Breast cancer, particularly metastatic breast cancer (MBC), presents aggressive clinical challenges with limited treatment success. Immunotherapy has emerged as a promising approach, however, discrepancies between preclinical animal models and human cancers complicate translation to clinical outcomes. This systematic review and meta-analysis evaluated the effect of immunotherapy on primary and metastatic tumor regression in animal models of MBC and assessed the models' appropriateness and reproducibility to improve future preclinical study design. Following a preregistered protocol in PROSPERO (CRD42021207033), we conducted searches in MEDLINE, Embase, and Web of Science databases, yielding 2255 studies for title/abstract screening and 108 studies included after full-text screening. All included studies used mouse models, assessing primary outcomes through tumor volume or weight and metastatic outcomes via nodule count or bioluminescence. Only 14% of studies fully reported experimental animal characteristics, and 43% provided detailed experimental procedures. Of 105 articles (293 comparisons) included in the meta-analysis, pooled effect sizes indicated significant reductions in both primary and metastatic tumors. However, high heterogeneity across studies and wide prediction intervals suggested substantial variability in model responses to immunotherapy. Univariable and multivariable meta-regressions failed to significantly explain this heterogeneity, suggesting additional factors may influence outcomes. Trim-and-fill and Egger's regression tests indicated funnel plot asymmetry, implying potential publication bias and small study effects. While our analysis demonstrated positive effects of immunotherapy on MBC and highlighted variability in animal tumor models, addressing model-related heterogeneity and enhancing methodological transparency are essential to improve reproducibility and clinical translatability.

Pancreatic cancer (PC) is an aggressive malignant disease with poor prognosis, often diagnosed late, progressing rapidly, and resistant to chemotherapy. Although small molecule inhibitors (SMIs) show promise in preclinical PC models, translation into clinics remains challenging. In this systematic review and meta-analysis, we screened literature according to predefined criteria to identify preclinical PC mouse models used for SMI therapy in primary tumors, assess reporting quality, and evaluate tumor reduction, heterogeneity and publication bias. Following a pre-registered PROSPERO protocol (CRD42022314932), literature searches in PubMed and Embase yielded 2972 articles, of which 297 were included for data extraction. Most studies used PDX models or MiaPaCa-2 and PANC-1 cell lines as heterotopic xenografts, with Foxn1nu mice representing the predominant genetic background. Reporting quality, assessed using the ARRIVE guidelines, revealed substantial gaps, particularly in blinding (94% not reported), inclusion/exclusion criteria (49% not reported), and randomization (34% not reported). Meta-regression accounted for part of the observed heterogeneity and funnel plot asymmetry was consistent with publication bias. This study emphasized the large variability among preclinical PC mouse models used to investigate SMI candidates and the complexity of model choice highlighting the critical need for improved reporting practices to enhance reproducibility and reliability of preclinical tumor models.

Article 23(2) of EU Directive 2010/63 on the protection of animals used for scientific purposes requires staff involved in the care and use of animals to be adequately educated and trained before carrying out procedures. Therefore, the 3Rs (refinement, reduction, and replacement) and knowledge of alternative methods should be part of the education and training itself. For this purpose, the digital learning concept "Virtual Reality (VR) in Biomedical Education" evolved, which successfully combines VR components with classical learning content. Procedures, such as anesthesia induction, substance application, and blood sampling in rats, as well as aspects of the laboratory environment were recorded in 360° videos. The generated VR teaching/learning modules (VR modules) were used to better prepare participants for hands-on training (refinement) or as a complete replacement for a live demonstration; thus, reducing the number of animals used for hands-on skills training (reduction). The current study evaluated users' experience of the VR modules. Despite little previous VR experience, participants strongly appreciated the VR modules and indicated that they believed VR has the potential to enhance delivery of procedures and demonstrations. Interestingly, participants with previous experience of laboratory animal science were more convinced about VR's potential to support the 3Rs principle, and endorsed its use for further educational purposes. In conclusion, VR appeared to be highly accepted as a learning/teaching method, indicating its great potential to further replace and reduce the use of animals in experimental animal courses.

PDF file 309K, Results of repeated dose acute toxicity study

XLS file 28K, List of top 50 interferon alpha induced gene across five humane melanoma cell lines

Animal experiments in biomedical research are debated in public, within the scientific community and among students. Despite increased efforts to reduce, refine and replace animal experiments, they remain integral components of the job of a biomedical scientist. In Germany, persons must have a university degree and adequate education and training to perform and direct animal experiments. Therefore, training courses such as FELASA (Federation of European Laboratory Animal Science Associations) courses are provided. However, in our experience, students become aware of this very late in their studies when decisions about their future careers have already been made. We initiated this study to have a better understanding of when and how animal experiments should be discussed during university education. We evaluated the knowledge, self-evaluation and attitudes of biology and medical students of different semesters regarding animal experiments at the RWTH Aachen University, Germany. An online survey was conducted to assess demographic information, knowledge about animal experiments, self-evaluation and attitudes towards animal experiments. Students of both fields showed limited knowledge of animal experiments. Biology students showed significantly better knowledge and self-evaluated their knowledge higher than medical students. The field of the study correlated with their knowledge and self-evaluation but did not predict participants’ attitudes towards animal experiments. In conclusion, the current study showed that there is still room for improvement to raise awareness about laboratory animal science in the biomedical research field.

Tissue adhesives as a physical barrier to microorganism penetration provide an alternative method with many advantages for wound closure in surgical settings compared to the clinical standard. This raises the need of developing and conducting in vitro methods that are sensitive and reproducible to assess their microbial barrier properties. In this study, three different polyurethane-based tissue adhesives with different physicochemical properties were evaluated in comparison to Dermabond® as a clinical gold standard for topical wound closure. Here, physicochemical properties varied in lactide concentration, viscosity, processing, and the full polymerization time. To evaluate the microbial barrier function, a 5 μl aliquot of E. coli Lux inoculum containing at least 1 × 109 CFU/ml was applied to the surface of each test adhesive and sterile filter paper as the control that was placed on an agar plate and incubated at 37°C. Plates were observed for bacterial growth (morphology), the adhesion of the adhesive/filter paper, and bioluminescence after 24, 48, and 72 hours. The data presented in this in vitro model indicated that polyurethane-based tissue adhesives with lactide concentration ≥ 5% provided a suitable barrier against microbial penetration with 95% confidence of 99% efficacy for 72 h along with Dermabond®. Interestingly, the here described method was able to discriminate between the different physicochemical properties showing a better microbial barrier function with increasing lactide concentration of the adhesive. Overall, the results of this study showed the noninferiority between Dermabond® and the two abovementioned polyurethane-based tissue adhesives.

Oncogenic drivers such as mutated EGFR are the preferred targets in modern drug development. However, restoring the lost function of tumor suppressor proteins could also be a valid approach to combatting cancer. ITIH5 has been revealed as a potent metastasis suppressor in both breast and pancreatic cancer. Here, we show that ITIH5 overexpression in MDA-MB-231 breast cancer cells can also locally suppress tumor growth by 85%, when transplanted into the mammary fat pad of nude mice. For a potential drug development approach, we further aimed to define downsized ITIH5 polypeptides that still are capable of mediating growth inhibitory effects. By cloning truncated and His-tagged ITIH5 fragments, we synthesized two recombinant N-terminal polypeptides (ITIH5681aa and ITIH5161aa), both covering the ITI heavy chain specific “vault protein inter-alpha-trypsin” (VIT) domain. Truncated ITIH5 variants caused dose-dependent cell growth inhibition by up to 50% when applied to various cancer cell lines (e.g., MDA-MB-231, SCaBER, A549) reflecting breast, bladder and lung cancer in vitro. Thus, our data suggest the substantial role of the ITIH5-specific VIT domain in ITIH5-mediated suppression of tumor cell proliferation. As extracellularly administered ITIH5 peptides mimic the growth-inhibitory effects of the full-length ITIH5 tumor suppressor protein, they may constitute the basis for developing anticancer drugs in the future.

Background. Tissue glues can minimize treatment invasiveness, mitigate the risk of infection, and reduce surgery time; ergo, they have been developed and used in surgical procedures as wound closure devices beside sutures, staples, and metallic grafts. Regardless of their structure or function, tissue glues should show an acceptable microbial barrier function before being used in humans. This study proposes a novel in vitro method using Escherichia coli Lux and bioluminescence imaging technique to assess the microbial barrier function of tissue glues. Different volumes and concentrations of E. coli Lux were applied to precured or cured polyurethane-based tissue glue placed on agar plates. Plates were cultured for 1 h, 24 h, 48 h, and 72 h with bioluminescence signal measurement subsequently. Herein, protocol established a volume of 5 μL of a 1 : 100 dilution of E. coli Lux containing around 2 × 107 CFU/mL as optimal for testing polyurethane-based tissue glue. Measurement of OD600nm, determination of CFU/mL, and correlation with the bioluminescence measurement in p/s unit resulted in a good correlation between CFU/mL and p/s and demonstrated good reproducibility of our method. In addition, this in vitro method could show that the tested polyurethane-based tissue glue can provide a reasonable barrier against the microbial penetration and act as a bacterial barrier for up to 48 h with no penetration and up to 72 h with a low level of penetration through the material. Overall, we have established a novel, sensitive, and reproducible in vitro method using the bioluminescence imaging technique for testing the microbial barrier function of new tissue glues.

Background Intra-bone marrow injection (IBMI) in rats is adopted in many studies for stem cell and hematopoietic cell transplantation. IBMI in the tibia or the femur results in severe distress to the animal. Therefore, this study aims to evaluate intra-iliac injections as an alternative approach for IBMI. Methods Twenty-seven Sprague Dawley rats were assigned into 3 groups, 9 rats each, for 4 weeks. The control group rats were not injected. Tibia group rats were injected intra-tibial and the iliac group rats were injected intra-iliac with saline. Behavioral, radiological, histopathological, and stress evaluation was performed. Total bilirubin, cortisol, and insulin-like growth factor-1 (IGF1) were measured. Results Behavioral measurements revealed deviation compared to control, in both injected groups, on the 1st and 2nd week. By the 3rd week, it was equivalent to control in the iliac group only. Bilirubin and cortisol levels were increased by intra-tibial injection compared to intra-iliac injection. The IGF-1 gene expression increased compared to control at 1st and 2nd weeks in intra-iliac injection and decreased by intra-tibial injection at 2nd week. The thickness of the iliac crest was not different from the control group, whereas there were significant differences between the control and tibia groups. Healing of the iliac crest was faster compared to the tibia. In the 3rd week, the tibia showed fibrosis at the site of injection whereas the iliac crest showed complete bone reconstruction. Conclusion Intra-iliac injections exert less distress on animals, and by 3 weeks, they regained their normal activity in comparison to intra-tibial injections.

Results of dose escalation study using 3 different doses of Oncaspar.

Because of its size, anatomical similarities, and now also accessibility to genetic manipulations, pigs are used as animal models for human diseases and immune system development. However, expression and function of CD28, the most important costimulatory receptor expressed by T cells, so far is poorly understood in this species. Using a newly generated mAb (mAb 3D11) with specificity for pig CD28, we detected CD28 on CD8+ and CD4+ αβ T cells. Among γδ T cells, CD28 expression was restricted to a small CD2+ subpopulation of phenotypically naive cells. Functionally, CD28 ligation with mAb 3D11-costimulated porcine T cells, enhanced proliferation and cytokine secretion in vitro. We used a second, likewise newly generated but superagonistic, anti-CD28 mAb (CD28-SA; mAb 4D12) to test the function of CD28 on porcine T cells in a pilot study in vivo. Injection of the CD28-SA into pigs in vivo showed a very similar dose-response relationship as in humans (i.e., 100 µg/kg body weight [BW]) of CD28-SA induced a cytokine release syndrome that was avoided at a dose of 10 µg/kg BW and below. The data further suggest that low-dose (10 µg/kg BW) CD28-SA infusion was sufficient to increase the proportion of Foxp3+ regulatory T cells among CD4+ T cells in vivo. The pig is thus a suitable animal model for testing novel immunotherapeutics. Moreover, data from our pilot study in pigs further suggest that low-dose CD28-SA infusion might allow for selective expansion of CD4+ regulatory T cells in humans.

The aim of this study was to compare two variants of a novel polycarbonate polyurethane prosthesis with polyethylene terephthalate (Dacron) prosthesis in an established sheep model of carotid patch angioplasty. Two variants of the polycarbonate polyurethane prosthesis were used: (1) Polycarbonate polyurethane equal (PCU-e) prosthesis consisted of a multilayered porous structure with equal internal and external layers; (2) Polycarbonate polyurethane with more porous inner layers (PCU-mp) prosthesis had more porous inner layers than external layers. Carotid patch angioplasty was performed in 12 sheep: in six sheep, the PCU-e variant and in the other six sheep, the PCU-mp variant was implanted. Dacron patches were implanted in the contralateral carotid artery of all sheep as a control. Half of the animals with each polycarbonate polyurethane variant were euthanized after two weeks and the other half after eight weeks. Cellular infiltration, endothelialization, and neointimal hyperplasia were examined. All grafts were patent, and no thrombus was seen in any of the harvested arteries. The pores of all the three patch materials allowed infiltration of inflammatory cells, capillaries, and connective tissue. After eight weeks, a nearly complete endothelialization was visible in all patch groups without an obvious difference between the three patch materials. The neointima was thinner in the PCU patches (PCU-e: 56 ± 13 µm, PCU-mp: 119 ± 60 µm) when compared to Dacron patches (156 ± 64 µm) after two weeks. After 8 weeks, a further neointimal growth was detectable, without any significant difference of neointimal thickness between the three patch materials (Dacron: 274 ± 82 µm, PCU-e: 324 ± 98 µm, PCU-mp: 235 ± 59 µm). With the novel polycarbonate polyurethane patch materials, we achieved promising functional and morphological results with 100% patency and nearly complete endothelialization. Our findings showed at least a non-inferiority of the novel polycarbonate polyurethane patch material compared to Dacron. There were no significant differences detected between the two polycarbonate polyurethane patch variants.

The development of new contrast agents (CAs) for magnetic resonance imaging (MRI) is of high interest, especially because of the increased concerns of patient safety and quick clearance of clinically used gadolinium and iron oxide-based CAs, respectively. Here, a two-step synthesis of superparamagnetic water-soluble iron platinum (FePt) nanoparticles (NPs) with core sizes between 2 and 8 nm for use as CAs in MRI is reported. First, wet-chemical organometallic NPs are synthesized by thermal decomposition in the presence of stabilizing oleic acid and oleylamine. Second, the hydrophobic NPs are coated with an amphiphilic polymer and transferred into aqueous media. Their magnetization values and relaxation rates exceed those published for CAs already used for clinical application. Their saturation magnetization increases with the core size to approximately 82 A·m2/kgFe. For 8 nm NPs, the T2 relaxivity of approximately 221 (mM·s)-1 is 5 times larger than that for the ferumoxides, and for 6 nm NPs, the T1 relaxivity of approximately 12 (mM·s)-1 is slightly higher than that of ultrasmall gadolinium oxide NPs. The 6 nm FePt NPs are identified as excellent CAs for both T1 and T2 imaging. Most importantly, because of their coating, significantly low cytotoxicity is achieved. FePt NPs prove to be a promising alternative to gadolinium and iron oxide NPs showing high-quality CA characteristics for both T1- and T2-weighted images.

The Pringle maneuver (PM) has been widely used to control blood loss during liver resection. However, hepatic inflow occlusion can also result in hepatic ischemia–reperfusion injury (IRI), especially in patients with a cholestatic, fibrotic, or cirrhotic liver. Here we investigate a nitric oxide synthase (NOS) inhibitor N-Nitroarginine methyl ester (L-NAME) on IRI after the PM and partial hepatectomy of cholestatic livers induced by bile duct ligation (BDL) in rats. Control group (non-BDL/no treatment), BDL + T group (BDL/L-NAME treatment) and BDL group (BDL/no treatment) were analyzed. Cholestasis was induced by BDL in the L-NAME and BDL group and a 50% partial hepatectomy with PM was performed. L-NAME was injected before PM in the BDL + T group. Hepatocellular damage, portal venous flow, microcirculation, endothelial lining, and eNOS, iNOS, interleukin (IL)-6, and transforming growth factor-β (TGF-β) were evaluated. Microcirculation of the liver in the BDL + T group tended to be higher. Liver damage and apoptotic index were significantly lower and Ki-67 labeling index was higher in the BDL + T group while iNOS and TGF-β expression was decreased. This was corroborated by a better preserved endothelial lining. L-NAME attenuated IRI following PM and improved proliferation/regeneration of cholestatic livers. These positive effects were considered as the result of improved hepatic microcirculation, prevention of iNOS formation, and TGF-β mRNA upregulation.

In vascular surgery, novel synthetic prosthesis materials for patch-angioplasties, interpositions, bypasses and shunts are continuously under development and optimization. The characteristics of an ideal vascular prosthesis would display long-term patency, biocompatibility, durability, low porosity, lack of stich hole bleeding, ease of handling, kink resistance, infection resistance and reasonable costs. The aim of this study was to establish and report a reliable sheep model including potential pitfalls where those parameters could be analyzed. Before surgery, sheep were acclimatized for 4–8 weeks, during which parasite infections were treated and blood and serum parameters monitored. Twenty-four sheep underwent surgery, and carotid patch-angioplasties (n = 12), graft interpositions (n = 6) or arteriovenous prosthetic shunts (n = 6) were implanted. Half of the animals in each group were sacrificed after 2 weeks and the other half after 8 weeks. The implants were analyzed for patency, endothelialization, thrombogenicity and biocompatibility by clinical observation, blood flow measurement and pathological and histopathological (H&E, EvG) as well as immunohistochemical (Ki67, CD31) evaluations.

BACKGROUND:The pig is one of the most frequently used large animal models for biomedical research, especially in the field of translational research and surgical models. While standard livestock breeds are used in short-term and acute studies, minipig breeds are the preferred breeds in long-term and chronic studies due to their limited growth and body weight.OBJECTIVE:In consideration of the 3R principle (refinement, reduction, replacement) and the increasing demand, the aim of this study was to generate a new, robust, non-specific-pathogen-free minipig breed, the Aachen minipig.METHODS:Phenotype, genotype, and hematological as well as clinical chemistry parameters were characterized, and reference values of the Aachen minipig were generated and compared to the values in the commonly used Göttingen minipig. Organ weights of the heart, kidney, liver, lung, spleen, and brain were determined using a laboratory balance. Blood samples were collected for hematology and clinical chemistry. Assessment of genetic diversity was performed by microsatellite markers. Nasal swabs were collected from 11 individual minipigs representing 6 races for DNA extraction. DNA was quantified and the identity and origin of the Aachen minipigs at the genomic level was determined by microsatellites.RESULTS:The Aachen minipig established here is based on the Mini-LEWE breed and consists of the Vietnamese potbelly pig, the Schwäbisch Hällisch Landpig, the German Landrace, and the Minnesota minipig. Relative organ weights (lung, heart, kidneys, brain), hematology (hemoglobin, hematocrit, platelet count, mean corpuscular hemoglobin concentration, segmented neutrophils, lymphocytes, eosinophils, basophils), and clinical chemistry parameters (sodium, calcium, chloride, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, creatine kinase) of the Aachen minipigs and the Göttingen minipigs were not significantly different. Significant differences where only seen in relative organ weights (liver, spleen), hematology (red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, banded neutrophils, monocytes), and clinical chemistry parameters (inorganic phosphorus, potassium, glucose, cholesterol, albumin, amylase).CONCLUSION:The Aachen minipig is a suitable model for research due to its similarity to other minipig breeds, especially the Göttingen minipig. The reference values established in this study may be used for the comparison of scientific data and encourage the use of the Aachen minipig as an animal model for biomedical research.

Overlap of hypo-and hypermethylated CpG sites with published gene sets (GSEA). This table summarizes an overlap of significantly hyper- and hypomethylated CpG sites between ΔpBK-ITIH5 and ΔpBK-mock clones with gene set data bases. (XLS 72 kb)

BACKGROUND:Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive.METHODS:ITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement.RESULTS:ITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards β1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility.CONCLUSIONS:Our results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.