Caftaric Acid Blunts MMP9 Activity and Expression in CAM Angiogenesis Model: In vitro and In vivo Evaluation by Targeting NFkB/VEGF/MMP-9 Signaling in Diabetic Retinopathy

(1) Background: The chorioallantoic membrane (CAM) model has the potential to contribute to the development of personalized medicine based on individual cancer patients. We previously established the CAM model using patient-derived CIC-DUX4 sarcoma cells. We also used the CAM model for characterization and a comparison with the mouse model by examining the tumor accumulation of small-size, highly dispersive mesoporous silica nanoparticles (MSNs). (2) Method: In this study, we transplanted a variety of cancer cell lines, including patient-derived osteosarcoma (OS) and extraskeletal osteosarcoma (ESOS) cells. Patient-derived OS, ESOS and other cell lines were transplanted onto CAMs. The proliferation of cancer cells within CAM tumors was confirmed using H&E staining. For the comparison of the CAM and mouse models, rhodamine B-labeled MSNs were administered intravenously to CAMs and to xenograft mice. Tumor accumulation was evaluated by examining fluorescence and by confocal microscopy. The biodistribution of MSNs was examined by measuring the Si content by ICP. (3) Results: H&E staining demonstrated the proliferation of cancer cells of OS, ESOS and others on CAMs. While growth patterns and morphologies varied among different cancer types, H&E staining confirmed the establishment of tumors. As for the tumor accumulation, both the CAM and mouse models showed that MSNs were selectively accumulated in the tumors in both the CAM and mouse models. (4) Conclusions: We have expanded the range of CAM models by using a variety of cancer cells, including patient-derived cell lines. We also report that the small-size, highly dispersive MSNs exhibit excellent tumor accumulation in both the CAM and mouse models. These results point to the usefulness of the CAM model for patient-derived cancer cells as well as for evaluating drug carriers for tumor targeting.

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.

Ginsenoside Rg_2(GRg2) is a triterpenoid compound found in Panax notoginseng. This study explored its effects and mechanisms on diabetic retinopathy and angiogenesis. The study employed endothelial cell models induced by glucose or vascular endothelial growth factor(VEGF), the chorioallantoic membrane(CAM) model, the oxygen-induced retinopathy(OIR) mouse model, and the db/db mouse model to evaluate the therapeutic effects of GRg2 on diabetic retinopathy and angiogenesis. Transwell assays and endothelial tube formation experiments were conducted to assess cell migration and tube formation, while vascular area measurements were applied to detect angiogenesis. The impact of GRg2 on the retinal structure and function of db/db mice was evaluated through retinal thickness and electroretinogram(ERG) analyses. The study investigated the mechanisms of GRg2 by analyzing the activation of Yes-associated protein(YAP) and Toll-like receptors(TLRs) pathways. The results indicated that GRg2 significantly reduced cell migration numbers and tube formation lengths in vitro. In the CAM model, GRg2 exhibited a dose-dependent decrease in the vascular area ratio. In the OIR model, GRg2 notably decreased the avascular and neovascular areas, ameliorating retinal structural disarray. In the db/db mouse model, GRg2 increased the total retinal thickness and enhanced the amplitudes of the a-wave, b-wave, and oscillatory potentials(OPs) in the ERG, improving retinal structural disarray. Transcriptomic analysis revealed that the TLR signaling pathway was significantly down-regulated following YAP knockdown, with PCR results consistent with the transcriptome sequencing findings. Concurrently, GRg2 downregulated the expression of Toll-like receptor 4(TLR4), TNF receptor-associated factor 6(TRAF6), and nuclear factor-kappaB(NF-κB) proteins in high-glucose-induced endothelial cells. Collectively, GRg2 inhibits cell migration and tube formation and significantly reduces angiogenesis in CAM and OIR models, improving retinal structure and function in db/db mice, with its pharmacological mechanism likely involving the down-regulation of YAP expression.

To improve (pre)malignant lesion identification in Barrett’s esophagus (BE), recent research focuses on new developments in fluorescence imaging and spectroscopy to enhance tissue contrast. Our aim was to validate the chorioallantoic membrane (CAM) model as a preclinical tool to study the fluorescence characteristics such as autofluorescence and exogenously induced fluorescence of human Barrett’s tissue. Therefore, esophageal biopsy specimens from Barrett’s patients were freshly grafted onto the CAM of fertilized hen’s eggs to simulate the in vivo situation. The BE biopsy specimens stayed between 1 and 9 days on the CAM to study the persistence of vitality. Fluorescence spectroscopy was performed using six excitation wavelengths (369, 395, 400, 405, 410, 416 nm). Obtained autofluorescence spectra were compared with in vivo spectra of an earlier study. Exogenous administration of 5-aminolevulinic-acid to the biopsy specimens was followed by fluorescence spectroscopy at several time points. Afterwards, the biopsy specimens were harvested and histologically evaluated. In total, 128 biopsy specimens obtained from 34 patients were grafted on the CAM. Biopsy specimens which stayed on average 1.7 days on the CAM were still vital. Autofluorescence spectra of the specimens correlated well with in vivo spectra. Administered 5-aminolevulinic-acid to the biopsy specimens showed conversion into protoporphyrin-IX. In conclusion, we showed that grafting freshly collected human BE biopsy specimens on the CAM is feasible. Our results suggest that the CAM model might be used to study the fluorescence behavior of human tissue specimens. Therefore, the CAM model might be a preclinical research tool for new photosensitizers.

Simple SummaryThe 5-year overall survival rate for all stages of pancreatic cancer is relatively low at about only 6%. As a result of this exceedingly poor prognosis, new research models are necessary to investigate this highly malignant cancer. One model that has been used extensively for a vast variety of different cancers is the chorioallantoic membrane (CAM) model. It is based on an exceptionally vascularized membrane that develops within fertilized chicken eggs and can be used for the grafting and analysis of tumor tissue. The aim of the study was to summarize already existing works on pancreatic ductal adenocarcinoma (PDAC) and the CAM model. The results were subdivided into different categories that include drug testing, angiogenesis, personalized medicine, modifications of the model, and further developments to help improve the unfavorable prognosis of this disease.Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with adverse outcomes that have barely improved over the last decade. About half of all patients present with metastasis at the time of diagnosis, and the 5-year overall survival rate across all stages is only 6%. Innovative in vivo research models are necessary to combat this cancer and to discover novel treatment strategies. The chorioallantoic membrane (CAM) model represents one 3D in vivo methodology that has been used in a large number of studies on different cancer types for over a century. This model is based on a membrane formed within fertilized chicken eggs that contain a dense network of blood vessels. Because of its high cost-efficiency, simplicity, and versatility, the CAM model appears to be a highly valuable research tool in the pursuit of gaining more in-depth insights into PDAC. A summary of the current literature on the usage of the CAM model for the investigation of PDAC was conducted and subdivided into angiogenesis, drug testing, modifications, personalized medicine, and further developments. On this comprehensive basis, further research should be conducted on PDAC in order to improve the abysmal prognosis of this malignant disease.

Simple SummaryThe chorioallantoic membrane (CAM) model is an alternative in vivo test system for the study of hallmarks of cancer such as angiogenesis, tumor growth, immune escape, metastasis, and drug resistance. It is suitable for tumor cell lines and patient-derived xenografts (PDX), allowing for studying treatment protocols for cancer patients. The purpose of this review is to give answers to the most challenging questions around the CAM model, which is not only bridging in vitro and mouse in vivo studies but also has an interesting role in itself.The chick chorioallantoic membrane (CAM), as an extraembryonic tissue layer generated by the fusion of the chorion with the vascularized allantoic membrane, is easily accessible for manipulation. Indeed, grafting tumor cells on the CAM lets xenografts/ovografts develop in a few days for further investigations. Thus, the CAM model represents an alternative test system that is a simple, fast, and low-cost tool to study tumor growth, drug response, or angiogenesis in vivo. Recently, a new era for the CAM model in immune-oncology-based drug discovery has been opened up. Although there are many advantages offering extraordinary and unique applications in cancer research, it has also disadvantages and limitations. This review will discuss the pros and cons with experts in the field.

The chick embryo chorioallantoic membrane (CAM) model is gaining increasing attention from cancer researchers worldwide. Its affordability, short experimental duration, robustness, and ease of tumor xenograft visualization make it a valuable tool in cancer research. This review explores recent advancements and potential applications of the avian CAM model, including the following: (1) studying tumor growth and metastasis, (2) investigating mechanisms of tumor chemoresistance, (3) optimizing drug delivery methods, (4) improving bioimaging techniques, (5) evaluating immuno-oncology drug efficacy, (6) examining tumor-extracellular matrix interactions, (7) analyzing tumor angiogenesis, and (8) exploring the roles of microRNAs in cancer. Additionally, we compare the in ovo CAM model with other in vivo animal models and in vitro cell culture systems. Positioned between in vitro and in vivo models in terms of cost-effectiveness and accuracy in cancer recapitulation, the CAM model enhances both preclinical and translational research. Its expanding use in cancer studies and therapy development is expected to continue growing.

Chick embryo chorioallantoic membrane (CAM) model: a useful tool to study short-term transplantation of cryopreserved human ovarian tissue

Simple SummaryIn the development of new targeted radiopharmaceuticals, it is mandatory to demonstrate their target-specific binding. Rodents are still primarily used for these experiments. With respect to the 3Rs principles, the demand for alternative methods to reduce the number of animal experiments is continuously increasing. In the present study, we investigated whether radiotracer uptake specificity can be evaluated by blocking studies in the CAM model. PET and MR imaging were used to visualize and quantify ligand accumulation. It was demonstrated that the CAM model could be used to evaluate the target-specific binding of a radiopharmaceutical. Due to intrinsic limitations of the CAM model, animal testing will still be required at more advanced stages of compound development. Still, the CAM model could significantly reduce the number of experiments through early compound pre-selection.Inhibition studies in small animals are the standard for evaluating the specificity of newly developed drugs, including radiopharmaceuticals. Recently, it has been reported that the tumor accumulation of radiotracers can be assessed in the chorioallantoic membrane (CAM) model with similar results to experiments in mice, such contributing to the 3Rs principles (reduction, replacement, and refinement). However, inhibition studies to prove receptor-specific binding have not yet been performed in the CAM model. Thus, in the present work, we analyzed the feasibility of inhibition studies in ovo by PET and MRI using the PSMA-specific ligand [18F]siPSMA-14 and the corresponding inhibitor 2-PMPA. A dose-dependent blockade of [18F]siPSMA-14 uptake was successfully demonstrated by pre-dosing with different inhibitor concentrations. Based on these data, we conclude that the CAM model is suitable for performing inhibition studies to detect receptor-specific binding. While in the later stages of development of novel radiopharmaceuticals, testing in rodents will still be necessary for biodistribution analysis, the CAM model is a promising alternative to mouse experiments in the early phases of compound evaluation. Thus, using the CAM model and PET and MR imaging for early pre-selection of promising radiolabeled compounds could significantly reduce the number of animal experiments.

Tumor angiogenesis is one essential aspect for the growth and metastasis of cancer cells, which means that adequate in vivo angiogenesis models are of utmost importance for the investigation of such diseases. The chick chorioallantoic membrane (CAM) model is one established method for this purpose and has already been used for research on multiple cancer types. One important part of the evaluation of tumors grafted onto the CAM is the measurement of tumor-induced angiogenesis. In order to address this central aspect, we utilized the novel PeriCam perfusion speckle imager (PSI) system high resolution (HR) model (Perimed AB, Järfälla, Sweden), which is based on laser speckle contrast imaging (LSCI) for the semiquantitative measurement of blood flow in the CAM model. This method enables a fast and accurate analysis of the angiogenesis of cell line tumors and primary tumors that are grafted onto the CAM. The proposed model can be regarded as a precursor model for personalized cancer therapy.

“Evaluation of anti-breast cancer, anti-angiogenic and antioxidant properties of selected medicinal plants”

(1) Background: angiogenesis plays an important role in the growth and metastasis of tumors. We established the CAM assay application, an image analysis software of the IKOSA platform by KML Vision, for the quantification of blood vessels with the in ovo chorioallantoic membrane (CAM) model. We added this proprietary deep learning algorithm to the already established laser speckle contrast imaging (LSCI). (2) Methods: angiosarcoma cell line tumors were grafted onto the CAM. Angiogenesis was measured at the beginning and at the end of tumor growth with both measurement methods. The CAM assay application was trained to enable the recognition of in ovo CAM vessels. Histological stains of the tissue were performed and gluconate, an anti-angiogenic substance, was applied to the tumors. (3) Results: the angiosarcoma cells formed tumors on the CAM that appeared to stay vital and proliferated. An increase in perfusion was observed using both methods. The CAM assay application was successfully established in the in ovo CAM model and anti-angiogenic effects of gluconate were observed. (4) Conclusions: the CAM assay application appears to be a useful method for the quantification of angiogenesis in the CAM model and gluconate could be a potential treatment of angiosarcomas. Both aspects should be evaluated in further research.

The chick embryo chorioallantoic membrane (CAM) model is extensively used in human medicine in preclinical oncological studies. The CAM model has several advantages: low cost, simple experimental approach, time saving and following “3R principles”. Research has shown that the human osteosarcoma cell lines U2OS, MMNG-HOS, and SAOS can form tumors on the CAM. In veterinary medicine, this has been described only for feline fibrosarcomas, feline mammary carcinomas and canine osteosarcomas. However, in case of canine osteosarcomas, it has been shown that only non-adherent osteosarcoma stem cells isolated from KTOSA5 and CSKOS cell lines have the ability to form microtumors on the CAM after an incubation period of 5 days, in contrast to adherent KTOSA5 and CSKOS cells. In the presented study, we have proven that the commercial adherent canine osteosarcoma cell line (D-17) can form vascularized tumors on the CAM after the incubation period of 10 days.

Simple SummaryThe chorioallantoic membrane (CAM) is a highly vascularized membrane found in avian eggs. Tumor cell lines can be grown on the CAM, which allows for the further analyses of the tumor grafts afterwards. We investigated the biological and growth characteristics of two breast cancer cell lines that resemble two biologically different breast cancer subgroups. Known biological features of the more aggressive breast cancer cell line were clearly confirmed in vitro and in the CAM model. Furthermore, the tissue-based pathological variables assessed in the CAM model were similar to those of the mouse xenografts and human patient tumor tissue. We suggest this in vivo model to be a reliable alternative for breast cancer research to reduce murine animal experiments.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.

The traditional Chinese medicine, “Hong-Qu”, also call “red mold rice” in the United States and Europe, has been identified and used for the treatment of blood stasis, a disorder related to hyperlipidemia and atherosclerosis. In addition to its value in improving metabolism syndrome, extracts from Monascus fermented rice inhibited proliferation of various cancer cells in vitro and in vivo have demonstrated previously. The objective of the current study was to examine correlation of red mold rice ethanol extract (RMRE) on the process of angiogenesis, invasion and metastasis during tumor progression. In the current study, we examined the inhibitory effects of SW480 and SW620 human colorectal carcinoma cells by RMRE using in vitro MTT assay. RMRE significantly inhibited the proliferation of SW480 and SW620 cells in dose- and time-dependent manner. However, human umbilical vein endothelial cells (HUVECs) showed a minor reduction effect of cell viability up to 20 µg/mL of RMRE for 48 hours using crystal violet staining assay. The capillary-like network morphology was observed following 20 ng/mL VEGF or SW620 culture-conditional medium (CM) addition. Nevertheless, capillary-like tube formation have failed to show when RMRE participation. Moreover, SW620 cells could growth in Matrigel grafts and spontaneously intravasation from the upper to the lower of chick embryo chorioallantoic membrane (CAM) model, which detected human Alu genomic DNA by PCR amplification from the lower CAMs at RMRE-untreated group. The percentage of neovascularization was increased 75.3±11.6% by SW620 cells onplant with Matrigel grafts on the CAM model. However, addition of RMRE significantly reduced CAM neovascularization in dose-dependent effect. Finally, we have finding that RMRE effectively decreased activity of matrix metalloproteinase (MMP)-7 determined by RT-PCR, Western blotting, and casein zymography assays. In summary Monascus fermented products exerted a potent effect on tumor growth and activation, suggesting that it may useful serves as a supplementary agent in adjuvant cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 467.