid polymorphism: Different agitation and solution conditions promote distinct amyloid polymorphs of alpha-synuclein

Aggregation of neuronal protein -synuclein is implicated in synucleinopathies, including Parkinsons disease. Despite abundant in vitro studies, the mechanism of -synuclein assembly process remains ambiguous. In this work, -synuclein aggregation was induced by its constant mixing in two separate modes, either by agitation in a 96-well microplate reader (MP) or in microcentrifuge tubes using a shaker incubator (SI). Aggregation in both modes occurred through a sigmoidal growth pattern with a well-defined lag, growth, and saturation phase. The end-stage MP- and SI-derived aggregates displayed distinct differences in morphological, biochemical, and spectral signatures as discerned through AFM, proteinase-K digestion, FTIR, Raman, and CD spectroscopy. The MP-derived aggregates showed irregular morphology with a significant random coil conformation, contrary to SI-derived aggregates, which showed typical {beta}-sheet fibrillar structures. The end-stage MP aggregates convert to {beta}-rich SI-like aggregates upon 1) seeding with SI-derived aggregates and 2) agitating in SI. We conclude that end-stage MP aggregates were in a kinetically trapped conformation, whose kinetic barrier was bypassed upon either seeding by SI-derived fibrils or shaking in SI. We further show that MP-derived aggregates that form in the presence of sorbitol, an osmolyte, displayed a {beta}-rich signature, indicating that the preferential exclusion effect of osmolytes helped overcome the kinetic barrier. Our findings help in unravelling the kinetic origin of different -synuclein aggregated polymorphs (strains) that encode diverse variants of synucleinopathies. We demonstrate that kinetic control shapes the polymorphic landscape of -synuclein aggregates, both through de novo generation of polymorphs, and by their interconversion.

biophysics↗

bioRxiv2022

Mutations in a set of ancient matrisomal glycoprotein genes across neoplasia predispose to disruption of morphogenetic transduction

Misexpression and remodeling of the extracellular matrix is a canonical hallmark of cancer, although the extent of cancer-associated aberrations in the genes coding for ECM proteins and consequences thereof, are not well understood. In this study, we examined the alterations in core matrisomal genes across a set of nine cancers. These genes, especially the ones encoding for ECM glycoproteins, were observed to be more susceptible to mutations than copy number variations across cancers. We classified the glycoprotein genes based on the ubiquity of their mutations across the nine cancer groups and estimated their evolutionary age using phylostratigraphy. To our surprise, the ECM glycoprotein genes commonly mutated across all cancers were predominantly unicellular in origin, whereas those commonly showing mutations in specific cancers evolved mostly during and after the unicellular-multicellular transition. Pathway annotation for biological interactions revealed that the most pervasively mutated glycoprotein set regulated a larger set of inter-protein interactions and constituted more cohesive interaction networks relative to the cancer-specific mutated set. In addition, ontological prediction revealed the pervasively mutated set to be strongly enriched for basement membrane dynamics. Our results suggest that ancient unicellular-origin ECM glycoproteins were canalized into playing critical tissue morphogenetic roles, and when disrupted through matrisomal gene mutations, associate with neoplastic transformation of a wide set of human tissues.

bioinformatics↗

bioRxiv2022

Prolactin-induced AMPK stabilizes alveologenesis and lactogenesis through regulation of STAT5 signaling

AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that regulates energy homeostasis at cellular and organismal levels. It has been shown to affect several steps of breast cancer progression in a context-dependent manner. However, its role in normal mammary gland development and physiology remains ill-explored. Here, we show that AMPK expression and activity increased within murine mammary epithelia from puberty to pregnancy with highest levels during lactation, and then declined during involution. In ex vivo cultures of mammary epithelial cells (MECs) in organotypic scaffolds, treatment with lactogenic hormone prolactin (PRL) enhanced AMPK expression and activity. To understand the role of AMPK on mammary morphogenesis in vivo, we generated mice with conditional knockout of AMPK isoforms 1 and 2 (AMPK KO) in MECs. AMPK KO mammary glands showed accelerated alveolar development with increased epithelial content of both luminal and myoepithelial lineages, suggestive of hyperproliferation. AMPK KO mice also showed elevated beta-casein expression during pregnancy and lactation. These observations were phenocopied upon treatment of ex vivo cultivated wild-type MECs with a cognate AMPK inhibitor. AMPK null MECs showed increased phosphorylated STAT5 which is known to drive alveologenesis downstream of prolactin signaling. Our study identifies a novel interplay between AMPK and PRL-STAT5 signaling that determines mammary alveologenesis and differentiation.

developmental biology↗

bioRxiv2022

Matrix-driven jamming dynamics mediates transition of ovarian cancer spheroids to stable morphologies

Cancer metastasis through a confining peritoneal fluid microenvironment is mediated by spheroids: clusters of disseminated transformed cells. Ovarian cancer spheroids are frequently cavitated and their blastuloid morphology is correlated with an extracellular matrix (ECM) coat. Here, we investigate the effects of such morphology on the mechanical integrity of confined cancer spheroids. Atomic force microscopy showed higher elastic modulus for blastuloid spheroids relative to their prefiguring non-lumen moruloid counterparts. Subsequently, spheroids were flowed through microfluidic conditions mimicking peritoneal confinement. Traversing moruloids exhibited asymmetric cell flows during entry, often deformed and disintegrated through travel, and showed an incomplete- and slow shape recovery upon exit. In contrast, blastuloids traveled faster, exhibited rapid and efficient shape recovery upon exit, symmetric vector flows, and lesser disintegration. A multiscale computational model predicted higher intercellular adhesion and a dynamical lumen make blastuloids resilient. Although, E-cadherin overexpression in moruloids did not affect their resilience, blastuloid ECM-debridement decreased E-cadherin membrane localization, obliterated the lumen, and reversed the rheological properties of blastuloids to those typifying moruloids. The ECM-induced lumen therefore drives spheroidal transition from a labile viscoplastic to a resilient elastic state allowing them to survive spatially-constrained peritoneal flows.

biophysics↗

bioRxiv2021

Galectin-9 signaling drives breast cancer invasion through matrix

Aberration in expression and function of glycans and their binding proteins (lectins) in transformed cells constitutes one of the earliest discovered hallmarks of cancer. Galectins are a conserved family of lectins that can bind to {beta}-galactosides. Among them, the role of Galectin-9, a galectin with two carbohydrate binding domains in immune-tumor cell interactions has been well-established, although its effect on cancer cell behavior remains as yet unclear. In this study, we used a spectrum of cell lines from homeostatic breast cells to transformed non-invasive and invasive cell lines cultured in microenvironment-diverse conditions to show that Galectin-9 expression shows an elevation in association with invasiveness of breast cancer epithelia. Our observations were supported by immunohistochemical studies of breast tumors and adjacent normal-tissues from patients. Genetic perturbation of Galectin-9 as well as the pharmacological inhibition of activity using cognate inhibitors confirmed a positive correlation between Galectin-9 levels and the adhesion of the aggressive triple negative breast cancer cells MDA-MB-231 to- and their invasion through-extracellular matrices (ECM). Within a constituted organomimetic multiECM microenvironment, Galectin-9 enhanced both the solitary and the collective invasion of cancer cells. Quantitative proteomics led us to uncover the inductive role of Galectin-9 in the expression of the proinvasive protein S100A4. In addition, Galectin-9 expression correlated with FAK signaling, the inhibition of which decreased S100A4 mRNA levels. Our results provide crucial signaling insights into how the elevation in Galectin-9 expression in breast cancer cells potentiates their invasiveness through ECM during early steps of metastasis.

cancer biology↗

bioRxiv2021

IRE1/XBP1 signaling promotes skeletal muscle regeneration through a cell non-autonomous mechanism

Skeletal muscle regeneration is regulated by coordinated activation of multiple signaling pathways activated in both injured myofibers and satellite cells. The unfolded protein response (UPR) is a major mechanism that detects and alleviates protein-folding stresses in ER. However, the role of individual arms of the UPR in skeletal muscle regeneration remains less understood. In the present study, we demonstrate that IRE1 (also known as ERN1) and its downstream target, XBP1, are activated in skeletal muscle of mice upon injury. Myofiber-specific ablation of IRE1 or XBP1 in mice diminishes skeletal muscle regeneration that is accompanied with reduced number of satellite cells and their fusion to injured myofibers. Ex vivo cultures of myofiber explants demonstrate that ablation of IRE1 reduces the proliferative capacity of myofiber- associated satellite cells. Myofiber-specific deletion of IRE1 dampens Notch signaling and canonical NF-{kappa}B pathway in skeletal muscle of mice. Our results also demonstrate that targeted ablation of IRE1 reduces skeletal muscle regeneration in the mdx mice, a model of Duchenne muscular dystrophy. Collectively, our results reveal that the IRE1-mediated signaling promotes muscle regeneration through augmenting the proliferation of satellite cells in a cell non- autonomous manner.

cell biology↗

bioRxiv2021

Low mammalian species richness increases Kyasanur Forest Disease outbreak risk in deforested landscapes in the Western Ghats, India

Kyasanur forest disease virus (KFDV) is a rapidly expanding tick-borne zoonotic virus with natural foci in the forested region of the Western Ghats of South India. The Western Ghats is one of the worlds most important biodiversity hotspots and, like many such areas of high biodiversity, is under significant pressure from anthropogenic landscape change. The current study sought to quantify mammalian species richness using ensemble models of the distributions of a sample of species extant in the Western Ghats and to explore its association with KFDV outbreaks, as well as the modifying effects of deforestation on this association. Species richness was quantified as a composite of individual species distributions, as derived from ensembles of boosted regression tree, random forest, and generalised additive models. Species richness was further adjusted for the potential biotic constraints of sympatric species. Both species richness and forest loss demonstrated strong positive associations with KFDV outbreaks, however forest loss substantially modified the association between species richness and outbreaks. High species richness was associated with increased KFDV risk but only in areas of low forest loss. In contrast, lower species richness was associated with increased KFDV risk in areas of greater forest loss. This relationship persisted when species richness was adjusted for biotic constraints at the taluk-level. In addition, the taluk-level species abundances of three monkey species (Macaca radiata, Semnopithecus hypoleucus, and Semnopithecus priam) were also associated with outbreaks. These results suggest that increased monitoring of wildlife in areas of significant habitat fragmentation may add considerably to critical knowledge gaps in KFDV epidemiology and infection ecology and should be incorporated into novel One Health surveillance development for the region. In addition, the inclusion of some primate species as sentinels of KFDV circulation into general wildlife surveillance architecture may add further value. HighlightsO_LILocal mammalian species richness is estimated across the entire Western Ghats region C_LIO_LILow species richness is associated with high KFDV risk in deforested landscapes C_LIO_LIThis work identifies key landscapes for wildlife disease surveillance development C_LI

ecology↗

bioRxiv2021

Modeling the bistable transition between cell phenotypes during limb precartilage condensation

The tetrapod limb skeleton is initiated in unpatterned limb bud mesenchyme by the formation of precartilage condensations. Here, based on time-lapse videographic analysis of a forming condensation in a high-density culture of chicken limb bud mesenchyme, we observe a phase transition to a more fluidized state for cells within spatial compacted foci (protocondensations that will progress to condensations), as reflected in their spatial confinement, cell-substratum interaction and speed of motion. Previous work showed that galectin-8 and galectin-1A, two proteins of the galactoside-binding galectin family, are the earliest determinants of this process in the chicken limb bud, and that their interactions in forming skeletogenic patterns of condensations can be interpreted mathematically through a reaction-diffusion-adhesion framework. Based on this framework, we use an ordinary differential equation-based approach to analyze the core switching modality of the galectin reaction network and characterize the states of the network independent of the diffusive and adhesive arms of the patterning mechanism. We identify two steady states where the concentrations of both galectins are respectively, negligible, and very high. An explicit Lyapunov function shows that there are no periodic solutions. For sigmoidal galectin production terms, the model exhibits a bistable switch that arises from a monostable state via saddle-node bifurcation. Our model therefore predicts that the galectin network exists in low and high expression states separated in space or time without any intermediate states. This provides a causal basis for the observed outside vs. inside transition observed in the in vitro video data. We performed a quantitative analysis of the distribution of galectin-1A in cultures of condensing chick limb mesenchymal cells and found that the interior of the protocondensations had concentrations of this protein (compared to the immediate exterior) over and above that expected from its higher cell density, consistent with the models predictions. The galectin-based patterning network is thus suggested, on theoretical grounds, to incorporate a core switch independent of any spatial or temporal dynamics, that drives the chondrogenic cell state transition in limb skeletogenesis.

developmental biology↗

bioRxiv2020

Distinct evolutionary trajectories in asexual populations through an interplay of their size, resource availability and mutation rates

Asexually reproducing populations of single cells evolve through mutation, natural selection, and genetic drift to enhance their reproductive fitness. The environment provides the contexts that allow and regulate their fitness dynamics. In this work, we used Avida - a digital evolution framework - to uncover the effect of mutation rates, maximum size of the population, and the relative abundance of resources, on evolutionary outcomes in asexually reproducing populations of digital organisms. We observed that over extended simulations, the population evolved predominantly to one of several discrete fitness classes, each with distinct sequence motifs and/or phenotypes. For a low mutation rate, the organisms acquired either of four fitness values through an enhancement in the rate of genomic replication. Evolution at a relatively higher mutation rate presented a more complex picture. While the highest fitness values at a high mutation rate were achieved through enhanced genome replication rates, a suboptimal one was achieved through organisms sharing information relevant to metabolic tasks with each other. The information sharing capacity was vital to fitness acquisition and frequency of the genotype associated with it increased with greater resource levels and maximum population size. In addition, populations optimizing their fitness through such means exhibited a greater degree of genotypic heterogeneity and metabolic activity than those that improved replication rates. Our results reveal a minimal set of conditions for the emergence of interdependence within evolving populations with significant implications for biological systems in appropriate environmental contexts.

evolutionary biology↗

bioRxiv2020

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

Label-free separation of rare cells (e.g. circulating tumor cells (CTCs)) based on their size is attractive due to its wider applicability, simpler sample preparation, faster turnaround, better efficiency and higher purity. Amongst cognate protocols for the same, 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) flow velocities. Fluid flowing through the chip is constrained to exit the trapping chamber at right angles to that of its entry. Such a flow configuration leads to the formation of vortex in the chamber. Above a critical flow velocity, larger particles are trapped in the vortex whereas smaller particles get ejected with the flow: we call this phenomenon the turn-effect. We have characterized the critical velocities for trapping of cells and particles of different sizes on chips with distinct entry-exit configurations. Optimal architectures for stable vortex trapping at low flow velocities are identified. We explain how shear-gradient lift, centrifugal and Dean flow drag forces contribute to the turn-effect by acting on cells which pushes them into specific vortices in a size- and velocity-dependent fashion. Finally, we demonstrate selective trapping of human breast cancer cells mixed with whole blood at low-concentration. Our findings suggest that the device shows promise for the gentle isolation of rare cells from blood.

bioengineering↗

bioRxiv2020

A dynamical extracellular matrix coat regulates moruloid-blastuloid transitions of ovarian cancer spheroids

SummaryOvarian cancer metastasizes into the peritoneum through dissemination of transformed epithelia as multicellular spheroids 1, 2. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues3: lumen surrounded by smoothly contoured, adhered, and immotile epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid ‘moruloid’ clusters that permit intercellular movement and penetration by new cells. Moruloid clusters can coalesce to form bigger clusters. Upon further culture, moruloid clusters mature into ‘blastuloid’ spheroids with smooth contours, lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow penetration by new cells. Ultrastructural examination reveals a basement membrane-like matrix coat on the surface of blastuloid, but not moruloid, spheroids: immunocytochemistry confirms the presence of extracellular matrix proteins: Collagen IV and Laminin-322. Enzymatic debridement of the coat results in a reversible loss of lumen and contour. Debridement also allows spheroidal coalescence and cell intrusion in blastuloid spheroids and enhances adhesion to peritoneal substrata. Therefore, the dynamical matrix coat regulates both the morphogenesis of cancer spheroids and their adhesive interaction with their substrata, affecting ultimately the progression of the disease.Results Survival of women afflicted with epithelial ovarian cancer (EOC) trails behind other gynecological malignancies, despite improvements in surgical-pharmacological approaches4,5. The morbidity associated with the disease is a consequence of its transcoelomic route of metastasis: transformed epithelia of the fallopian tubes and ovaries in the form of spheroids, eventually home and adhere to the mesothelial lining of the peritoneum, occasionally invade through the underlying collagenous extracellular matrix and form secondary metastatic foci around abdominal organs1, 6, 7. EOC spheroids impede the drainage of the fluid from the peritoneal cavity and alter its composition; in turn the fluid, now known as malignant ascites serves as a pro-tumorigenic milieu for the spheroids8, 9The formation and presence of spheroids within ascites of an ovarian cancer patient is strongly associated with recurrence of cancer and greater resistance to chemotherapy10. Therefore, in order to develop novel strategies to target spheroidal metastatic niche, it is essential to investigate mechanisms that underlie their morphogenesis. Several proteins have been proposed to mediate the adhesion between ovarian cancer epithelia that give rise to spheroids. These include transmembrane receptors such as CD4411, cell adhesion molecules, such as E-cadherin and N-cadherin12, matrix adhesion-inducing proteins such as integrins13, 14. Remarkably, a phase-contrast microscopic examination of spheroids from patients, or from aggregated epithelia of immortalized cancer lines cultured on low attachment substrata, shows features of morphogenetic organization: presence of a central lumen, radially arranged apposed epithelia and compacted surfaces. Such traits are cognate to organized morphogenesis within the glandular epithelial organs,15 which are built through principles that include, but are not limited to, cell-cell adhesion16, 17. In fact, loss of tissue architecture seen in tumorigenesis involves the disappearance of such morphogenetic traits (such as matrix adhesion and polarity)18, 19.In this manuscript, we investigate how these traits are recapitulated in a fluid metastatic context. Using spheroids from patients with high grade serous adenocarcinoma and ovarian cancer cell lines, we show that the development of a basement membrane (BM)-like coat of extracellular matrix is responsible for the compaction and stability of cancer spheroids, for decreasing the motility of cells within it and for generation of lumen. The coat, which is rapidly replenished by cells upon enzymatic debridement, also prevents the attachment of spheroids to matrix substrata. This may have significant implications for the build-up of the massive cellular fraction within the malignant ascites of patients afflicted with ovarian cancer.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Differential levels of dermatan sulfate generate distinct Collagen I gel architectures

Collagen I is the most abundant extracellular matrix (ECM) protein in vertebrate tissues. As an endogenously synthesized fibrillar biopolymer or as a synthetic hydrogel, it provides mechanical durability to tissue microenvironments and regulates cell function. Predominant regulators of its fibrillogenesis are dermatan sulfate proteoglycans (DSPGs), proteins conjugated with iduronic acid containing DS glycosaminoglycans (GAGs). Although DS is known to regulate tissue function through its modulation of Collagen I architecture, a precise quantifiable understanding of the latter remains elusive. We investigate this problem by visualizing the pattern of structural elements within fixed Collagen I gels polymerized in the presence of varying concentrations of DS (50-, 200- and 400- g/mL) using second harmonic generation microscopy (SHG). Measuring four independent imaging parameters: fibril density, mean SHG signal (which estimates the ordering of the fibrils), surface occupancy (which estimates the space occupied by fibrils), and the fibril width allows us to construct an informative model of the effects of DS on Collagen I element architecture. Supported by confocal microscopy, our observations indicate that the effect on collagen fibril pattern of DS is contextual upon its concentrations. Lower levels of DS result in more numerous disorganized fibrils; higher levels restore organization, but at lower fibril densities. Such Collagen I gel pattern-tuning of DS is likely of relevance for understanding its functions in disease progression and biomaterial applications.

physiology↗