Chorioallantoic Membrane Model Research Articles

Abstract 6791: Inovative in vivo (in ovo) CAM model to predict efficacy and mode of action of a new antitumor vaccine stc-1010 on human colorectal adenocarcinoma

Abstract BACKGROUND: Colorectal cancer (CRC) is the second-most deadly cancer. Therapeutic resistance to immuno-oncology drives the need for new treatments. Stimulated tumor cell (STC) vaccine (Brenus Pharma) is composed of selected tumor cell lines, stimulated to overexpress tumor-associated or tumor-specific antigens and neoantigens including resistance factors. Haptenization of these proteins forms an immunogenic complex which stimulate the immune system to recognize and target the patient’s tumor cells expressing same resistance factors. We report in vivo results of STC-1010 vaccine, on human CRC adenocarcinoma from HT29 cell using chorioallantoic membrane (CAM) assay developed by Inovotion in immune reactive model. METHODS: This study was carried out in 2 steps: Firstly, three batches of naïve chicken embryos were stimulated by injections of STC1010 at Embryo Development Day (EDD) 11 and EDD13. At EDD18 of each batch, chicken peripheral blood mononuclear cells (PBMCs) were collected and used as anti-tumor reagent to treat respectively, at EDD 11, EDD 13 and EDD16 chicken embryos xenografted with HT29 cells. Activation of PBMCs was evaluated by IL2 and IL12 secretion quantified by ELISA. At EDD18, i.e., 9 days post-graft, in ovo anti-tumor efficacy was evaluated by tumor weight, metastatic invasion (qPCR analysis of human Alu sequence in lower CAM) and quantification of tumor-infiltrating by CD8, CD4, IFN-gamma, Perforin and TNF-alpha. RESULTS: Compared to negative control, STC-1010 vaccine induced: 1) significant increase of IL-12 and IL-2 secretion in peripheric blood during the generation of all three batches of PBMCs, confirming previous results (IL-12: +52%, p=0.0003 ; IL-2: +482%, p=0.0033); 2) a significant expression of IFN-gamma in tumor (+130,83%, p=0.0185); 3) a tendency to increase infiltrating cells: CD4+: +79,2%, CD8+: +29,4% , Perforin: +105,5%, TNFα : +78,63% confirmed by immunohistochemistry and translated into 4) a significant increase of tumor necrosis (p = 0.0267); and 5) a tendency of metastasis regression (-49%); with 6) no embryonic toxicity/mortality (daily evaluation of embryonic viability) induced by STC-1010. CONCLUSION: This in ovo study confirms efficacy of the STC-1010 observed in previous CRC syngeneic models and gives more insight about STC mechanism of action with the activation and maturation of dendritic cells, induction of CD8+ and LTh1 against tumor as the main driver of the response, all without toxicity. Inovotion’s CAM model could be used for indication screening and as a pre-proof-of-concept before syngeneic model study. Citation Format: Yan Wang, Arnaud Peyronnier, Benoit Pinteur, Lionel Chalus, Corinne Tortorelli, Paul Bravetti, Jean Viallet, François Ghiringhelli, Philippe Fornies. Inovative in vivo (in ovo) CAM model to predict efficacy and mode of action of a new antitumor vaccine stc-1010 on human colorectal adenocarcinoma [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 6791.

Abstract 4866: Pharmacological mechanisms of osimertinib in advanced non-small lung cancer bearing the deletion exon 19 of EGFR In an original chicken chorioallantoic membrane (CAM) Model

Abstract Introduction: NSCLC accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor EGFR activating mutations, such as in-frame deletions in exon 19 (Ex19del). Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of anti-EGFR inhibitors such as Osimertinib. The CAM xenograft offers a rich environment in nutrients and embryonic growth factors, favorable for aggressive tumor development, with its highly vascularized membrane and natural immunodeficiency at engraftment (at EDD9). Methods: HCC827 Ex19del cells have been engrafted in ovo, and treated with various doses of osimertinib for seven days (4 treatments). The xenografts were collected and immunohistopathological, transcriptional and genetic analyses were carried out. Based on human transcriptional data, supervised multivariate discrimination between conditions and ontology analysis was performed through GSEA. Results: The xenograft growth was inversely dependent on the exposure dose of osimertinib, with a reduction in tumor weight of 58% for 10 µM (corresponding to 8.3 µg/Kg, T1/2 =4hr). The histological analysis showed epithelial tumor cells composed of solid sheets. The quantity of tumor cells was significantly lower in the treated group, with an area of tumor necrosis. The immunohistochemistry of tumor cells confirmed the human pulmonary epithelial origin, with TTF1 positive in treated and control groups. As expected, the ontology analysis of transcriptomic findings highlighted a downregulation of the EGFR pathway and its downstream effectors. Furthermore, the transcriptomic responses associated with chemotaxis, immune cell recruitment, and angiogenesis were dampened in the presence of osimertinib. This suggests that therapeutic efficacy was not uniquely guided through cell-autonomous mechanisms, but also takes place at the tissue level. We confirmed this in an independent experiment of in ovo HCC827 engraftment, showing a statically significant reduction of 15% in the number of vessels surrounding the xenograft in the osimertinib-treated condition. This demonstrates the advantage of using the in ovo model to decipher the regulations associated with therapeutics in a complex tissue context. Conclusions: For the first time, we have observed that osimertinib modulates angiogenesis and chemotaxis, which apparently reduces immune system recruitment and immune checkpoint responses. The ontologic analysis of transcriptomic findings in the CAM model strongly support the clinical observations of the pharmacological tumoral response to targeted therapy. The fact that immune infiltration was reduced in osimertinib-treated tumors may partially explain the lower response to immunotherapy regimens in osimertinib resistance contexts. Citation Format: David Barthelemy, Arnaud Vigneron, Xavier Rousset, Jerome Guitton, Emmanuel Grolleau, Margaux Raffin, Julie Balandier, Gaelle Lescuyer, Florence Geiguer, Sebastien Couraud, Jean Vaillet, Nazim Benzerdjeb, Lea Payen-Gay. Pharmacological mechanisms of osimertinib in advanced non-small lung cancer bearing the deletion exon 19 of EGFR In an original chicken chorioallantoic membrane (CAM) Model. [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 4866.

Abstract 3620: N-myristoylation inhibition reduces angiogenesis and cancer cell migration

Abstract Myristoylation is the modification of proteins by the 14-carbon fatty acid myristate. In humans, two N-myristoyltransferases (NMT1 and NMT2) catalyze myristate transfer onto >200 proteins to regulate membrane binding and signal transduction. The pan-NMT inhibitor PCLX-001 has been in phase I clinical trials over a year for the treatment of lymphoma and advanced solid malignancies. Lack of myristoylation promotes the degradation of numerous normally myristoylated proteins like the proto-oncogenic Src family kinases (SFKs). SFKs like Src and Lyn are essential for downstream signaling of Vascular Endothelial Growth Factor (VEGF) and Epidermal Growth Factor (EGF) receptor tyrosine kinases. Thus, SFKs are critical for angiogenesis and cell motility. Angiogenesis is essential for tumor growth and dissemination of cancer cells to distal locations. VEGF-induced angiogenesis requires Src activation, suggesting that PCLX-001 may have antiangiogenic properties. SFKs also promote cell migration/metastasis, the leading cause of cancer-associated mortality. Lyn SFK knockdown was shown to reduce breast cancer migration and neighbouring tissue invasion, suggesting that PCLX-001 may also inhibit cell migration/metastasis. We investigated the antiangiogenic properties of PCLX-001 in vitro using HUVEC umbilical cord cells. PCLX-001 treatment significantly reduced HUVEC sprouting and tube-formation to levels lower than two FDA-approved angiogenesis inhibitors Sorafenib and Sunitinib. PCLX-001 also significantly reduced neo-vascularisation in vivo at lower concentrations than these two drugs using a chicken chorioallantoic membrane model, demonstrating the high potential of myristoylation inhibitors to reduce angiogenesis. We then validated our observations in vivo using a HT-1376 bladder mouse xenograft model. Tumors from mice treated for 28 days at increasing PCLX-001 concentrations showed significant reduction of Src, Lyn, and VEGF receptor protein levels as measured by immunohistochemistry, further validating PCLX-001 effect in vivo. We hypothesize that PCLX-001 acts downstream of VEGF/EGF receptors by inducing degradation of un-myristoylated SFKs. We confirmed this by knocking down NMT1 or treating breast cancer cells with PCLX-001 and found that both significantly reduced SFK protein levels. In a wound-healing assay using PCLX-001, we observed significant dose-dependent cell migration inhibition. PCLX-001 also dose-dependently reduced migration and invasion in transwell migration/invasion assays, indicating that myristoylation is required for optimal migration and potentially cancer cell metastasis. Overall, by lowering SFK levels, PCLX-001 reduces angiogenesis, cell migration/invasion, and thus potentially cancer metastasis. Reducing these classical cancer hallmarks in malignant cells may benefit a wide array of cancer patients and ultimately improve cancer treatment outcomes. Citation Format: Rony Pain, Erwan Beauchamp, Katia Carmine-Simmen, Jay Gamma, Rebecca Reif, Abul Azad, Allan Murray, John Lewis, Luc Berthiaume. N-myristoylation inhibition reduces angiogenesis and cancer cell migration. [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 3620.

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.

The Chorioallantoic Membrane Xenograft Assay as a Reliable Model for Investigating the Biology of Breast Cancer

The chorioallantoic membrane (CAM) assay is an alternative in vivo model that allows for minimally invasive research of cancer biology. Using the CAM assay, we investigated phenotypical and functional characteristics (tumor grade, mitosis rate, tumor budding, hormone receptor (HR) and HER2 status, Ki-67 proliferation index) of two breast cancer cell lines, MCF-7 and MDA-MB-231, which resemble the HR+ (luminal) and triple-negative breast cancer (TNBC) subgroups, respectively. Moreover, the CAM results were directly compared with murine MCF-7- and MDA-MB-231-derived xenografts and human patient TNBC tissue. Known phenotypical and biological features of the aggressive triple-negative breast cancer cell line (MDA-MB-231) were confirmed in the CAM assay, and mouse xenografts. Furthermore, the histomorphological and immunohistochemical variables assessed in the CAM model were similar to those in human patient tumor tissue. Given the confirmation of the classical biological and growth properties of breast cancer cell lines in the CAM model, we suggest this in vivo model to be a reliable alternative test system for breast cancer research to reduce murine animal experiments.

Abstract P3-08-11: Establishing Breast Cancer Patient-derived Xenografts from circulating cancer stem cells in the chorioallantoic membrane (CAM) Model

Abstract Background: Circulating cancer stem cells (cCSCs) are a small aggressive subset of circulating tumor cells with cancer stem cells features: resistance to diverse cancer treatments and the capacity for generating new metastases. Patient-derived xenografts (PDX) are an increasingly accepted tool in oncology, providing biologically meaningful models of many cancer types, and potential platforms for the development of precision oncology approaches. Commonly, mouse models are used for the xenotransplant assessment of potential new therapeutic targets in cancers. However, animal models do not necessarily represent the real world scenario and are costly and time consuming. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. CAM assay is increasingly used as a rapid cost-effective in vivo drug-testing platform that recapitulates many aspects of human cancers. Methods: In this study, primary cultures from circulating cancer stem cells were established using sphere-forming assays. Subsequently, tumorspheres were transplanted onto the CAM membrane of fertilized chicken eggs to form secondary microtumors. Histopathological analyses were performed to confirm that the CAM tumor had the original morphological profile of the patient tumor. Results: We have developed an innovative reliable in vitro platform for cultivation of CSCs from peripheral blood of breast cancer patients. The number of tumorspheres increased significantly with tumor progression. Patients with metastatic disease had statistically more tumorspheres as compared to patients without metastasis (30 vs 10/100µl blood, p< 0.05). Patients with multiple metastases had more tumorspheres compared to patients with single metastases (60 vs 30/100µl blood, p< 0.05). The number of tumorspheres was positively correlated with Ki-67, Her2 status and grade score in primary breast tumors. Tumorspheres showed self-renewal, growth potential, invasion and differentiation in vivo. Tumorspheres could be successfully grafted onto the CAM and grafting positively correlated with aggressiveness and proliferation capacity of the primary tumor. These tumors growing on the CAM pathologically closely resembled the primary tumor. Conclusion: The number of tumorspheres cultured from peripheral blood of cancer patients and the success rate of establishing PDX directly reflect the aggressiveness and proliferation capacity of the primary tumor. Our results support the CAM model using cCSC as a valuable alternative for xenotransplant models. It allows the establishment of new PDXs and provides a fast, cost-effective, and easy to use preclinical platform for cancer biology research, new drug development, treatment individualization, and biomarker discovery. Citation Format: Monika PIZON, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. Establishing Breast Cancer Patient-derived Xenografts from circulating cancer stem cells in the chorioallantoic membrane (CAM) Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-11.

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.

Establishment of patient-derived xenografts of retinoblastoma and choroidal melanoma on the avian chorioallantoic membrane.

To develop a viable in vivo chorioallantoic membrane (CAM) model to study the growth and invasion of patient-derived retinoblastoma (RB) and choroidal melanoma (CM) xenografts (PDXs). The study utilizes primary tumor samples instead of cancer cell lines, which provides a more authentic representation of tumors due to conserved morphology and heterogeneity. Fertilized chicken eggs were procured, windowed, and their CAM layers were dropped. On embryonic development day (EDD) 10, freshly cut patient-derived CM and RB tumors were implanted on the CAM layer and the setup was incubated for 7 days. The tumor-embedded CAM layer was harvested on EDD 17, and the extracted tumor samples were subjected to hematoxylin and eosin staining and immunohistochemical analysis to evaluate the extent of tumor invasion. Significant changes in the vascularity around the RB and CM PDXs were observed, indicating an angiogenic environment. The cross-sectional histological view of the tumor implant site revealed the invasion of both the tumors into the CAM mesoderm. Invasion of CM into CAM mesoderm was visualized in the form of pigmented nodules, and that of RB was indicated by synaptophysin and Ki-67 positivity in Immunohistochemistry (IHC). The CAM xenograft model was successfully able to support the growth of CM and RB PDXs and their invasion in CAM, thus presenting as a feasible alternative to mammalian models for studying tumorigenicity and invasiveness of ocular tumors. Moreover, this model can further be utilized to develop personalized medicine by inoculating patient-specific tumors for preclinical drug screening.

A Study of Ungernia trisphaera Bunge and Thymus transcaspicus Cytotoxicity in Cancer Cell Lines

Objective: Thymus transcaspicus (Lamiaceae) is an Iranian species of Thymus, and Ungernia trisphaera Bunge belongs to the family Amaryllidaceae. Cytotoxic properties of total ethanolic extract of T. transcaspicus and U. trisphaera were investigated against different cell lines including B16F10, PC3, U87, and MCF-7. Methods: The proliferation, cell cycle, and migration of the cells were determined by thiazolyl blue tetrazolium bromide (MTT) test, propidium iodide (PI) staining, and scratch assay respectively. The effects of U. trisphaera extract (UTE) on the activity of matrix metalloproteinases (MMPs) and angiogenesis were evaluated by the gelatin zymography method and chicken chorioallantoic membrane model, respectively. The GSH, SOD, and MDA were evaluated by colorimetric method. Results: The results showed that UTE could inhibit the development of malignant cells in a concentration- dependent manner, while the inhibitory effect of T. transcaspicus extract (TTE) was not significant compared to the control group. The UTE-induced sub-G1 peak apoptosis compared to the control group indicated that apoptotic cell death is involved in UTE-induced cytotoxicity. MMPs activity was significantly decreased 48 hours after treatment. Moreover, GSH level and SOD activity were significantly decreased while MDA and ROS levels were significantly increased after 24 hours treatment. In addition, combination of UTE (1.5-25 μg/mL) with doxorubicin (6 μg/mL) showed an additive cell growth inhibitory effect. Conclusion: UTE demonstrated cytotoxic and apoptogenic effects in different cancer cell lines, and it was found that apoptosis plays a crucial role in the cytotoxicity of UTE. Thus, U. trisphaera can be considered a potential medicinal herb in cancer treatment after comprehensive pharmacological and toxicological studies.

Exploring RAB11A Pathway to Hinder Chronic Myeloid Leukemia-Induced Angiogenesis In Vivo.

Neoangiogenesis is generally correlated with poor prognosis, due to the promotion of cancer cell growth, invasion and metastasis. The progression of chronic myeloid leukemia (CML) is frequently associated with an increased vascular density in bone marrow. From a molecular point of view, the small GTP-binding protein Rab11a, involved in the endosomal slow recycling pathway, has been shown to play a crucial role for the neoangiogenic process at the bone marrow of CML patients, by controlling the secretion of exosomes by CML cells, and by regulating the recycling of vascular endothelial factor receptors. The angiogenic potential of exosomes secreted by the CML cell line K562 has been previously observed using the chorioallantoic membrane (CAM) model. Herein, gold nanoparticles (AuNPs) were functionalized with an anti-RAB11A oligonucleotide (AuNP@RAB11A) to downregulate RAB11A mRNA in K562 cell line which showed a 40% silencing of the mRNA after 6 h and 14% silencing of the protein after 12 h. Then, using the in vivo CAM model, these exosomes secreted by AuNP@RAB11A incubated K562 did not present the angiogenic potential of those secreted from untreated K562 cells. These results demonstrate the relevance of Rab11 for the neoangiogenesis mediated by tumor exosomes, whose deleterious effect may be counteracted via targeted silencing of these crucial genes; thus, decreasing the number of pro-tumoral exosomes at the tumor microenvironment.

Evaluation of the EPR Effect in the CAM-Model by Molecular Imaging with MRI and PET Using 89Zr-Labeled HSA

Mouse models are commonly used to study the biodistribution of novel radioligands, but alternative models corresponding to the 3Rs principles, such as the chorioallantoic membrane (CAM) model, are highly required. While there are promising data from the CAM model regarding target-specific radiolabeled compounds, its utility for assessing macromolecule biodistribution and analyzing the EPR effect remains to demonstrated. Using 89Zr-labeled human serum albumin, the accumulation of nontarget-specific macromolecules in CAM and mouse xenograft models was studied using PET and MRI. Therefore, the radioligand [89Zr]Zr-DFO-HSA was analyzed in both chicken embryos (n = 5) and SCID mice (n = 4), each with TZM-bl and PC-3 tumor entities. Dynamic PET and anatomical MRI, as well as ex vivo biodistribution analyses, were performed to assess ligand distribution over 24 h. Histological staining and autoradiography verified the intratumoral accumulation. The tumors were successfully visualized for CAM and mouse models by PET, and the albumin influx from the blood into the respective tumors did not differ significantly. The accumulation and retention of HSA in tumors due to the EPR effect was demonstrated for both models. These results highlight that the CAM model is a potential alternative to the mouse model for initial studies with novel radiolabeled macromolecules with respect to the 3Rs principles.

Multi-level optical angiography for photodynamic therapy.

Blood flow imaging is widely applied in photodynamic therapy (PDT) to provide vascular morphological and statistical parameters. This approach relies on the intensity of time-domain signal differences between blood vessels and background tissues; therefore, it often ignores differences within the vasculature and cannot accommodate abundant structural information. This study proposes a multi-level optical angiography (MOA) method for PDT. It can enhance capillaries and image vessels at different levels by measuring the signal frequency shift associated with red blood cell motion. The experimental results regarding the PDT-induced chorioallantoic membrane model showed that the proposed method could not only perform multi-level angiography but also provide more accurate quantitative information regarding various vascular parameters. This MOA method has potential applications in PDT studies.

Evaluation of Human Mesenchymal Stromal Cells as Carriers for the Delivery of Oncolytic HAdV-5 to Head and Neck Squamous Cell Carcinomas.

Human multipotent mesenchymal stromal cells (hMSCs) are of significant therapeutic interest due to their ability to deliver oncolytic adenoviruses to tumors. This approach is also investigated for targeting head and neck squamous cell carcinomas (HNSCCs). HAdV-5-HexPos3, a recently reported capsid-modified vector based on human adenovirus type 5 (HAdV-5), showed strongly improved infection of both hMSCs and the HNSCC cell line UM-SCC-11B. Given that, we generated life cycle-unmodified and -modified replication-competent HAdV-5-HexPos3 vector variants and analyzed their replication within bone marrow- and adipose tissue-derived hMSCs. Efficient replication was detected for both life cycle-unmodified and -modified vectors. Moreover, we analyzed the migration of vector-carrying hMSCs toward different HNSCCs. Although migration of hMSCs to HNSCC cell lines was confirmed in vitro, no homing of hMSCs to HNSCC xenografts was observed in vivo in mice and in ovo in a chorioallantoic membrane model. Taken together, our data suggest that HAdV-5-HexPos3 is a potent candidate for hMSC-based oncolytic therapy of HNSCCs. However, it also emphasizes the importance of generating optimized in vivo models for the evaluation of hMSC as carrier cells.

Sesamin Attenuates VEGFA-Induced Angiogenesis via Inhibition of Src and FAK Signaling in Chick Chorioallantoic Membrane Model and Human Endothelial EA.hy926 Cells.

Sesamin, a major phytochemical in sesame seeds and oil, has been reported to have effects on physiological and pathological angiogenesis in several studies. Nevertheless, the underlying mechanisms of sesamin's effect on angiogenesis are not understood well enough. This study aimed to investigate its effect on both physiological and pathological angiogenesis using the in vivo chick chorioallantoic membrane (CAM) model and the in vitro human endothelial cell line, EA.hy926, model. Sesamin inhibited the VEGFA-induced pathological angiogenesis significantly, although no effect was seen on angiogenesis without induction. It reduced the formation of vascular branches in the VEGFA-treated CAMs and also the proliferation and migration of EA.hy926 endothelial cells induced by VEGFA. Sesamin impeded the VEGF-mediated activation of Src and FAK signaling proteins, which may be responsible for sesamin-mediated reduction of pathological angiogenesis. Moreover, the effect of sesamin on the expressions of angiogenesis-related genes was then investigated and it was found that both mRNA and protein expressions of Notch1, the key pathway in vascular development, induced by VEGFA, were significantly reduced by sesamin. Our results altogether suggested that sesamin, by inhibiting pathological angiogenesis, has the potential to be employed in the prevention or treatment of diseases with over-angiogenesis, such as cancers.

Opposing MMP-9 Expression in Mesenchymal Stromal Cells and Head and Neck Tumor Cells after Direct 2D and 3D Co-Culture

Bone marrow-derived mesenchymal stromal cells (BMSCs) respond to a variety of tumor cell-derived signals, such as inflammatory cytokines and growth factors. As a result, the inflammatory tumor microenvironment may lead to the recruitment of BMSCs. Whether BMSCs in the tumor environment are more likely to promote tumor growth or tumor suppression is still controversial. In our experiments, direct 3D co-culture of BMSCs with tumor cells from the head and neck region (HNSCC) results in strong expression and secretion of MMP-9. The observed MMP-9 secretion mainly originates from BMSCs, leading to increased invasiveness. In addition to our in vitro data, we show in vivo data based on the chorioallantoic membrane (CAM) model. Our results demonstrate that MMP-9 induces hemorrhage and increased perfusion in BMSC/HNSCC co-culture. While we had previously outlined that MMP-9 expression and secretion originate from BMSCs, our data showed a strong downregulation of MMP-9 promoter activity in HNSCC cells upon direct contact with BMSCs using the luciferase activity assay. Interestingly, the 2D and 3D models of direct co-culture suggest different drivers for the downregulation of MMP-9 promoter activity. Whereas the 3D model depicts a BMSC-dependent downregulation, the 2D model shows cell density-dependent downregulation. In summary, our data suggest that the direct interaction of HNSCC cells and BMSCs promotes tumor progression by significantly facilitating angiogenesis via MMP-9 expression. On the other hand, data from 3D and 2D co-culture models indicate opposing regulation of the MMP-9 promoter in tumor cells once stromal cells are involved.

Deciphering Brain Metastasis Stem Cell Properties From Colorectal Cancer Highlights Specific Stemness Signature and Shared Molecular Features

Brain metastases (BMs) from colorectal cancer (CRC) are associated with significant morbidity and mortality, with chemoresistance and short overall survival. Migrating cancer stem cells with the ability to initiate BM have been described in breast and lung cancers. In this study, we describe the identification and characterization of cancer stem cells in BM from CRC. Four brain metastasis stem cell lines from patients with colorectal cancer (BM-SC-CRC1 to BM-SC-CRC4) were obtained by mechanical dissociation of patient's tumors and selection of cancer stem cells by appropriate culture conditions. BM-SC-CRCs were characterized invitro by clonogenic and limiting-dilution assays, as well as immunofluorescence and Western blot analyses. In ovo, a chicken chorioallantoic membrane (CAM) model and invivo, xenograft experiments using BALB/c-nude mice were realized. Finally, a whole exome and RNA sequencing analyses were performed. BM-SC-CRC formed metaspheres and contained tumor-initiating cells with self-renewal properties. They expressed stem cell surface markers (CD44v6, CD44, and EpCAM) in serum-free medium and CRC markers (CK19, CK20 and CDX-2) in fetal bovine serum-enriched medium. The CAM model demonstrated their invasive and migratory capabilities. Moreover, mice intracranial xenotransplantation of BM-SC-CRCs adequately recapitulated the original patient BM phenotype. Finally, transcriptomic and genomic approaches showed a significant enrichment of invasiveness and specific stemness signatures and highlighted KMT2C as a potential candidate gene to potentially identify high-risk CRC patients. This original study represents the first step in CRC BM initiation and progression comprehension, and further investigation could open the way to new therapeutics avenues to improve patient prognosis.

Antiangiogenic Potential of Troxerutin and Chitosan Loaded Troxerutin on Chorioallantoic Membrane Model.

Angiogenesis is crucial to the development of cancer because it allows the transport of oxygen, nutrients, and growth factors as well as the spread of tumors to distant organs. Inhibitors of angiogenesis prevent the formation of blood vessels that allow tumor cells to shrink, rather than promote tumor growth. Chitosan acts as a carrier for many drugs, since the compound has various properties such as biodegradable, less toxicity, more stable, simple, easy to prepare, and biocompatible. The aim of the current study was to evaluate the efficacy of chitosan nanoparticles encapsulated with troxerutin (Chi-Trox NPs) against angiogenesis and cancer in ova chick chorioallantoic membrane (CAM) model. Chi-Trox NPs were synthesized using a nanoprecipitation method and were characterized by various analyses. 24 hours' fertilized eggs (6 eggs/group) were treated with native Trox and Chi-Trox NPs for 5 days. The antiangiogenic activity was evaluated by morphometric, histopathological, immunohistochemical (CD104 and vimentin), and mRNA expression of MMP and FGF2 using RT-PCR. The anticancer activity was evaluated by histopathological, immunohistochmical (CD44), and mRNA expression of FGF2 and MMP. The synthesized chitosan NPs were successfully encapsulated with troxerutin, and the loading efficiency of chitosan NPs was found to be 86.4 ± 0.12% and 13.2 ± 0.16% respectively. Morphometric analysis of Chi-Trox NPs showed a considerable decrease in the number of blood vessels compared with control and native Trox. The histopathological observation of CAM confirmed that Chi-Trox NPs induce a significant reduction in inflammatory cells and the thickness of blood capillaries compared to control and native Trox. The immunohistochemical evaluation of CAM revealed Chi-Trox decreased CD104, vimentin and CD44 protein levels were compared with control and native Trox. Furthermore, the mRNA expression levels of FGF2 and MMP were significantly downregulated compared to their native forms. From the obtained results, Chi-Trox NPs possess significant inhibition of angiogenesis and can be used as therapeutic agents for cancer in the future.

Chick chorioallantoic membrane model to investigate role of migrasome in angiogenensis.

The development of the vascular system is essential for embryonic development, including processes such as angiogenesis. Angiogenesis plays a critical role in many normal physiological and pathological processes. It is driven by a set of angiogenic proteins, including angiogenic growth factors, chemokines, and extracellular matrix proteins. Among various animal model systems, the chorioallantoic membrane (CAM), a specialized and highly vascularized tissue of the avian embryo, has proven to be a valuable tool for analyzing the angiogenic potential of candidate cells or factors. In this protocol, we provide detailed procedures for establishing the CAM model to evaluate the function and mechanism of migrasomes in embryonic angiogenesis. This includes the CAM nylon mesh assay and CAM ex vivo sprouting assay to assess CAM angiogenesis, as well as the observation, purification, and delivery of migrasomes. Additionally, we describe the generation of T4-KO-mCherry-KI embryos using the CRISPR system within the CAM tissue to investigate the role of migrasomes in angiogenesis.

Interventional effect of processing temperature on anti-angiogenesis of Coptis chinensis and screening of active components by UPLC-MS/MS on quail chick chorioallantoic membrane model

Ethnopharmacological relevanceCoptis chinensis Franch. (CC), as a commonly used heat-clearing and toxin-resolving traditional Chinese herbal medicine, has gained increased attention for its anti-tumor activity. However, little is known about the anti-tumor angiogenesis effect of CC and its possible bioactive components. Also, it has been shown that temperature affects the quality of CC, albeit whether and how it affects the anti-angiogenic activity of CC is currently unknown. Aim of the studyTo determine the processing temperatures (40, 60, 80, 120, 140, 150, 160 and 200 °C) at which CC has the strongest anti-angiogenic effect and speculate the possible bioactive components. Materials and methodsThe q-CAM model was constructed to explore the anti-angiogenesis agents of CC. The angiogenesis inhibition effects of CC samples at different processing temperatures and its seven alkaloids were determined based on morphological observation and vascular area proportion analysis. UPLC-MS/MS was employed to screen the potent active components of CC on anti-angiogenesis. ResultsAll the intervention by CC at different processing temperatures and its seven alkaloids could inhibit angiogenesis on q-CAM vessels, as evidenced by a poor vasular development in morphological observation and a low vascular area proportion in vascular quantitative analysis, most evident in CC processed at 40 °C and palmatine. LC-MS revealed that palmatine displayed strongest inhibitory effect on q-CAM vessels with a high absorption due to its stable structure. And the maternal nucleus transformation phenomenon of CC alkaloids was found in the quail embryo metabolism. ConclusionsThe q-CAM models in conjunction with the UPLC-MS/MS technique could be a useful tool for assessing tumor angiogenesis and screening tumor-targeted medicines. Processing temperature can affect the anti-angiogenesis effect of CC because of its function on the content of alkaloids, and palmatine can be considered as a prospective anti-angiogenic drug.