Chick Embryo Chorioallantoic Membrane Research Articles

PEG-liposomal doxorubicin as a potential agent for canine metastatic osteosarcoma - in vitro and ex ovo studies.

Appendicular osteosarcoma (OSA) is a highly aggressive and metastatic primary bone tumour in dogs. Standard therapy is amputation and adjuvant chemotherapy (e.g. with doxorubicin). Liposomal drug delivery may augment therapeutic efficacy and reduce negative side effects. Polyethylene glycol (PEG)-liposomal doxorubicin treats human metastatic cancers effectively. The study aimed was to evaluate PEG-liposomal doxorubicin's inhibitory effect on canine metastatic proliferation and migration in vitro. It also aimed to appraise the drug's extravasation inhibition in vivo using the human medicine-proven chick embryo chorioallantoic membrane ex ovo model. The canine D-17 OSA cell line was cultured and inoculated with decreasing concentrations of PEG-liposomal doxorubicin and conventional doxorubicin in a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test of cell viability, proliferation and cytotoxicity. Flow cytometry with Annexin V and Draq 7 staining confirmed the MTT test results, indicating dead, early and late apoptotic, and live cells. The inhibitory effect of the two preparations on cancer cell migration was investigated with a wound-healing assay. Culture plates seeded with cells were prepared. The cell monolayer was scratched and images of cells migrating to the scratch were captured at 0 h, 12 h and 24 h. Also, embryos were removed from three-day-incubated fertilised chicken eggs. On the 12th day, labelled D-17 cells were injected into each embryo. Embryos in one group received 100 μL of phosphate-buffered saline as controls, those in another group 30 μg/mL of PEG-liposomal doxorubicin, and those in the last group 6 μg/mL of conventional doxorubicin. The effectiveness of the intravascular administration of the D-17 cells was confirmed under a microscope. PEG-liposomal doxorubicin inhibited the migration of canine OSA cells more effectively than conventional doxorubicin (P ≤ 0.05). The ex ovo model showed that both drugs had similar impacts on canine metastatic OSA. The liposomal form of the drug may be considered a potentially effective compound in canine metastatic OSA; nevertheless, further in vivo studies are essential to confirm this hypothesis.

Prolonged cell cycle arrest by the CDK4/6 antagonist narazaciclib restores ibrutinib response in preclinical models of BTKi‐resistant mantle cell lymphoma

Introduction: Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of mantle cell lymphoma (MCL); however, primary and acquired resistance to these agents remains a challenge. Previous studies have suggested that narazaciclib (ON123300), a second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor (CDKi), may trigger cell cycle arrest and significant tumor growth inhibition (TGI) in BTKi-resistant MCL models. Methods: We compared the efficacy and safety of narazaciclib with the health authority approved CDKi in association with different BTKi, in a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib. We used the CellTiter-Glo proliferation assay, FACS-mediated quantification of cell cycle and apoptosis, and RNA sequencing followed by gene set enrichment analysis (GSEA), RT-PCR and western blot validations. In addition, we evaluated the safety and efficacy of narazaciclib/ibrutinib combo in vivo in an immune-competent chicken embryo chorioallantoic membrane (CAM) MCL xenograft. Results: Narazaciclib exhibited the highest antitumor activity among MCL cell lines (mean IC50: 3.61 ± 2.1 µM), regardless of their sensitivity to ibrutinib. Although there was no correlation between CDKi sensitivity and activation of the CDK4/CDK6-pRb pathway in MCL, transcriptomic and phenotypic analyses revealed a predominant downregulation of E2F target genes and G2/M checkpoint response (NES > 2.5) upon narazaciclib treatment. This feature was associated to intracellular accumulation of p21, p16, and phospho-p27, decreased mitotic index, G1 cell cycle blockade, and apoptosis onset. When combined with ibrutinib, but not with the second generation therapeutic acalabrutinib, narazaciclib achieved significant synergistic antitumor activity in both BTK-sensitive and BTK-resistant cells. The combination was not associated with improved apoptosis, but rather with a slight but constant (+10%–15%) augmentation in G1 phase blockade and the down-modulation of cell cycle-associated transcriptome. Both the downregulation of phospho-histone H3 and the upregulation of p-p27/p27 and p16, also underwent a 10%–15% improvement in combination-treated cells. In vivo, while narazaciclib single agent achieved a 28% TGI in the CAM model, the narazaciclib-ibrutinib combination reduced tumor spreading by 65% and allowed a 50% reduction in malignant B cell infiltration into the bone marrow, with no detectable toxicity. Conclusions: Narazaciclib, due to its completely distinct MoA from BTKi involving the direct modulation of the cell cycle, can achieve significant synergistic activity with ibrutinib in vitro and in vivo, especially in BTKi-resistant models of MCL. Ongoing phospho-proteomics and genetic edition assays will help deciphering the molecular bases of this unique drug cooperation at the cell cycle level. Encore Abstract - previously submitted to AACR 2023 The research was funded by: This study was financially supported by Onconova Therapeutics, Spanish Ministry of Science and Innovation (grant PID2021-123039OB-C21) (to GR). MFS holds a predoctoral fellowship (FI19/00338) from Instituto de Salud Carlos III. This work was carried out under the CERCA Program (Generalitat de Catalunya). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. A. Makovski-Silverstein Employment or leadership position: Onconova Therapeutics S. Cosenza Employment or leadership position: Onconova Therapeutics G. Roué Research funding: Onconova Therapeutics

E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways.

E7050 is an inhibitor of VEGFR2 with anti-tumor activity; however, its therapeutic mechanism remains incompletely understood. In the present study, we aim to evaluate the anti-angiogenic activity of E7050 in vitro and in vivo and define the underlying molecular mechanism. It was observed that treatment with E7050 markedly inhibited proliferation, migration, and capillary-like tube formation in cultured human umbilical vein endothelial cells (HUVECs). E7050 exposure in the chick embryo chorioallantoic membrane (CAM) also reduced the amount of neovessel formation in chick embryos. To understand the molecular basis, E7050 was found to suppress the phosphorylation of VEGFR2 and its downstream signaling pathway components, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. Moreover, E7050 suppressed the phosphorylation of VEGFR2, FAK, Src, Akt, JNK, and p38 MAPK in HUVECs exposed to MES-SA/Dx5 cells-derived conditioned medium (CM). The multidrug-resistant human uterine sarcoma xenograft study revealed that E7050 significantly attenuated the growth of MES-SA/Dx5 tumor xenografts, which was associated with inhibition of tumor angiogenesis. E7050 treatment also decreased the expression of CD31 and p-VEGFR2 in MES-SA/Dx5 tumor tissue sections in comparison with the vehicle control. Collectively, E7050 may serve as a potential agent for the treatment of cancer and angiogenesis-related disorders.

CYD0281, a Bcl-2 BH4 domain antagonist, inhibits tumor angiogenesis and breast cancer tumor growth

BackgroundB-cell lymphoma 2 (Bcl-2) family proteins are key regulators of apoptosis, which possess four conserved Bcl-2 homologies (BH) domains. Among the BH domains, the BH3 domain is considered as a potent ‘death domain’ while the BH4 domain is required for anti-apoptotic activity. Bcl-2 can be converted to a pro-apoptotic molecule through the removal or mutation of the BH4 domain. Bcl-2 is considered as an inducer of angiogenesis, which can promote tumor vascular network formation and further afford nutrients and oxygen to promote tumor progression. However, whether disrupting the function of the BH4 domain to convert Bcl-2 into a pro-apoptotic molecule could make Bcl-2 possess the potential for anti-angiogenic therapy remains to be defined.MethodsCYD0281 was designed and synthesized according to the lead structure of BDA-366, and its function on inducing a conformational change of Bcl-2 was further evaluated via immunoprecipitation (IP) and immunofluorescence (IF) assays. Moreover, the function of CYD0281 on apoptosis of endothelial cells was analyzed via cell viability, flow cytometry, and western blotting assays. Additionally, the role of CYD0281 on angiogenesis in vitro was determined via endothelial cell migration and tube formation assays and rat aortic ring assay. Chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models, breast cancer cell xenograft tumor on CAM and in mouse models as well as the Matrigel plug angiogenesis assay were used to explore the effects of CYD0281 on angiogenesis in vivo.ResultsWe identified a novel potent small-molecule Bcl-2-BH4 domain antagonist, CYD0281, which exhibited significant anti-angiogenic effects both in vitro and in vivo, and further inhibited breast cancer tumor growth. CYD0281 was found to induce conformational changes in Bcl-2 through the exposure of the BH3 domain and convert Bcl-2 from an anti-apoptotic molecule into a cell death inducer, thereby resulting in the apoptosis of vascular endothelial cells.ConclusionsThis study has revealed CYD0281 as a novel Bcl-2-BH4 antagonist that induces conformational changes of Bcl-2 to convert to a pro-apoptotic molecule. Our findings indicate that CYD0281 plays a crucial role in anti-angiogenesis and may be further developed as a potential anti-tumor drug candidate for breast cancer. This work also provides a potential anti-angiogenic strategy for breast cancer treatment.

Human Monocytes Are Suitable Carriers for the Delivery of Oncolytic Herpes Simplex Virus Type 1 In Vitro and in a Chicken Embryo Chorioallantoic Membrane Model of Cancer.

Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for the treatment of unresectable melanoma. T-VEC, like most OVs, is administered via intratumoral injection, underlining the unresolved problem of the systemic delivery of the oncolytic agent for the treatment of metastases and deep-seated tumors. To address this drawback, cells with a tropism for tumors can be loaded ex vivo with OVs and used as carriers for systemic oncolytic virotherapy. Here, we evaluated human monocytes as carrier cells for a prototype oHSV-1 with a similar genetic backbone as T-VEC. Many tumors specifically recruit monocytes from the bloodstream, and autologous monocytes can be obtained from peripheral blood. We demonstrate here that oHSV-1-loaded primary human monocytes migrated in vitro towards epithelial cancer cells of different origin. Moreover, human monocytic leukemia cells selectively delivered oHSV-1 to human head-and-neck xenograft tumors grown on the chorioallantoic membrane (CAM) of fertilized chicken eggs after intravascular injection. Thus, our work shows that monocytes are promising carriers for the delivery of oHSV-1s in vivo, deserving further investigation in animal models.

Effect of Melittin Complexes with Graphene and Graphene Oxide on Triple-Negative Breast Cancer Tumors Grown on Chicken Embryo Chorioallantoic Membrane.

One of the components of bee venom is melittin (M), which has strong lysing properties on membranes. M has high toxicity to cancer cells, but it also affects healthy cells, making it necessary to use methods for targeted delivery to ensure treatment. This research is a continuation of previous studies using graphene nanomaterials as M carriers to breast cancer cells. The studies described below are conducted on a more organized biological structure than what is found in vitro cells, namely, cancerous tumors grown on a chicken embryo chorioallantoic membrane. Caspase 3 and 8 levels are analyzed, and the level of oxidative stress markers and changes in protein expression for cytokines are examined. The results show that M complexes with nanomaterials reduce the level of oxidative stress more than M alone does, but the use of graphene (GN) as a carrier increases the level of DNA damage to a greater extent than the increase caused by M alone. An analysis of cytokine levels shows that the use of the M and GN complex increases the level of proteins responsible for inhibiting tumor progression to a greater extent than the increase occasioned by a complex with graphene oxide (GO). The results suggest that the use of GN as an M carrier may increase the toxic effect of M on structures located inside a cell.

Adipose Tissue-Derived Stem Cell Extracellular Vesicles Suppress Glioblastoma Proliferation, Invasiveness and Angiogenesis.

Extracellular vesicles (EVs) are attractive anticancer drug delivery candidates as they confer several fundamental properties, such as low immunogenicity and the ability to cross biological barriers. Mesenchymal stem cells (MSCs) are convenient producers for high EV yields, and patient-derived adipose tissue MSC-EVs could serve as personalised carriers. However, MSC-EV applications raise critical concerns as their natural cargo can affect tumour progression in both inducing and suppressing ways. In this study, we investigated the effect of adipose tissue-derived mesenchymal stem cell EVs (ASC-EVs) on several glioblastoma (GBM) cell lines to define their applicability for anticancer therapies. ASC-EVs were isolated from a cell-conditioned medium and characterised by size and specific markers. The internalisation of fluorescently labelled ASC-EVs by human GBM cells HROG36, U87 MG, and T98G was evaluated by fluorescent microscopy. Changes in GBM cell proliferation after ASC-EV application were determined by the metabolic PrestoBlue assay. Expression alterations in genes responsible for cell adhesion, proliferation, migration, and angiogenesis were evaluated by quantitative real-time PCR. ASC-EV effects on tumour invasiveness and neoangiogenesis in ovo were analysed on the chicken embryo chorioallantoic membrane model (CAM). ASC-EV treatment reduced GBM proliferation in vitro and significantly downregulated invasiveness-related genes ITGα5 (in T98G and HROG63) and ITGβ3 (in HROG36) and the vascularisation-inducing gene KDR (in all GBM lines). Additionally, an approximate 65% reduction in the GBM invasion rate was observed in CAM after ASC-EV treatment. Our study indicates that ASC-EVs possess antitumour properties, reducing GBM cell proliferation and invasiveness, and can be applied as anticancer therapeutics and medicine carriers.

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Abstract 5974: Synergistic activity of the CDK4/6 antagonist narazaciclib (ON123300) with irreversible BTK inhibition in ibrutinib-resistant mantle cell lymphoma

Abstract Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of B-cell lymphoma including the aggressive mantle cell lymphoma (MCL) subtype, although primary and acquired resistance is frequently observed. Interestingly, preliminary results have suggested that the second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor, ON123300 (narazaciclib), may be effective in preclinical models of BTKi-resistant MCL. We compared the efficacy and safety of ON123300 vs the approved CDK inhibitors palbociclib, abemaciclib or ribociclib, in association with various BTKi, in a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib, or the second generation acalabrutinib. We evaluated the effects of these combinations by CellTiter-Glo proliferation assay, FACS-mediated analysis of cell cycle and apoptosis, RT-PCR and western blot. Efficacy and safety of ON123300 in vivo was evaluated in a chicken embryo chorioallantoic membrane (CAM) xenograft model of MCL. We observed that ON123300 exhibited a significant antitumor activity in MCL cell lines independently of their sensitivity to ibrutinib, with IC50 at 72h ranging from 0.7 to 7.1µM (mean: 3.61±2.1µM), while it achieved a 32% tumor growth inhibition, with no detectable toxicity, in the CAM-MCL model. In vitro, ON123300 demonstrated a superior activity than palbociclib and ribociclib, and a similar effect that abemaciclib. Interestingly, ON123300 exhibited an antitumor activity comparable to ibrutinib but was more effective than acalabrutinib. ON123300 single agent treatment repressed several positive regulators of the G2/M cell cycle phase and triggered the accumulation of the CDK inhibitors p21, p16, and phospho-p27, with the consequent loss of phospho-Histone H3 and phospho-CDK2. Accordingly, ON123300 treatment evoked a 20-35% increase in G1 cell cycle fraction at 24h, which preceded the ignition of mitochondrial apoptosis. When combined with ibrutinib, rather than with acalabrutinib, ON123300 exhibited a significant antitumor activity with synergistic combination indexes in both BTK-sensitive and BTK-resistant cell lines. This phenomenon was associated with a slight but constant (+10-15%) augmentation of G1 blockade, histone H3/CDK2 dephosphorylation and p27/p16 accumulation in cells exposed to ibrutinib-ON123300 combination, with no detectable potentiation of apoptosis. In conclusion, ON123300 is safe and effective as single agent in preclinical models of MCL, including BTKi-resistant cases. Due to its complete distinct MoA from BTKi involving the direct modulation of cell cycle, ON123300, but not abemaciclib or palbociclib, can achieve significant synergistic activity in combination with ibrutinib, and in lower extent with acalabrutinib, especially in BTKi-resistant MCL cases. Citation Format: Nuria Profitos-Peleja, Marcelo L. Ribeiro, Adar Makovski Silverstein, Stephen Cosenza, Gaël Roué. Synergistic activity of the CDK4/6 antagonist narazaciclib (ON123300) with irreversible BTK inhibition in ibrutinib-resistant mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5974.

Abstract 4980: Design and functional characterization of a first-in-class irreversible inhibitor of HOIL-1-interacting protein (HOIP) with selective antitumor activity against B-cell non-Hodgkin lymphoma

Abstract Activating single-nucleotide polymorphisms of HOIL-1-interacting protein (HOIP), the catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC), have recently been shown to promote myeloid differentiation primary response 88 (MYD88)-mediated B-cell lymphomagenesis. To assess the relevance of targeting HOIP in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) with MYD88 mutation, we used systems biology to build a mathematical model aimed at evaluating the impact of HOIP depletion on DLBCL. From this model, an AI-mediated query demonstrated that HOIP blockade was associated with the suppression of three main pathophysiological motifs in malignant B cells, i.e., cell growth and proliferation, apoptosis evasion and deregulated metabolism. We then undertook a computational study that consisted of a combinatorial substitution of several α-β unsaturated moieties to be used as covalent binding warheads to the catalytic cysteine residue of HOIP. The resulting chemical library was used on subsequent molecular docking to assess the best HOIP binding candidates. Out of the four candidates synthesized from this library, we isolated compound A (Cpd A), a covalent irreversible inhibitor of HOIP with a pyrido[2,3-d]pyrimidine core, which exerted selective antitumor activity in a panel of ABCL-DLBCL cell lines (mean IC50 at 48hours: 90.7± 13.09 µM) while sparing normal B cells. Immunoprecipitation studies demonstrated that Cpd A was able to modulate the interaction between HOIP and the LUBAC component, SHARPIN, leading to the blockade of NF-κB signaling and to the downregulation of several downstream effectors, including CCL3, IL6 and IRF4. Specificity of Cpd A towards HOIP was confirmed by a drug affinity responsive target stability (DARTS) assay based on the immuno-detection of persistent HOIP peptides after Cpd A-mediated blockade of enzymatic proteolysis, and thereafter in a CRISPR-engineered HOIP-knockout ABC-DLBCL model. Finally, efficacy of the compound was confirmed in vivo in a chicken embryo chorioallantoic membrane (CAM)-derived model of ABCL-DLBCL subjected to a twice weekly dosing with Cpd A, in which the compound achieved a 25% tumor growth inhibition, associated with a 60.5% and 89% reduction in brain and bone marrow infiltration by lymphoma cells, respectively. Altogether, our results confirm that HOIP represents a promising therapeutic target for ABC-DLBCL with activating MYD88 mutation, and that a pyrido[2,3-d]pyrimidine derivative can successfully and specifically block HOIP, resulting in NF-κB disruption and selective antitumor activity in this aggressive subtype of B-cell lymphoma. Citation Format: Ana Maria Montagut, Marc Antoni Armengol Cubillos, Gema Gorjón-de-Pablo, Judith Farrés, Laia Huguet Garcia, Núria Profitós-Pelejà, Juliana Carvalho Santos, Marcelo Lima Ribeiro, Miranda Fernández-Serrano, Roger Estrada Tejedor, José I. Borrell, Gaël Roué. Design and functional characterization of a first-in-class irreversible inhibitor of HOIL-1-interacting protein (HOIP) with selective antitumor activity against B-cell non-Hodgkin lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4980.

YAP inhibitor verteporfin suppresses tumor angiogenesis and overcomes chemoresistance in esophageal squamous cell carcinoma.

Targeting angiogenesis is an attractive strategy for the effective treatment of cancer. This study aimed to investigate the anti-cancer activities of YAP inhibitor verteporfin (VP) in esophageal squamous cell carcinoma (ESCC) cells through its inhibitory effect on tumor angiogenesis. Cell proliferation, apoptosis, migration and invasion abilities were estimated by MTT, colony formation, DAPI staining, wound healing and transwell assays, respectively. Human umbilical vein endothelial cell (HUVEC) tube formation assay and chick embryo chorioallantoic membrane (CAM) model were used to observe angiogenesis in vitro and in vivo. The interactions between ESCC cells and HUVECs were assessed by cell chemotactic migration and adhesion assays. The expression levels of angiogenesis-related molecules were detected by Western blot. We found that VP was potential to inhibit ESCC cell proliferation, migration, invasion and induce apoptosis in the dose-dependent fashion. VP also significantly suppressed proliferation, migration, and tube formation of HUVECs and promoted apoptosis of HUVECs, and reduced angiogenesis in CAM. Moreover, VP inhibited ESCC cell-induced angiogenesis in vitro by decreasing HUVEC chemotactic migration, adhesion and tube formation, and also reduced ESCC cell-induced neovascularization of the CAM in vivo. In addition, VP suppressed the expression of pro-angiogenic molecules such as VEGFA, MMP-2 and β-catenin in ESCC cells. Furtherly, VP increased the chemosensitivity of ESCC-resistant cells to paclitaxel (PTX). The combination of VP and PTX attenuated the resistant cell-mediated angiogenesis in vitro and in vivo. These results reveal for the first time that VP potently inhibits malignant progression and overcomes chemoresistance of ESCC cells via inhibition of tumor angiogenesis. It provides insight into a new strategy for the treatment of ESCC that VP could be a potential drug candidate for targeting tumor angiogenesis.

PD-1 Independent Role of PD-L1 in Triple-Negative Breast Cancer Progression

Triple-negative breast cancer (TNBC) is a type of breast malignancy characterized by a high proliferative rate and metastatic potential leading to treatment failure, relapse, and poor prognosis. Therefore, efforts are continuously being devoted to understanding its biology and identifying new potential targets. Programmed death-ligand 1 (PD-L1) is an immunosuppressive protein that inactivates T cells by binding to the inhibitory receptor programmed death-1 (PD-1). PD-L1 overexpression in cancer cells contributes to immune evasion and, subsequently, poor survival and prognosis in several cancers, including breast cancer. Apart from its inhibitory impact on T cells, this ligand is believed to have an intrinsic role in cancer cells. This study was performed to clarify the PD-1 independent role of PD-L1 in TNBC MDA-MB-231 cells by knocking out the PD-L1 using three designs of CRISPR-Cas9 lentiviral particles. Our study revealed that PD-L1 knockout significantly inhibited MDA-MB-231 cell proliferation and colony formation in vitro and tumor growth in the chick embryo chorioallantoic membrane (CAM) model in vivo. PD-L1 knockout also decreased the migration and invasion of MDA-MB-231 cells in vitro. We have shown that PD-L1 knockout MDA-MB-231 cells have low levels of p-Akt and p-ERK in addition to some of their downstream proteins, c-Fos, c-Myc, p21, survivin, and COX-2. Furthermore, PD-L1 knockout significantly decreased the expression of Snail and RhoA. This study shows the intrinsic role of PD-L1 in TNBC independently of its binding to PD-1 receptors on T cells. It may pave the way for developing novel therapeutic strategies using PD-L1 inhibitors alone and in combination to treat TNBC more effectively.

Ischemia and vasculogenesis after transplantation of frozen and vitrified human ovarian tissue onto Chick Embryo Chorioallantoic Membrane (CAM)

ObjectivesThe first days of post-ovarian transplantation are critical periods, as the ischemic injury can diminish the success rate. In this study, the first day’s events of ovarian transplantation in two dimensions of structure and ultrastructure following slow freezing and vitrification were assessed. Study designOvarian tissues (OTs) from 10 cancerous patients were frozen in two methods of slow freezing and vitrification. Tissues were transplanted onto the CAM and then retrieved at 5 and 10 days of culture. Nine groups were assigned as follows; I-III; fresh, 5 and 10 days culture, IV-VI; vitrification, 5 and 10 days culture, and VII-IX; slow freezing, 5 and 10 days culture. Structural and ultra-structural studies were done to assess the tissue viability and integrity following CAM transplantation. Image J software was used to measure the amounts of fibrosis and necrosis. ResultsThe first sign of successful transplantation was found on day 3 post-transplantation. Vitrified tissues showed higher viability and transplantation rate compared to the slow frozen group (65% vs 57.5%) (p = 0.7). Tissue fibrosis and areas didn’t increase significantly after cryopreservation using two methods (p > 0.05). The areas of fibrosis and necrosis and avian vessels increased significantly after 5 and 10 days of culture (p < 0.05). Large ultra-structural follicular deformities were noticed after 10 days of CAM transplantation. Better stromal ultrastructure features can be found after vitrified tissue culture. Also, the CAM transplantation technique had negative effects on the integrity of follicles, independent of the freezing procedure. ConclusionEvaluation of early events of the ovarian post-transplantation is of amount importance, since the hypoxia during this period may accelerate follicular pool depletion, before the tissue stability. Vitrification can be considered a reliable alternative for slow freezing. CAM transplantation is a good technique for confirmation of tissue viability after warming but damaged the follicle ultrastructure in a short period.

STIM1-regulated exosomal EBV-LMP1 empowers endothelial cells with anaggressive phenotype by activating the Akt/ERK pathway in nasopharyngeal carcinoma.

Stromal interaction molecule 1 (STIM1)-mediated Ca2+ signaling regulates tumor angiogenesis in nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-related human malignancy. However, the mechanism by which STIM1 modulates endothelial functional phenotypes contributing to tumor angiogenesis remains elusive. NPC cell-derived exosomes were isolated via differential centrifugation and observed using transmission electron microscopy. Exosome particle sizes were assessed by nanoparticle tracking analysis (NTA). Uptake of exosomes by recipient ECs was detected by fluorescent labeling of the exosomes with PKH26. Tumor angiogenesis-associated profiles were characterized by determining cell proliferation, migration, tubulogenesis and permeability in human umbilical vein endothelial cells (HUVECs). Activation of the Akt/ERK pathway was assessed by detecting the phosphorylation levels using Western blotting. A chick embryo chorioallantoic membrane (CAM) xenograft model was employed to study tumor-associated neovascularization in vivo. We found that NPC cell-derived exosomes harboring EBV-encoded latent membrane protein 1 (LMP1) promoted proliferation, migration, tubulogenesis and permeability by activating the Akt/ERK pathway in ECs. STIM1 silencing reduced LMP1 enrichment in NPC cell-derived exosomes, thereby reversing its pro-oncogenic effects in an Akt/ERK pathway-dependent manner. Furthermore, STIM1 knockdown in NPC cells blunted tumor-induced vascular network formation and inhibited intra-tumor neovascularization in the chorioallantoic membrane (CAM) xenograft model. STIM1 regulates tumor angiogenesis by controlling exosomal EBV-LMP1 delivery to ECs in the NPC tumor microenvironment. Blocking exosome-mediated cell-to-cell horizontal transfer of EBV-associated oncogenic signaling molecules may be an effective therapeutic strategy for NPC.

Hyperthermia Reduces Irradiation-Induced Tumor Repopulation in an In Vivo Pancreatic Carcinoma Model.

Pancreatic cancer has a poor prognosis due to its aggressive nature and ability to metastasize at anearly stage. Currently, its management is still a challenge because this neoplasm is resistant to conventional treatment approaches, among which is chemo-radiotherapy (CRT), due to the abundant stromal compartment involved in the mechanism of hypoxia. Hyperthermia, among other effects, counteracts hypoxia by promoting blood perfusion and thereby can enhance the therapeutic effect of radiotherapy (RT). Therefore, the establishment of integrated treatments would be a promising strategy for the management of pancreatic carcinoma. Here, the effects of joint radiotherapy/hyperthermia (RT/HT) on optimized chick embryo chorioallantoic membrane (CAM) pancreatic tumor models are investigated. This model enables a thorough assessment of the tumor-arresting effect of the combined approach as well as the quantitative evaluation of hypoxia and cell cycle-associated mechanisms by both gene expression analysis and histology. The analysis of the lower CAM allows to investigate the variation of the metastatic behaviors of the cancer cells associated with the treatments. Overall, this study provides a potentially effective combined strategy for the non-invasive management of pancreatic carcinoma.

The chick embryo chorioallantoic membrane patient-derived xenograft (PDX) model.

The chick embryo chorioallantoic membrane (CAM) CAM is an extraembryonic membrane generated by the fusion of the chorion with the vascularized allantoic membrane. It performs multiple functions during embryonic development, including respiration, calcium transport from the eggshell, acid-base homeostasis, and ion/water reabsorption from the allantoic fluid. The CAM is a widely used model for the study of angiogenesis, anti-angiogenesis, tumor growth, and metastasis as well as drug efficacy. Ethical approval is omitted if experiments are terminated at embryonic day 14 in most countries, facilitating screenings of pharmacological or physics-based therapies with high reproducibility at large scales supporting the 3Rs principle. Being naturally immunodeficient, the chick embryo accepts transplantation from various tissues and species without immune response. This review article is focused on the analysis of the literature and personal data concerning the effects of patient-derived xenografts (PDX) on the CAM.

Cyclophilin A Inhibitors Suppress Proliferation and Induce Apoptosis of MKN45 Gastric Cancer Stem-like Cells by Regulating CypA/CD147-Mediated Signaling Pathway

Gastric cancer stem cells (GCSCs) are a subgroup of gastric cancer (GC) cells with high self-renewal and multi-lineage differentiation abilities that lead to tumor initiation, metastasis, drug resistance, and tumor relapse. Therefore, the eradication of GCSCs can contribute to the effective treatment of advanced or metastatic GC. In our previous study, compound 9 (C9), a novel derivative of nargenicin A1, was identified as a potential natural anticancer agent that specifically targeted cyclophilin A (CypA). However, its therapeutic effect and molecular mechanisms of action on GCSC growth have not been assessed. In this study, we investigated the effects of natural CypA inhibitors, including C9 and cyclosporin A (CsA), on the growth of MKN45-derived GCSCs. Compound 9 and CsA effectively suppressed cell proliferation by inducing cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade in MKN45 GCSCs. In addition, C9 and CsA potently inhibited tumor growth in the MKN45 GCSC-grafted chick embryo chorioallantoic membrane (CAM) model. Furthermore, the two compounds significantly decreased the protein expression of key GCSC markers including CD133, CD44, integrin α6, Sox2, Oct4, and Nanog. Notably, the anticancer activities of C9 and CsA in MKN45 GCSCs were associated with the regulation of CypA/CD147-mediated AKT and mitogen-activated protein kinase (MAPK) signaling pathways. Collectively, our findings suggest that the natural CypA inhibitors C9 and CsA could be novel anticancer agents used to combat GCSCs by targeting the CypA/CD147 axis.

The development of a rapid patient-derived xenograft model to predict chemotherapeutic drug sensitivity/resistance in malignant glial tumors.

High-grade gliomas (HGG) are aggressive brain tumors associated with short median patient survival and limited response to therapies, driving the need to develop tools to improve patient outcomes. Patient-derived xenograft (PDX) models, such as mouse PDX, have emerged as potential Avatar platforms for personalized oncology approaches, but the difficulty for some human grafts to grow successfully and the long time required for mice to develop tumors preclude their use for HGG. We used a rapid and efficient ex-ovo chicken embryo chorioallantoic membrane (CAM) culture system to evaluate the efficacy of oncologic drug options for HGG patients. Implantation of fresh glioma tissue fragments from 59 of 60 patients, that include difficult-to-grow IDH-mutated samples, successfully established CAM tumor xenografts within 7 days, with a tumor take rate of 98.3%. These xenografts faithfully recapitulate the histological and molecular characteristics of the primary tumor, and the ability of individual fragments to form tumors was predictive of poor patient prognosis. Treatment of drug-sensitive or drug-resistant xenografts indicates that the CAM-glioma assay enables testing tumor sensitivity to temozolomide and carboplatin at doses consistent with those administered to patients. In a proof-of-concept study involving 14 HGG patients, we observed a correlation of 100% between the CAM xenograft response to temozolomide or carboplatin and the clinical response of patients. The CAM-glioma model is a fast and reliable assay that has the potential to serve as a complementary model to drug discovery and a real-time Avatar platform to predict the best treatment for HGG patients.

Plasmid containing VEGF-165 and ANG-1 dual genes packaged with fibroin-modified PEI to promote the regeneration of vascular network and dermal tissue

Reducing the cytotoxicity of cationic polymers is the major issue to their use as a gene delivery carrier. In this study, plasmids containing encoding vascular endothelial cell growth factor 165 and angiopoietin-1 were packaged with the conjugates of cationic fibroin (CSF) and polyethylenimine (PEI), instead of packaging pDNA with PEI alone, to prepare nanocomplexes (CSF+PEI)/pDNA. The complexes were loaded into a silk fibroin scaffold to enhance its function to induce microvascular network generation and dermal tissue regeneration. The results of transfecting EA.hy926 cells with the complexes in vitro showed that (CSF+PEI)/pDNA had a stronger transfection ability than PEI/pDNA. Importantly, compared with PEI as the gene carrier alone, the cell viability was significantly increased and the cytotoxicity was effectively reduced after the conjugate of CSF and PEI was used as the gene carrier. The results of angiogenesis in chick embryo chorioallantoic membranes showed that compared with scaffolds loaded with PEI/pDNA, the neovascularization ratio in scaffolds loaded with (CSF+PEI)/pDNA was significantly increased. In vivo experimental results of scaffolds implantation for full-thickness skin defects in SD rats showed that, compared with loading PEI/pDNA complex, loading (CSF+PEI)/pDNA complex in the scaffold more effectively promoted the formation of vascular network in the scaffold and accelerated the regeneration of dermal tissue. The gene delivery system established in this study has application potential not only in the regeneration of vascular-containing tissues, but also in tumor gene therapy.

Partial Decellularization as a Method to Improve the Biocompatibility of Heart Tissue Implants.

Increasing the biocompatibility of some biological implants through tissue engineering is important for regenerative medicine, which recently has a rapid development dynamic. In this study we used tree different washing protocols, respectively with Sodium Lauryl Sulfate (SLS), with Sodium Deoxycholate (SD), and with saline (Sa) to achieve partial decellularization of 2-3mm thick cross-sections through Wistar rat hearts. Pieces of the heart tissue were either histologically analyzed to evaluate the decellularization processes or implanted for 5 days on 9-day-old chick embryo chorioallantoic membrane (CAM) and then histologically analyzed to evaluate CAM-implant interactions. Histological analysis of SLS or SD washed tissues showed different microscopic features of the decellularization processes, SLS-washing leading to the formation of a completely decellularized ECM layer at the periphery of the heart tissue. Both detergents induced changes in the spatial arrangement of collagen fibers of the heart tissue. Histological analysis of the CAM implants shoved that the peripheral zone with complete decellularization induced by SLS increased the biocompatibility of heart tissue implants by favoring neovascularization and cell migration. These results suggested that the biocompatibility of the heart tissue implant can be modulated by the appropriate use of a SLS-based decellularization protocol.