Co-culture Of Cells Research Articles

In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma

BackgroundChimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immunosuppression in the tumor microenvironment. CAR-T cell exhaustion is a major cause of relapse. Epigenetic modifications can regulate T cell activation, maturation and depletion; they can be applied to reduce T cell depletion, improve infiltration, and promote memory phenotype formation to reduce relapse after CAR-T cell therapy.PurposeWe propose to develop and validate in vitro the function of novel CAR-T cells for the treatment of DLBCL, which simultaneously express an anti-CD19 CAR with lysine-specific demethylase 1 (LSD1) short hairpin (sh)RNA to prevent depletion and prolong the survival of CAR-T cells.MethodsWe designed an shRNA sequence targeting LSD1 mRNA, and created a vector with the following elements: the U6 promoter driving expression of the LSD1 shRNA sequence, the EF1a promoter driving a second-generation anti-CD19 CAR sequence encoding an anti-CD19 single-chain variable fragment (FMC63), the CD8 hinge and transmembrane structural domains, the CD28 co-stimulatory structural domain, and the CD3ζ-activating structural domain. The MFG-LSD1 shRNA anti-CD19 CAR plasmid was first constructed, then packaged in retroviral vectors and transduced into human primary peripheral blood mononuclear cell-derived T cells to generate the corresponding CAR-T cells. We examined by flow cytometry the efficiency of two CAR-T cells in killing U-2932 cells (a human DLBCL line) upon co-culture with RNAU6 anti-CD19 CAR-T cells or LSD1 shRNA anti-CD19 CAR-T cells. We analyzed Ki-67 staining of the CAR-T cells by flow cytometry on days 0, 5, and 10, and counted the cells to assess expansion. We also used flow cytometry to detect the central memory T cell (TCM) proportion.ResultsWe detected the expression of the CAR in the CAR-T cells by flow cytometry, and observed transduction rates of 31.5% for RNAU6 anti-CD19 CAR-T cells and 60.7% for LSD1 shRNA anti-CD19 CAR-T cells. The killing efficiency of LSD1 shRNA anti-CD19 CAR-T cells was significantly higher than that of RNAU6 anti-CD19 CAR-T cells at the low effector target ratio. We further found that LSD1 shRNA anti-CD19 CAR-T cells secreted more IFN-γ and granzyme B than RNAU6 anti-CD19 CAR-T cells. CAR-T cells proliferated after U-2932 cell stimulation and were able to sustain proliferation. After stimulation via U-2932 cell co-culture, both RNAU6 anti-CD19 CAR-T and LSD1 shRNA anti-CD19 CAR-T populations had increased proportions of cells with the TCM phenotype, with a higher percentage among LSD1 shRNA anti-CD19 CAR-T cells.ConclusionWe developed a novel, feasible CD19-LSD1 shRNA CAR-T cell strategy for the treatment of DLBCL. Our in vitro assay results showed that LSD1 shRNA anti-CD19 CAR-T cells more effectively killed target cells than RNAU6 anti-CD19 CAR-T cells, and developed a higher proportion of TCM phenotype cells. LSD1 shRNA anti-CD19 CAR-T cells may represent a potential treatment for DLBCL.

Schisanhenol Inhibits the Proliferation of Hepatocellular Carcinoma Cells by Targeting Programmed Cell Death-ligand 1 via the STAT3 Pathways.

Programmed cell death-ligand 1 (PD-L1) is overexpressed in tumor cells, which promotes tumor cell survival and cell proliferation and causes tumor cells to escape T-cell killing. Schisanhenol, a biphenyl cyclooctene lignin-like compound, was extracted and isolated from the plant named Schisandra rubriflora (Franch.). In this work, we studied the anticancer potential of schisanhenol and explored whether schisanhenol mediated its effect by inhibiting the expression of PD-L1 in vitro and in vivo. In vitro, we performed western blot, immunofluorescence, immunoprecipitation, and colony formation assays to study the proteins, genes, and pathways related to the anti-tumour activity of schisanhenol. In vivo, we explored the antitumor activity of schisanhenol through orthotopic liver transplantation and subcutaneous transplantation tumor models of hepatocellular carcinoma (HCC) cells. We found that schisanhenol decreased the viability of HCC cells. It inhibited the expression of programmed cell death ligand-1 (PD-L1), which plays a pivotal role in tumorigenesis. Subsequently, schisanhenol suppressed the expression of PD-L1 by decreasing the activation of STAT3. Furthermore, we found that schisanhenol inhibited the activation of STAT3 via JAK/STAT3 (T705), Src/STAT3 (T705), and PI3K/AKT/mTOR/STAT3 (S727) pathways. Colony formation tests showed that schisanhenol suppressed cell proliferation by inhibiting PD-L1. Schisanhenol also enhanced cytotoxic T lymphocytes (CTL) activity and regained their ability to kill tumour cells in co-culture. Finally, in vivo observation confirmed the antitumor activity of schisanhenol. Schisanhenol inhibits the proliferation of HCC cells by targeting PD-L1 via the STAT3 pathways. These findings prove that schisanhenol is a valuable candidate for HCC therapeutics and reveal previously unknown characteristics of schisanhenol.

Stimulator of Interferon Genes Signal in Lung Cancer Regulates Differentiation of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment Via the Interferon Regulatory Factor 3/NF-κB Pathway.

Background: This study was designed to explore the action mechanism of stimulator of interferon genes (STING) on the differentiation of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment of lung cancer. Methods: Bioinformatics analysis yielded a potential pathway for STING to regulate MDSC differentiation, the interferon regulatory factor 3 (IRF3)/NF-κB axis. The transfection efficiency of STING overexpression plasmid and small interfering RNA against IRF3 (siIRF3) was examined by quantitative real-time polymerase chain reaction (qRT-PCR). After transfection, A9 cells were co-cultured with extracted bone marrow cells (BMCs). MDSC differentiation, protein expression of the IRF3/NF-κB pathway, and changes in nuclear translocation of NF-κB were analyzed by flow cytometry, Western blot, and immunofluorescence staining experiments. A transplanted tumor mouse model was used for invivo experiments. After cyclic diadenyl monophosphate (CDA; STING agonist) treatment, changes in MDSC differentiation and protein expression of the IRF3/NF-κB axis in transplanted tumors were verified by immunohistochemical staining, qRT-PCR, and Western blot. Results: Coculture of A9 cells and BMCs promoted MDSC differentiation, inhibited activation of IRF3/NF-κB signal in A9 cells, and boosted nuclear translocation of NF-κB. However, after the upregulation of STING, IRF3/NF-κB signal was activated, while MDSC differentiation and nuclear translocation of NF-κB were inhibited. SiIRF3 reversed the effects of STING overexpression. In vivo, CDA dampened MDSC differentiation and promoted protein expression of the IRF3/NF-κB axis. Conclusion: STING signal in lung cancer cells inhibits MDSC differentiation through activation of the IRF3/NF-κB pathway.

MAP4 kinase-regulated reduced CLSTN1 expression in medulloblastoma is associated with increased invasiveness

De-regulated protein expression contributes to tumor growth and progression in medulloblastoma (MB), the most common malignant brain tumor in children. MB is associated with impaired differentiation of specific neural progenitors, suggesting that the deregulation of proteins involved in neural physiology could contribute to the transformed phenotype in MB. Calsynthenin 1 (CLSTN1) is a neuronal protein involved in cell-cell interaction, vesicle trafficking, and synaptic signaling. We previously identified CLSTN1 as a putative target of the pro-invasive kinase MAP4K4, which we found to reduce CLSTN1 surface expression. Herein, we explored the expression and functional significance of CLSTN1 in MB. We found that CLSTN1 expression is decreased in primary MB tumors compared to tumor-free cerebellum or brain tissues. CLSTN1 is expressed in laboratory-established MB cell lines, where it localized to the plasma membrane, intracellular vesicular structures, and regions of cell-cell contact. The reduction of CLSTN1 expression significantly increased growth factor-driven invasiveness. Pharmacological inhibition of pro-migratory MAP4 kinases caused increased CLSTN1 expression and CLSTN1 accumulation in cell-cell contacts. Co-culture of tumor cells with astrocytes increased CLSTN1 localization in cell-cell contacts, which was further enhanced by MAP4K inhibition. Our study revealed a repressive function of CLSTN1 in growth-factor-driven invasiveness in MB, identified MAP4 kinases as repressors of CLSTN1 recruitment to cell-cell contacts, and points towards CLSTN1 implication in the kinase-controlled regulation of tumor-microenvironment interaction.

A Novel In Vitro Model of the Bone Marrow Microenvironment in Acute Myeloid Leukemia Identifies CD44 and Focal Adhesion Kinase as Therapeutic Targets to Reverse Cell Adhesion-Mediated Drug Resistance.

Acute myeloid leukemia (AML) is an aggressive neoplasm. Although most patients respond to induction therapy, they commonly relapse due to recurrent disease in the bone marrow microenvironment (BMME). So, the disruption of the BMME, releasing tumor cells into the peripheral circulation, has therapeutic potential. Using both primary donor AML cells and cell lines, we developed an in vitro co-culture model of the AML BMME. We used this model to identify the most effective agent(s) to block AML cell adherence and reverse adhesion-mediated treatment resistance. We identified that anti-CD44 treatment significantly increased the efficacy of cytarabine. However, some AML cells remained adhered, and transcriptional analysis identified focal adhesion kinase (FAK) signaling as a contributing factor; the adhered cells showed elevated FAK phosphorylation that was reduced by the FAK inhibitor, defactinib. Importantly, we demonstrated that anti-CD44 and defactinib were highly synergistic at diminishing the adhesion of the most primitive CD34high AML cells in primary autologous co-cultures. Taken together, we identified anti-CD44 and defactinib as a promising therapeutic combination to release AML cells from the chemoprotective AML BMME. As anti-CD44 is already available as a recombinant humanized monoclonal antibody, the combination of this agent with defactinib could be rapidly tested in AML clinical trials.

Influence of Seven Ion Species on Osteogenic Differentiation of Mesenchymal Stem Cells Stimulated by Macrophages in Indirect and Direct Coculture Systems.

Implanted biomaterials release inorganic ions that trigger inflammatory responses, which recruit immune cells whose biochemical signals affect bone tissue regeneration. In this study, we evaluated how mouse macrophages (RAW264, RAW) and mesenchymal stem cells (KUSA-A1, MSCs) respond to seven types of ions (silicon, calcium, magnesium, zinc, strontium, copper, and cobalt) that reportedly stimulate cells related to bone formation. The collagen synthesis, alkaline phosphatase activity, and osteocalcin production of the MSCs varied by ion dose and type after culture in the secretome of RAW cells. However, DNA production was relatively unaffected. The MSC secretome may also stimulate RAW cells in coculture and, therefore, affect osteogenic differentiation of MSCs. Overall, the ions often exerted different effects on each cell type. This study guides future work that explores the mechanisms behind ion-dependent osteogenic differentiation and cell functions.

Protocol for Immune Cell Isolation, Organoid Generation, and Co-culture Establishment from Cryopreserved Whole Human Intestine.

The human intestine plays a pivotal role in nutrient absorption and immune system regulation. Along the longitudinal axis, cell-type composition changes to meet the varying functional requirements. Therefore, our protocol focuses on the processing of the whole human intestine to facilitate the analysis of region-specific characteristics such as tissue architecture and changes in cell populations. We describe how to generate a biobank that can be used to isolate specific immune cell subtypes, generate organoid lines, and establish autologous immune cell-organoid co-cultures. Key features • Dissection and tissue analysis of whole human intestines. • Cryopreservation for biobank generation. • Optimized protocols for the isolation of epithelial and immune cells. • Autologous co-culture of organoids and lamina propria-derived immune cells.

Research on curcumin mediating immunotherapy of colorectal cancer by regulating cancer associated fibroblasts.

The objective was to investigate curcumin's (Cur) function and associated molecular mechanisms in regulating tumor immunity in colon cancer. Primary cancer-associated fibroblasts (CAFs) from mouse CT26 colon cancer tumors were isolated. Validation of primary CAFs using immunofluorescence assay was done. Cell Counting Kit-8 experiments, real-time quantitative PCR (qPCR), and enzyme linked immunosorbent assay experiments were conducted to investigate how curcumin affected the growth and cytokine secretion functions of CAFs. The effect of curcumin on regulating PD-L1 expression on CT26 cells through CAFs in vitro was explored through coculture of CAFs and tumor cells, qPCR, and western blot experiments. A mouse colon cancer cell model was established in Balb/c nude mice to explore the effect of curcumin on colon tumor cells. Changes in the tumor microenvironment were detected by flow cytometry to explore the synergistic effect of curcumin combined with anti-PD-1 monoclonal antibody in the treatment of mouse colon cancer. In vitro, curcumin prevented the growth and TGF-β secretion of CT26 cells. At the same time, curcumin inhibited the secretion of TGF-β by CAFs, thereby downregulating the PD-L1 expression of CT26 cells. In vivo, curcumin combined with anti-PD-1 antibodies can further enhance the inhibitory effect of PD-1 antibodies on tumors and increase the number of tumor-suppressing immune cells in the tumor microenvironment, such as M1 macrophages and CD8 T cells, thus inhibiting tumors. Immune M2 macrophages, regulatory T cells, and other cells were reduced. In conclusion, curcumin reduces the expression of PD-L1 in colon cancer cells and improves the tumor immune microenvironment by inhibiting the proliferation of CAFs and the secretion of TGF-β. Curcumin and anti-PD-1 treatment have synergistic inhibitory effects on colon cancer.

Chitosan/Alginate-Based Hydrogel Loaded With VE-Cadherin/FGF as Scaffolds for Wound Repair in Different Degrees of Skin Burns.

Burns are complex traumatic injuries that lead to severe physical and psychological problems due to the prolonged healing period and resulting physical scars. Owing to their versatility, hydrogels can be loaded with various functional factors, making them promising wound dressings. However, many hydrogel dressings cannot support cell survival for a long time, thereby delaying the process of tissue repair. Herein, based on chitosan (CS)/alginate (SA)/poly(ethylene glycol) diacrylate (PEGDA), a basic hydrogel with hemostasis and antibacterial properties was prepared, and loaded with vascular endothelial cadherin (VE-cadherin) and fibroblast growth factors (FGF) to promote the co-culture of various skin cells, suitable for treating various skin injury types: (1) Construct a three-dimensional microenvironment conducive to the release of drugs and factors using natural biological macromolecules CS/SA. (2) Promote the cell growth by loading growth factors. (3) Establish skin burn models of different degrees and observe the repair process. From the results, the 3D microenvironment provided by hydrogel could support the active growth of cells for 12 days. Furthermore, deep burns with full-thickness skin were substantially repaired within about 24 days. Collectively, CS/SA hydrogel containing VE-cadherin and FGF can promote tissue healing in wounds with necrotic tissue, making it an ideal candidate for burn treatment.

Vascularized tumor on a microfluidic chip to study mechanisms promoting tumor neovascularization and vascular targeted therapies.

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature, as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of cancer spheroids and endothelial cells in a three dimensional environment. Methods: We investigated both, tumor neovascularization and therapy, via co-culture of human derived endothelial cells and adjacently localized metastatic renal cell carcinoma spheroids on a commercially available microfluidic chip system. Metastatic renal cell carcinoma spheroids adjacent to primary vessels model tumor, and induce vessels to sprout neovasculature towards the tumor. We monitored real time changes in vessel formation, probed the interactions of tumor and endothelial cells, and evaluated the role of important effectors in tumor vasculature. In addition to wild type endothelial cells, we evaluated endothelial cells that overexpress Prostate Specific Membrane Antigen (PSMA), that has emerged as a marker of tumor associated neovasculature. We characterized the process of neovascularization on the microfluidic chip stimulated by enhanced culture medium and the investigated metastatic renal cell carcinomas, and assessed endothelial cells responses to vascular targeted therapy with bevacizumab via confocal microscopy imaging. To emphasize the potential clinical relevance of metastatic renal cell carcinomas on chip, we compared therapy with bevacizumab on chip with an in vivo model of the same tumor. Results: Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprouted towards the tumor and mimicked a vascular network. Bevacizumab, an antiangiogenic agent, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Conclusion: Observations in the vascularized tumor on chip permitted direct and conclusive quantification of vascular targeted therapies in weeks as opposed to months in a comparable animal model, and bridged the gap between in vitro and in vivo models.

Intranasal insulin administration affecting perioperative neurocognitive dysfunction by regulating calcium transport protein complex IP3R/GRP75/VDAC1 on MAMs.

Male C57BL/6J mice underwent partial hepatectomy to induce PND and were subsequently treated with either intranasal insulin or saline. Cognitive function was evaluated using the Morris water maze test, and hippocampal tissue was analyzed for calcium transport protein complex IP3R/GRP75/VDAC1 expression and apoptosis markers. In vitro, HT22 and BV2 cell co-cultures were utilized to simulate surgical injury, with IP3R knockdown employed to assess its effects on oxidative stress and apoptosis. Intranasal insulin effectively alleviated cognitive impairment as demonstrated by improved performance in the Morris water maze. It significantly reduced neuronal apoptosis and modulated the expression of the IP3R/GRP75/VDAC1 complex, enhancing mitochondrial ATP production and stabilizing MAMs. Furthermore, insulin administration also increased PI3K/AKT signaling, counteracting the impact of surgical stress. In vitro experiments confirmed that IP3R knockdown mitigated inflammation-induced oxidative stress and neuronal apoptosis, while insulin's beneficial effects were blocked by inhibition of the PI3K/AKT pathway. Intranasal insulin mitigates PND by modulating the IP3R/GRP75/VDAC1 complex and enhancing mitochondrial function through the PI3K/AKT signaling pathway. This study supports the potential of intranasal insulin as a promising therapeutic strategy for preventing and managing PND, potentially leading to improved surgical outcomes for elderly patients.

EFFECTIVENESS OF STEM CELL THERAPY IN MYOCARDIAL INFARCTION BASED ON ENZYME ACTIVITY AND LIVER HISTOPATOLOGY

This research aims to study the effectiveness of single placenta stem cell therapy (ST) or a combination of cardiomyocyte co-cultures in pigs with myocardial infarction based on enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity as well as liver histopathology. Nine pigs were divided into three treatment groups (K1, K2, and K3) consisting of three pigs per group. Blood sampling was performed one-hour pre-surgery, post-ST-elevation, and eight weeks post-therapy. The experimental animals were treated for eight weeks, then euthanized and necropsied for liver removal. ALT and AST values were analyzed using one-way analysis of variance (ANOVA) and followed by Tukey's test. Histopathological changes in liver tissue were analyzed using Image J 1.53 software, and the measured data were analyzed using oneway ANOVA. The results showed an increase in the ALT and AST values of the three treatment groups on post-ST-elevation blood sampling but were still within normal limits except for K1. ALT and AST values decreased significantly (P0.05) in the group treated with single placental stem cell (K2) or combined cardiomyocyte co-culture (K3). The ALT value of K3 decreased but not significantly (P0.05) compared to K2. The AST value of K3 decreased significantly (P0.05) but was not significantly different (P0.05) compared to K2. The liver tissue showed circulatory disturbances in the central venous congestion, portal vein, sinusoidal dilatation and congestion, and hepatocyte changes in the form of centrilobular necrosis and hydropic degeneration. Stem cell therapy alone (K2) or in combination with cardiomyocyte co-culture (K3) had similar percentage of centrilobular necrosis which was significantly lower than K1 (P0.05). Hepatocyte hydropic degeneration on K2 was classified as mild, whereas on K3 was classified as moderate. As conclusion, administration of placental stem cell therapy alone or in combination with cardiomyocyte co-culture can reduce ALT and AST values and could repair liver tissue.

CircENTPD7 affects the immune escape of non‑small cell lung cancer cells by modulating the IGF2BP2/PD‑L1 axis.

Programmed death ligand 1 (PD-L1), an important immune checkpoint molecule, is abnormally activated in non-small cell lung cancer (NSCLC), which can interact with programmed death 1 to aid cancer cells in evading immune surveillance. Furthermore, tumor driver genes may be involved in the occurrence and development of NSCLC and have a potential role in PD-L1-mediated immune escape mechanisms. Therefore, the present study aimed to assess the behavioral and regulatory mechanisms by which circular RNA ENTPD7 (circENTPD7; hsa_circ_0019421) induces an immune response in the progression of NSCLC cells. In brief, circENTPD7, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and PD-L1 expression were assessed using reverse transcription-quantitative PCR, and IGF2BP2 and PD-L1 protein expression levels were evaluated using western blotting. Subsequently, the proliferation, migration, invasion and immune escape of NSCLC cells were assessed using a Cell Counting Kit-8 assay, Transwell assay, T cell co-culture assay, FITC ester assay, flow cytometry and ELISA. The experimental results revealed that circENTPD7, IGF2BP2 and PD-L1 levels were significantly elevated in NSCLC cells. CircENTPD7 knockdown suppressed the malignant phenotype of NSCLC cells, whereas overexpression of IGF2BP2 counteracted these effects, indicating that circENTPD7 contributed to the proliferation and metastasis of NSCLC cells by upregulating IGF2BP2. Furthermore, overexpression of IGF2BP2 accelerated the proliferation, metastasis and immune escape processes of NSCLC cells by upregulating PD-L1. Collectively, the results indicate that circENTPD7 contributes to the malignant progression of NSCLC cells by modulating the IGF2BP2/PD-L1 axis-mediated immune escape.

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria.

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

Botox-A Induced Apoptosis and Suppressed Cell Proliferation in Fibroblasts Pre-Treated with Breast Cancer Exosomes.

breast cancer-associated fibroblast (CAF) is linked to metastasis and is poor for breast cancer prognosis. Since Clostridium Toxin A (Botox-A) had represented a cytotoxic effect on fibroblasts, this study aims to assess Botox-A cytotoxicity in both normal fibroblasts and exosome-induced CAFs. the serum exosomes of 40 BC patients and 30 healthy individuals were isolated and lncRNA H19 (lnch19) levels were assessed by qRT-PCR method. After that, Breast Cancer (BC) exosomes co-cultured with Human foreskin fibroblasts (HFF) and qRT-PCR were applied to evaluate α-SMA, Vimentin, BCL-2, and BAX expression. Both Normal and malignant HFFs co-cultured with Botox-A, and Botox-A loaded exosome for 24 and 48 hours and their apoptosis, Cell proliferation, and viability were monitored by MTT assay, Annexin V-FITC and PI staining and qRT-PCR for BCL-2, BAX, and cyclin D1 mRNAs. Serum exosomes of BC patients had significantly higher levels of lncRNA H19 than healthy individuals. MTT assay results showed Botox-A decreased vital Human foreskin fibroblasts in a dose-dependent manner. BC exosomes significantly increased α-SMA, Vimentin, and BCL-2 mRNA levels in Human foreskin fibroblasts, on the other hand, BAX decreased meaningfully. Co-culture of exosome-treated HFF cells with both Botox-A and Botox-A loaded exosomes significantly boosted BCL-2 mRNA levels, completely contrary to BAX and cyclid d1 expression. Meanwhile, flow cytometry results confirmed a high rate of apoptosis in malignant Human foreskin fibroblasts treated with Botox-A loaded exosome. The findings of this study indicate that exosomal lncRNA H19 could be a diagnostic marker for Breast Cancer and these Breast cancer exosomes can induce malignant phenotype in fibroblasts and turn them into CAFs. Botox-A could be toxic for both normal fibroblasts and CAFs, inducing apoptosis and suppressing cell proliferation among them.

Ecteinascidin synthetic analogues: a new class of selective inhibitors of transcription, exerting immunogenic cell death in refractory malignant pleural mesothelioma

BackgroundMalignant pleural mesothelioma (MPM) is a highly chemo-refractory and immune-evasive tumor that presents a median overall survival of 12–14 months when treated with chemotherapy and immunotherapy. New anti-tumor therapies as well as the concomitant reactivation of immune destruction are urgently needed to treat patients with this tumor. The aim of this work is to investigate the potential effect of ecteinascidin derivatives as lurbinectedin as new first-line treatment option in MPM, alone and in combination with immunotherapy.MethodsThe antitumor activity of ecteinascidin synthetic analogues: lurbinectedin, ecubectedin and PM54 was evaluated in an array of patient-derived MPM cells in terms of cell proliferation, cell cycle, apoptosis, DNA damage and repair. Immunoblot was used to assess the cGAS/STING pathway. ELISA and flow cytometry-based assays were used to evaluate immunogenic cell death parameters and the effect on the immunophenotype in autologous peripheral blood monocyte-MPM cells co-cultures. Patient-derived xenografts (PDX) in humanized mice were used to evaluate the efficacy of ecteinascidins in vivo.ResultsLurbinectedin, ecubectedin, and PM54 were effective in reducing cell proliferation and migration, as well as inducing S-phase cell cycle arrest and DNA damage in malignant pleural mesothelioma cells. These effects were more pronounced compared to the standard first-line treatment (platinum-based plus pemetrexed). Mechanistically, the drugs downregulated DNA repair genes, activated the cGAS/STING pathway, and promoted the release of pro-inflammatory cytokines. They also induced immunogenic cell death of mesothelioma cells, enhancing the activation of anti-tumor CD8+T-cells and natural killer cells while reducing tumor-tolerant T-regulatory cells and myeloid-derived suppressor cells in ex vivo co-cultures. These promising results were also observed in humanized patient-derived xenograft models, where the drugs were effective in reducing tumor growth and increasing the ratio anti-tumor/pro-tumor infiltrating immune populations, either alone or combined with the anti-PD-1L atezolizumab.ConclusionsCollectively, these findings reveal a previously unknown mechanism of action of ecteinascidins that merits further investigation for potential clinical applications in the treatment of MPM, as new first line treatment in monotherapy or in association with immunotherapy.

MiR-139-5p regulates the Notch/RBP-J/Hes1 axis to promote homing of bone mesenchymal stem cells in bronchial asthma

To observe the role of miR-139-5p and Notch1 signaling pathway in regulation of homing of bone mesenchymal stem cells (BMSCs) of asthmatic rats. Normal rat BMSCs were co-cultured with bronchial epithelial cells from normal or asthmatic rats, followed by transfection with miR-139-5p mimics or a negative control sequence. The changes in cell viability and cell cycle were analyzed, and the cellular expressions of CXCR4 and SDF-1 were detected using immunofluorescence staining. The changes of BMSC homing after the transfection were observed, and the expressions of Notch1, RBP-J, and Hes1 mRNAs and proteins and Th1/Th2 cytokines were detected with RT-qPCR, Western blotting or ELISA. The co-cultures of BMSCs and asthmatic bronchial epithelial cells showed significantly decreased expressions of miR-139-5p, IL-2 and IL-12 and increased expressions of CXCR4, SDF-1, IL-5, IL-9, Notch1, RBP-J, and Hes1. Transfection with miR-139-5p mimics significantly increased the expressions of miR-139-5p, IL-2, CXCR4 and SDF-1 and lowered the expression levels of IL-5, IL-9, Notch1, activated Notch1, and Hes1 in the co-cultured cells. Correlation analysis showed that BMSC homing was positively correlated with miR-139-5p and IL-12 and negatively correlated with IL-5 expression. The expression of CXCR4 was negatively correlated with activated Notch1, and SDF-1 was positively correlated with miR-139-5p but negatively correlated with Notch1 expression. High expression of miR-139-5p promotes homing of BMSCs in asthma by targeting the Notch1 signaling pathway to regulate the expressions of Th1/Th2 cytokines, thereby alleviating airway inflammation.

In Vitro Infant Fecal Fermentation Metabolites of Osteopontin and 2'-Fucosyllactose Support Intestinal Barrier Function.

In this study, we investigated the effects of infant fecal fermentation-derived metabolites of digested osteopontin (OPN) and 2'-fucosyllactose (2'-FL), either individually or in combination, on intestinal barrier function using a Caco-2/HT-29 coculture cell model. Our results suggested that the OPN/2'-FL (1:36-1:3) cofermentation metabolites improved epithelial barrier integrity by supporting the mRNA and protein expression of occludin, claudin-1, claudin-2, ZO-1, and ZO-2. All of the OPN/2'-FL treatments decreased the production of IL-1β, IL-6, and TNF-α, while the OPN/2'-FL ratio increased IL-10 production by inhibiting activation of the MyD88/IκB-α/NF-κB signaling pathway. OPN/2'-FL cofermentation altered the metabolic pathways, and the protective effect of fermentation metabolites on intestinal barrier function was related to differential metabolite expression such as short-chain fatty acids, deoxycholic acid, and 4-aminobutyric acid. Our findings provide in vitro evidence to support the application of the OPN/2'-FL combination in infant formula for the advancement of formulation functionality, including intestinal barrier function.

A spheroid whole mount drug testing pipeline with machine-learning based image analysis identifies cell-type specific differences in drug efficacy on a single-cell level

BackgroundThe growth and drug response of tumors are influenced by their stromal composition, both in vivo and 3D-cell culture models. Cell-type inherent features as well as mutual relationships between the different cell types in a tumor might affect drug susceptibility of the tumor as a whole and/or of its cell populations. However, a lack of single-cell procedures with sufficient detail has hampered the automated observation of cell-type-specific effects in three-dimensional stroma-tumor cell co-cultures.MethodsHere, we developed a high-content pipeline ranging from the setup of novel tumor-fibroblast spheroid co-cultures over optical tissue clearing, whole mount staining, and 3D confocal microscopy to optimized 3D-image segmentation and a 3D-deep-learning model to automate the analysis of a range of cell-type-specific processes, such as cell proliferation, apoptosis, necrosis, drug susceptibility, nuclear morphology, and cell density.ResultsThis demonstrated that co-cultures of KP-4 tumor cells with CCD-1137Sk fibroblasts exhibited a growth advantage compared to tumor cell mono-cultures, resulting in higher cell counts following cytostatic treatments with paclitaxel and doxorubicin. However, cell-type-specific single-cell analysis revealed that this apparent benefit of co-cultures was due to a higher resilience of fibroblasts against the drugs and did not indicate a higher drug resistance of the KP-4 cancer cells during co-culture. Conversely, cancer cells were partially even more susceptible in the presence of fibroblasts than in mono-cultures.ConclusionIn summary, this underlines that a novel cell-type-specific single-cell analysis method can reveal critical insights regarding the mechanism of action of drug substances in three-dimensional cell culture models.

Indoleamine 2, 3-dioxygenase Regulates the Differentiation of T Lymphocytes to Promote the Growth of Gastric Cancer Cells through the PI3K/Akt/mTOR Pathway.

To investigate the regulatory mechanism of indoleamine 2, 3-dioxygenase (IDO) in T lymphocyte differentiation and its role in promoting the growth of gastric cancer (GC) cells through the PI3K/Akt/mTOR pathway. GC cell lines (MFC and NCI-N87) and PBMC cells were co-cultured and IDO inhibitor 1-methyl-tryptophan (1-MT) was added. The proliferation was detected by CCK-8, the apoptosis was detected by flow cytometry, and the contents of TNF-α, IL-1β, IL-6, IL-8, and INF-γ were detected by ELISA. The expression levels of PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR were tested using Western blot, and the proportion of CD4+/CD8+, CD4+CD25+Foxp3+Treg cells was detected by flow cytometry. C57BL/6 mice were used to establish the MFC GC mouse model and treated with 1-MT. The changes in body weight and tumor diameter were measured. Ki-67, CD4+, CD8+, and CD25+ expressions were detected by immunohistochemistry. IDO promoted the proliferation of MFC and NCI-N87 cells, inhibited apoptosis, and decreased the levels of TNF-α, IL-1β, IL-6, IL-8, and INF-γ in the supernatant after co-culture with BPMC. The expressions of p-AKT, p-mTOR, and p-PI3K increased after 1-MT treatment. The proportion of CD4+/CD8+ cells was increased and the proportion of Treg cells was decreased in PBMC cells after the addition of 1-MT. Overexpression of IDO suppressed T cells differentiation by inhibiting the PI3K/Akt/mTOR pathway. In vivo, 1-MT treatment reduced the tumor size and weight, increased CD4+ and CD8+ positive area proportion, and decreased Ki-67 and CD25+ positive area proportion. Co-culture of GC cells and immune cells promotes the proliferation of GC cells and inhibits apoptosis, which can be reversed by 1-MT. IDO may suppress the proliferation of T lymphocyte through inhibiting the PI3K/Akt/mTOR signaling pathway. This provides new evidence for the potential of exploiting IDO inhibitors for GC treatment.