Cell geometry distinguishes migration-associated heterogeneity in two-dimensional systems

Background: In vitro migration assays are a cornerstone of cell biology and have found extensive utility in research. Over the past decade, several variations of the two-dimensional (2D) migration assay have improved our understanding of this fundamental process. However, the ability of these approaches to capture the functional heterogeneity during migration and their accessibility to inexperienced users has been limited. Methods: We downloaded published time-lapse 2D cell migration datasets and subjected them to feature extraction with the Fiji software. We used the 'Analyze Particles' tool to extract ten cell geometry features (CGFs), which were grouped into 'shape, 'size and 'position' descriptors. Next, we defined the migratory status of cells using the 'MTrack2' plugin. All data obtained from Fiji were further subjected to rigorous statistical analysis with R version 4.0.2. Results: We observed consistent associative trends between size and shape descriptors and validated the robustness of our observations across four independent datasets. We used these descriptors to resolve the functional heterogeneity during migration by identifying and characterizing 'non-migrators (NM)' and 'migrators (M)'. Statistical analysis allowed us to identify considerable heterogeneity in the NM subset, that has not been previously reported. Interestingly, differences in 2D-packing appeared to affect CGF trends and heterogeneity of the migratory subsets for the datasets under investigation. Conclusion: We developed an analytical pipeline using open source tools, to identify and morphologically characterize functional migratory subsets from label-free, time-lapse migration data. Our quantitative approach identified a previously unappreciated heterogeneity of non-migratory cells and predicted the influence of 2D-packing on migration.

CENPU Promotes Growth and Metastasis in Nasopharyngeal Carcinoma by Inhibiting DUSP6

Background: Centromere protein U (CENPU), a centromere component, is key for mitosis and involved in the carcinogenesis of cancers. The role and mechanisms of CENPU in nasopharyngeal carcinoma (NPC) has not been described. Methods: CENPU expression in NPC cells and tissues was evaluated by RT-PCR and western blotting. Clinical significance of CENPU was evaluated by Immunohistochemistry. Biological functions of CENPU were evaluated by cell growth assay, colony formation assay, apoptosis assay, migration assay and invasion assays. Xenograft growth and lung metastasis model were conducted to investigate the effect of CENPU in vivo. Gene chip analysis, Ingenuity Pathway Analysis (IPA), and co-immunoprecipitation (Co-IP) experiments were used to explore the mechanisms of CENPU in NPC.Results: CENPU was highly expressed in NPC cells and samples. Patients with CENPU positive expression were closely associated with poor overall survival. Knockdown of CENPU inhibited proliferation and migration in vitro and in vivo in NPC. Gene chip analysis and IPA suggested that differentially expressed genes (DEGs) were significantly enriched in cancer and functions, including cellular movement, cellular development, cell growth and death, and proliferation when CENPU was downregulated. Dual specificity phosphatase 6 (DUSP6) was one of the DEGs and significantly decreased in NPC samples, and inversely correlated with expression with CENPU. Mechanism studies confirmed that CENPU increased the activation of ERK1/2 and p38 signal pathways by suppressing the expression of DUSP6. Therefore, our results suggested that CENPU might act as an oncogene in NPC and promote the development of NPC via inhibition of DUSP6, resulting in the inactivation of Erk1/2 and p38 pathways. Conclusions: CENPU facilitated cell proliferation and invasion by interacting with DUSP6. CENPU might be a promising prognostic biomarker and a potential target for NPC therapy.

ITGB1 Drives Hepatocellular Carcinoma Progression by Modulating Cell Cycle Process Through PXN/YWHAZ/AKT Pathways

Integrin β1 (ITGB1), which acts as an extracellular matrix (ECM) receptor, has gained increasing attention as a therapeutic target for the treatment of hepatocellular carcinoma (HCC). However, the underpinning mechanism of how ITGB1 drives HCC progression remains elusive. In this study, we first found that ITGB1 expression was significantly higher in HCC tissues than in normal controls by bioinformatics analysis. Furthermore, bioinformatics analysis revealed that paxillin (PXN) and 14-3-3 protein zeta (YWHAZ) are the molecules participating in ITGB1-regulated HCC tumor cell cycle progression. Indeed, immunohistochemistry (IHC) revealed that ITGB1, paxillin, and YWHAZ were strongly upregulated in paired HCC tissue compared with adjacent normal tissues. Notably, the inhibition of ITGB1 expression by small interfering RNA (siRNA) resulted in the downregulated expression of PXN and YWHAZ in primary HCC cells, as assessed by western blot and immunostaining. In addition, ITGB1 knockdown markedly impaired the aggressive behavior of HCC tumor cells and delayed cell cycle progression as determined by cell migration assay, drug-resistance analysis, colony formation assay, quantitative real-time polymerase chain reaction (qRT-PCR), and cell cycle analysis as well as cell viability measurements. More importantly, we proved that xenograft ITGB1high tumors grew more rapidly than ITGB1low tumors. Altogether, our study showed that the ITGB1/PXN/YWHAZ/protein kinase B (AKT) axis enhances HCC progression by accelerating the cell cycle process, which offers a promising approach to halt HCC tumor growth.

NDUFC1 Is Upregulated in Gastric Cancer and Regulates Cell Proliferation, Apoptosis, Cycle and Migration

Gastric cancer is one of the most common primary tumors of the digestive system. NADH: ubiquinone oxidoreductase subunit C1 (NDUFC1), which is an accessory subunit of the NADH dehydrogenase (complex I), is responsible for the transportation of electrons from NADH to the respiratory chain essential for the oxidative phosphorylation. However, little is known about the roles of NDUFC1 in carcinogenesis. In this study, NDUFC1 protein level in NSCLC tissues was tested by immunohistochemistry (IHC) staining. NDUFC1 mRNA level in gastric cancer cell lines was determined by qRT-PCR. MGC-803 and SGC-7901 cells were transfected with shNDUFC1 lentivirus designed to silence NDUFC1. MTT assay, CCK8 assay, wound healing assay and transwell migration assay were conducted. Cell cycle and apoptosis were detected by flow cytometry. In vivo experiments were performed using nude mice. The results indicated that overexpressed NDUFC1 in gastric cancer was related to more serious tumor infiltrates, a higher risk of lymphatic metastasis, a higher proportion of positive lymph nodes, and a more advanced tumor stage. Compared with shCtrl groups, MGC-803 and SGC-7901 of shNDUFC1 groups had lower abilities of proliferation and migration, higher levels of apoptosis. NDUFC1 knockdown also inhibited SGC-7901 cell growth in vivo and suppressed Ki67 expression in xenograft tumors. More importantly, we found that NDUFC1 downregulation made the levels of P-Akt, P-mTOR, CCND1, CDK6, PIK3CA, Bcl-2, Survivin, and XIAP decreased, and that PI3K/AKT signaling pathway agonist SC79 rescued the inhibitory effects on cell proliferation and migration, reversed the promoted effects on cell apoptosis caused by NDUFC1 knockdown. More importantly, compared with NDUFC1 knockdown group, the expression of P-Akt, Bcl-2, Survivin, and XIAP was raised in shNDUFC1 + SC79 group. Thus, our suspicion was that NDUFC1 exacerbates NSCLC progression via PI3K/Akt pathway. Taken together, our study indicated that targeting NDUFC1 could open innovative perspectives for new multi-targeting approaches in the treatment of gastric cancer.

Effect of Hydroxyapatite Microspheres, Amoxicillin–Hydroxyapatite and Collagen–Hydroxyapatite Composites on Human Dental Pulp-Derived Mesenchymal Stem Cells

In this study, the preparation and characterization of three hydroxyapatite-based bioactive scaffolds, including hydroxyapatite microspheres (HAps), amoxicillin–hydroxyapatite composite (Amx–HAp), and collagen–hydroxyapatite composite (Col–HAp) were performed. In addition, their behavior in human dental pulp mesenchymal stem cell (hDPSC) culture was investigated. HAps were synthesized through the following methods: microwave hydrothermal, hydrothermal reactor, and precipitation, respectively. hDPSCs were obtained from samples of third molars and characterized by immunophenotypic analysis. Cells were cultured on scaffolds with osteogenic differentiation medium and maintained for 21 days. Cytotoxicity analysis and migration assay of hDPSCs were evaluated. After 21 days of induction, no differences in genes expression were observed. hDPSCs highly expressed the collagen IA and the osteonectin at the mRNA. The cytotoxicity assay using hDPSCs demonstrated that the Col–HAp group presented non-viable cells statistically lower than the control group (p = 0.03). In the migration assay, after 24 h HAps revealed the same migration behavior for hDPSCs observed compared to the positive control. Col–HAp also provided a statistically significant higher migration of hDPSCs than HAps (p = 0.02). Migration results after 48 h for HAps was intermediate from those achieved by the control groups. There was no statistical difference between the positive control and Col–HAp. Specifically, this study demonstrated that hydroxyapatite-based bioactive scaffolds, especially Col-Hap, enhanced the dynamic parameters of cell viability and cell migration capacities for hDPSCs, resulting in suitable adhesion, proliferation, and differentiation of this osteogenic lineage. These data presented are of high clinical importance and hold promise for application in therapeutic areas, because Col–HAp can be used in ridge preservation, minor bone augmentation, and periodontal regeneration. The development of novel hydroxyapatite-based bioactive scaffolds with clinical safety for bone formation from hDPSCs is an important yet challenging task both in biomaterials and cell biology.

Tumor-associated macrophages induced spheroid formation by CCL18-ZEB1-M-CSF feedback loop to promote transcoelomic metastasis of ovarian cancer

BackgroundOvarian cancer (OvCa)-tumor-associated macrophages (TAMs) spheroids are abundantly present within ascites of high malignant patients. This study investigated the mutual interaction of OvCa cells and TAMs in the spheroids.MethodsThree-dimensional coculture system and transwell coculture system were created to mimic the OvCa and TAMs in spheroids and in disassociated state. Transwell-migration assay and scratch wound healing assay were used to measure the invasive and migratory capacity. Western blot, quantitative reverse transcription-PCR and immunostaining were used to measure the mesenchymal and epithelial markers. Flow cytometry was used to assess the polarization of TAMs. Also, the differential gene expression profile of OvCa cells and OvCa cells from spheroids were tested by RNA-sequence. Finally, the ovarian mice models were constructed by intraperitoneal injection of ID8 or OvCa-TAMs spheroids.ResultsOur results indicated that the formation of OvCa-TAMs spheroids was positive related to the malignancy of OvCa cells. M2-TAMs induced the epithelial-mesenchymal transition of OvCa cells by releasing chemokine (C-C motif) ligand 18 (CCL18) in the spheroids. While, CCL18 induced macrophage colony-stimulating factor (M-CSF) transcription in OvCa cells through zinc finger E-box-binding homeobox 1 (ZEB1). This study further indicated that M-CSF secreted by OvCa cells drived the polarization of M2-TAMs. Therefore, a CCL18-ZEB1-M-CSF interacting loop between OvCa cells and TAMs in the spheroids was identified. Moreover, with blocking the expression of ZEB1 in the OvCa cell, the formation of OvCa-TAMs spheroids was impeded. In the ovarian mice models, the formation of OvCa-TAMs spheroids in the ascites was promoted by overexpressing of ZEB1 in OvCa cells, which resulted in faster and earlier transcoelomic metastasis.ConclusionThese findings suggested that the formation of OvCa-TAMs spheroids resulted in aggressive phenotype of OvCa cells, as a specific feedback loop CCL18-ZEB1-M-CSF in it. Inhibition of ZEB1 reduced OvCa-TAMs spheroids in the ascites, impeding the transcoelomic metastasis and improving the outcome of ovarian patients.

Genistein Mitigates High Glucose-Induced Podocyte Adhesion Injury by Modulating Autophagy via Mtor Signal

Purpose: The study aimed to investigate the characteristics of autophagy on podocyte under high glucose (HG) conditions and further explore the effect of Genistein on podocyte autophagy，adhesion and the potential mechanism.Materials and methods: CCK-8 was used to detect the viability of podocyte. The level of autophagy was mainly detected by western blot and immunofluorescence. The expression of autophagy related factors and podocyte adhesion markers, including LC3-II, p62, p-mTOR and integrin β1-MF, were detected by immunofluorescence at 0，6，12，24，36，48，72h. The expression levels of proteins in the LC3-II, p62, p-mTOR/mTOR, integrin β1-MF were further investigated by western blot. Wound healing test and cell migration assay were used to detect podocyte adhesion ability.Results: The present study showed that HG-induced podocyte viability was reduced significantly for 6 h. Decreased integrin β1-MF, LC3-II, increased p62 and abnormal activation of the mTOR signal was detected in podocyte under HG conditions. Genistein restored podocyte viability and up-regulated integrin β1-MF, LC3-II expression, down-regulated p62, p-mTOR expression. Moreover, the HG-induced podocyte adhesion injury was abrogated by treatment with Genistein.Conclusion：Our results demonstrated that podocyte adhesion injury in HG environment was related to the decrease of autophagy level. Genistein activated podocyte autophagy by inhibiting the mTOR signaling pathway, regulated the renewal expression of integrin β1-MF, and finally reduced HG-induced podocyte adhesion injury.

Antitumor Effect of Sclerostin against Osteosarcoma

Various risk factors and causative genes of osteosarcoma have been reported in the literature; however, its etiology remains largely unknown. Bone formation is a shared phenomenon in all types of osteosarcomas, and sclerostin is an extracellular soluble factor secreted by osteocytes that prevents bone formation by inhibiting the Wnt signaling pathway. We aimed to investigate the antitumor effect of sclerostin against osteosarcoma. Osteosarcoma model mice were prepared by transplantation into the dorsal region of C3H/He and BALB/c-nu/nu mice using osteosarcoma cell lines LM8 (murine) and 143B (human), respectively. Cell proliferations were evaluated by using alamarBlue and scratch assays. The migratory ability of the cells was evaluated using a migration assay. Sclerostin was injected intraperitoneally for 7 days to examine the suppression of tumor size and extension of survival. The administration of sclerostin to osteosarcoma cells significantly inhibited the growth and migratory ability of osteosarcoma cells. Kaplan–Meier curves and survival data demonstrated that sclerostin significantly inhibited tumor growth and improved survival. Sclerostin suppressed the proliferative capacity and migratory ability of osteosarcoma cells. Osteosarcoma model mice inhibited tumor growth and prolonged survival periods by the administration of sclerostin. The effect of existing anticancer drugs such as doxorubicin should be investigated for future clinical applications.

Coating of Polystyrene Surface with REDV-Gelatin Conjugate for Selective Isolation of Endothelial Cells

Endothelial cells (EC), which line the internal surface of blood vessels, play various essential roles in controlling vascular function. The mouse is an important animal model for the study of vascular biology and cardiovascular diseases. However, the isolation of primary EC from the murine aorta is challenging because they are readily contaminated by smooth muscle cells (SMC). A previous study developed a simple method to isolate murine EC from SMC. By taking advantage of the differential sedimentation rate between the two cells, the EC was selectively enriched with collagen-coated polystyrene surfaces. Our study further improved this method by introducing a biomimetic peptide REDV (Arg-Glu-Asp-Val), which may bind specifically to EC but not to SMC or fibroblasts. Firstly, REDV-gelatin conjugate was synthesized by using the amine-to-sulfhydryl crosslinker SMCC. REDV-gelatin coating was then prepared on polystyrene surfaces, and their affinities to EC and SMC were subsequently investigated. Fluorescence microscopy and flow cytometric analysis showed that EC adhesion to the gelatin coating was significantly promoted by REDV peptide conjugation. Moreover, cell migration assay and cell viability assay also showed that the conjugation of REDV does not affect EC migration, and this coating did not show cytotoxicity against EC. This gelatin-REDV coating provides a cost-effective and straightforward tool for isolating EC from SMC, which may facilitate in vitro investigations of EC from mice.