Standardisation and future of preclinical echocardiography.

Echocardiography is a non-invasive imaging technique providing real-time information to assess the structure and function of the heart. Due to advancements in technology, ultra-high-frequency transducers have enabled the translation of ultrasound from humans to small animals due to resolutions down to 30 µm. Most studies are performed using mice and rats, with ages ranging from embryonic, to neonatal, and adult. In addition, alternative models such as zebrafish and chicken embryos are becoming more frequently used. With the achieved high temporal and spatial resolution in real-time, cardiac function can now be monitored throughout the lifespan of these small animals to investigate the origin and treatment of a range of acute and chronic pathological conditions. With the increased relevance of in vivo real-time imaging, there is still an unmet need for the standardisation of small animal echocardiography and the appropriate cardiac measurements that should be reported in preclinical cardiac models. This review focuses on the development of standardisation in preclinical echocardiography and reports appropriate cardiac measurements throughout the lifespan of rodents: embryonic, neonatal, ageing, and acute and chronic pathologies. Lastly, we will discuss the future of cardiac preclinical ultrasound.

The Ultrasound Window Into Vascular Ageing: A Technology Review by the VascAgeNet COST Action.

Non-invasive ultrasound (US) imaging enables the assessment of the properties of superficial blood vessels. Various modes can be used for vascular characteristics analysis, ranging from radiofrequency (RF) data, Doppler- and standard B/M-mode imaging, to more recent ultra-high frequency and ultrafast techniques. The aim of the present work was to provide an overview of the current state-of-the-art non-invasive US technologies and corresponding vascular ageing characteristics from a technological perspective. Following an introduction about the basic concepts of the US technique, the characteristics considered in this review are clustered into: 1) vessel wall structure; 2) dynamic elastic properties, and 3) reactive vessel properties. The overview shows that ultrasound is a versatile, non-invasive, and safe imaging technique that can be adopted for obtaining information about function, structure, and reactivity in superficial arteries. The most suitable setting for a specific application must be selected according to spatial and temporal resolution requirements. The usefulness of standardization in the validation process and performance metric adoption emerges. Computer-based techniques should always be preferred to manual measures, as long as the algorithms and learning procedures are transparent and well described, and the performance leads to better results. Identification of a minimal clinically important difference is a crucial point for drawing conclusions regarding robustness of the techniques and for the translation into practice of any biomarker.

Coronary Microvascular Dysfunction in Acute Cholestasis-Induced Liver Injury.

BACKGROUND: Previous studies have shown cardiac abnormalities in acute liver injury, suggesting a potential role in the associated high mortality. METHODS: We designed an experimental study exploring the short-term effects of acute cholestasis-induced liver injury on cardiac function and structure in a rodent bile duct ligation (BDL) model to elucidate the potential interplay. Thirty-seven male Sprague-Dawley rats were subjected to BDL surgery (n = 28) or served as sham-operated (n = 9) controls. Transthoracic echocardiography, Doppler evaluation of the left anterior descending coronary artery, and myocardial contrast echocardiography were performed at rest and during adenosine and dobutamine stress 5 days after BDL. Immunohistochemical staining of myocardial tissue samples for hypoxia and inflammation as well as serum analysis were performed. RESULTS: BDL animals exhibited acute liver injury with elevated transaminases, bilirubin, and total circulating bile acids (TBA) 5 days after BDL (TBA control: 0.81 ± 2.54 µmol/L vs. BDL: 127.52 ± 57.03 µmol/L; p < 0.001). Concurrently, cardiac function was significantly impaired, characterized by reduced cardiac output (CO) and global longitudinal strain (GLS) in the echocardiography at rest and under pharmacological stress (CO rest control: 120.6 ± 24.3 mL/min vs. BDL 102.5 ± 16.6 mL/min, p = 0.041; GLS rest control: -24.05 ± 3.8% vs. BDL: -18.5 ± 5.1%, p = 0.01). Myocardial perfusion analysis revealed a reduced myocardial blood flow at rest and a decreased coronary flow velocity reserve (CFVR) under dobutamine stress in the BDL animals (CFVR control: 2.1 ± 0.6 vs. BDL: 1.7 ± 0.5 p = 0.047). Immunofluorescence staining indicated myocardial hypoxia and increased neutrophil infiltration. CONCLUSIONS: In summary, acute cholestasis-induced liver injury can lead to impaired cardiac function mediated by coronary microvascular dysfunction, suggesting that major adverse cardiac events may contribute to the mortality of acute liver failure. This may be due to endothelial dysfunction and direct bile acid signaling.

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.

Frequent detection of human cytomegalovirus in neuroblastoma: a novel therapeutic target?

Neuroblastoma is the most common and deadly tumor of childhood, where new therapy options for patients with high-risk disease are highly warranted. Human cytomegalovirus (HCMV) is prevalent in the human population and has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumor cell immune evasion. Here we show that the majority of primary neuroblastomas and neuroblastoma cell lines are infected with HCMV. Our analysis show that HCMV immediate-early protein was expressed in 100% of 36 primary neuroblastoma samples, and HCMV late protein was expressed in 92%. However, no infectious virus was detected in primary neuroblastoma tissue extracts. Remarkably, all six human neuroblastoma cell lines investigated contained CMV DNA and expressed HCMV proteins. HCMV proteins were expressed in neuroblastoma cells expressing the proposed stem cell markers CD133 and CD44. When engrafted into NMRI nu/nu mice, human neuroblastoma cells expressed HCMV DNA, RNA and proteins but did not produce infectious virus. The HCMV-specific antiviral drug valganciclovir significantly reduced viral protein expression and cell growth both in vitro and in vivo. These findings indicate that HCMV is important for the pathogenesis of neuroblastoma and that anti-viral therapy may be a novel adjuvant treatment option for children with neuroblastoma.

Comparative assessment of coronary physiology using transthoracic pulsed-wave Doppler and myocardial contrast echocardiography in rats.

BACKGROUND: Coronary physiology assessment in rodents by ultrasound is an excellent noninvasive and easy to perform technique, including pulsed-wave Doppler (PWD) and myocardial contrast echocardiography (MCE). Both techniques and the corresponding calculated parameters were investigated in this study at rest as well as their response to pharmacologically induced stress. METHODS: Left ventricular myocardial function was assessed in eight anaesthetised rats using transthoracic echocardiography. Coronary physiology was assessed by both PWD of the left coronary artery and MCE using a bolus method. Measurements were performed at rest and under stimulation with adenosine and dobutamine. Effects of stimulation on the calculated parameters were evaluated and rated by effect size (η2). RESULTS: Changes could be demonstrated by selected parameters of PWD and MCE. The clearest effect in PWD was found for diastolic peak velocity (η2 = 0.58). It increased from 528 ± 110 mm/s (mean ± standard deviation) at rest to 839 ± 342 mm/s (p = 0.001) with adenosine and 1093 ± 302 mm/s with dobutamine (p = 0.001). The most distinct effect from MCE was found for the normalised wash-in rate (η2 = 0.58). It increased from 1.95 ± 0.35% at rest to 3.87 ± 0.85% with adenosine (p = 0.001) and 3.72 ± 1.03% with dobutamine (p = 0.001). CONCLUSION: Induced changes in coronary physiology by adenosine and dobutamine could successfully be monitored using MCE and PWD in anaesthetised rats. Due to the low invasiveness of the measurements, this protocol could be used for longitudinal animal studies.

Functional Cardiovascular Characterization of the Common Marmoset (Callithrix jacchus).

Appropriate cardiovascular animal models are urgently needed to investigate genetic, molecular, and therapeutic approaches, yet the translation of results from the currently used species is difficult due to their genetic distance as well as their anatomical or physiological differences. Animal species that are closer to the human situation might help to bridge this translational gap. The common marmoset (Callithrix jacchus) is an interesting candidate to investigate certain heart diseases and cardiovascular comorbidities, yet a basic functional characterization of its hemodynamic system is still missing. Therefore, cardiac functional analyses were performed by utilizing the invasive intracardiac pressure-volume loops (PV loop) system in seven animals, magnetic resonance imaging (MRI) in six animals, and echocardiography in five young adult male common marmosets. For a direct comparison between the three methods, only data from animals for which all three datasets could be acquired were selected. All three modalities were suitable for characterizing cardiac function, though with some systemic variations. In addition, vena cava occlusions were performed to investigate the load-independent parameters collected with the PV loop system, which allowed for a deeper analysis of the cardiac function and for a more sensitive detection of the alterations in a disease state, such as heart failure or certain cardiovascular comorbidities.

Broad geographical distribution and high genetic diversity of shrew-borne Seewis hantavirus in Central Europe.

For a long time hantaviruses were believed to be exclusively rodent-borne pathogens. Recent findings of numerous shrew- and mole-borne hantaviruses raise important questions on their phylogenetic origin. The objective of our study was to prove the presence and distribution of shrew-associated Seewis virus (SWSV) in different Sorex species in Central Europe. Therefore, a total of 353 Sorex araneus, 59 S. minutus, 27 S. coronatus, and one S. alpinus were collected in Germany, the Czech Republic, and Slovakia. Screening by hantavirus-specific L-segment RT-PCR revealed specific amplification products in tissues of 49 out of 353 S. araneus and four out of 59 S. minutus. S-segment sequences were obtained for 45 of the L-segment positive S. araneus and all four L-segment positive S. minutus. Phylogenetic investigation of these sequences from Germany, the Czech Republic, and Slovakia demonstrated their similarity to SWSV sequences from Hungary, Finland, Austria, and other sites in Germany. The low intra-cluster sequence variability and the high inter-cluster divergence suggest a long-term SWSV evolution in isolated Sorex populations. In 28 of the 49 SWSV S-segment sequences, an additional putative open reading frame (ORF) on the opposite strand to the nucleocapsid protein-encoding ORF was identified. This is the first comprehensive sequence analysis of SWSV strains from Germany, the Czech Republic, and Slovakia, indicating its broad geographical distribution and high genetic divergence. Future studies have to prove whether both S. araneus and S. minutus represent SWSV reservoir hosts or spillover infections are responsible for the parallel molecular detection of SWSV in both species.

Ultrasound and Photoacoustic Imaging of the Kidney: Basic Concepts and Protocols.

Noninvasive, robust, and reproducible methods to image kidneys are provided by different imaging modalities. A combination of modalities (multimodality) can give better insight into structure and function and to understand the physiology of the kidney. Magnetic resonance imaging can be complemented by a multimodal imaging approach to obtain additional information or include interventional procedures. In the clinic, renal ultrasound has been essential for the diagnosis and management of kidney disease and for the guidance of invasive procedures for a long time. Adapting ultrasound to preclinical requirements and for translational research, the combination with photoacoustic imaging expands the capabilities to obtain anatomical, functional, and molecular information from animal models. This chapter describes the basic concepts of how to image kidneys using different and most appropriate modalities.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This introduction chapter is complemented by two separate chapters describing the experimental procedure and data analysis.

Metronomic administration of the drug GMX1777, a cellular NAD synthesis inhibitor, results in neuroblastoma regression and vessel maturation without inducing drug resistance.

High-risk neuroblastoma is a rapidly growing tumor with a survival rate below 50%. A new treatment strategy is to administer chemotherapeutic drugs metronomically, i.e., at lower doses and frequent intervals. The aim of the study was to investigate the effects of GMX1777, a chemotherapeutic drug affecting cellular energy metabolism, in a mouse model for high-risk neuroblastoma. Female SCID mice were injected s.c. with MYCN-amplified human neuroblastoma cells and randomized to either treatment with GMX1777 or vehicle. In some animals, treatment was discontinued allowing tumor relapse. Treatment response was evaluated using the pediatric preclinical testing program (PPTP). Immunohistochemistry and qRT-PCR was performed on tumor cryosections to investigate the microscopic and molecular changes in tumors in response to GMX1777. Despite an increase in vessel density, tumor regression and a high group response score according to PPTP criteria was induced by GMX1777 without inducing drug resistance. Treatment resulted in inhibition of tumor cell proliferation, vessel maturation, reduced hypoxia, increased infiltration of MHC class II negative macrophages and expansion of the nonvascular stromal compartment. Decreased stromal VEGF-A and PDGF-B mRNA in response to treatment together with the structural data suggest a "deactivation" or "silencing" of the tumor stroma as a paracrine entity. In conclusion, GMX1777 was highly efficient against high-risk neuroblastoma xenografts through modulation of both the tumor cell and stromal compartment.

Regression of orthotopic neuroblastoma in mice by targeting the endothelial and tumor cell compartments.

BACKGROUND: High-risk neuroblastoma has an overall five-year survival of less than 40%, indicating a need for new treatment strategies such as angiogenesis inhibition. Recent studies have shown that chemotherapeutic drugs can inhibit angiogenesis if administered in a continuous schedule. The aim of this study was primarily to characterize tumor spread in an orthotopic, metastatic model for aggressive, MYCN-amplified neuroblastoma and secondarily to study the effects of daily administration of the chemotherapeutic agent CHS 828 on tumor angiogenesis, tumor growth, and spread. METHODS: MYCN-amplified human neuroblastoma cells (IMR-32, 2 x 10(6)) were injected into the left adrenal gland in SCID mice through a flank incision. Nine weeks later, a new laparotomy was performed to confirm tumor establishment and to estimate tumor volume. Animals were randomized to either treatment with CHS 828 (20 mg/kg/day; p.o.) or vehicle control. Differences between groups in tumor volume were analyzed by Mann-Whitney U test and in metastatic spread using Fisher's exact test. Differences with p < 0.05 were considered statistically significant. RESULTS: The orthotopic model resembled clinical neuroblastoma in respect to tumor site, growth and spread. Treatment with CHS 828 resulted in tumor regression (p < 0.001) and reduction in viable tumor fraction (p < 0.001) and metastatic spread (p < 0.05) in correlation with reduced plasma levels of the putative tumor marker chromogranin A (p < 0.001). These effects were due to increased tumor cell death and reduced angiogenesis. No treatment-related toxicities were observed. CONCLUSION: The metastatic animal model in this study resembled clinical neuroblastoma and is therefore clinically relevant for examining new treatment strategies for this malignancy. Our results indicate that daily scheduling of CHS 828 may be beneficial in treating patients with high-risk neuroblastoma.

On caring and sharing-Addressing psychological, biographical, and spiritual aspects in integrative cancer care: A qualitative interview study on physicians' perspectives.

BACKGROUND: Patients confronted with a cancer diagnosis experience a variety of existential needs encompassing emotional, psychological, and spiritual areas of being. A patient-centered care approach addressing such existential issues is recognized as an essential aspect of health care. The aim of this study is to explore what role psychological, biographical, and spiritual factors play for experienced doctors working in integrative cancer care. METHOD: The qualitative study was based on in-depth interviews with 35 purposively sampled doctors, all practicing integrative oncology in the field of anthroposophic medicine in hospitals and/or office-based practices in Germany and other countries. Data were analyzed using structured content analysis. RESULTS: Psychological, biographical, and spiritual factors are important issues in integrative cancer care. Prevailing themes identified in this study were enabling patients to participate in life, promoting autonomy and coping, stabilizing patients emotionally and cognitively, overcoming the disease, and-primarily if addressed by patients-integrating spiritual issues. Doctors offered conversation, counseling, and time, but also referred to art, music, literature, and nature, so that patients' ongoing emotional, psychological, and spiritual needs could be explored and addressed. Doctors' attitudes with regard to existential issues were seen as important, as was maintaining an attitude of openness towards existential issues. CONCLUSION: Doctors in integrative cancer care utilize different methods to explore the needs of patients and employ a variety of treatment methods that address not just patients' medical issues but their existential concerns as well.

The Subjective Dimension of Integrative Cancer Care: A Qualitative Study Exploring the Perspectives, Themes, and Observations of Experienced Doctors from the Area of Anthroposophic Medicine.

BACKGROUND: Integrative cancer care (ICC) is used by approximately 50% of cancer patients to complement oncologic treatments and to address unmet needs. One ICC practice is anthroposophic medicine, integrating conventional and complementary cancer care. It specifically provides mistletoe therapy (MT), but also intensive counselling, natural remedies, creative and movement therapies, nursing procedures, nutrition, and others. The objective of this study was to explore perceptions, themes, goals, procedures, and observations of experienced AM doctors with regard to the subjective dimensions of ICC. METHOD: A guideline-based qualitative interview study was conducted with 35 AM doctors working in hospitals and office-based practices in Germany and other countries. Structured qualitative content analysis was applied to examine the data. Triangulation was done with published studies investigating patients' perspectives on AM and MT. RESULTS: The interviewed doctors integrated conventional and holistic cancer concepts. Overarching therapeutic themes were: to enable patients to live with or overcome their disease, to find their own way through their disease, and to possibly reframe their situation. A broad variety of therapeutic goals were pursued, depending on the situation and priorities of the particular patient. Doctors described varying levels of patients' improved strength; increased vitality, thermal comfort, and recovery; relief from suffering, particularly in the areas of fatigue, appetite, sleep, pain, infections, and reactions to toxic anti-cancer therapies. The doctors also described how they perceived changes of patients' emotional level, their coping, autonomy, functional abilities, and finding their own way to deal with the disease. This is consistent with patients' perspectives described in published studies. CONCLUSION: Themes, goals, and described benefit of ICC from doctors' and patients' perspective may be an important complementation of conventional cancer care, as it meets important needs, distresses and conditions of patients which often stay unmet and unrelieved. Further research should investigate these goals and procedures.

Angiogenesis can be reduced without significant reduction of tumor growth.

BACKGROUND: High risk neuroblastoma (NB) patients have an overall five-year survival of approximately 50%, indicating the need for new treatment strategies, such as angiogenesis inhibition. MATERIALS AND METHODS: The angiogenesis inhibitor TNP-470 (30 mg/kg, every other day, subcutaneously) was given to nude mice with subcutaneous human neuroblastoma xenografts. The plasma concentrations of the angiogenesis stimulators, i.e. vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2) and hepatocyte growth factor (HGF), were assayed longitudinally. Angiogenesis, proliferation and apoptosis were quantified on tumor tissue slides. RESULTS: Upon treatment with TNP-470, angiogenesis was significantly inhibited by the reduction of length and surface area of vessels per tumor volume, without having significant effect on tumor growth, tumor cell proliferation or apoptosis. Plasma concentrations of VEGF-A per tumor volume were significantly increased upon treatment. CONCLUSION: Angiogenesis inhibition must reach a threshold before significant tumor cell apoptosis and a reduction of the tumor growth rate occur.

Individualized Integrative Cancer Care in Anthroposophic Medicine: A Qualitative Study of the Concepts and Procedures of Expert Doctors.

Background Cancer patients widely seek integrative oncology which embraces a wide variety of treatments and system approaches. Objective To investigate the concepts, therapeutic goals, procedures, and working conditions of integrative oncology doctors in the field of anthroposophic medicine. Methods This qualitative study was based on in-depth interviews with 35 highly experienced doctors working in hospitals and office-based practices in Germany and other countries. Structured qualitative content analysis was applied to examine the data. Results The doctors integrated conventional and holistic cancer concepts. Their treatments aimed at both tumor and symptom control and at strengthening the patient on different levels: living with the disease, overcoming the disease, enabling emotional and cognitive development, and addressing spiritual or transcendental issues according to the patient's wishes and initiatives. Therapeutic procedures were conventional anticancer and symptom-relieving treatments, herbal and mineral remedies, mistletoe therapy, art therapies, massages and other external applications, nutrition and lifestyle advice, psychological support, and multiple forms of empowerment. The approach emphasised good patient-doctor relationships and sufficient time for patient encounters and decision-making. Individualization appeared in several dimensions and was interwoven with standards and mindlines. The doctors often worked in teams and cooperated with other cancer care-related specialists. Conclusion Integrative cancer care pursues an individualized and patient-centered approach, encompassing conventional and multimodal complementary interventions, and addressing, along with physical and functional needs, the emotional and spiritual needs of patients. This seems to be important for tumor and symptom control, and addresses major challenges and important goals of modern cancer care.

Intravenous Mistletoe Treatment in Integrative Cancer Care: A Qualitative Study Exploring the Procedures, Concepts, and Observations of Expert Doctors.

Background. Mistletoe therapy (MT) is widely used in patient-centered integrative cancer care. The objective of this study was to explore the concepts, procedures, and observations of expert doctors, with a focus on intravenous MT. Method. A qualitative interview study was conducted with 35 highly experienced doctors specialized in integrative and anthroposophic medicine. Structured qualitative content analysis was applied. For triangulation, the results were compared with external evidence that was systematically collected, reviewed, and presented. Results. Doctors perform individualized patient assessments that lead to multimodal treatment approaches. The underlying goal is to help patients to live with and overcome disease. Mistletoe infusions are a means of accomplishing this goal. They are applied to stabilize disease, achieve responsiveness, induce fever, improve quality of life, and improve the tolerability of conventional cancer treatments. The doctors reported long-term disease stability and improvements in patients' general condition, vitality, strength, thermal comfort, appetite, sleep, pain from bone metastases, dyspnea in pulmonary lymphangitis carcinomatosa, fatigue, and cachexia; chemotherapy was better tolerated. Also patients' emotional and mental condition was reported to have improved. Conclusion. Individualized integrative cancer treatment including MT aims to help cancer patients to live well with their disease. Further research should investigate the reported observations.

Advanced echocardiography in adult zebrafish reveals delayed recovery of heart function after myocardial cryoinjury.

Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

Crizotinib inhibits metabolic inactivation of gemcitabine in c-Met-driven pancreatic carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclinical tests fail in these patients, emphasizing the need of improved preclinical models to test novel anticancer strategies. Here, we developed four orthotopic mouse models using primary human PDAC cells genetically engineered to express firefly- and Gaussia luciferase, simplifying the ability to monitor tumor growth and metastasis longitudinally in individual animals with MRI and high-frequency ultrasound. In these models, we conducted detailed histopathologic and immunohistochemical analyses on paraffin-embedded pancreatic tissues and metastatic lesions in liver, lungs, and lymph nodes. Genetic characteristics were compared with the originator tumor and primary tumor cells using array-based comparative genomic hybridization, using frozen specimens obtained by laser microdissection. Notably, the orthotopic human xenografts in these models recapitulated the phenotype of human PDACs, including hypovascular and hypoxic areas. Pursuing genomic and immunohistochemical evidence revealed an increased copy number and overexpression of c-Met in one of the models; we examined the preclinical efficacy of c-Met inhibitors in vitro and in vivo. In particular, we found that crizotinib decreased tumor dimension, prolonged survival, and increased blood and tissue concentrations of gemcitabine, synergizing with a cytidine deaminase-mediated mechanism of action. Together, these more readily imaged orthotopic PDAC models displayed genetic, histopathologic, and metastatic features similar to their human tumors of origin. Moreover, their use pointed to c-Met as a candidate therapeutic target in PDAC and highlighted crizotinib and gemcitabine as a synergistic combination of drugs warranting clinical evaluation for PDAC treatment.

The anti-VEGF antibody bevacizumab potently reduces the growth rate of high-risk neuroblastoma xenografts.

Neuroblastoma (NB) is a rapidly growing, well-vascularized childhood cancer that often presents with metastases. The overall five-year survival in NB is approximately 45% despite multimodality treatment, and therefore there is a clinical need for new therapeutic strategies. NB frequently overexpresses the angiogenic factor VEGF (vascular endothelial growth factor). The aim of this study was to investigate the effect of bevacizumab (Avastin, Genentech/Roche), a humanized anti-VEGF-A antibody, on NB growth in three different xenograft models, chosen to resemble high-risk NB. The human NB cell lines SK-N-AS, IMR-32 and SH-SY5Y, which are poorly differentiated and overexpress VEGF-A, were injected s.c. in immunodeficient mice. Bevacizumab was given intraperitoneally twice weekly at 5 mg/kg body weight, starting at a tumor volume of 0.3 mL. Bevacizumab significantly (p < 0.01-0.05) reduced NB growth in vivo without toxicity by causing a 30-63% reduction of angiogenesis, but had no effect on NB cell survival in vitro. Serum concentrations of VEGF-A increased two- to six-fold during bevacizumab therapy which did not result in faster tumor growth compared with control animals. Based on our experimental data we suggest consideration of bevacizumab in treatment of high-risk NB that does not respond to conventional therapy and that overexpresses VEGF.