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.

Importance and implications of exosomes in nephrology and urology.

Exosomes are extracellular vesicles that are formed by two invaginations of the plasma membrane and can be released by all eukaryotic cells. Because of their bioactive contents, including nucleic acids and proteins, exosomes can activate a variety of functions in their recipient cells. Due to the plethora of physiological and pathophysiological functions, exosomes have received a lot of attention from researchers over the past few years. However, there is still no consensus regarding isolation and characterization protocols of exosomes and their subtypes. This heterogeneity poses a lot of methodical challenges but also offers new clinical opportunities simultaneously. So far, exosome-based research is still mostly limited to preclinical experiments and early-stage clinical trials since the translation of experimental findings remains difficult. Exosomes could potentially play an important role as future diagnostic and prognostic agents and might also be part of the development of new treatment strategies. Therefore, they have previously been investigated in a variety of nephrological and urological conditions such as acute kidney injury or prostate cancer.

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.

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.

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.

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.

The Role of Citrate Homeostasis in Merkel Cell Carcinoma Pathogenesis.

Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin with a poor prognosis. The factors driving this cancer must be better understood in order to discover novel targets for more effective therapies. In the search for targets, we followed our interest in citrate as a central and critical metabolite linked to fatty acid synthesis in cancer development. A key to citrate uptake in cancer cells is the high expression of the plasma membrane citrate transporter (pmCiC), which is upregulated in the different adenocarcinoma types tested so far. In this study, we show that the pmCiC is also highly expressed in Merkel cell carcinoma cell lines by western blot and human tissues by immunohistochemistry staining. In the presence of extracellular citrate, MCC cells show an increased proliferation rate in vitro; a specific pmCiC inhibitor (Na+-gluconate) blocks this citrate-induced proliferation. Furthermore, the 3D in vivo Chick Chorioallantoic Membrane (CAM) model showed that the application of Na+-gluconate also decreases Merkel cell carcinoma growth. Based on our results, we conclude that pmCiC and extracellular citrate uptake should be considered further as a potential novel target for the treatment of Merkel cell carcinoma.

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.

An Innovative Simulation Model for Microvascular Training.

SUMMARY: Preclinical/clinical microsurgical training is essential for clinical practice. Therefore, various training models have been established, such as synthetic and cadaveric models. The most common limitation of these models is the lack of circulation, which limits the simulation of real intraoperative circumstances. Thus, the authors aimed to create a novel model that provides blood circulation with an extracorporeal perfusion device that they attached to rat cadavers for the reestablishment of a circulatory system. Patent blue and heparin were added to the perfusion fluid to visualize circulation and to dissolve thrombosis, and indocyanine green fluorescent imaging was applied to show the perfusion of the entire body. The femoral and brachial vessels were dissected, and an end-to-end anastomosis was performed on the femoral artery. The patency of the operated vessel was visualized with indocyanine green fluorescent imaging. Indocyanine green fluorescent imaging showed appropriate vessel patency and extremity perfusion through the anastomosis. The use of this novel rat model enables a solution for ethical problems encountered when using rats for surgical training courses. By practicing on these animal-sparing models with intact circulation, microsurgical skills can be improved. Future studies on further microsurgical techniques and vascular perfusion of organs or tumors may benefit from our model.

The 3D in vivo chorioallantoic membrane model and its role in breast cancer research.

PURPOSE: We aimed to evaluate the role of the chorioallantoic membrane model (CAM) in breast cancer research. METHODS: The following is an overview of the use of the CAM in the field of breast cancer research based on a PubMed literature query. RESULTS: The CAM is a 3D in vivo model that can be used for the analysis of tumor growth, biology and angiogenesis of primary tumor tissue or tumor cell lines. The CAM model has been used in breast cancer research for drug testing, migration assays and the evaluation of vascularization, amongst others. The CAM model is a valuable method that offers a better imitation of the physiological phenomena compared to 2D or 3D in vitro models. CONCLUSION: The CAM model has primarily and successfully been utilized for the assessment of the tumor biology of established breast cancer cell lines. Further, the CAM model is a promising method to analyze patient derived primary tumor material and could be used as a "patient-specific 3D-tumor-therapy-model" for the cost-efficient evaluation of anti-cancer drugs to find the optimal treatment for breast cancer patients.

Indocyanine Green for Leakage Control in Isolated Limb Perfusion.

Sarcomas are characterized by a high metastatic potential and aggressive growth. Despite surgery, chemotherapy plays an important role in the treatment of these tumors. Optimal anti-cancer therapy with maximized local efficacy and minimized systemic side effects has been the object of many studies for a long time. To improve the local efficacy of anti-tumor therapy, isolated limb perfusion with high-dose cytostatic agents has been introduced in surgical oncology. In order to control the local distribution of substances, radiolabeled cytostatic drugs or perfusion solutions have been applied but often require the presence of specialized personnel and result in a certain exposure to radiation. In this study, we present a novel strategy using indocyanine green to track tumor perfusion with high-dose cytostatic therapy. In a rat cadaver model, the femoral vessels were cannulated and connected to a peristaltic pump to provide circulation within the selected limb. The perfusion solution contained indocyanine green and high-dose doxorubicin. An infrared camera enabled the visualization of indocyanine green during limb perfusion, and subsequent leakage control was successfully performed. Histologic analysis of sections derived proximally from the injection site excluded systemic drug dispersion. In this study, the application of indocyanine green was proven to be a safe and cost- and time-efficient method for precise leakage control in isolated limb perfusion with a high-dose cytostatic agent.

Use of adipose-derived stem cells in lymphatic tissue engineering and regeneration.

The potential to differentiate into different cell lines, added to the easy and cost-effective method of extraction, makes adipose-derived stem cells (ADSCs) an object of interest in lymphedema treatment. Our study's goal was to conduct a comprehensive systematic review of the use of ADSCs in lymphatic tissue engineering and regeneration. On July 23, 2019, using PubMed/MEDLINE, Cochrane Clinical Answers, Cochrane Central Register of Controlled Trials, and Embase databases, we conducted a systematic review of published literature on the use of ADSCs in lymphatic tissue engineering and regeneration. There were no language or time frame limitations, and the following search strategy was applied: ((Adipose stem cell) OR Adipose-derived stem cell)) AND ((Lymphedema) OR Breast Cancer Lymphedema). Only original research manuscripts were included. Fourteen studies fulfilled the inclusion criteria. Eleven studies were experimental (in vitro or in vivo in animals), and only three were clinical. Publications on the topic demonstrated that ADSCs promote lymphangiogenesis, and its effect could be enhanced by modulation of vascular endothelial growth factor-C, interleukin-7, prospero homeobox protein 1, and transforming growth factor-ß1. Pilot clinical studies included 11 patients with breast cancer-related lymphedema, and no significant side effects were present at 12-month follow-up. Literature on the use of ADSCs in lymphatic tissue engineering and regeneration demonstrated promising data. Clinical evidence is still in its infancy, but the scientific community agrees that ADSCs can be useful in regenerative lymphangiogenesis. Data collected in this review indicate that unprecedented advances in lymphedema treatment can be anticipated in the upcoming years.

Assessment of breast cancer primary tumor material in a 3D in vivo model.

BACKGROUND: Breast cancer is the most common malignant tumor in women and highly heterogeneous with a variety of different molecular subtypes. The analysis of the individual tumor biology is necessary to develop a specific and individualized treatment plan for every patient. The chick chorioallantoic membrane (CAM) model, a 3D-in-vivo-tumor-model, could potentially provide a methodology that facilitates the gain of additional information regarding the tumor biology as well as the testing of the tumor's individual sensitivity to different therapies. OBJECTIVE: The objective was to establish the grafting of different breast cancer primaries onto the CAM for tumor profiling and the investigation of different parameters. METHODS: Breast cancer primary tissue of different patients was grafted onto the CAM. Subsequently, 3D volume and perfusion measurements were performed during the engraftment period. Histological analyses of the tumors were carried out after the engraftment period. RESULTS: The grafting of the breast cancer primaries onto the CAM was successful. The tumors remained partially vital and displayed angiogenic development on the CAM. CONCLUSIONS: Breast cancer primary material can be grafted onto the CAM and we observed visible and measurable changes of perfusion over time.

How to Approach Secondary Breast Reduction: International Trends and a Systematic Review of the Literature.

BACKGROUND: Secondary breast reduction is complex and poses significant challenges to surgeons. Complication rates exceed those of primary reduction, commonly caused by impaired vascular supply of the nipple-areolar complex (NAC). Literature on the topic is scare and provides contradicting recommendations, especially with regard to pedicle choice in cases with unknown primary reduction technique. Aim of this study was to investigate international trends and to compare findings with literature. METHODS: A large-scale web-based questionnaire on international trends in mammaplasty (mastopexy and breast reduction) was designed and distributed to over five thousand surgeons in eight geographic regions. The presented manuscript evaluated information regarding pedicle choice in secondary breast reduction and compared data to literature identified in a systematic review. RESULTS: The survey was completed by 1431 participants. Overall, secondary procedures were performed in less than 5% or in 5 to 10% of cases. The preferred pedicle for secondary reductions differed significantly between geographic regions (p<0.001). The majority of respondents reported to use a superior or supero-medial pedicle (34.8% and 32.2%, respectively). Residual analysis revealed a strong association between the use of an inferior pedicle and procedures performed in North America. CONCLUSIONS: Secondary breast reduction is challenging and there remains international disparity with regard to pedicle choice for secondary procedures. Studies investigating outcome when the primary pedicle is unknown are scarce and provide incoherent recommendations. High-quality data is needed to provide evidence-based practice guidelines. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Cancer-associated cells release citrate to support tumour metastatic progression.

Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.

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.

pH sensing in skin tumors: Methods to study the involvement of GPCRs, acid-sensing ion channels and transient receptor potential vanilloid channels.

Solid tumors exhibit an inversed pH gradient with increased intracellular pH (pHi ) and decreased extracellular pH (pHe ). This inside-out pH gradient is generated via sodium/hydrogen antiporter 1, vacuolar-type H + ATPases, monocarboxylate transporters, (bi)carbonate (co)transporters and carboanhydrases. Our knowledge on how pHe -signals are sensed and what the respective receptors induce inside cells is scarce. Some pH-sensitive receptors (GPR4, GPR65/TDAG8, GPR68/OGR1, GPR132/G2A, possibly GPR31 and GPR151) and ion channels (acid-sensing ion channels ASICs, transient receptor potential vanilloid receptors TRPVs) transduce signals inside cells. As little is known on the expression and function of these pH sensors, we used immunostainings to study tissue samples from common and rare skin cancers. Our current and future work is directed towards investigating the impact of all the pH-sensing receptors in different skin tumors using cell culture techniques with selective knockdown/knockout (siRNA/CRISPR-Cas9). To study cell migration and proliferation, novel impedance-based wound healing assays have been developed and are used. The field of pH sensing in tumors and wounds holds great promise for the development of pH-targeting therapies, either against pH regulators or sensors to inhibit cell proliferation and migration.

Enhanced Resorption of Liposomal Packed Vitamin C Monitored by Ultrasound.

Vitamin C is an essential nutrient for humans and is involved in a plethora of health-related functions. Several studies have shown a connection between vitamin C intake and an improved resistance to infections that involves the immune system. However, the body cannot store vitamin C and both the elevated oral intake, and the intravenous application have certain disadvantages. In this study, we wanted to show a new formulation for the liposomal packaging of vitamin C. Using freeze etching electron microscopy, we show the formed liposomes. With a novel approach of post-processing procedures of real-time sonography that combines enhancement effects by contrast-like ultrasound with a transducer, we wanted to demonstrate the elevated intestinal vitamin C resorption on four participants. With the method presented in this study, it is possible to make use of the liposomal packaging of vitamin C with simple household materials and equipment for intake elevation. For the first time, we show the enhanced resorption of ingested liposomes using microbubble enhanced ultrasound imaging.