Ultra high frequency ultrasound enables real-time visualization of blood supply from chorioallantoic membrane to human autosomal dominant polycystic kidney tissue.

Ultra high frequency (UHF) ultrasound enables the visualization of very small structures that cannot be detected by conventional ultrasound. The utilization of UHF imaging as a new imaging technique for the 3D-in-vivo chorioallantoic membrane (CAM) model can facilitate new insights into tissue perfusion and survival. Therefore, human renal cystic tissue was grafted onto the CAM and examined using UHF ultrasound imaging. Due to the unprecedented resolution of UHF ultrasound, it was possible to visualize microvessels, their development, and the formation of anastomoses. This enabled the observation of anastomoses between human and chicken vessels only 12 h after transplantation. These observations were validated by 3D reconstructions from a light sheet microscopy image stack, indocyanine green angiography, and histological analysis. Contrary to the assumption that the nutrient supply of the human cystic tissue and the gas exchange happens through diffusion from CAM vessels, this study shows that the vasculature of the human cystic tissue is directly connected to the blood vessels of the CAM and perfusion is established within a short period. Therefore, this in-vivo model combined with UHF imaging appears to be the ideal platform for studying the effects of intravenously applied therapeutics to inhibit renal cyst growth.

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.

Application of Laser Speckle Contrast Imaging (LSCI) for the Angiogenesis Measurement of Tumors in the Chorioallantoic Membrane (CAM) Model.

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.

Modified Borggreve-Van Nes-Winkelmann rotationplasty for surgery in developing countries.

BACKGROUND: Amputation is still the most common therapy for patients suffering from osteosarcoma in Myanmar, despite the fact that limb salvage surgery e.g. Borggreve-Van Nes-Winkelmann rotationplasty for malignant tumors located within the distal femur or proximal tibia is the current state-of-the-art reconstructive procedure. A safe and reliable operation technique is crucial in order to perform a complex surgical procedure like the rotationplasty in lower-middle income economies with limited infrastructure and resources. The authors present seven cases of patients with osteosarcomas that received a Borggreve-Van Nes-Winkelmann rotationplasty with an evaluation of the procedures focusing on safety and sustainability. METHODS: From 2019 until 2020, seven young patients with osteosarcomas of the distal femur or proximal tibia were treated with Borggreve-Van Nes-Winkelmann rotationplasties in the Orthopaedic Hospital in Mandalay, Myanmar. As modification of the standard procedure the dissection and subsequent clamping of the femoral artery in order to minimize blood loss as well as the formation of an adipocutaneous flap that minimizes swelling and decreases the pressure on the vessels were successfully performed. This modified procedure resembles a safe and simplified surgical technique that is feasible under the circumstances of lower-middle income economies with good outcomes. RESULTS: All patients showed good functional and aesthetic results. One of the seven patients needed secondary wound closure due to wound dehiscence. CONCLUSIONS: A simplified and safe operation technique for the performance of the Van Nes-Borggreve rotationplasty was adapted to the given constraints in lower-middle income economies and proved to be successful. Trial registration All patients approved to participate in the study and have given consent to publication.

3D In Vivo Models for Translational Research on Pancreatic Cancer: The Chorioallantoic Membrane (CAM) Model.

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.

Deep Learning-Based Image Analysis for the Quantification of Tumor-Induced Angiogenesis in the 3D In Vivo Tumor Model-Establishment and Addition to Laser Speckle Contrast Imaging (LSCI).

(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.

A 3D In Vivo Model for Studying Human Renal Cystic Tissue and Mouse Kidney Slices.

(1) Background: Autosomal dominant polycystic kidney disease (ADPKD) is a frequent monogenic disorder that leads to progressive renal cyst growth and renal failure. Strategies to inhibit cyst growth in non-human cyst models have often failed in clinical trials. There is a significant need for models that enable studies of human cyst growth and drug trials. (2) Methods: Renal tissue from ADPKD patients who received a nephrectomy as well as adult mouse kidney slices were cultured on a chorioallantoic membrane (CAM) for one week. The cyst volume was monitored by microscopic and CT-based applications. The weight and angiogenesis were quantified. Morphometric and histological analyses were performed after the removal of the tissues from the CAM. (3) Results: The mouse and human renal tissue mostly remained vital for about one week on the CAM. The growth of cystic tissue was evaluated using microscopic and CT-based volume measurements, which correlated with weight and an increase in angiogenesis, and was accompanied by cyst cell proliferation. (4) Conclusions: The CAM model might bridge the gap between animal studies and clinical trials of human cyst growth, and provide a drug-testing platform for the inhibition of cyst enlargement. Real-time analyses of mouse kidney tissue may provide insights into renal physiology and reduce the need for animal experiments.

New, Innovative, Three-Dimensional In Vivo Model for High-Level Microsurgical and Supermicrosurgical Training: A Replacement for Animal Models.

SUMMARY: Microsurgery and supermicrosurgery are surgical subdomains necessary for a large variety of surgical disciplines. So far, there is no training model for lymphatic surgery or perforator flap surgery, and the most commonly used microsurgical training models are living animals. However, the ethical principles of replacement, refinement, and reduction (the three Rs) of living animals for training purposes were implemented, highlighting the necessity of an animal-sparing microsurgical training model. Formed during embryogenesis, the chick chorioallantoic membrane resembles a highly vascularized, noninnervated membrane within fertilized chicken eggs. The aim of this study was to utilize the chorioallantoic membrane model as an innovative and versatile training model for supermicrosurgery and microsurgery that can reduce the number of animals used for these purposes. The variety of different sized vessels for the implementation of an anastomosis proved the chorioallantoic membrane model as a well-functioning supermicrosurgical and microsurgical training model. The circulatory system is resilient enough to withstand the mechanical stress applied to the tissue, and the patency of the implemented anastomosis can be tested for the verification of the procedures. In summary, the integration of the chorioallantoic membrane model into a surgical training program can benefit its quality by representing a realistic anatomical and physiological model with a high variety of vascular structures. Moreover, the chorioallantoic membrane model satisfies the principles of replacement, refinement, and reduction as an animal-sparing model, indicating the potential of this model as an innovative microsurgical training model for the improvement of surgical skills.

Laser speckle contrast analysis (LASCA) technology for the semiquantitative measurement of angiogenesis in in-ovo-tumor-model.

BACKGROUND: The process of angiogenesis is a key element for tumor growth and proliferation and therefore one of the determining factors for aggressiveness and malignancy. A better understanding of the underlying processes of tumor induced angiogenesis is crucial for superior cancer treatment. Furthermore, the PeriCam perfusion speckle imager (PSI) system high resolution (HR) model by PERIMED presents a noninvasive method for semi-quantitative measurement of blood perfusion, based on laser speckle contrast analysis (LASCA). Aim of the present study was to utilize the chick chorioallantoic membrane (CAM) model as an in-ovo-tumor-model which enables rapid neovascularization of tumors while allowing real-time observation of the microcirculation via LASCA. METHODS: Fertilized chicken eggs were grafted with embryonal/alveolar rhabdomyosarcoma cells or primary sarcoma tumors. The blood perfusion was measured before and after tumor growth using LASCA. The procedure is accelerated and simplified through the integrated PIMSoft software which provides real-time graphs and color-coded images during the measurement. RESULTS: Sarcoma cells and primary sarcoma tumors exhibited satisfactory growth processes on the CAM. LASCA visualized microcirculation accurately and enabled an extensive investigation of the angiogenic potential of sarcoma cells on the CAM. We were able to show that sarcoma cells and primary sarcoma tumors induced larger quantities of neovasculature on the CAM than the controls. CONCLUSIONS: The utilization of LASCA for the investigation of tumor angiogenesis within the CAM model appears to be a highly beneficial, cost-efficient and easily practicable procedure. The proposed model can be used as a drug-screening model for individualized cancer therapy, especially with regards to anti-angiogenic agents.

Extended analysis of intratumoral heterogeneity of primary osteosarcoma tissue using 3D-in-vivo-tumor-model.

BACKGROUND: Osteosarcomas are a rare, heterogeneous and malignant group of bone tumors that have a high potential for metastasis and aggressive growth patterns. Treatment of metastasized osteosarcoma is often insufficient and research is compromised by problems encountered when culturing cells or analyzing genetic alterations due to the high level of intratumoral and intertumoral heterogeneity. The chick chorioallantoic membrane (CAM) model, a 3D-in-vivo-tumor-model, could potentially facilitate the investigation of osteosarcoma heterogeneity at an individual and highly specified level. OBJECTIVE: Objective was to establish the grafting and transplantation of different primary osteosarcoma tissue parts onto several consecutive CAMs for tumor profiling and investigation of osteosarcoma heterogeneity. METHODS: Various parts of primary osteosarcoma tissue were grafted onto CAMs and were transplanted onto another CAM for five to seven consecutive times, enabling further experimental analyzes. RESULTS: Primary osteosarcoma tissue parts exhibited satisfactory growth patterns and displayed angiogenic development on the CAM. It was possible to graft and transplant different tumor parts several times while the tissue viability was still high and tumor profiling was performed. CONCLUSIONS: Primary osteosarcoma tissue grew on several different CAMs for an extended time period and neovascularization of serial transplanted tumor parts was observed, improving the versatility of the 3D-in-vivo-tumor-model.

3D monitoring of tumor volume in an in vivo model.

BACKGROUND: The ability to evaluate tumor development within experimental oncology is of upmost importance. However, determining tumor volumes in 3D in vivo tumor models is challenging. The chick chorioallantoic membrane (CAM) model represents an optimized xenograft model that surpasses many disadvantages that are inherent to rodent models and provides the opportunity of real-time monitoring of tumor growth. OBJECTIVE: The objective of this study was to introduce a new method that enables monitoring of tumor growth within the CAM model throughout the course of the experiment. METHODS: Sarcoma cell lines and sarcoma primary tumors were grafted onto the CAM of fertilized chicken eggs. A digital microscope (Keyence VHX-6000) was used for 3D volume monitoring before and after tumor excision and compared it to tumor weight. RESULTS: Accuracy of tumor volumes was validated through correlation with tumor weight. In and ex ovo tumor volumes correlated significantly with tumor weight values. CONCLUSIONS: The described method can be used to assess the effects of chemotherapeutic agents on the growth of tumors that have been grafted onto the CAM and further advance personalized cancer therapy. In summary, we established a promising protocol that enables in vivo real-time tracking of tumor growth in the CAM model using a digital microscope.