Chick Embryo Chorioallantoic Membrane Research Articles

Phytochemical profiling, cytotoxic, anti-migration, and anti-angiogenic potential of phenolic-rich fraction from Peganum harmala: in vitro and in ovo studies.

Peganum harmala has been extensively employed in Algerian traditional medicine practices. This study aimed to explore the impact of n-butanol (n-BuOH) extract sourced from Peganum harmala seeds on cell proliferation, cell migration, and angiogenesis inhibition. Cytotoxic potential of n-BuOH extract was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide) assay against human breast adenocarcinoma MCF-7 cells, cell migration was determined using scratch assay, and anti-angiogenic effect was evaluated through macroscopic and histological examinations conducted on chick embryo chorioallantoic membrane. Additionally, this research estimated the phytochemical profile of n-BuOH extract. Fifteen phenolic compounds were identified using Ultra-performance liquid chromatography UPLC-ESI-MS-MS analysis. In addition, the n-BuOH extract of P. harmala exhibited potent antioxidant and free radical scavenging properties. The n-BuOH extract showed potent cytotoxicity against MCF-7 cell with an IC50 value of 8.68 ± 1.58 μg/mL. Furthermore, n-BuOH extract significantly reduced migration. A strong anti-angiogenic activity was observed in the groups treated with n-BuOH extract in comparison to the negative control. Histological analysis confirmed the anti-angiogenic effect of the n-BuOH extract. This activity is probably a result of the synergistic effects produced by different polyphenolic classes.

Effects of Resveratrol on In Vivo Ovarian Cancer Cells Implanted on the Chorioallantoic Membrane (CAM) of a Chicken Embryo Model.

Ovarian cancer poses a significant threat to patients in its advanced stages, often with limited treatment options available. In such cases, palliative management becomes the primary approach to maintaining a reasonable quality of life. Therefore, the administration of any medication that can benefit patients without a curative option holds potential. Resveratrol, a natural compound known for its in vitro anticancer activities, has generated contrasting results in vivo and human studies. In this study, we aimed to assess the anticancer effects of resveratrol on ovarian cancer cells grown on the chorioallantoic membrane (CAM) of chicken embryos. Two ovarian cancer cell lines, OVCAR-8 and SKOV-3, were cultured in collagen scaffolds for four days before being implanted on the CAM of chicken embryos on day 7. Different doses of resveratrol were applied to the CAM every two days for six days. Subsequently, CAM tissues were excised, fixed, and subjected to histological analysis. Some CAM tumours were extracted to analyse proteins through Western blotting. Our findings indicate that specific doses of resveratrol significantly reduce angiogenic activities, pNF-κB levels, and SLUG protein levels by using immunohistochemistry. These results suggest that resveratrol may have the potential to impact the behaviour of ovarian cancer CAM tumours, thereby warranting further consideration as a complementary treatment option for women with incurable ovarian cancer.

The biological characteristics of chicken embryo mesenchymal stem cells isolated from chorioallantoic membrane.

Mesenchymal stem cells (MSCs) derived from fetal membranes (FMs) have the potential to exhibit immunosuppression, improve blood flow, and increase capillary density during transplantation. In the field of medicine, opening up new avenues for disease treatment. Chicken embryo chorioallantoic membrane (CAM), as an important component of avian species FM structure, has become a stable tissue engineering material in vivo angiogenesis, drug delivery, and toxicology studies. Although it has been confirmed that chorionic mesenchymal stem cells (Ch-MSCs) can be isolated from the outer chorionic layer of FM, little is known about the biological characteristics of MSCs derived from chorionic mesodermal matrix of chicken embryos. Therefore, we evaluated the characteristics of MSCs isolated from chorionic tissues of chicken embryos, including cell proliferation ability, stem cell surface antigen, genetic stability, and in vitro differentiation potential. Ch-MSCs exhibited a broad spindle shaped appearance and could stably maintain diploid karyotype proliferation to passage 15 in vitro. Spindle cells were positive for multifunctional markers of MSCs (CD29, CD44, CD73, CD90, CD105, CD166, OCT4, and NANOG), while hematopoietic cell surface marker CD34, panleukocyte marker CD45, and epithelial cell marker CK19 were negative. In addition, chicken Ch-MSC was induced to differentiate into four types of mesodermal cells in vitro, including osteoblasts, chondrocytes, adipocytes, and myoblasts. Therefore, the differentiation potential of chicken Ch-MSC in vitro may have great potential in tissue engineering. In conclusion, chicken Ch-MSCs may be an excellent model cell for stem cell regenerative medicine and chorionic tissue engineering.

Abstract 1458: Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents a very aggressive type of pancreatic cancer (9% survival rate at 5 years). PDX1 is an important transcription factor for embryonic development of the pancreas, endocrine lineage differentiation and the maintenance of mature beta cells. Notably, in PDAC patients, the expression of PDX1 is downregulated in tumor cells compared to adjacent non-tumoral tissue. Interestingly, increasing tumor PDX1 expression has been shown to enhance the overall survival rate in PDAC patients. Therefore, our aim was to analyze the role of PDX1 on tumor aggressiveness of PDAC cells. To induce PDX1 expression, PANC1 cells were treated with BRD7552 (a PDX1 inducer) for 9 days. Overexpression of PDX1 was confirmed by Western Blot analysis and no cytotoxic effects were observed by MTT or Trypan Blue exclusion assays on treated cells compared to control. Wound healing and transwell migration assays showed a significant reduction in the migration rate compared to the control group. A higher proportion of cells in G1 phase was observed in treated cells compared to control by propidium iodide staining followed by flow cytometry assay. Furthermore, the cell confluence assay showed a significant reduction in proliferation rate in treated cells compared to control, but no differences were observed on the proportion of KI67+ and PH3+ cells by immunostaining. To assess the in ovo effects of BRD7552, treated PANC1 cells were implanted onto the chorioallantoic membrane of chick embryos and tumor growth was measured at different stages observing a significant reduction in tumor growth in PANC1 cells compared to control between days 3 and 8 post implantation. No significant differences in morphology or color were observed between treated and control implanted cells. Hematoxylin-eosin staining showed reduced invasion of treated cells into the chorioallantoic membrane compared to control. In conclusion, the overexpression of PDX1 affects cell cycle, reduces proliferation rate and inhibits migratory potential in vitro. Moreover, in an in ovo model this overexpression diminished tumor growth and invasion in PANC1 cells. In conclusion, the overexpression of PDX1 induced by BRD7552 drives pancreatic ductal adenocarcinoma to a less aggressive tumoral phenotype. Citation Format: Jimena Mosna, Federico Garde, Marcelo Stinson, Candela Pastore, Pablo Sanchis, Abel Carcagno. Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1458.

Alternating Current-Electric Field Inducing Chorio Allantoic Membrane (CAM) Angiogenesis through Exogenous Growth Factor Intervention

Angiogenesis is widely used in various therapies by promoting or inhibiting the formation of new blood vessels. The use of Alternating Current-Electric Fields (AC-EF) in Electro-Capacitive Cancer Therapy (ECCT) showed its potential as an anti-cancer device, and is characterized by its anti-proliferative and pro-apoptotic effects. However, the role of AC-EF in angiogenesis remains unclear. To investigate the effects of AC-EF on CAM angiogenesis, we used the ex ovo culture method of chorioallantoic membrane (CAM). A basic fibroblast growth factor (bFGF) dose of 30 ng/µL was administered as an exogenous growth factor. The ECCT device, generating AC-EF of 150 kHz and 18 Vpp, was exposed to the CAMs. Subsequently, the 24 CAMs of chick embryo were divided into four groups. Two groups were non-bFGF-induced CAM, while the other two were bFGF-induced CAM, and each group was exposed either with or without AC-EF. The vascularization was evaluated through macroscopic observation, while vascular endothelial growth factor A (VEGFA) gene expression was measured using qPCR. The data were statistically analyzed using ANOVA with GraphPad Prism 9.5. The results showed that an AC-EF exposure had no effects on normal CAM angiogenesis (P>0.05). Moreover, VEGFA gene expression did not show significant upregulation (P>0.05) in the bFGF-induced CAM with or without AC-EF exposure. Interestingly, the number of new blood vessels was significantly higher (P<0.05) in the bFGF-induced with AC-EF exposure than in the non-bFGF-induced group. In conclusion, AC-EF of ECCT did not affect normal angiogenesis. AC-EF may trigger CAM angiogenesis with bFGF induction. This observation suggested that AC-EF of intermediate frequency could enhance angiogenesis by administration of external growth factors, offering a potential avenue for addressing obstructive vascular conditions.

EPAC inhibitor suppresses angiogenesis and tumor growth of triple-negative breast cancer

AimsExchange protein directly activated by cAMP 1 (EPAC1), a major isoform of guanine nucleotide exchange factors, is highly expressed in vascular endothelia cells and regulates angiogenesis in the retina. High intratumor microvascular densities (MVD) resulting from angiogenesis is responsible for breast cancer development. Downregulation of EPAC1 in tumor cell reduces triple-negative breast cancer (TNBC)-induced angiogenesis. However, whether Epac1 expressed in vascular endothelial cells contributes to angiogenesis and tumor development of TNBC remains elusive. Main methodsWe employed NY0123, a previously identified potent EPAC inhibitor, to explore the anti-angiogenic biological role of EPAC1 in vitro and in vivo through vascular endothelial cells, rat aortic ring, Matrigel plug, and chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) assays, as well as the in vivo xenograft tumor models of TNBC in both chick embryo and mice. Key findingsInhibiting EPAC1 in vascular endothelial cells by NY0123 significantly suppresses angiogenesis and tumor growth of TNBC. In addition, NY0123 possesses a better inhibitory efficacy than ESI-09, a reported specific EPAC inhibitor tool compound. Importantly, inhibiting EPAC1 in vascular endothelia cells regulates the typical angiogenic signaling network, which is associated with not only vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) signaling, but also PI3K/AKT, MEK/ERK and Notch pathway. ConclusionsOur findings support that EPAC1 may serve as an effective anti-angiogenic therapeutic target of TNBC, and EPAC inhibitor NY0123 has the therapeutic potential to be developed for the treatment of TNBC.

Inhibition of PRMT1 Suppresses the Growth of U87MG-Derived Glioblastoma Stem Cells by Blocking the STAT3 Signaling Pathway.

Glioblastoma stem cells (GSCs) play a pivotal role in the initiation, progression, resistance to treatment, and relapse of glioblastoma multiforme (GBM). Thus, identifying potential therapeutic targets and drugs that interfere with the growth of GSCs may contribute to improved treatment outcomes for GBM. In this study, we first demonstrated the functional role of protein arginine methyltransferase 1 (PRMT1) in GSC growth. Furamidine, a PRMT1 inhibitor, effectively inhibited the proliferation and tumorsphere formation of U87MG-derived GSCs by inducing cell cycle arrest at the G0/G1 phase and promoting the intrinsic apoptotic pathway. Moreover, furamidine potently suppressed the in vivo tumor growth of U87MG GSCs in a chick embryo chorioallantoic membrane model. In particular, the inhibitory effect of furamidine on U87MG GSC growth was associated with the downregulation of signal transducer and activator of transcription 3 (STAT3) and key GSC markers, including CD133, Sox2, Oct4, Nanog, aldehyde dehydrogenase 1, and integrin α6. Our results also showed that the knockdown of PRMT1 by small interfering RNA significantly inhibited the proliferation of U87MG GSCs in vitro and in vivo through a molecular mechanism similar to furamidine. In addition, combined treatment with furamidine and berbamine, a calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ) inhibitor, inhibited the growth of U87MG GSCs more strongly than single-compound treatment. The increased antiproliferative effect of combining the two compounds resulted from a stronger downregulation of STAT3-mediated downstream GBM stemness regulators through dual PRMT1 and CaMKIIγ function blockade. In conclusion, these findings suggest that PRMT1 and its inhibitor, furamidine, are potential novel therapeutic targets and drug candidates for effectively suppressing GSC growth.

Dioclea violacea lectin inhibits tumorigenesis and tumor angiogenesis in vivo

Dioclea violacea seed mannose-binding lectin (DvL) has attracted considerable attention because of its interesting biological activities, including antitumor, antioxidant, and anti-inflammatory activities. This study evaluated the cytotoxic effect of DvL on tumor and normal cells using the mitochondrial activity reduction (MTT) assay, the carcinogenic and anti-carcinogenic activity by the epithelial tumor test (ETT) in Drosophila melanogaster, and the anti-angiogenic effect by the chick embryo chorioallantoic membrane (CAM) assay. Data demonstrated that DvL promoted strong selective cytotoxicity against tumor cell lines, especially A549 and S180 cells, whereas normal cell lines were weakly affected. Furthermore, DvL did not promote carcinogenesis in D. melanogaster at any concentration tested, but modulated DXR-induced carcinogenesis at the highest concentrations tested. In the CAM and immunohistochemical assays, DvL inhibited sarcoma 180-induced angiogenesis and promoted the reduction of VEGF and TGF-β levels at all concentrations tested. Therefore, our results demonstrated that DvL is a potent anticancer, anti-angiogenic, and selective cytotoxic agent for tumor cells, suggesting its potential application as a prototype molecule for the development of new drugs with chemoprotective and/or antitumor effects.

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants. This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed. No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Multi-parametric investigations on the effects of vascular disrupting agents based on a platform of chorioallantoic membrane of chick embryos.

Vascular disrupting agents (VDAs) are known to specifically target preexisting tumoural vasculature. However, systemic side effects as safety or toxicity issues have been reported from clinical trials, which call for further preclinical investigations. The purpose is to gain insights into their non-specific off-targeting effects on normal vasculature and provide clues for exploring underlying molecular mechanisms. Based on a recently introduced platform consisting laser speckle contrast imaging (LSCI), chick embryo chorioallantoic membrane (CAM), and assisted deep learning techniques, for evaluation of vasoactive medicines, hemodynamics on embryonic day 12 under constant intravascular infusion of two VDAs were qualitatively observed and quantitatively measured in real time for 30 min. Blood perfusion, vessel diameter, vessel density, and vessel total length were further analyzed and compared between blank control and medicines dose groups by using multi-factor analysis of variance (ANOVA) analysis with factorial interactions. Conventional histopathology and fluorescent immunohistochemistry (FIHC) assays for endothelial cytoskeleton including ß-tubulin and F-actin were qualitatively demonstrated, quantitatively analyzed and further correlated with hemodynamic and vascular parameters. The normal vasculature was systemically negatively affected by VDAs with statistical significance (P<0.0001), as evidenced by four positively correlated parameters, which can explain the side-effects observed among clinical patients. Such effects appeared to be dose dependent (P<0.0001). FIHC assays qualitatively and quantitatively verified the results and exposed molecular mechanisms. LSCI-CAM platform combining with deep learning technique proves useful in preclinical evaluations of vasoactive medications. Such new evidences provide new reference to clinical practice.

FS145, the first flea-derived disintegrin, inhibits angiogenesis through specifically binding integrin αvβ3

FS145, a protein containing a WGD motif, was previously described from the salivary transcriptome of the flea Xenopsylla cheopis. Nevertheless, its biological function and complete structure are still uncertain. Herein, FS145 was confirmed to adopt a common αββ structure with the WGD motif exposed on its surface and located right at the top of a loop composed of residues 72–81. Furthermore, FS145 dose-dependently inhibited the proliferation, adhesion, migration, and tube formation of HUVECs by not only binding to integrin αvβ3 but also by subsequently inactivating the FAK/Src/MAPK pathway along with the reduction of the expression of MMP-2, MMP-9, VEGFA, bFGF, Ang2, Tie2, HIF-1α, and FAK. Moreover, FS145 also inhibited aortic vessel sprout and showed strong anti-angiogenic activities as assessed ex vivo, by employing the rat aortic ring assay, chick embryo chorioallantoic membrane, and zebrafish embryo models. Altogether, our results suggest that FS145 suppresses angiogenesis ex vivo and in vitro by blocking integrin αvβ3. The current study reveals the first anti-angiogenesis disintegrin with WGD motif from invertebrates and provides a beneficial pharmacological activity to inhibit abnormal angiogenesis.

Moraea sisyrinchium inhibits proliferation, cell cycle, and migration of cancerous cells, and decreases angiogenesis in chick chorioallantoic membrane.

Experimental studies reported that some plants in the genus of Moraea (Iridaceae family) show anticancer potential. This study aimed to evaluate the effects of Moraea sisyrinchium on U87 glioblastoma multiforme and HepG2 liver cancer cells. The cells were incubated for 24 hr with hydroalcoholic extract of the stem, flower, and bulb of M. sisyrinchium. Then, the cell proliferation (MTT) assay, cell cycle analysis (propidium iodide staining), cell migration test (scratch), Western blotting (Bax and Bcl-2 expression), and gelatin zymography (for matrix metalloproteinases, MMPs) were performed. Oxidative stress was evaluated by determining the levels of reactive oxygen species and lipid peroxidation. Angiogenesis was evaluated on chick embryo chorioallantoic membrane. The extracts of the flower, stem, and bulb significantly decreased the proliferation of HepG2 and U87 cells. This effect was more for U87 than HepG2 and for the bulb and stem than the flower. In U87 cells, the bulb extract increased oxidative stress, cell cycle arrest, and the Bax/Bcl-2 ratio. Also, this extract suppressed the migration ability of HepG2 and U87 cells, which was associated with the inhibition of MMP2 activity. In addition, it significantly reduced the number and diameter of vessels in the chorioallantoic membrane. Liquid chromatography-mass spectrometry revealed the presence of xanthones (bellidifolin and mangiferin), flavonoids (quercetin and luteolin), isoflavones (iridin and tectorigenin), and phytosterols (e.g., stigmasterol) in the bulb. M. sisyrinchium bulb decreased the proliferation and survival of cancer cells by inducing oxidative stress. It also reduced the migration ability of the cells and inhibited angiogenesis.

Artificial Intelligence (AI) Based Analysis of In Vivo Polymers and Collagen Scaffolds Inducing Vascularization.

Biomaterials are essential in modern medicine, both for patients and research. Their ability to acquire and maintain functional vascularization is currently debated. The aim of this study was to evaluate the vascularization induced by two collagen-based scaffolds (with 2D and 3D structures) and one non-collagen scaffold implanted on the chick embryo chorioallantoic membrane (CAM). Classical stereomicroscopic image vascular assessment was enhanced with the IKOSA software by using two applications: the CAM assay and the Network Formation Assay, evaluating the vessel branching potential, vascular area, as well as tube length and thickness. Both collagen-based scaffolds induced non-inflammatory angiogenesis, but the non-collagen scaffold induced a massive inflammation followed by inflammatory-related angiogenesis. Vessels branching points/Region of Interest (Px^2) and Vessel branching points/Vessel total area (Px^2), increased exponentially until day 5 of the experiment certifying a sustained and continuous angiogenic process induced by 3D collagen scaffolds. Collagen-based scaffolds may be more suitable for neovascularization compared to non-collagen scaffolds. The present study demonstrates the potential of the CAM model in combination with AI-based software for the evaluation of vascularization in biomaterials. This approach could help to reduce and replace animal experimentation in the pre-screening of biomaterials.

DFMG decreases angiogenesis to uphold plaque stability by inhibiting the TLR4/VEGF pathway in mice.

The aim of this study was to elucidate the specific mechanism through which 7-difluoromethoxy-5,4'-dimethoxygenistein (DFMG) inhibits angiogenesis in atherosclerosis (AS) plaques, given its previously observed but poorly understood inhibitory effects. In vitro, a model using Human Umbilical Vein Endothelial (HUVEC-12) cells simulated the initial lesion in the atherosclerotic pathological process, specifically oxidative stress injury, by exposing cells to 30 μmol/L LPC. Additionally, an AS mouse model was developed in ApoE knockout mice through a 16-week period of high-fat feeding. DFMG demonstrated a reduction in tubule quantities in the tube formation assay and neovascularization induced by oxidative stress-damaged endothelial cells in the chicken embryo chorioallantoic membrane assay. Furthermore, DFMG decreased lipid levels in the blood of ApoE knockout mice with AS, along with a decrease in atherosclerotic plaques and neovascularizations in the aortic arch and descending aorta of AS animal models. DFMG treatment upregulated microRNA140 (miR-140) expression and suppressed VEGF secretion in HUVEC-12 cells. These effects were counteracted by Toll-like receptor 4 (TLR4) overexpression in HUVEC-12 cells subjected to oxidative injury or in a mouse model of AS. Dual-luciferase reporter assays demonstrated that miR-140 directly targeted TLR4. Immunohistochemical assay findings indicated a significant inverse relationship between miR-140 expression and TLR4 expression in ApoE knockout mice subjected to a high-fat diet. The study observed a close association between DFMG inhibitory effects on angiogenesis and plaque stability in AS, and the inhibition of the TLR4/NF-κB/VEGF signaling pathway, negatively regulated by miR-140.

HET-CAM test in evaluation of irritating action of adhesives used in shoe making industry

The global tendency of the contemporary scientific studies is using alternative biologic models as substitutes of experimental animals. HET-CAM (The Hen's Egg Test on the Chorioallantoie Membrane Assay) is an alternative to in vivo tests involving experimental animals. This test is actively used in different biomedical studies. The aim of our work was to study the irritating potential of adhesives used in shoe making industry in experimental setting using the alternative HET-CAM method. Polyurethane, polychloroprene, rubber and styrene-butadiene adhesives that are widely used in shoe-making industry were studied . HET-CAM test was used for the evaluation of the irritating action of the aforementioned adhesives. All adhesives were applied directly onto the chorioallantoic membrane of chick embryos with reactions and changes (hemorrhages, vascular lysis and coagulation) observed and registered in 30, 120 and 300 seconds after the application of the adhesive. Irritating potential of the adhesives was evaluated according to a calculated irritating index. The most pronounced signs of irritating action were caused by polyurethane adhesives, namely hemorrhages and coagulation (30 sec) – two-component adhesive and hemor­rhages (30 sec) and coagulation (120-300 sec) – one-component adhesive. Vascular reactions from application of sty­rene-butadiene adhesives manifested predominantly with lysis and hemorrhages (30 sec), in some samples these reactions were observed at a later time-point (120-300 sec). Irritating action of rubber adhesives manifested mostly with hemor­rhages (30 sec), additionally one observation showed some lysis (120 sec). Polychloroprene adhesive caused hemorrhages (30-120 sec) and also lysis (30 sec) in one of the samples. According to the irritating index, polyurethane (one- and two-component) and styrene-butadiene adhesives were estimated to be strong irritants, while rubber and poly­chloroprene ones of moderate irritating action. НЕТ-САМ test can be used as a component in the evaluation of evidence of irritating action of shoe adhesives.

Hovenia dulcis Suppresses the Growth of Huh7-Derived Liver Cancer Stem Cells by Inducing Necroptosis and Apoptosis and Blocking c-MET Signaling.

Liver cancer stem cells (LCSCs) contribute to the initiation, metastasis, treatment resistance, and recurrence of hepatocellular carcinoma (HCC). Therefore, exploring potential anticancer agents targeting LCSCs may offer new therapeutic options to overcome HCC treatment failure. Hovenia dulcis Thunberg (HDT), a tree from the buckthorn family found in Asia, exhibits various biological activities, including antifatigue, antidiabetic, neuroprotective, hepatoprotective, and antitumor activities. However, the therapeutic effect of HDT in eliminating LCSCs remains to be confirmed. In this study, we evaluated the inhibitory activity of ethanol, chloroform, and ethyl acetate extracts from HDT branches on the growth of Huh7-derived LCSCs. The ethyl acetate extract of HDT (EAHDT) exhibited the most potent inhibitory activity against the growth of Huh7 LCSCs among the three HDT extracts. EAHDT suppressed the in vitro self-renewal ability of Huh7 LCSCs and reduced tumor growth in vivo using the Huh7 LCSC-transplanted chick embryo chorioallantoic membrane model. Furthermore, EAHDT not only arrested the cell cycle in the G0/G1 phase but also induced receptor-interacting protein kinase 3/mixed-lineage kinase domain-like protein-mediated necroptosis and caspase-dependent apoptosis in Huh7 LCSCs in a concentration-dependent manner. Furthermore, the growth inhibitory effect of EAHDT on Huh7 LCSCs was associated with the downregulation of c-MET-mediated downstream signaling pathways and key cancer stemness markers. Based on these findings, we propose that EAHDT can be used as a new natural drug candidate to prevent and treat HCC by eradicating LCSCs.

Haemostatic potency of sodium alginate/aloe vera/sericin composite scaffolds – preparation, characterisation, and evaluation

Fabrication of haemostatic materials with excellent antimicrobial, biocompatible and biodegradable properties remains as a major challenge in the field of medicine. Haemostatic agents play vital role in protecting patients and military individuals during emergency situations. Natural polymers serve as promising materials for fabricating haemostatic compounds due to their efficacy in promoting hemostasis and wound healing. In the present work, sodium alginate/aloe vera/sericin (SA/AV/S) scaffold has been fabricated using a simple cost-effective casting method. The prepared SA/AV/S scaffolds were characterised for their physicochemical properties such as scanning electron microscope, UV–visible spectroscopy and Fourier transform infra-red spectroscopy. SA/AV/S scaffold showed good mechanical strength, swelling behaviour and antibacterial activity. In vitro experiments using erythrocytes proved the hemocompatible and biocompatible features of SA/AV/S scaffold. In vitro blood clotting assay performed using human blood demonstrated the haemostatic and blood absorption properties of SA/AV/S scaffold. Scratch wound assay was performed to study the wound healing efficacy of prepared scaffolds. Chick embryo chorioallantoic membrane assay carried out using fertilised embryos proved the angiogenic property of SA/AV/S scaffold. Thus, SA/AV/S scaffold could serve as a potential haemostatic healthcare product due to its outstanding haemostatic, antimicrobial, hemocompatible, biocompatible and angiogenic properties.

A novel patient-derived immortalised cell line of myxofibrosarcoma: a tool for preclinical drugs testing and the generation of near-patient models

BackgroundMyxofibrosarcoma is a rare malignant soft tissue sarcoma characterised by multiple local recurrence and can become of higher grade with each recurrence. Consequently, myxofibrosarcoma represents a burden for patients, a challenge for clinicians, and an interesting disease to study tumour progression. Currently, few myxofibrosarcoma preclinical models are available.MethodsIn this paper, we present a spontaneously immortalised myxofibrosarcoma patient-derived cell line (MF-R 3). We performed phenotypic characterization through multiple biological assays and analyses: proliferation, clonogenic potential, anchorage-independent growth and colony formation, migration, invasion, AgNOR staining, and ultrastructural evaluation.ResultsMF-R 3 cells match morphologic and phenotypic characteristics of the original tumour as 2D cultures, 3D aggregates, and on the chorioallantoic membrane of chick embryos. Overall results show a clear neoplastic potential of this cell line. Finally, we tested MF-R 3 sensitivity to anthracyclines in 2D and 3D conditions finding a good response to these drugs.ConclusionsIn conclusion, we established a novel patient-derived myxofibrosarcoma cell line that, together with the few others available, could serve as an important model for studying the molecular pathogenesis of myxofibrosarcoma and for testing new drugs and therapeutic strategies in diverse experimental settings.

Exploring the Anticancer Potential of Origanum majorana Essential Oil Monoterpenes Alone and in Combination against Non-Small Cell Lung Cancer

Lung cancer is the second most commonly diagnosed cancer and has the highest mortality rate worldwide despite the remarkable advances in its treatment. Origanum majorana Essential Oil (OMEO) has been shown to be effective against non-small cell lung cancer (NSCLC) cells, decreasing their viability and colony growth in vitro, as well as inhibiting tumor growth in chick embryo chorioallantoic membranes (CAM) and nude mice in vivo. OMEO is mainly composed of four monoterpenes, namely terpinen-4-ol, sabinene hydrate, α-terpinene, and γ-terpinene. In this study, we aimed to investigate the potential anticancer effects of these monoterpenes, either alone or in combination, on NSCLC. Our findings indicate that these four monoterpenes significantly decreased NSCLC cell viability in a concentration-dependent manner, reduced their colony growth in vitro, and also downregulated survivin expression in these cells. Moreover, different combined mixtures of these monoterpenes further enhanced their anticancer effects on cellular viability, with a terpinen-4-ol and sabinene hydrate combination being the most potent. We also found that terpinen-4-ol, in combination with sabinene hydrate, markedly enhanced the anticancer effect of the individual monoterpenes on NSCLC viability within a shorter treatment duration through, at least in part, survivin downregulation. Furthermore, this combination enhanced the inhibition of colony growth in vitro and the tumor growth of NSCLC cells xenografted onto chick embryo CAM in vivo. Altogether, our study highlights the potential of these monoterpenes for use in further pre-clinical investigations against various cancer hallmarks.

Multiomic Characterization of Myelodysplastic Neoplasms (MDS) with Micromegakaryocytes Highlights the Role of EZH2-RUNX1 deregulation in Disease Physiopathology and Response to Targeted Therapies

Micromegakaryocytes (micro-MKs), an unequivocal myeloid dysplastic feature in adults, are present in 15-20% of myelodysplastic neoplasms (MDS). We recently associated the detection of these small megakaryocytes with a mono- or bi-lobated nucleus in bone marrow (BM) smears of MDS patients with an unfavorable prognosis. The aim of the present study was to elucidate the molecular bases of micro-MK formation and its impact on MDS response to targeted therapies. We selected retrospectively a cohort of 40 MDS patient samples subjected to NGS myeloid panel and RNA sequencing and carried out a morphological re-evaluation that included micro-MKs recounting. Cases with isolated del(5q) or SF3B1 mutation were excluded. MDS cases were selected according to the following morphological findings: no dismegakaryopoiesis, dismegakaryopoiesis without micro-MK, low rate of micro-MK (&amp;lt;10%; micro-MK low) and high rate of micro-MK (&amp;gt;40%; micro-MK high). No patient harbored an intermediate (10-40%) rate of micro-MK. A comparative characterization of 8 representative patients was carried out subjecting diagnosis BM samples to Tapestri and Chromium single cell proteogenomic and transcriptomic platforms, gene set enrichment analysis, and deep cytogenetic characterization by optical genome mapping. Functional validation studies were performed using the megakaryoblastic cell line MEG01 differentiated to a mature MK phenotype by exposure to phorbol myristate acetate (PMA), as well as using the CRISPR-Cas9 tool for gene editing. Finally, activity of investigational drugs including 5-azacytidine, lenalidomide, venetoclax, rigosertib and pevonedistat, was evaluated in MEG01-activated cells and in vivo using a chicken embryo chorioallantoic membrane (CAM) model of MDS patient-derived xenograft (PDX). The most frequently mutated genes across all cohorts were ASXL1 (58%), SRSF2 (38%), RUNX1 (30%), TET2 (30%), STAG2 (20%), EZH2 (18%), DDX41 (10%), U2AF1 (10%), and ZRSR2 (10%) . Interestingly, EZH2 and RUNX1 mutations were enriched in the micro-MK high group ( EZH2: 0%, 10%, 14.3% vs. 66.7%, p 0.006; RUNX1: 0%, 20%, 35.7% vs. 83.3%, p 0.003), and both mutations co-occurred in 85.7% (6/7) of the EZH2 mutated cases. Single cell analyses revealed an enrichment of RUNX1 alterations in three populations within micro-MK high BM samples, including hematopoietic stem and progenitor cells, non-classical monocytes and immature erythroid cells. These samples were also characterized by a 5-fold increase in MK progenitor abundance and by the upregulation of a set of 10 EZH2-repressed genes in neutrophil-like cells and myeloid progenitors, with 7q loss presumably affecting the EZH2 locus in 50% of the cases with micro-MK. Using the in vitro MK differentiation model, we further demonstrated that EZH2 downregulation hampered the acquisition of MK surface markers CD41 and CD61, and that venetoclax was the sole agent retaining notable cytotoxic activity in differentiated cells when compared to undifferentiated megakaryoblasts. Accordingly, in a first-in-kind in vivo CAM-MDS transplant model, venetoclax was able to impair BM infiltration by human cells in a PDX model of micro-MK+ MDS. In summary, supporting the notion that EZH2 and RUNX1 exert a crucial role in the regulation of megakaryopoiesis, our present findings suggest that deregulation of EZH2-RUNX1 activity by loss-of-function mutation and/or genetic deletion affecting myeloid precursors, impairs a proper megakaryocytic differentiation process in MDS. Thus, this phenomenon may be considered as a characteristic feature of MDS cases with an elevated proportion of micro-MK. Of note, preliminary in vitro and in vivo results support the use of venetoclax for the treatment of this subgroup of patients with dismal outcome.