The chemicals between us-First results of the cluster analyses on anatomy embalming procedures in the German-speaking countries.

Hands-on courses utilizing preserved human tissues for educational training offer an important pathway to acquire basic anatomical knowledge. Owing to the reevaluation of formaldehyde limits by the European Commission, a joint approach was chosen by the German-speaking anatomies in Europe (Germany, Austria, Switzerland) to find commonalities among embalming protocols and infrastructure. A survey comprising 537 items was circulated to all anatomies in German-speaking Europe. Clusters were established for "ethanol"-, formaldehyde-based ("FA"), and "other" embalming procedures, depending on the chemicals considered the most relevant for each protocol. The logistical framework, volumes of chemicals, and infrastructure were found to be highly diverse between the groups and protocols. Formaldehyde quantities deployed per annum were three-fold higher in the "FA" (223 L/a) compared to the "ethanol" (71.0 L/a) group, but not for "other" (97.8 L/a), though the volumes injected per body were similar. "FA" was strongly related to table-borne air ventilation and total fixative volumes ≤1000 L. "Ethanol" was strongly related to total fixative volumes >1000 L, ceiling- and floor-borne air ventilation, and explosion-proof facilities. Air ventilation was found to be installed symmetrically in the mortuary and dissection facilities. Certain predictors exist for the interplay between the embalming used in a given infrastructure and technical measures. The here-established cluster analysis may serve as decision supportive tool when considering altering embalming protocols or establishing joint protocols between institutions, following a best practice approach to cater toward best-suited tissue characteristics for educational purposes, while simultaneously addressing future demands on exposure limits.

Detection of Spontaneous Bone Metastases of Solid Human Tumor Xenografts in Mice.

The formation of bone metastases from solid primary tumors comprises several processes following each other in a sequential order in terms of the metastatic cascade. The most widely used preclinical models of bone metastasis formation do not reflect this pathophysiological situation as they are based on intracardiac (left ventricle) or intracaudal artery injection of tumor cells. These attempts circumvent all early steps of the metastatic cascade taking place within primary tumors (e.g., epithelial-mesenchymal transition), the passage of circulating tumor cells through upstream organ "filters" like the lung, and the initial establishment of single disseminated tumor cells/cell clusters within the bone marrow. In this chapter, we describe how the entire cascade of bone metastasis formation can be modelled in vivo using bioluminescence techniques. The cascade ranges from the formation of a primary tumor to the outgrowth of single disseminated tumor cells to micro-metastases within the bone marrow. In addition, we describe how the disseminated tumor cells and bone metastases can be visualized by histological and immunohistochemical staining. The described methodology provides the opportunity to investigate the basic mechanisms of spontaneous bone metastasis formation of solid human tumors in partly immunodeficient hosts in vivo.

Tumor cell E-selectin ligands determine partialefficacy of bortezomib on spontaneous lung metastasis formation of solid human tumors in vivo.

Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.

Modeling Spontaneous Bone Metastasis Formation of Solid Human Tumor Xenografts in Mice.

The majority of cancer-related deaths are due to hematogenous metastases, and the bone marrow (BM) represents one of the most frequent metastatic sites. To study BM metastasis formation in vivo, the most efficient approach is based on intracardiac injection of human tumor cells into immunodeficient mice. However, such a procedure circumvents the early steps of the metastatic cascade. Here we describe the development of xenograft mouse models (balb/c rag2-/- and severe combined immunodeficient (SCID)), in which BM metastases are spontaneously derived from subcutaneous (s.c.) primary tumors (PTs). As verified by histology, the described methodology including ex vivo bioluminescence imaging (BLI) even enabled the detection of micrometastases in the BM. Furthermore, we established sublines from xenograft primary tumors (PTs) and corresponding BM (BM) metastases using LAN-1 neuroblastoma xenografts as a first example. In vitro "metastasis" assays (viability, proliferation, transmigration, invasion, colony formation) partially indicated pro-metastatic features of the LAN-1-BM compared to the LAN-1-PT subline. Unexpectedly, after s.c. re-injection into mice, LAN-1-BM xenografts developed spontaneous BM metastases less frequently than LAN-1-PT xenografts. This study provides a novel methodologic approach for modelling the spontaneous metastatic cascade of human BM metastasis formation in mice. Moreover, our data indicate that putative bone-metastatic features get rapidly lost upon routine cell culture.

Selectin Binding Sites Are Involved in Cell Adhesive Properties of Head and Neck Squamous Cell Carcinoma.

The formation of distant metastases often determines the fate of patients with head and neck squamous cell carcinoma (HNSCC). The expression of cell adhesion molecules (CAMs) and their ligands of the leukocyte adhesion cascade has been associated with metastatic competence in several malignant entities. In this study, human HNSCC cell lines were analyzed in vitro and in a spontaneous metastatic xenograft model. Immunohistochemical analyses of several CAMs were performed on xenograft tumors and tissue microarrays (TMA) from 453 patients with head and neck squamous cell carcinomas with full histo-pathological and clinical follow-up data. UTSCC 24A and 24B cells bind to E-selectin in vitro, show E-selectin dependent binding to human umbilical vein endothelial cells (HUVECs), and express sLeX. All HNSCC cells engrafted into severe combined immunodeficient (SCID) mice, and UTSCC 24A cells formed sporadically spontaneous lung metastases. The expression of CAMs varied between the cell lines, but a correlation between tumor growth and metastatic potential did not exist. None of the CAMS or their ligands could be identified to be of prognostic relevance in the TMA study. The in vitro results indicate that E-selectin and sLeX are involved in the adhesion of HNSCC cells to endothelium. However, specific prognostic markers chosen from the leukocyte adhesion cascade for HNSCC were not identified.

Differential Proteome Analysis of Human Neuroblastoma Xenograft Primary Tumors and Matched Spontaneous Distant Metastases.

Metastasis formation is the major cause for cancer-related deaths and the underlying mechanisms remain poorly understood. In this study we describe spontaneous metastasis xenograft mouse models of human neuroblastoma used for unbiased identification of metastasis-related proteins by applying an infrared laser (IR) for sampling primary tumor and metastatic tissues, followed by mass spectrometric proteome analysis. IR aerosol samples were obtained from ovarian and liver metastases, which were indicated by bioluminescence imaging (BLI), and matched subcutaneous primary tumors. Corresponding histology proved the human origin of metastatic lesions. Ovarian metastases were commonly larger than liver metastases indicating differential outgrowth capacities. Among ~1,900 proteins identified at each of the three sites, 55 proteins were differentially regulated in ovarian metastases while 312 proteins were regulated in liver metastases. There was an overlap of 21 and 7 proteins up- and down-regulated at both metastatic sites, respectively, most of which were so far not related to metastasis such as LYPLA2, EIF4B, DPY30, LGALS7, PRPH, and NEFM. Moreover, we established in vitro sublines from primary tumor and metastases and demonstrate differences in cellular protrusions, migratory/invasive potential and glycosylation. Summarized, this work identified several novel putative drivers of metastasis formation that are tempting candidates for future functional studies.

Locally Ablative Radiation Therapy of a Primary Human Small Cell Lung Cancer Tumor Decreases the Number of Spontaneous Metastases in Two Xenograft Models.

PURPOSE: To investigated the influence of radiation therapy (RT), surgery (OP), radio-chemotherapy (RChT), or chemotherapy (ChT) on small cell lung cancer metastases in 2 xenograft models. METHODS AND MATERIALS: A total of 1 × 106 human small cell lung cancer cells (OH1, H69) were subcutaneously injected into severe combined immunodeficiency mice to form a local primary tumor node at the lower trunk. Radiation therapy, OP, RChT, or ChT were started after development of palpable tumors. Chemotherapy was given as a single intraperitoneal injection of cisplatin. Radiation therapy was 5 × 10 Gy on the local tumor node. Two additional groups were implemented to assess primary tumors and distant metastases in untreated mice at the beginning (control group A) and at the end of the experiment (control group B). Proapoptotic, antiproliferative, antiangiogenic, and hypoxic effects were assessed by Feulgen, Ki67, S1P1 receptor, and hypoxia-inducible factor 1α staining, respectively. Quantitative Alu-polymerase chain reaction was used to determine circulating tumor cells in the blood, and disseminated tumor cells in the lungs, bone marrow, liver, and brain. RESULTS: In both xenograft models, RT and RChT abrogated local tumor growth, indicated by increased apoptosis, decreased cell proliferation, and reduced microvessel density (equally affecting vessels of all diameters). Regarding metastases, RT and RChT not only counteracted the time-dependent increase of dissemination but also decreased the metastatic load pre-existing at therapy induction in the blood, lungs, and liver. Only in the case of relapse-free surgery could similar effects be achieved by OP. CONCLUSIONS: Our models provide evidence that RT and RChT ablate the primary tumor and inhibit metastasis development over time. Upon local recurrence, RT showed beneficial effects compared with OP with regard to suppression of circulating tumor cells and disseminated tumor cells.

Cell adhesion molecules in metastatic neuroblastoma models.

Several cell adhesion molecules (CAMs) including selectins, integrins, cadherins and immunoglobulin-like CAMs are involved in leukocyte adhesion especially at sites of inflammation. In cancer cells, these CAMs have been associated with the growth and metastatic behavior in several malignant entities. In this study adhesion of LAN 1 and SK-N-SH neuroblastoma cells to selectins, hyaluronan and endothelial cells were determined under flow conditions. Furthermore cells were injected subcutaneously into wildtype and selectin deficient scid mice and their growth and metastatic behavior were analyzed. Under shear stress SK-N-SH cells firmly adhered to E-selectin-Fc-fusion protein, hyaluronan and endothelial cells, while LAN 1 cells showed less or hardly any adhesive events by comparison. In the SK-N-SH xenograft model metastasis formation was slightly dependent on the expression of selectins, while LAN 1 cells developed metastases completely independent of selectin expression. The different adhesive and metastatic properties of LAN 1 and SK-N-SH cells are reflected by a different expression profile of several CAMs. The results indicate that endothelial selectins are not essential for metastasis formation of human LAN 1 and SK-N-SH cells. However, other CAMs namely CD44, N-cadherin, NCAM and integrins were upregulated or downregulated, respectively, in SK-N-SH and LAN 1 cells and are potential adhesion molecules involved in the metastatic cascade of these cells.

Lectin histochemistry of metastasizing and non-metastasizing breast and colon cancer cells.

BACKGROUND: Glycosylation of the tumour cell surface is of importance in metastasis formation as indicated by lectin-binding studies. In particular, binding of the lectin HPA is associated with metastasis formation, both in clinical studies and in xenograft models of breast and colon cancer. Here we examined if there is an association between the HPA-positive glycotopes of metastasizing cancer cells and selectin-binding properties. MATERIALS AND METHODS: Glycotope expression of human breast and colon cancer cells (MCF7, T47D, HBL100, HT29, SW480) grown in culture and xenografted into SCID mice were investigated by histochemical analysis. RESULTS: HPA binding was observed in metastasizing breast and colon cancers and not in non-metastasizing ones. In colon cancer, E-selectin binding and expression of the selectin ligands CD15s and CA19-9 was higher in metastatic HT29 than in non-metastatic SW480 cells, especially when cells were grown in vitro. In breast cancer, E-selectin binding, CD15s and CA19-9 expression were independent of the metastatic potential. P-Selectin binding was slightly higher in metastasizing breast cancer cells (MCF7, T47D) than in non-metastasizing HBL100 cells. CONCLUSION: Binding to E-selectin and expression of E-selectin ligands of colon cancer cells grown in vitro is associated with metastasis formation in a xenograft model. However, analysis of selectin ligands is of limited predictive value for the metastatic potential of breast cancer cells in our xenograft model.

Expression of the neural cell adhesion molecule and polysialic acid in human neuroblastoma cell lines.

The neural cell adhesion molecule NCAM is a cell surface glycoprotein of the immunoglobulin superfamily and is widely expressed in tumours of neuroectodermal origin such as neuroblastoma. NCAM can be decorated by the carbohydrate polymer polysialic acid (polySia), which attenuates NCAM-mediated cell adhesion and increases cellular motility. The key enzymes in the biosynthesis of polySia are the two polysialyltransferases ST8SiaII and ST8SiaIV. In the present study, expression of NCAM, polySia-NCAM, ST8SiaII and ST8SiaIV was investigated in five human neuroblastoma cell lines before and after xenografting into SCID mice by immunohistochemistry, Western blot analysis and real-time PCR. Results were correlated with the metastatic potential. In vitro, three cell lines (LAN-1, LAN-5 and SH-SY5Y) were positive for polySia attached to the transmembrane isoforms NCAM-140 and NCAM-180, whereas Kelly and SK-N-SH cells were negative for NCAM and polySia. In the presence of NCAM, the level of polySia correlated with the amount of polysialyltransferase transcripts, which were highest in LAN-1, LAN­5 and SH-SY5Y cells. In the respective primary tumours grown in SCID mice, the expression patterns of NCAM, polySia and polysialyl-transferases were similar to those observed in vitro. After subcutaneous engraftment, polySia-NCAM-positive neuroblastoma developed disseminated micrometastases, a metastatic pattern that was not observed for tumours derived from NCAM-negative cell lines. Together, this indicates that the presence of polySia reduces the adhesiveness of tumour cells and promotes dissemination.

The effect of the PPAR-gamma agonist rosiglitazone on neuroblastoma SK-N-SH cells in a metastatic xenograft mouse model.

Rosiglitazone, a peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonist used in clinical practice to treat type 2 diabetes, has been shown to inhibit neuroblastoma cell proliferation in vitro. In the present study, SK-N-SH neuroblastoma cells were subcutaneously injected into SCID mice and their growth and metastatic behavior under the treatment with rosiglitazone was analyzed. Therapeutic effects were evaluated comparing primary tumor weight, cell proliferation, apoptosis, and number of pulmonary metastasis. Rosiglitazone significantly decreased cell proliferation of the SK-N-SH neuroblastomas from 57.0% in the solvent control to 45.0% and 47.0% in the two treatment groups, respectively. However, primary tumor weight, apoptosis, and metastasis were not considerably influenced. These results indicate that the PPAR-gamma agonist rosiglitazone has only slight antitumor effects on SK-N-SH neuroblastoma growth in vivo in contrast to in vitro.

Effects of bortezomib on human neuroblastoma cells in vitro and in a metastatic xenograft model.

BACKGROUND: Inhibition of the proteasome-ubiquitin pathway has shown to exert growth inhibitory effects on several human carcinoma cell lines. In this study, the influence of bortezomib on human neuroblastoma cells was investigated. MATERIALS AND METHODS: Cell proliferation of seven human neuroblastoma cell lines under bortezomib treatment was assessed by a colorimetric XTT-based assay. Subsequently, the influence of bortezomib on SK-N-SH neuroblastoma cell growth was examined in a spontaneous metastatic SCID mouse model. RESULTS: In vitro, bortezomib inhibited proliferation of all cell lines in a dose-dependent manner. In the xenograft model, bortezomib treatment did not have an effect on the tumour weight, but induced apoptosis and reduced mitosis and angiogenesis, as well as the formation of pulmonary metastases. CONCLUSION: Bortezomib has anticancer effects on neuroblastoma cells in vitro and in a metastatic xenograft model. These findings provide a basis for further investigations of bortezomib in the treatment of metastasising neuroblastoma.

[18F]FDG and [18F]FLT PET-CT and MR imaging of human neuroblastomas in a SCID mouse xenograft model.

BACKGROUND: Finding new therapeutic agents is of great clinical interest in neuroblastoma research because prognosis of children with disseminated stages of disease is still poor. As xenograft mouse models are frequently used for studying anticancer drugs in vivo, small animal imaging is an important method of monitoring in anticancer research. MATERIALS AND METHODS: SCID mice inoculated with human neuroblastoma SK-N-SH cells were examined with positron-emission tomography-computed tomography (PET-CT) using [18F]fluorodeoxyglucose (FDG) or [18F]fluoro-L-thymidine (FLT) and with magnetic resonance imaging (MRI). RESULTS: All neuroblastomas were detected by MRI. In PET-CT imaging, no tumour was visualized with [18F]FDG, but 13 out of 14 (93%) were found with [18F]FLT. Uptake of [18F]FLT was significantly associated with tumour weight. Necrotic areas could not be identified either by MR imaging or on PET-CT scans. CONCLUSION: Both MR and PET-CT imaging with [18F]FLT are highly qualified for the detection of neuroblastomas grown in SCID mice. However, [18F]FDG, which is the standard tracer in clinical PET-CT imaging, is not suited for PET-CT imaging in the neuroblastoma model.

Expression of CD44 is associated with a metastatic pattern of human neuroblastoma cells in a SCID mouse xenograft model.

Expression of CD44, a transmembrane glycoprotein involved in cell-cell and cell-matrix interactions, has been associated with growth and metastatic behavior in several malignant tumors. In contrast to most other malignancies, in which up-regulation of CD44 is related to tumor progression, the absence of CD44 expression characterizes the aggressiveness of neuroblastomas in clinical studies. In this study, cells of human neuroblastoma cell lines (IMR-32, Kelly, LAN-1, LAN-5, LS, SH-SY5Y and SK-N-SH) were injected subcutaneously into SCID mice, and their growth behavior and CD44 expression were analyzed. All neuroblastoma cells engrafted in the SCID mouse, but primary tumor growth and metastatic potential varied considerably. Expression of CD44 was associated with a metastatic pattern of the neuroblastoma cell lines. CD44-positive neuroblastomas produced multicellular metastases predominantly located in the intra- and periarterial space of the lung. CD44-negative neuroblastomas developed numerous micrometastases in the lung interstitium. In conclusion, the entire spectrum of metastatic patterns can be modeled in SCID mice using the human neuroblastoma cell lines employed in this study. Our xenograft model provides a platform for investigating the complex processes involved in metastasis formation and for testing new anti-metastatic drugs. In particular, the role of CD44 in the formation of metastasis can be evaluated.

Visualisation of neuroblastoma growth in a Scid mouse model using [18F]FDG and [18F]FLT-PET.

BACKGROUND: Tumor therapy has been monitored using the metabolic indicator [18F]fluorodeoxyglucose ([18F]FDG). However, the nucleotide precursor [18F]fluoro-thymidine ([18F]FLT) is in principle more specific as it is incorporated into DNA. Thus, the [18F]FDG and [18F]FLT uptake by human neuroblastomas grown in Scid mice are compared in this study. MATERIALS AND METHODS: Scid mice were inoculated with human neuroblastoma cells. Tumor imaging was performed with a human whole-body full-ring PET scanner. Furthermore, the tumor weight and the cell proliferation rate were determined. RESULTS: Neuroblastomas could be visualised using [18F]FDG in 40% and with [18F]FLT in 70% of the cases. [18F]FDG or [18F]FLT uptake could not be visualised in neuroblastomas less than 1.0 g in weight. No correlation between the cell proliferation rate and tracer uptake could be detected. CONCLUSION: [18F]FLT showed a higher uptake than [18F]FDG and, therefore, might be more suitable for monitoring anticancer therapy, at least in this tumor model.

In vivo xenograft models of breast cancer metastasis.

If breast cancer patients are not cured, it is largely because of the fact that the cancer has spread beyond its primary site--the breast--to distant sites, such as, e.g., bone marrow, lung, brain, and/or liver. These secondary tumors are called metastases, and the underlying mechanisms leading to these secondary tumor deposits are complex and still ill understood. In this chapter, we report on how to develop clinically relevant human breast cancer cell line xenografts in severe combined immunodeficient mice. In severe combined immunodeficient mice, metastasizing human breast cancer cell lines were identified by their ability to bind the lectin Helix pomatia agglutinin, which was identified as a marker of metastasis in clinical studies. This model system was created to help to define the rate-limiting steps of the metastatic cascade.

Ligands for the peroxisome proliferator-activated receptor-gamma have inhibitory effects on growth of human neuroblastoma cells in vitro.

The thiazolidinedione (TZD) or glitazone class of peroxisome proliferator-activated-gamma (PPAR-gamma) ligands not only induce adipocyte differentiation and increase insulin sensitivity, but also exert growth inhibitory effects on several carcinoma cell lines in vitro as well as in vivo. In the current study the in vitro effect of four PPAR-gamma agonists (ciglitazone, pioglitazone, troglitazone, rosiglitazone) on the cell growth of seven human neuroblastoma cell lines (Kelly, LAN-1, LAN-5, LS, IMR-32, SK-N-SH, SH-SY5Y) was investigated. Growth rates were assessed by a colorimetric XTT-based assay kit. Expression of PPAR-gamma protein was examined by immunohistochemistry and Western blot analysis. All glitazones inhibited in vitro growth and viability of the human neuroblastoma cell lines in a dose-dependent manner showing considerable effects only at high concentrations (10 microM and 100 microM). Effectiveness of the glitazones on neuroblastoma cell growth differed depending on the cell line and the agent. The presence of PPAR-gamma protein was demonstrated in all cell lines. Our findings indicate that ligands for PPAR-gamma may be useful therapeutic agents for the treatment of neuroblastoma. Thus the effect of glitazones on the growth of neuroblastoma should now be investigated in an in vivo animal model.

HPA binding and metastasis formation of human breast cancer cell lines transplanted into severe combined immunodeficient (scid) mice.

Six human breast cancer cell lines were injected subcutaneously into scid mice and their in vivo growth behaviour and HPA binding pattern were analysed. Furthermore, the role of HPA binding glycoconjugates concerning the adhesion to endothelial cells in vitro was investigated. Four of the tested cell lines engrafted in the scid mouse model but they showed considerable variations concerning their growth behaviour, their metastatic potential and their HPA binding pattern. HPA inhibited adhesive interactions between cell lines derived from metatstatic sources and tumour necrosis factor (TNF)alpha stimulated endothelial cells. The transplantation of HPA defined breast cancer cell lines into scid mice is a useful animal model for the research of breast cancer and its metastasis. The HPA binding glycoconjugates appear to be associated with adhesive interactions between metastasising tumour cells and endothelial cells.

The influence of dietary lectins on the cell proliferation of human breast cancer cell lines in vitro.

BACKGROUND: Lectins, carbohydrate proteins, bind non-covalently to glycoconjugate of normal and malignant cells. If used in cell culture, they can influence cellular proliferation. In this study the in vitro effects of six dietary lectins on the cell proliferation of human breast cancer cell lines were investigated. MATERIALS AND METHODS: Cell proliferation was assessed by a colorimetric XTT-based assay kit. Lectin binding was characterized by lectin histochemistry. RESULTS: WGA considerably influenced the cell growth of all tested cell lines (MCF-7, T 47D, HBL 100, BT 20), whereas the effects of PHA-L, SBA and HPA were smaller, began at higher concentrations and were restricted to three cell lines (MCF-7, T 47D and HBL 100 for PHA-L; MCF-7, T 47D and BT 20 for SBA, respectively) and to one cell line (HBL 100 for HPA). STA and PNA had no effect at all. CONCLUSION: The present data suggested that some dietary lectins can inhibit cell growth of human breast cancer cells in vitro. These findings would argue for a protective effect of these plant lectins for breast cancer.