Boyden Chamber Research Articles

NOD1 cooperates with HAX-1 to promote cell migration in a RIPK2- and NF-ĸB-independent manner.

The human Nod-like receptor protein NOD1 is a well-described pattern-recognition receptor (PRR) with diverse functions. NOD1 associates with F-actin and its protein levels are upregulated in metastatic cancer cells. A hallmark of cancer cells is their ability to migrate, which involves actin remodelling. Using chemotaxis and wound healing assays, we show that NOD1 expression correlated with the migration rate and chemotactic index in the cervical carcinoma cell line HeLa. The effect of NOD1 in cell migration was independent of the downstream kinase RIPK2 and NF-ĸB activity. Additionally, NOD1 negatively regulated the phosphorylation status of cofilin, which inhibits actin turnover. Co-immunoprecipitation assays identified HCLS1-associated protein X-1 (HAX-1) as a previously unknown interaction partner of NOD1. Silencing of HAX-1 expression reduced the migration behaviour to similar levels as NOD1 knockdown, and simultaneous knockdown of NOD1 and HAX-1 showed no additive effect, suggesting that both proteins act in the same pathway. In conclusion, our data revealed an important role of the PRR NOD1 in regulating cell migration as well as chemotaxis in human cervical cancer cells and identified HAX-1 as a protein that interacts with NOD1 and is involved in this signalling pathway.

Comprehensive analysis of E47‑like factors and verification of ELF4 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most prominent subtype of renal cancer and E47-like factors (ELFs) are important in tumorigenesis; however, the specific role of key ELFs in ccRCC remains unclear. The present study comprehensively analyzed RNA sequencing and clinical data from multiple databases, and identified differentially expressed ELFs (ELF3-5) in ccRCC. The DNA promoter methylation, genetic variation and clinical significance of ELF3-5 in ccRCC were analyzed using the cBioPortal and UALCAN databases. The association between ELF3-5 and multiple immune cell infiltration was analyzed using Tumor Immune Estimation Resource. Subsequently, ELF4 was selected and its association with biological functions was assessed. Cell counting kit-8 (CCK-8), colony formation, Transwell, macrophage chemotaxis and polarization assays were conducted to validate the functions of ELF4. Notably, the mRNA expression levels of ELF4 were significantly upregulated in ccRCC, whereas ELF3 and ELF5 mRNA expression levels were significantly downregulated. Clinical significance analysis revealed that ELF4 showed a high clinical significance with tumor grade, clear cell type A and B subtypes, and incidence rates of amplification in genetic variation. Further analyses indicated that ELF4 may be involved in multiple immune cell differentiation. Additionally, cell experiments revealed that ELF4 inhibition downregulated 769-P and 786-O proliferation, migration and invasion. Knockdown of ELF4 in cancer cells also inhibited M2 macrophage polarization and chemotaxis towards 769-P and 786-O cells. Conclusively, the present findings indicated the clinical significance of ELF4 in ccRCC, and verified its key role in driving cell proliferation, migration and invasion, and promoting M2 macrophage polarization and chemotaxis in ccRCC.

Microtubule-associated septin complexes modulate kinesin and dynein motility with differential specificities

Long-range membrane traffic is guided by microtubule-associated proteins and posttranslational modifications, which collectively comprise a traffic code. The regulatory principles of this code and how it orchestrates the motility of kinesin and dynein motors are largely unknown. Septins are a large family of GTP-binding proteins, which assemble into complexes that associate with microtubules. Using single-molecule invitro motility assays, we tested how the microtubule-associated SEPT2/6/7, SEPT2/6/7/9, and SEPT5/7/11 complexes affect the motilities of the constitutively active kinesins KIF5C and KIF1A and the dynein-dynactin-bicaudal D (DDB) motor complex. We found that microtubule-associated SEPT2/6/7 is a potent inhibitor of DDB and KIF5C, preventing mainly their association with microtubules. SEPT2/6/7 also inhibits KIF1A by obstructing stepping along microtubules. On SEPT2/6/7/9-coated microtubules, KIF1A inhibition is dampened by SEPT9, which alone enhances KIF1A, showing that individual septin subunits determine the regulatory properties of septin complexes. Strikingly, SEPT5/7/11 differs from SEPT2/6/7, in permitting the motility of KIF1A and immobilizing DDB to the microtubule lattice. In hippocampal neurons, filamentous SEPT5 colocalizes with somatodendritic microtubules that underlie Golgi membranes and lack SEPT6. Depletion of SEPT5 disrupts Golgi morphology and polarization of Golgi ribbons into the shaft of somato-proximal dendrites, which is consistent with the tethering of DDB to microtubules by SEPT5/7/11. Collectively, these results suggest that microtubule-associated complexes have differential specificities in the regulation of the motility and positioning of microtubule motors. We posit that septins are an integral part of the microtubule-based code that spatially controls membrane traffic.

Multiplexed microfluidic screening of bacterial chemotaxis.

Microorganism sensing of and responding to ambient chemical gradients regulates a myriad of microbial processes that are fundamental to ecosystem function and human health and disease. The development of efficient, high-throughput screening tools for microbial chemotaxis is essential to disentangling the roles of diverse chemical compounds and concentrations that control cell nutrient uptake, chemorepulsion from toxins, and microbial pathogenesis. Here, we present a novel microfluidic multiplexed chemotaxis device (MCD) which uses serial dilution to simultaneously perform six parallel bacterial chemotaxis assays that span five orders of magnitude in chemostimulant concentration on a single chip. We first validated the dilution and gradient generation performance of the MCD, and then compared the measured chemotactic response of an established bacterial chemotaxis system (Vibrio alginolyticus) to a standard microfluidic assay. Next, the MCD's versatility was assessed by quantifying the chemotactic responses of different bacteria (Psuedoalteromonas haloplanktis, Escherichia coli) to different chemoattractants and chemorepellents. The MCD vastly accelerates the chemotactic screening process, which is critical to deciphering the complex sea of chemical stimuli underlying microbial responses.

Dissecting the Therapeutic Mechanisms of Sphingosine-1-Phosphate Receptor Agonism during Ischaemia and Reperfusion.

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PR) regulate many cellular processes, including lymphocyte migration and endothelial barrier function. As neutrophils are major mediators of inflammation, their transendothelial migration may be the target of therapeutic approaches to inflammatory conditions such as ischaemia-reperfusion injury (IRI). The aim of this project was to assess whether these therapeutic effects are mediated by S1P acting on neutrophils directly or indirectly through the endothelial cells. First, our murine model of peritoneum cell recruitment demonstrated the ability of S1P to reduce CXCL8-mediated neutrophil recruitment. Mechanistic in vitro studies revealed that S1P signals in neutrophils mainly through the S1PR1 and 4 receptors and induces phosphorylation of ERK1/2; however, this had no effect on neutrophil transmigration and adhesion. S1P treatment of endothelial cells significantly reduced TNF-α-induced neutrophil adhesion under flow (p < 0.01) and transendothelial migration towards CXCL8 during in vitro chemotaxis assays (p < 0.05). S1PR1 agonist CYM5442 treatment of endothelial cells also reduced neutrophil transmigration (p < 0.01) and endothelial permeability (p < 0.005), as shown using in vitro permeability assays. S1PR3 agonist had no effects on chemotaxis or permeability. In an in vivo mouse model of renal IRI, S1PR agonism with CYM5442 reduced endothelial permeability as shown by reduced Evan's Blue dye extravasation. Western blot was used to assess phosphorylation at different sites on vascular endothelial (VE)-cadherin and showed that CYM5442 reduced VEGF-mediated phosphorylation. Taken together, the results of this study suggest that reductions in neutrophil infiltration during IRI in response to S1P are mediated primarily by S1PR1 signalling on endothelial cells, possibly by altering phosphorylation of VE-cadherin. The results also demonstrate the therapeutic potential of S1PR1 agonist during IRI.

RNA m5C methylation orchestrates BLCA progression via macrophage reprogramming.

Recently, epigenetics showed essential roles in tumour microenvironment (TME) and immunotherapy response, however, the functions of RNA 5-methylcytosine (m5C) modification in TME remains unknown. According to 13 m5C regulators, we evaluated 412 BLCA patients from The Cancer Genome Atlas (TCGA) database. The m5C score was constructed by unsupervised clustering analysis and principal component analysis (PCA) algorithms. Gene set variation analysis (GSVA), ESTIMATE algorithm, and immunohistochemical (IHC) staining were performed. Macrophage chemotaxis assay was used to assess the M2 macrophages. Among the 412 patients, the frequency of mutation was 13%. m5C regulators was expressed significantly in BLCA tissue compared with normal tissue. Then, two m5C methylation modification patterns were identified with dissimilar TME cell infiltration patterns. The C1 alteration pattern in the m5C cluster was connected with better survival. In addition, we found that NSUN6 was highly correlated with recruitment of macrophages via bioinformatics and IHC. Further experiment validated that NSUN6 promoted HDAC10 expression by mediating m5C methylation, inhibited the transcription of macrophage-associated chemokines and thus inhibited the recruitment of M2 macrophages. The m5C score constructed by m5C modification pattern showed that high m5C score group had a better prognosis. This study uncovered the significant roles of m5C modifications in modulating the TME and indicated that NSUN6 could inhibit the recruitment of M2 macrophages via m5C methylation, which provided novel insight into epigenetic regulation of TME and clinical suggestions for immunotherapeutic strategies.

Cell surface nucleocapsid protein expression: A betacoronavirus immunomodulatory strategy.

We recently reported that SARS-CoV-2 nucleocapsid (N) protein is abundantly expressed on the surface of both infected and neighboring uninfected cells, where it enables activation of Fc receptor-bearing immune cells with anti-N antibodies (Abs) and inhibits leukocyte chemotaxis by binding chemokines (CHKs). Here, we extend these findings to N from the common cold human coronavirus (HCoV)-OC43, which is also robustly expressed on the surface of infected and noninfected cells by binding heparan sulfate/heparin (HS/H). HCoV-OC43 N binds with high affinity to the same set of 11 human CHKs as SARS-CoV-2 N, but also to a nonoverlapping set of six cytokines. As with SARS-CoV-2 N, HCoV-OC43 N inhibits CXCL12β-mediated leukocyte migration in chemotaxis assays, as do all highly pathogenic and common cold HCoV N proteins. Together, our findings indicate that cell surface HCoV N plays important evolutionarily conserved roles in manipulating host innate immunity and as a target for adaptive immunity.

Lack of Short-term Memory Improvement in Caenorhabditis elegans after Petunidin-3-glucoside Supplementation

BACKGROUND: Learning and memory can be enhanced by antioxidants by protecting the neurons against oxidative damage. Phytochemicals, like anthocyanins, may hold the potential to improve cognitive function by ameliorating neuronal damage induced by reactive oxygen species. Petunidin-3-glucoside (P3G) is one of the different classes of anthocyanin found in various plants. AIMS AND OBJECTIVES: This study investigated the effects of P3G on the cognitive function in Caenorhabditis elegans (C. elegans) exposed to glyphosate or ethanol. MATERIALS AND METHODS: P3G was purchased from AS Polyphenols (Norway). This compound was dissolved in distilled water to obtain the desired concentrations. The solutions were stored at 4°C until use. Worms were supplemented with various concentrations of P3G prior to massed training and chemotaxis assays. The collected data were presented as mean ± standard error of the mean, and results were analyzed as biological replicates with three trials. The statistical analysis was done through the SPSS 21.0 (IBM) software, and the results were considered statistically significant when P &lt; 0.05. RESULTS: We found out that P3G did not affect the learning and short-term memory of C. elegans. P3G did not also salvage the decline in learning and memory of the nematodes after exposure to glyphosate or ethanol. CONCLUSION: Taken together, the antioxidant activity of P3G may not be enough to enhance learning and short-term memory in C. elegans nor protect their cognitive function against glyphosate nor ethanol-induced neuronal toxicity.

Osimertinib Induces the Opposite Effect of Proliferation and Migration in the Drug Resistance of EGFR-T790M Non-small Cell Lung Cancer Cells.

Lung cancer has become one of the leading causes of cancer incidence and mortality worldwide. Non-small cell lung carcinoma (NSCLC) is the most common type among all lung cancer cases. NSCLC patients contained high levels of activating epidermal growth factor receptor (EGFR) mutations, such as exon 19 deletion, L858R and T790M. Osimertinib, a third-generation of EGFR tyrosine kinase inhibitor (EGFR-TKI), has therapeutic efficacy on the EGFR-T790M mutation of NSCLC patients; however, treatment of osimertinib still can induce drug resistance in lung cancer patients. Therefore, investigation of the drug resistance mechanisms of osimertinib will provide novel strategies for lung cancer therapy. The H1975OR osimertinib-resistant cell line was established by prolonged exposure with osimertinib derived from the H1975 cells. The cell proliferation ability was evaluated by the cell viability and cell growth assays. The cell migration ability was determined by the Boyden chamber assays. The differential gene expression profile was analyzed by genome-wide RNA sequencing. The protein expression and location were analyzed by western blot and confocal microscopy. In this study, we established the osimertinib-resistant H1975 (T790M/L858R) cancer cells, named the H1975OR cell line. The cell growth ability was decreased in the H1975OR cells by comparison with the H1975 parental cells. Conversely, the cell migration ability was elevated in the H1975OR cells. We found the differential gene expression profile of cell proliferation and migration pathways between the H1975OR and H1975 parental cells. Interestingly, the protein levels of phospho-EGFR, PD-L1, E-cadherin and β-catenin were decreased, but the survivin and N-cadherin proteins were increased in the H1975OR drug-resistant cells. Osimertinib induces the opposite effect of proliferation and migration in the drug resistance of EGFRT790M lung cancer cells. We suggest that differential gene and protein expressions in the cell proliferation and migration pathways may mediate the drug resistance of osimertinib in lung cancer cells. Understanding the molecular drugresistant mechanisms of proliferation and migration pathways of osimertinib may provide novel targets and strategies for the clinical treatment of EGFR-TKIs in lung cancer patients.

INVESTIGATION INTO P2Y RECEPTOR FUNCTION IN PLATELETS FROM PATIENTS WITH SEPSIS.

Key underlying pathological mechanisms contributing to sepsis are hemostatic dysfunction and overwhelming inflammation. Platelet aggregation is required for hemostasis, and platelets are also separately involved in inflammatory responses that require different functional attributes. Nevertheless, P2Y receptor activation of platelets is required for this dichotomy of function. The aim of this study was to elucidate whether P2YR-dependent hemostatic and inflammatory functions were altered in platelets isolated from sepsis patients, compared with patients with mild sterile inflammation. Platelets from patients undergoing elective cardiac surgery (20 patients, 3 female) or experiencing sepsis after community-acquired pneumonia (10 patients, 4 female) were obtained through the IMMunE dysfunction and Recovery from SEpsis-related critical illness in adults (IMMERSE) Observational Clinical Trial. In vitro aggregation and chemotaxis assays were performed with platelets after stimulation with ADP and compared with platelets isolated from healthy control subjects (7 donors, 5 female). Cardiac surgery and sepsis both induced a robust inflammatory response with increases in circulating neutrophil counts with a trend toward decreased circulating platelet counts being observed. The ability of platelets to aggregate in response to ex vivo ADP stimulation was preserved in all groups. However, platelets isolated from patients with sepsis lost the ability to undergo chemotaxis toward N -formylmethionyl-leucyl-phenylalanine, and this suppression was evident at admission through to and including discharge from hospital. Our results suggest that P2Y 1 -dependent inflammatory function in platelets is lost in patients with sepsis resulting from community-acquired pneumonia. Further studies will need to be undertaken to determine whether this is due to localized recruitment to the lungs of a platelet responsive population or loss of function as a result of dysregulation of the immune response.

Investigation of Anticancer Properties of Monoterpene-Aminopyrimidine Hybrids on A2780 Ovarian Cancer Cells

The present study aimed to characterize the antiproliferative and antimetastatic properties of two recently synthesized monoterpene-aminopyrimidine hybrids (1 and 2) on A2780 ovary cancer cells. Both agents exerted a more pronounced cell growth inhibitory action than the reference agent cisplatin, as determined by the MTT assay. Tumor selectivity was assessed using non-cancerous fibroblast cells. Hybrids 1 and 2 induced changes in cell morphology and membrane integrity in A2780 cells, as evidenced by Hoechst 33258–propidium iodide fluorescent staining. Cell cycle analysis by flow cytometry revealed substantial changes in the distribution of A2780 ovarian cancer cells, with an increased rate in the subG1 and G2/M phases, at the expense of the G1 cell population. Moreover, the tested molecules accelerated tubulin polymerization in a cell-free in vitro system. The antimetastatic properties of both tested compounds were investigated by wound healing and Boyden chamber assays after 24 and 48 h of incubation. Treatment with 1 and 2 resulted in time- and concentration-dependent inhibition of migration and invasion of A2780 cancer cells. These results support that the tested agents may be worth of further investigation as promising anticancer drug candidates.

Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions. MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry. ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14+ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells. ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.

Downregulation of VEGFR2 signaling by cedrol abrogates VEGF‑driven angiogenesis and proliferation of glioblastoma cells through AKT/P70S6K and MAPK/ERK1/2 pathways.

Cedrol is a sesquiterpene alcohol isolated from Cedrus atlantica, which has been traditionally used in aromatherapy and has anticancer, antibacterial and antihyperalgesic effects. One characteristic of glioblastoma (GB) is the overexpression of vascular endothelial growth factor (VEGF), which induces a high degree of angiogenesis. Although previous studies have reported that cedrol inhibits GB growth by inducing DNA damage, cell cycle arrest and apoptosis, its role in angiogenesis remains unclear. The aim of the present study was to investigate the effects of cedrol on VEGF-induced angiogenesis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 0-112 µM cedrol and 20 ng/ml VEGF for 0-24 h, and then anti-angiogenic activation of cedrol was determined by MTT assay, wound healing assay, Boyden chamber assay, tube formation assay, semi-quantitative reverse transcription-PCR and western blotting. These results demonstrated that cedrol treatment inhibited VEGF-induced cell proliferation, migration and invasion in HUVECs. Furthermore, cedrol prevented VEGF and DBTRG-05MG GB cells from inducing capillary-like tube formation in HUVECs and decreased the number of branch points formed. Moreover, cedrol downregulated the phosphorylation of VEGF receptor 2 (VEGFR2) and the expression levels of its downstream mediators AKT, ERK, VCAM-1, ICAM-1 and MMP-9 in HUVECs and DBTRG-05MG cells. Taken together, these results demonstrated that cedrol exerts anti-angiogenic effects by blocking VEGFR2 signaling, and thus could be developed into health products or therapeutic agents for the prevention or treatment of cancer and angiogenesis-related diseases in the future.

The Role of Ascorbate-Glutathione System and Volatiles Emitted by Insect-Damaged Lettuce Roots as Navigation Signals for Insect and Slug Parasitic Nematodes.

The effect of wireworm-damaged lettuce roots on the antioxidative defense system (ascorbate-glutathione cycle, photosynthetic pigments) and movement of insect/slug parasitic nematodes towards determined root exudates was studied in a glasshouse experiment. Lettuce seedlings were grown in a substrate soil in the absence/presence of wireworms (Elateridae). The ascorbate-glutathione system and photosynthetic pigments were analyzed by HPLC, while volatile organic compounds (VOC) emitted by lettuce roots were investigated by GC-MS. Herbivore-induced root compounds, namely 2,4-nonadienal, glutathione, and ascorbic acid, were selected for a chemotaxis assay with nematodes Steinernema feltiae, S. carpocapsae, Heterorhabditis bacteriophora, Phasmarhabditis papillosa, and Oscheius myriophilus. Root pests had a negative effect on the content of photosynthetic pigments in the leaves of infested plants, indicating that they reacted to the presence of reactive oxygen species (ROS). Using lettuce as a model plant, we recognized the ascorbate-glutathione system as a redox hub in defense response against wireworms and analyzed its role in root-exudate-mediated chemotaxis of nematodes. Infected plants also demonstrated increased levels of volatile 2,4-nonadienal. Entomopathogenic nematodes (EPNs, S. feltiae, S. carpocapsae, and H. bacteriophora) proved to be more mobile than parasitic nematodes O. myriophilus and P. papillosa towards chemotaxis compounds. Among them, 2,4-nonadienal repelled all tested nematodes. Most exudates that are involved in belowground tritrophic interactions remain unknown, but an increasing effort is being made in this field of research. Understanding more of these complex interactions would not only allow a better understanding of the rhizosphere but could also offer ecologically sound alternatives in the pest management of agricultural systems.

Comprehensive analysis the diagnosis, malignant progression and immune infiltrate of ANXA6 in prostate cancer.

Annexins (ANXAs) play a crucial role in the development and progression of tumors. However, their specific involvement in prostate cancer (PCa) remains unclear. To investigate the function and clinical significance of key ANXAs in PCa. Multiple databases were used to analyze the expression levels, genetic variations, potential prognostic value and clinical significance of ANXAs in PCa. Then, the co-expressed genes of ANXA6 were identified, and the correlation between ANXA6 and immune cell infiltration was validated using the Tumor Immune Estimation Resource (TIMER) database. Additionally, in vitro assays such as Cell Counting Kit-8 (CCK-8), Colony Formation, Transwell and T-cell Chemotaxis assays were conducted to validate the functions of ANXA6. Moreover, multiple types of in vivo assays were performed to further validate the identified ANXA6 functions. The results demonstrated that ANXA2, ANXA6 and ANXA8 were significantly downregulated in PCa. ANXA6 upregulation was significantly associated with improved PCa patients' overall survival. Enrichment analysis revealed that ANXA6 and its co-expressed genes were involved in tumor progression, and ANXA6 overexpression could effectively inhibit the proliferation, migration and invasion of PC-3 cells. In vivo studies also demonstrated that ANXA6 overexpression suppressed tumor growth. Importantly, ANXA6 was found to promote the chemotaxis of CD4+ T cells and CD8+ T cells towards PC-3 cells, and the overexpression of ANXA6 in PC-3 cells promoted the polarization of macrophages into M1 macrophages in the supernatant of PCa cells. ANXA6 demonstrated promising potential for consideration as a prognostic biomarker in PCa as it was found to play key roles in regulating immune cell infiltration and the malignant progression to PCa.

ETV2 Enhances CXCL5 Secretion from Endothelial Cells, Leading to the Promotion of Vascular Smooth Muscle Cell Migration.

Abnormal communication between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) promotes vascular diseases, including atherogenesis. ETS variant transcription factor 2 (ETV2) plays a substantial role in pathological angiogenesis and the reprogramming of ECs; however, the role of ETV2 in the communication between ECs and VSMCs has not been revealed. To investigate the interactive role of ETV2 in the EC to VSMC phenotype, we first showed that treatment with a conditioned medium from ETV2-overexpressed ECs (Ad-ETV2 CM) significantly increased VSMC migration. The cytokine array showed altered levels of several cytokines in Ad-ETV2 CM compared with those in normal CM. We found that C-X-C motif chemokine 5 (CXCL5) promoted VSMC migration using the Boyden chamber and wound healing assays. In addition, an inhibitor of C-X-C motif chemokine receptor 2 (CXCR2) (the receptor for CXCL5) significantly inhibited this process. Gelatin zymography showed that the activities of matrix metalloproteinase (MMP)-2 and MMP-9 increased in the media of VSMCs treated with Ad-ETV2 CM. Western blotting revealed a positive correlation between Akt/p38/c-Jun phosphorylation and CXCL5 concentration. The inhibition of Akt and p38-c-Jun effectively blocked CXCL5-induced VSMC migration. In conclusion, CXCL5 from ECs induced by ETV2 promotes VSMC migration via MMP upregulation and the activation of Akt and p38/c-Jun.

Characterization of chimeric antigen receptor modified T cells expressing scFv-IL-13Rα2 after radiolabeling with 89Zirconium oxine for PET imaging

BackgroundChimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products.MethodsTo determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with 89Zirconium-oxine (89Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The 89Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells.ResultsWe observed that radiolabeling of CAR-T cells with 89Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13Rα2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells.ConclusionsImportantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with 89Zr-oxine retain critical product attributes and suggest 89Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.

A Triazaspirane Derivative Inhibits Migration and Invasion in PC3 Prostate Cancer Cells.

Cancer is a serious health problem due to the complexity of establishing an effective treatment. The purpose of this work was to evaluate the activity of a triazaspirane as a migration and invasion inhibitor in PC3 prostatic tumor cells through a possible negative regulation of the FAK/Src signal transduction pathway and decreased secretion of metalloproteinases 2 and 9. Molecular docking analysis was performed using Moe 2008.10 software. Migration (wound-healing assay) and invasion (Boyden chamber assay) assays were performed. In addition, the Western blot technique was used to quantify protein expression, and the zymography technique was used to observe the secretion of metalloproteinases. Molecular docking showed interactions in regions of interest of the FAK and Src proteins. Moreover, the biological activity assays demonstrated an inhibitory effect on cell migration and invasion, an important suppression of metalloproteinase secretion, and a decrease in the expression of p-FAK and p-Src proteins in treated PC3 cells. Triazaspirane-type molecules have important inhibitory effects on the mechanisms associated with metastasis in PC3 tumor cells.

RHOA deficiency drives decreased CD19 expression and immune dysregulation in CAR‐T resistant diffuse large B‐cell lymphoma

Background: CD19-directed chimeric antigen receptor T-cell (CAR-19) therapies are promising new options in relapsed or refractory diffuse large B-cell lymphoma (DLBCL), but over half of patients still progress. Recent whole-genome sequencing data published by our lab revealed that deletion of RHOA (3p21.31) is strongly associated with poor response (p = 0.0013). In newly diagnosed DLBCL, 20% of patients harbor a RHOA deletion and another 5% have loss-of-function mutations. To date, no work has comprehensively identified the role of RHOA in DLBCL or how its inactivation may protect CAR-19 resistant tumors. Methods: RHOA loss-of-function (LoF) lymphoma cell lines were generated using shRNA and CRISPR approaches. Diminished RHOA activity was confirmed via active RHO pulldowns. RNA-sequencing in conjunction with western blotting and flow cytometry was conducted to uncover deregulated pathways in RHOA-deficient lymphoma. Cell proliferation, migration, and viability were assessed to phenotype RHOA LoF and uncover signaling pathways activated in RHOA-deficient lymphoma. CD19-directed murine and human CAR-T cells were co-cultured with corresponding RHOA LoF systems to assess CAR-T engagement and killing. Results: RNA-seq analysis of RHOA-deficient cell lines revealed enrichment of gene sets associated with cell-cycle progression and interferon signaling pathways, and cell proliferation and chemotaxis assays both demonstrated increased activation. Western blotting revealed upregulated pAKT, but strikingly RHOA LoF systems showed enhanced sensitivity to PI3K, AKT, and mTOR inhibition. We assessed if RHOA LoF associates with previously described decreased CD19 driving CAR-19 resistance and found consistent downregulation by flow cytometry in both A20 shRhoa (p = 0.0002) murine lymphomas and RIVA human cell line CRISPR het RHOA knockouts (p = 0.0031), confirmed by western blotting in both systems. RNA-seq analysis of 74 DLBCL patients (PCAWG data) revealed a strong positive correlation between expression CD19 and RHOA (p < 0.001, R2 = 0.236). Impedance-based serial cell viability assessments and flow cytometry cytotoxicity assays with CAR-19 and RHOA LoF cells revealed a statistically significant decrease in CAR-19 killing of RHOA-deficient lymphomas (p = 0.0005 and p < 0.0001). Conclusion: RHOA-deficient lymphoma relies on PI3K/AKT/mTOR signaling for proliferation that works in parallel with decreased CD19 to decrease CAR-19 killing. Upregulation of interferon signaling pathways gives evidence of increased immunoinhibitory ligands that could promote exhausted CAR19 products. Further research will challenge in vivo RHOA-deficient tumors with CAR-19 cells and employ scRNA-seq to reveal transcriptomic changes within RHOA-deficient lymphoma responsible for CAR-19 resistance. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Immunotherapy, Microenvironment No conflicts of interests pertinent to the abstract.

Shugoshin as a key mediator in the metastasis of triple negative breast cancer.

e13098 Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the lack of estrogen, progesterone, or human epidermal growth factor 2 (HER2) receptor expression. TNBC has poor prognosis due to its high metastatic potential and limited treatment options. Shugoshin-1 (Sgo1), a centromeric protein that maintains stability in chromatids during mitosis, has been thought to play an important role in the progression of TNBC. Previous publications from our group have shown that the transient downregulation of Sgo1 in TNBC cells decrease the epithelial-to-mesenchymal transition (EMT), which is a precursor of metastasis. Our purpose with this study is to evaluate the role of long-term effects of Sgo1 downregulation in cell invasion, cell migration, tumor growth, and metastasis. Methods: To achieve our goal, MDA-MB-231 cells were stably transfected with a lentivirus containing a shRNA Sgo1 sequence or a scramble shRNA control. Cell invasion and migration were evaluated by Boyden chamber assays. Meanwhile, tumor growth and metastasis were evaluated using an immunocompromised orthotopic mice model. Female NOD-SCID Gamma (NSG) mice were injected either with 2.5 X 106 shRNA Control (n= 10) or shRNA Sgo1 (n= 10) MDA-MB-231 cells. Tumor growth was measured for eight weeks using a vernier caliper. Afterwards, primary, and metastatic tumors as well as primary organs were harvested. Lung and liver samples were embedded in paraffin and stained with hematoxylin and eosin. A Student t test was used to evaluate the differences between groups. A p-value &lt; 0.05 was considered significant. Results: Our results show a significant reduction in cell invasion and migration in MDA-MB-231 cells treated with shRNA Sgo1. Moreover, we observed a significant reduction in tumor growth and metastasis to lymph nodes, liver, and lungs in the mice treated with shRNA Sgo1 when compared to shRNA Control group. Conclusions: These results suggest that Sgo1 is an important regulator in TNBC tumor growth and metastasis through the modulation of EMT genes. Thus, targeting Sgo1 early in the course of TNBC can be a potential therapeutic treatment of TNBC.