Effect of passage number of genetically modified TGF-β3 expressing primary chondrocytes on the chondrogenesis of ATDC5 cells in a 3D coculture system

Due to the lack of blood vessels, nerves and lymphatics, articular cartilage is difficult to repair once damaged. Tissue engineering is considered to be a potential strategy for cartilage regeneration. Successful tissue engineering strategies depend on the effective combination of biomaterials, seed cells and biological factors. In our previous study, a genetically modified coculture system with chondrocytes and ATDC5 cells in an alginate hydrogel has exhibited a superior ability to enhance chondrogenesis. In this study, we further evaluated the influence of chondrocytes at various passages on chondrogenesis in the coculture system. The results demonstrated that transfection efficiency was hardly influenced by the passage of chondrocytes. The coculture system with passage 5 (P5) chondrocytes had a better effect on chondrogenesis of ATDC 5 cells, while chondrocytes in this coculture system presented higher levels of dedifferentiation than other groups with P1 or P3 chondrocytes. Therefore, P5 chondrocytes were shown to be more suitable for the coculture system, as they accumulated in sufficient cell numbers with more passages and had a higher level of dedifferentiation, which was prone to form a favorable niche for chondrogenesis of ATDC5 cells. This study may provide fresh insights for future cartilage tissue engineering strategies with a combination of a coculture system and advanced biomaterials.

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.

Analysis of the Influence of Jaw Periosteal Cells on Macrophages Phenotype Using an Innovative Horizontal Coculture System

Jaw periosteum-derived mesenchymal stem cells (JPCs) represent a promising cell source for bone tissue engineering in oral and maxillofacial surgery due to their high osteogenic potential and good accessibility. Our previous work demonstrated that JPCs are able to regulate THP-1-derived macrophage polarization in a direct coculture model. In the present study, we used an innovative horizontal coculture system in order to understand the underlying paracrine effects of JPCs on macrophage phenotype polarization. Therefore, JPCs and THP-1-derived M1/M2 macrophages were cocultured in parallel chambers under the same conditions. After five days of horizontal coculture, flow cytometric, gene and protein expression analyses revealed inhibitory effects on costimulatory and proinflammatory molecules/factors as well as activating effects on anti-inflammatory factors in M1 macrophages, originating from multiple cytokines/chemokines released by untreated and osteogenically induced JPCs. A flow cytometric assessment of DNA synthesis reflected significantly decreased numbers of proliferating M1/M2 cells when cocultured with JPCs. In this study, we demonstrated that untreated and osteogenically induced JPCs are able to switch macrophage polarization from a classical M1 to an alternative M2-specific phenotype by paracrine secretion, and by inhibition of THP-1-derived M1/M2 macrophage proliferation.

Metformin Downregulates PD-L1 Expression in Esophageal Squamous Cell Catrcinoma by Inhibiting IL-6 Signaling Pathway

PurposeTo characterize the mechanism by which metformin inhibits PD-L1 expression in esophageal squamous cell carcinoma (ESCC) and to evaluate the effect of metformin on the antitumor immune response.MethodsThe Cancer Genome Atlas (TCGA) database was used to analyze the correlations between IL-6 and prognosis and between IL-6 and PD-L1 gene expression in esophageal cancer. Reverse transcription-quantitative polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence were used to study the mechanism by which metformin affects PD-L1 expression. Additionally, T cell function was assessed in a coculture system containing ESCC cells and peripheral blood mononuclear cells (PBMCs) treated with metformin or IL-6. In an in vivo assay, we used a model established with NPIdKO™ mice, which have a reconstituted immune system generated by transplanting PBMCs through intravenous injection, to evaluate the effect of metformin on tumors.ResultsThe TCGA esophageal cancer data showed that IL-6 expression was positively correlated with PD-L1 expression and that patients with high IL-6 expression had a significantly lower overall survival rate than patients with low IL-6 expression. PD-L1 expression in ESCC cell lines was significantly inhibited by metformin via the IL-6/JAK2/STAT3 signaling pathway but was not correlated with the canonical AMPK pathway. In the coculture system, the metformin pretreatment group showed higher T cell activation and better T cell killing function than the control group. Animal experiments confirmed that metformin downregulated PD-L1 expression and that combination treatment with metformin and PD-1 inhibitors synergistically enhanced the antitumor response.ConclusionsMetformin downregulated PD-L1 expression by blocking the IL-6/JAK2/STAT3 signaling pathway in ESCC, which enhanced the antitumor immune response.

Flagella, Type I Fimbriae and Curli of Uropathogenic Escherichia coli Promote the Release of Proinflammatory Cytokines in a Coculture System

Background. Urinary tract infections (UTIs) are a public health problem in Mexico, and uropathogenic Escherichia coli (UPEC) is one of the main etiological agents. Flagella, type I fimbriae, and curli promote the ability of these bacteria to successfully colonize its host. Aim. This study aimed to determine whether flagella-, type I fimbriae-, and curli-expressing UPEC induces the release of proinflammatory cytokines in an established coculture system. Methods. The fliC, fimH, and csgA genes by UPEC strain were disrupted by allelic replacement. Flagella, type I fimbriae, and curli were visualized by transmission electron microscopy (TEM). HTB-5 (upper chamber) and HMC-1 (lower chamber) cells cocultured in Transwell® plates were infected with these UPEC strains and purified proteins. There was adherence to HTB-5 cells treated with different UPEC strains and they were quantified as colony-forming units (CFU)/mL. Results. High concentrations of IL-6 and IL-8 were induced by the FimH and FliC proteins; however, these cytokines were detected in low concentrations in presence of CsgA. Compared with UPEC CFT073, CFT073ΔfimH, CFT073ΔfimHΔfliC, and CFT073ΔcsgAΔfimH strains significantly reduced the adherence to HTB-5 cells. Conclusion. The FimH and FliC proteins are involved in IL-6 and IL-8 release in a coculture model of HTB-5 and HMC-1 cells.

Neuroblastoma and DIPG Organoid Coculture System for Personalized Assessment of Novel Anticancer Immunotherapies

Cancer immunotherapy has transformed the landscape of adult cancer treatment and holds a great promise to treat paediatric malignancies. However, in vitro test coculture systems to evaluate the efficacy of immunotherapies on representative paediatric tumour models are lacking. Here, we describe a detailed procedure for the establishment of an ex vivo test coculture system of paediatric tumour organoids and immune cells that enables assessment of different immunotherapy approaches in paediatric tumour organoids. We provide a step-by-step protocol for an efficient generation of patient-derived diffuse intrinsic pontine glioma (DIPG) and neuroblastoma organoids stably expressing eGFP-ffLuc transgenes using defined serum-free medium. In contrast to the chromium-release assay, the new platform allows for visualization, monitoring and robust quantification of tumour organoid cell cytotoxicity using a non-radioactive assay in real-time. To evaluate the utility of this system for drug testing in the paediatric immuno-oncology field, we tested our in vitro assay using a clinically used immunotherapy strategy for children with high-risk neuroblastoma, dinutuximab (anti-GD2 monoclonal antibody), on GD2 proficient and deficient patient-derived neuroblastoma organoids. We demonstrated the feasibility and sensitivity of our ex vivo coculture system using human immune cells and paediatric tumour organoids as ex vivo tumour models. Our study provides a novel platform for personalized testing of potential anticancer immunotherapies for aggressive paediatric cancers such as neuroblastoma and DIPG.

The Adipose-Derived Stem Cell and Endothelial Cell Coculture System—Role of Growth Factors?

Adequate vascularization is a fundamental prerequisite for bone regeneration, formation and tissue engineering applications. Endothelialization of scaffold materials is a promising strategy to support neovascularization and bone tissue formation. Besides oxygen and nutrition supply, the endothelial network plays an important role concerning osteogenic differentiation of osteoprogenitor cells and consecutive bone formation. In this study we aimed to enhance the growth stimulating, proangiogenic and osteogenic features of the ADSC and HUVEC coculture system by means of VEGFA165 and BMP2 application. We were able to show that sprouting phenomena and osteogenic differentiation were enhanced in the ADSC/HUVEC coculture. Furthermore, apoptosis was unidirectionally decreased in HUVECs, but these effects were not further enhanced upon VEGFA165 or BMP2 application. In summary, the ADSC/HUVEC coculture system per se is a powerful tool for bone tissue engineering applications.

Liver Organoid and T Cell Coculture Models Cytotoxic T Cell Responses against Hepatitis C Virus

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV nonstructural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

Acinetobacter tandoii ZM06 Assists Glutamicibacter nicotianae ZM05 in Resisting Cadmium Pressure to Preserve Dipropyl Phthalate Biodegradation

Dipropyl phthalate (DPrP) coexists with cadmium as cocontaminants in environmental media. A coculture system including the DPrP-degrading bacterium Glutamicibacter nicotianae ZM05 and the nondegrading bacterium Acinetobacter tandoii ZM06 was artificially established to degrade DPrP under Cd(II) stress. Strain ZM06 relieved the pressure of cadmium on strain ZM05 and accelerated DPrP degradation in the following three ways: first, strain ZM06 adsorbed Cd(II) on the cell surface (as observed by scanning electron microscopy) to decrease the concentration of Cd(II) in the coculture system; second, the downstream metabolites of ZM05 were utilized by strain ZM06 to reduce metabolite inhibition; and third, strain ZM06 supplied amino acids and fatty acids to strain ZM05 to relieve stress during DPrP degradation, which was demonstrated by comparative transcriptomic analysis. This study provides an elementary understanding of how microbial consortia improve the degradation efficiency of organic pollutants under heavy metals contamination.