Crocetin Enhances Temozolomide Efficacy in Glioblastoma Therapy Through Multiple Pathway Suppression.

BACKGROUND: Glioblastoma multiforme (GBM) is an aggressive type of brain tumor that is difficult to remove surgically. Research suggests that substances from saffron, namely crocetin and crocin, could be effective natural treatments, showing abilities to kill cancer cells. METHODS: Our study focused on evaluating the effects of crocetin on glioma using the U87 cell line. We specifically investigated how crocetin affects the survival, growth, and spread of glioma cells, exploring its impact at concentrations ranging from 75-150 µM. The study also included experiments combining crocetin with the chemotherapy drug Temozolomide (TMZ) to assess potential synergistic effects. RESULTS: Crocetin significantly reduced the viability, proliferation, and migration of glioma cells. It achieved these effects by decreasing the levels of Matrix Metallopeptidase 9 (MMP-9) and Ras homolog family member A (RhoA), proteins that are critical for cancer progression. Additionally, crocetin inhibited the formation of cellular structures necessary for tumor growth. It blocked multiple points of the Ak Strain Transforming (AKT) signaling pathway, which is vital for cancer cell survival. This treatment led to increased cell death and disrupted the cell cycle in the glioma cells. When used in combination with TMZ, crocetin not only enhanced the reduction of cancer cell growth but also promoted cell death and reduced cell replication. This combination therapy further decreased levels of high mobility group box 1 (HMGB1) and Receptor for Advanced Glycation End-products (RAGE), proteins linked to inflammation and tumor progression. It selectively inhibited certain pathways involved in the cellular stress response without affecting others. CONCLUSION: Our results underscore the potential of crocetin as a treatment for glioma. It targets various mechanisms involved in tumor growth and spread, offering multiple avenues for therapy. Further studies are essential to fully understand and utilize crocetin's benefits in treating glioma.

Actomyosin forces in cell migration: Moving beyond cell body retraction.

In textbook illustrations of migrating cells, actomyosin contractility is typically depicted as the contraction force necessary for cell body retraction. This dogma has been transformed by the molecular clutch model, which acknowledges that actomyosin traction forces also generate and transmit biomechanical signals at the leading edge, enabling cells to sense and shape their migratory path in mechanically complex environments. To fulfill these complementary functions, the actomyosin system assembles a gradient of contractile energy along the front-rear axis of migratory cells. Here, we highlight the hierarchic assembly and self-regulatory network structure of the actomyosin system and explain how the kinetics of different nonmuscle myosin II (NM II) paralogs synergize during contractile force generation. Our aim is to emphasize how protrusion formation, cell adhesion, contraction, and retraction are spatiotemporally integrated during different modes of migration, including chemotaxis and durotaxis. Finally, we hypothesize how different NM II paralogs might tune aspects of migration in vivo, highlighting future research directions.

Tata-box-binding protein-associated factor 15 as a new potential marker in gastrointestinal tumors.

In this editorial, the roles of tata-box-binding protein-associated factor 15 (TAF15) in oncogenesis, tumor behavior, and as a therapeutic target in cancers in the context of gastrointestinal (GI) tumors are discussed concerning the publication by Guo et al. TAF15 is a member of the FET protein family with a comprehensive range of cellular processes. Besides, evidence has shown that TAF15 is involved in many diseases, including cancers. TAF15 contributes to carcinogenesis and tumor behavior in many tumors. Besides, its relationship with the mitogen-activated protein kinases (MAPK) signaling pathway makes TAF15 a new target for therapy. Although, the fact that there is few studies investigating the expression of TAF15 constitutes a potential limitation in GI system, the association of TAF15 expression with aggressive tumor behavior and, similar to other organ tumors, the influence of TAF15 on the MAPK signaling pathway emphasize that this protein could serve as a new molecular biomarker to predict tumor behavior and target therapeutic intervention in GI cancers. In conclusion, more studies should be performed to better understand the prognostic and therapeutic role of TAF15 in GI tumors, especially in tumors resistant to therapy.

Engineered intrinsically fluorescent galectin-8 variants with altered valency, ligand recognition and biological activity.

Galectin-8 is a small soluble lectin with two carbohydrate recognition domains (CRDs). N- and C-terminal CRDs of Gal-8 differ in their specificity for glycan ligands. Here, we wanted to find out whether oligomerization of individual CRDs of galectin-8 affects its biological activity. Using green fluorescent protein polygons (GFPp) as an oligomerization scaffold, we generated intrinsically fluorescent CRDs with altered valency. We show that oligomers of C-CRD are characterized by significant cell surface affinity. Furthermore, the multivalency of the resulting variants has an impact on cellular activities such as cell signaling, heparin binding and proliferation. Our data indicates that tunable valence is a useful tool for modifying the biological activity of CRDs of galectins.

The prognostic and immune significance of SLAMF9 in pan-cancer and validation of its role in colorectal cancer.

SLAMF9, a member of the conserved lymphocyte activation molecules family (SLAMF), has been less investigated compared to other SLAMs, especially concerning its implications across various cancer types. In our systematic pan-cancer investigation, we observed elevated SLAMF9 expression in various tumor tissues, which was correlated with reduced patient survival across most malignancies. Correlation analyses further revealed significant associations between SLAMF9 expression and immune cell infiltrates, immune checkpoint inhibitors, tumor mutation load, microsatellite instability, and epithelial-mesenchymal transition (EMT) scores. Cell-based assays demonstrated that SLAMF9 knockdown attenuated the proliferative, motile, and invasive capacities of colorectal cancer (CRC) cells. In a nude mouse xenograft model, suppression of SLAMF9 expression substantially inhibited tumor growth. These findings highlight the potential of SLAMF9 as a prognostic and therapeutic biomarker across tumors, with notable implications for CRC cell proliferation and migration.

Aberrant migration features in primary skin fibroblasts of Huntington's disease patients hold potential for unraveling disease progression using an image based machine learning tool.

Huntington's disease (HD) is a complex neurodegenerative disorder with considerable heterogeneity in clinical manifestations. While CAG repeat length is a known predictor of disease severity, this heterogeneity suggests the involvement of additional genetic and environmental factors. Previously we revealed that HD primary fibroblasts exhibit unique features, including distinct nuclear morphology and perturbed actin cap, resembling characteristics seen in Hutchinson-Gilford Progeria Syndrome (HGPS). This study establishes a link between actin cap deficiency and cell motility in HD, which correlates with the HD patient disease severity. Here, we examined single-cell motility imaging features in HD primary fibroblasts to explore in depth the relationship between cell migration patterns and their respective HD patients' clinical severity status (premanifest, mild and severe). The single-cell analysis revealed a decline in overall cell motility in correlation with HD severity, being most prominent in severe HD subgroup and HGPS. Moreover, we identified seven distinct spatial clusters of cell migration in all groups, which their proportion varies within each group becoming a significant HD severity classifier between HD subgroups. Next, we investigated the relationship between Lamin B1 expression, serving as nuclear envelope morphology marker, and cell motility finding that changes in Lamin B1 levels are associated with specific motility patterns within HD subgroups. Based on these data we present an accurate machine learning classifier offering comprehensive exploration of cellular migration patterns and disease severity markers for future accurate drug evaluation opening new opportunities for personalized treatment approaches in this challenging disorder.

Long-Term In Vitro Culture Alters Gene Expression Pattern of Genes Involved in Ontological Groups Representing Cellular Processes.

The oviduct provides an optimal environment for the final preparation, transport, and survival of gametes, the fertilization process, and early embryonic development. Most of the studies on reproduction are based on in vitro cell culture models because of the cell's accessibility. It creates opportunities to explore the complexity of directly linked processes between cells. Previous studies showed a significant expression of genes responsible for cell differentiation, maturation, and development during long-term porcine oviduct epithelial cells (POECs) in vitro culture. This study aimed at establishing the transcriptomic profile and comprehensive characteristics of porcine oviduct epithelial cell in vitro cultures, to compare changes in gene expression over time and deliver information about the expression pattern of genes highlighted in specific GO groups. The oviduct cells were collected after 7, 15, and 30 days of in vitro cultivation. The transcriptomic profile of gene expression was compared to the control group (cells collected after the first day). The expression of COL1A2 and LOX was enhanced, while FGFBP1, SERPINB2, and OVGP1 were downregulated at all selected intervals of cell culture in comparison to the 24-h control (p-value < 0.05). Adding new detailed information to the reproductive biology field about the diversified transcriptome profile in POECs may create new future possibilities in infertility treatments, including assisted reproductive technique (ART) programmes, and may be a valuable tool to investigate the potential role of oviduct cells in post-ovulation events.

Design, synthesis, and evaluation of benzodioxolane compounds for antitumor activity.

This study reports the design, synthesis, and comprehensive biological evaluation of 13 benzodioxolane derivatives, derived from the core structure of piperine, a natural product with established antitumor properties. Piperine, primarily found in black pepper, has been noted for its diverse pharmacological activities, including anti-inflammatory, antioxidant, and anticancer effects. Leveraging piperine's antitumor potential, we aimed to enhance its efficacy through structural modifications. Among the synthesized compounds, HJ1 emerged as the most potent, exhibiting a 4-fold and 10-fold increase in inhibitory effects on HeLa and MDA-MB-231 cell lines, respectively, compared to piperine. Furthermore, HJ1 demonstrated a favorable safety profile, characterized by significantly lower cytotoxicity towards the human normal cell line 293T. Mechanistic investigations revealed that HJ1 markedly inhibited clonogenicity, migration, and adhesion of HeLa cells. In vivo studies utilizing the chick embryo chorioallantoic membrane (CAM) model substantiated the robust antitumor activity of HJ1, evidenced by its ability to suppress tumor angiogenesis and reduce tumor weight. These results suggest that HJ1 holds significant promise as a lead compound for the development of novel antitumor therapies.

RBM10 loss induces aberrant splicing of cytoskeletal and extracellular matrix mRNAs and promotes metastatic fitness.

RBM10 modulates transcriptome-wide cassette exon splicing. Loss-of-function RBM10 mutations are enriched in thyroid cancers with distant metastases. Analysis of transcriptomes and genes mis-spliced by RBM10 loss showed pro-migratory and RHO/RAC signaling signatures. RBM10 loss increases cell velocity. Cytoskeletal and ECM transcripts subject to exon-inclusion events included vinculin (VCL), tenascin C (TNC) and CD44. Knockdown of the VCL exon inclusion transcript in RBM10-null cells reduced cell velocity, whereas knockdown of TNC and CD44 exon-inclusion isoforms reduced invasiveness. RAC1-GTP levels were increased in RBM10-null cells. Mouse Hras G12V /Rbm1O KO thyrocytes develop metastases that are reversed by RBM10 or by combined knockdown of VCL, CD44 and TNC inclusion isoforms. Thus, RBM10 loss generates exon inclusions in transcripts regulating ECM-cytoskeletal interactions, leading to RAC1 activation and metastatic competency. Moreover, a CRISPR-Cas9 screen for synthetic lethality with RBM10 loss identified NFkB effectors as central to viability, providing a therapeutic target for these lethal thyroid cancers.

MCP-1 exerts the inflammatory response via ILK activation during endometriosis pathogenesis.

AIMS: MCP-1 has been shown to be elevated in endometriosis. ILK functions in several cellular events and interacts with MCP-1-signaling. In the current study, we evaluated the role of MCP-1-ILK signaling in human endometriotic cell's (Hs832(C).TCs) potential for colonization, invasion, adhesion, etc. and differentiation of macrophage along with inflammation in an endometriosis mouse model. MATERIALS AND METHODS: A mouse model of endometriosis with elevated levels of MCP-1 was developed by injecting MCP-1. We examined the migration, adhesion, colonization and invasion of Hs832(C).TCs in response to MCP-1-ILK signaling. We also examined the differentiation of THP-1 cells to macrophage in response to MCP-1-ILK signaling. KEY FINDINGS: We observed that MCP-1 increased Ser246 phosphorylation of ILK in Hs832(C).TCs and enhanced the migration, adhesion, colonization, and invasion of Hs832(C).TCs. In the mouse model of endometriosis, we found elevated chemokines (CCL-11, CCL-22 and CXCL13) levels. An increased level of MCP-1 mediated ILK activation, leading to increased inflammatory reaction and infiltration of residential and circulatory macrophages, and monocyte differentiation, but suppressed the anti-inflammatory reaction. The inhibitor (CPD22) of ILK reversed the MCP-1-mediated action by restoring Hs832(C).TCs and THP-1 phenotype. ILK inhibition in a mouse model of endometriosis reduced the effects of MCP-1 mediated pro-inflammatory cytokines, but increased anti-inflammatory response along with T-regulatory and T-helper cell restoration. SIGNIFICANCE: Targeting ILK restores MCP-1 milieu in the peritoneal cavity and endometrial tissues, reduces the inflammatory response, improves the T-regulatory and T-helper cells in the endometriosis mouse model and decreases the migration, adhesion, colonization and invasion of endometriotic cells.

Biophysical modeling identifies an optimal hybrid amoeboid-mesenchymal phenotype for maximal T cell migration speeds.

Despite recent experimental progress in characterizing cell migration mechanics, our understanding of the mechanisms governing rapid cell movement remains limited. To effectively limit tumor growth, antitumoral T cells need to rapidly migrate to find and kill cancer cells. To investigate the upper limits of cell speed, we developed a new hybrid stochastic-mean field model of bleb-based cell motility. We first examined the potential for adhesion-free bleb-based migration and show that cells migrate inefficiently in the absence of adhesion-based forces, i.e., cell swimming. While no cortical contractility oscillations are needed for cells to swim in viscoelastic media, high-to-low cortical contractility oscillations are necessary for cell swimming in viscous media. This involves a high cortical contractility phase with multiple bleb nucleation events, followed by an intracellular pressure buildup recovery phase at low cortical tensions, resulting in modest net cell motion. However, our model suggests that cells can employ a hybrid bleb- and adhesion-based migration mechanism for rapid cell motility and identifies conditions for optimality. The model provides a momentum-conserving mechanism underlying rapid single-cell migration and identifies factors as design criteria for engineering T cell therapies to improve movement in mechanically complex environments.

Human ARMC6 binds in vitro to both cancer genes and telomeric RNA, favoring G-quadruplex structure recognition.

Armadillo repeat-containing proteins (ARMCs) are a large family found throughout eukaryotes, which play prominent roles in cell adhesion, signaling and cytoskeletal regulation. The ARMC6 protein is highly conserved in primates, including humans, but to date does not have a clear function beyond initial hints of a link to cancer and telomerase activity. We report here in vitro experiments showing ARMC6 binding to DNA promoter sequences from several cancer-related genes (e.g., EGFR, VEGF and c-MYC), and also to the telomeric RNA repeat (TERRA). ARMC6 binding activity appears to recognize G-quadruplex motifs, which are being increasingly implicated as structure-based protein binding sites in chromosome maintenance and repair. In vivo investigation of ARMC6 function revealed that when this protein is overexpressed in human cell lines, there is different expression of genes connected with oncogenic pathways and those implicated in downstream non-canonical telomerase pathways (e.g., VEGF, hTERT, c-MYC, ESM1, MMP3). ARMC6 is already known to interact with human shelterin protein TRF2 and telomerase. The protein binds G-quadruplex structures and does so preferentially to RNA over DNA. As such, this protein may be an example of how a non-canonical nucleic acid structural motif allows mediation between gene regulation and telomeric chromatin rearrangement pathways.

Spheroid Model of Mammary Tumor Cells: Epithelial-Mesenchymal Transition and Doxorubicin Response.

Breast cancer is the most prevalent cancer among women worldwide. Therapeutic strategies to control tumors and metastasis are still challenging. Three-dimensional (3D) spheroid-type systems more accurately replicate the features of tumors in vivo, working as a better platform for performing therapeutic response analysis. This work aimed to characterize the epithelial-mesenchymal transition and doxorubicin (dox) response in a mammary tumor spheroid (MTS) model. We evaluated the doxorubicin treatment effect on MCF-7 spheroid diameter, cell viability, death, migration and proteins involved in the epithelial-mesenchymal transition (EMT) process. Spheroids were also produced from tumors formed from 4T1 and 67NR cell lines. MTSs mimicked avascular tumor characteristics, exhibited adherens junction proteins and independently produced their own extracellular matrix. Our spheroid model supports the 3D culturing of cells isolated from mice mammary tumors. Through the migration assay, we verified a reduction in E-cadherin expression and an increase in vimentin expression as the cells became more distant from spheroids. Dox promoted cytotoxicity in MTSs and inhibited cell migration and the EMT process. These results suggest, for the first time, that this model reproduces aspects of the EMT process and describes the potential of dox in inhibiting the metastatic process, which can be further explored.

De novo monoallelic Reelin missense variants act in a dominant-negative manner causing neuronal migration disorders.

Reelin (RELN) is a secreted glycoprotein essential for cerebral cortex development. In humans, recessive RELN variants cause cortical and cerebellar malformations, while heterozygous variants were associated to epilepsy, autism and mild cortical abnormalities. However, their functional effects remain unknown. We identified inherited and de novo RELN missense variants in heterozygous patients with neuronal migration disorders (NMDs) as diverse as pachygyria and polymicrogyria. We investigated in culture and in the developing mouse cerebral cortex how different variants impacted RELN function. Polymicrogyria-associated variants behaved as gain-of-function showing an enhanced ability to induce neuronal aggregation, while those linked to pachygyria as loss-of-function leading to defective neuronal aggregation/migration. The pachygyria-associated de novo heterozygous RELN variants acted as dominant-negative by preventing wild-type RELN secretion in culture, animal models and patients, thereby causing dominant NMDs. We demonstrated how mutant RELN proteins in vitro and in vivo predict cortical malformation phenotypes, providing valuable insights into the pathogenesis of such disorders.

Reduced Cell Migration in Human Chondrocyte Sheets Increases Tissue Stiffness and Cartilage Protein Production.

BACKGROUND: Chondrogenic differentiation medium (CDM) is usually used to maintain chondrogenic activity during chondrocyte sheet production. However, tissue qualities remain to be determined as to what factors improve cell functions. Moreover, the relationship between CDM and cell migration proteins has not been reported. METHOD: In this study, the effect of CDM on the behavior of chondrocyte sheets was investigated. Structural analysis, mechanical testing and proteomics were performed to observe tissue qualities. The relationship between CDM and cell migration proteins were investigated using time-lapse observations and bioinformatic analysis. RESULTS: During 48 h, CDM affected the chondrocyte behaviors by reducing cell migration. Compared to the basal medium, CDM impacted the contraction of monolayered chondrocyte sheets. At day 7, the contracted sheets increased tissue thickness and improved tissue stiffness. Cartilage specific proteins were also upregulated. Remarkedly, the chondrocyte sheets in CDM displayed downregulated proteins related to cell migration. Bioinformatic analysis revealed that TGFß1 was shown to be associated with cartilage functions and cell migration. Pathway analysis of chondrocyte sheets in CDM also revealed the presence of a TGFß pathway without activating actin production, which might be involved in synthesizing cartilage-specific proteins. Cell migration pathway showed MAPK signaling in both cultures of the chondrocyte sheets. CONCLUSION: Reduced cell migration in the chondrocyte sheet affected the tissue quality. Using CDM, TGFß1 might trigger cartilage protein production through the TGFß pathway and be involved in cell migration via the MAPK signaling pathway. Understanding cell behaviors and their protein expression would be beneficial for developing high-quality tissue-engineered cartilage.

Microliter Whole Blood Neutrophil Assay Preserving Physiological Lifespan and Functional Heterogeneity.

For in vitro neutrophil functional assays, neutrophils are typically isolated from whole blood, having the target cells exposed to an artificial microenvironment with altered kinetics. Isolated neutrophils exhibit limited lifespans of only a few hours ex vivo, significantly shorter than the 3-5 day lifespan of neutrophils in vivo. In addition, due to neutrophils' inherently high sensitivity, neutrophils removed from whole blood exhibit stochastic non-specific activation that contributes to assay variability. Here, a method - named "µ-Blood" - is presented that enables functional neutrophil assays using a microliter of unprocessed whole blood. µ-Blood allows multiple phenotypic readouts of neutrophil function (including cell/nucleus morphology, motility, recruitment, and pathogen control). In µ-Blood, neutrophils show sustained migration and limited non-specific activation kinetics (<0.1% non-specific activation) over 3-6 days. In contrast, neutrophils isolated using traditional methods show increased and divergent activation kinetics (10-70% non-specific activation) in only 3 h. Finally, µ-Blood allows the capture and quantitative comparison of distinct neutrophil functional heterogeneity between healthy donors and cancer patients in response to microbial stimuli with the preserved physiological lifespan over 6 days.

FAM122A functions as a tumor suppressor in oral squamous cell carcinoma.

Family with sequence similarity 122a (FAM122A), identified as an endogenous inhibitor of protein phosphatase 2A (PP2A) previously, is involved in multiple important physiological processes, and essential for the growth of acute myeloid leukemia and hepatocellular carcinoma cells. However, the function of FAM122A in oral squamous cell carcinoma (OSCC) is undetermined. In this study, by analyzing TCGA and GEO databases, we found that the expression of FAM122A was significantly down-regulated in head and neck squamous cell carcinoma and OSCC patients, meanwhile this low expression was tightly associated with the poor prognosis and advanced clinical stage during OSCC development. The similar low expression pattern of FAM122A could also been seen in OSCC cell lines compared with normal human oral keratinocytes. Further, we demonstrated that FAM122A knockdown significantly promoted the growth, clonogenic potential as well as migration capabilities of OSCC cells, while these alterations could be rescued by the re-expression of FAM122A. Over-expression of FAM122A suppressed OSCC cell proliferation and migration. FAM122A also inhibited the epithelial-mesenchymal transition (EMT) in OSCC cells by the up-regulation of epithelial marker E-cadherin and down-regulation of mesenchymal markers Fibronectin and Vimentin, which is presumably mediated by transforming growth factor ß receptor 3 (TGFBR3), a novel tumor suppressor. In addition, FAM122A could induce T cell infiltration in OSCC, indicating that FAM122A might influence the immune cell activity of tumor environment and further interfere the tumor development. Collectively, our results suggest that FAM122A functions as a tumor suppressor in OSCC and possibly acts as a predictive biomarker for the diagnosis and/or treatment of OSCC.

TAZ DOWNREGULATES ANXA1 EXPRESSION TO MODULATE MYELOMA CELL INTERACTION WITH BONE MARROW MESENCHYMAL STROMAL CELLS.

We and others have previously shown that TAZ plays a tumor suppressive role in multiple myeloma. However, recent reports suggest that molecular crosstalk between the myeloma cells and bone marrow stromal components contributes to the myeloma cell survival and drug resistance. These reports further point to reciprocal interaction via adhesion molecules as the most prominent mechanism of intercellular crosstalk between myeloma cells and BM-MSCs. YAP/TAZ silencing/expression has been shown to correlate across all cancers with a set of adhesion/extracellular matrix proteins. Therefore, we hypothesized that TAZ may regulate myeloma cell interaction with BM stromal cells by influencing the expression of distinct cell adhesion signatures. We used previously established TAZ myeloma cell line models: DELTA47-pLENTI or TAZ knockout DELTA47 cells cocultured with or without BM-MSCs as our study models. Using RNA sequencing analysis, we performed the first comprehensive screen for cell adhesion-related transcriptional targets of TAZ in MM. In doing so, we uncovered an enrichment of cell adhesion-related genes in TAZ knockout DELTA47 cells relatively to pLENTI-DELTA47 cells, including 11 genes with log2 fold change>2 (P<0.05): ANXA1, ADGRL2, NCAM1, NCAM2, ADGRL3, CXADR, ALCAM, JAM2, KIRREL1, KIRREL2, and ADGRG7, suggesting possible relationship with TAZ. We validated ANXA1 as a bona fide target of TAZ in MM. We show that TAZ represses myeloma cell migration and interaction with BM-MSCs by transcriptionally downregulating ANXA1 expression via TEAD-dependent mechanism. Our data provide new insights into the understanding of the role of TAZ in the intercellular communication signals between myeloma cells and BM-MSCs. Our findings also suggest that ANXA1 represents a putative cell adhesion target to attenuate BM-MSC driven, tumour-promoting interaction with myeloma cells.

Alpha-synuclein shapes monocyte and macrophage cell biology and functions by bridging alterations of autophagy and inflammatory pathways.

Introduction: Abnormal spreading of alpha-synuclein (αS), a hallmark of Parkinson's disease, is known to promote peripheral inflammation, which occurs in part via functional alterations in monocytes/macrophages. However, underlying intracellular mechanisms remain unclear. Methods: Herein we investigate the subcellular, molecular, and functional effects of excess αS in human THP-1 monocytic cell line, THP-1-derived macrophages, and at least preliminarily, in primary monocyte-derived macrophages (MDMs). In cells cultured w/wo recombinant αS (1 µM) for 4 h and 24 h, by Confocal microscopy, Western Blot, RT-qPCR, Elisa, and Flow Cytometry we assessed: i) αS internalization; ii) cytokine/chemokine expression/secretion, and C-C motif chemokine receptor 2 (CCR2) levels; iii) autophagy (LC3II/I, LAMP1/LysoTracker, p62, pS6/total S6); and iv) lipid droplets (LDs) accumulation, and cholesterol pathway gene expression. Transwell migration assay was employed to measure THP-1 cell migration/chemotaxis, while FITC-IgG-bead assay was used to analyze phagocytic capacity, and the fate of phagocytosed cargo in THP-1-derived macrophages. Results: Extracellular αS was internalized by THP-1 cells, THP-1-derived macrophages, and MDMs. In THP1 cells, αS induced a general pro-inflammatory profile and conditioned media from αS-exposed THP-1 cells potently attracted unstimulated cells. However, CCL2 secretion peaked at 4 h αS, consistent with early internalization of its receptor CCR2, while this was blunted at 24 h αS exposure, when CCR2 recycled back to the plasma membrane. Again, 4 h αS-exposed THP-1 cells showed increased spontaneous migration, while 24 h αS-exposed cells showed reduced chemotaxis. This occurred in the absence of cell toxicity and was associated with upregulation of autophagy/lysosomal markers, suggesting a pro-survival/tolerance mechanism against stress-related inflammation. Instead, in THP-1-derived macrophages, αS time-dependently potentiated the intracellular accumulation, and release of pro-inflammatory mediators. This was accompanied by mild toxicity, reduced autophagy-lysosomal markers, defective LDs formation, as well as impaired phagocytosis, and the appearance of stagnant lysosomes engulfed with phagocytosed cargo, suggesting a status of macrophage exhaustion reminiscent of hypophagia. Discussion: In summary, despite an apparently similar pro-inflammatory phenotype, monocytes and macrophages respond differently to intracellular αS accumulation in terms of cell survival, metabolism, and functions. Our results suggest that in periphery, αS exerts cell- and context-specific biological effects bridging alterations of autophagy, lipid dynamics, and inflammatory pathways.

Modeling the mechanosensitive collective migration of cells on the surface and the interior of morphing soft tissues.

Cellular contractility, migration, and extracellular matrix (ECM) mechanics are critical for a wide range of biological processes including embryonic development, wound healing, tissue morphogenesis, and regeneration. Even though the distinct response of cells near the tissue periphery has been previously observed in cell-laden microtissues, including faster kinetics and more prominent cell-ECM interactions, there are currently no models that can fully combine coupled surface and bulk mechanics and kinetics to recapitulate the morphogenic response of these constructs. Mailand et al. (Biophys J 117(5):975-986, 2019) had shown the importance of active elastocapillarity in cell-laden microtissues, but modeling the distinct mechanosensitive migration of cells on the periphery and the interior of highly deforming tissues has not been possible thus far, especially in the presence of active elastocapillary effects. This paper presents a framework for understanding the interplay between cellular contractility, migration, and ECM mechanics in dynamically morphing soft tissues accounting for distinct cellular responses in the bulk and the surface of tissues. The major novelty of this approach is that it enables modeling the distinct migratory and contractile response of cells residing on the tissue surface and the bulk, where concurrently the morphing soft tissues undergo large deformations driven by cell contractility. Additionally, the simulation results capture the changes in shape and cell concentration for wounded and intact microtissues, enabling the interpretation of experimental data. The numerical procedure that accounts for mechanosensitive stress generation, large deformations, diffusive migration in the bulk and a distinct mechanism for diffusive migration on deforming surfaces is inspired from recent work on bulk and surface poroelasticity of hydrogels involving elastocapillary effects, but in this work, a two-field weak form is proposed and is able to alleviate numerical instabilities that were observed in the original method that utilized a three-field mixed finite element formulation.