The chorioallantoic-membrane (CAM)-assay is an established model for in vivo tumor research. Contrary to rodent-xenograft-models, the CAM-assay does not require breeding of immunodeficient strains due to native immunodeficiency. This allows xenografts to grow on the non-innervated CAM without pain or impairment for the embryo. Considering multidirectional tumor growth, limited monitoring capability of tumor size is the main methodological limitation of the CAM-assay for tumor research. Enclosure of the tumor by the radiopaque eggshell and the small structural size only allows monitoring from above and challenges established imaging techniques. We report the eligibility of ultrasonography for repetitive visualization of tumor growth and vascularization in the CAM-assay. After tumor ingrowth, ultrasonography was repetitively performed in ovo using a commercial ultrasonographic scanner. Finally, the tumor was excised and histologically analyzed. Tumor growth and angiogenesis were successfully monitored and findings in ultrasonographic imaging significantly correlated with results obtained in histological analysis. Ultrasonography is cost efficient and widely available. Tumor imaging in ovo enables the longitudinal monitoring of tumoral development, yet allowing high quantitative output due to the CAM-assays simple and cheap methodology. Thus, this methodological novelty improves reproducibility in the field of in vivo tumor experimentation emphasizing the CAM-assay as an alternative to rodent-xenograft-models.

Ki-ras gene mutations that specifically occur in codons 12, 13 and 61 are involved in the carcinogenesis of acute myeloid leukemia, melanoma and different carcinomas. In order to define potential mutation-specific therapeutic targets, stable transfectants of NIH3T3 cells carrying different Ki-ras4B gene mutations were generated. Wild type Ki-ras transformants, mock transfectants and parental cells served as controls. These in vitro model systems were systematically analyzed for their protein expression pattern using two-dimensional gel electrophoresis followed by mass spectrometry and/or protein sequencing. Using this approach, a number of target molecules that are differentially but coordinately expressed in the ras transfectants were identified next to other proteins that exhibit a distinct regulation pattern in the different cell lines analyzed. The differentially expressed proteins predominantly belong to the families of cytoskeletal proteins, heat shock proteins, annexins, metabolic enzymes and oxidoreductases. Their validation was assessed by real-time quantitative RT-PCR and/or Western blot analysis. Our results suggest that the Ki-ras-transformed cells represent a powerful tool to study Ki-ras gene mutation-driven protein expression profiles. In addition, this approach allows the discovery of ras-associated cellular mechanisms, which might lead to the identification of physiological targets for pharmacological interventions of the treatment of Ki-ras-associated human tumors.

Radiotherapy is an important treatment option in the therapy of multiple tumor entities among them head and neck squamous cell carcinoma (HNSCC). However, the success of radiotherapy is limited by the development of radiation resistances. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a cofactor of p53 and represents a potential target for radio sensitization of tumor cells. In this study, we analyzed the impact of hnRNPK on the DNA damage response after gamma irradiation. By yH2AX foci analysis, we found that hnRNPK knockdown increases DNA damage levels in irradiated cells. Tumor cells bearing a p53 mutation showed increased damage levels and delayed repair. Knockdown of hnRNPK applied simultaneously with irradiation reduced colony-forming ability and survival of tumor cells. Taken together, our data shows that hnRNPK is a relevant modifier of DNA damage repair and tumor cell survival. We therefore recommend further studies to evaluate the potential of hnRNPK as a drug target for improvement of radiotherapy success.

Due to the demographic change, medicine faces a growing demand for tissue engineering solutions and implants. Often, satisfying tissue regeneration is difficult to achieve especially when co-morbidities hamper the healing process. As a novel strategy, we propose the incorporation of zinc oxide nanoparticles (ZnO NPs) into biomaterials to improve tissue regeneration. Due to their wide range of biocompatibility and their antibacterial properties, ZnO NPs are already discussed for different medical applications. As there are versatile possibilities of modifying their form, size, and function, they are becoming increasingly attractive for tissue engineering. In our study, in addition to antibacterial effects of ZnO NPs, we show for the first time that ZnO NPs can foster the metabolic activity of fibroblasts as well as endothelial cells, both cell types being crucial for successful implant integration. With the gelatin sponge method performed on the chicken embryo’s chorioallantoic membrane (CAM), we furthermore confirmed the high biocompatibility of ZnO NPs. In summary, we found ZnO NPs to have very favorable properties for the modification of biomaterials. Here, incorporation of ZnO NPs could help to guide the tissue reaction and promote complication-free healing.

The best studied costimulatory pathway is represented by the CD28/CTLA4-B7 system (2, 3) where B7-1 (CD80) and B7-2 (CD86) bind either to an activating (CD28) or to an inhibitory (CTLA4) receptor on T cells (4). A variety of studies demonstrate the significance of CD28 costimulation in the clonal expansion of naïve T cells, the regulation of long term survival of T cells, induction of cell cycle progression, and cytokine secretion (5). The elucidation of the CD28-mediated functions has facilitated the development of therapeutic strategies, particularly vaccination studies, that target this pathway. Most efforts to induce antigen-specific immunity by application of cancer vaccines have focused on the generation of tumor-reactive CD8 ϩ cytotoxic T cells (CTLs) (6). Whereas B7-negative tumor cells most often fail to induce an efficient immune response, B7-1 and/or B7-2 expression on tumor cells augments the anti-tumor response by inducing T cell proliferation, cytokine secretion, and an enhanced generation of tumor-specific CD8 ϩ CTLs (7-10). B7-transduced renal cell carcinoma cells are an example of the strategy to stimulate tumor-infiltrating T cells to promote an efficient antitumor response (11,12).The efficacy of an antitumor reaction is usually monitored by in vitro determination of the number of antigen-specific T cells that secrete cytokines in response to antigen challenge. The monitoring can be achieved by enzyme-linked immunospot techniques (13,14), flow cytometry (15), multiplex-based immunoassays (16) or . Such assays, however, do not provide any information regarding the various signaling and metabolic pathways activated upon CD28 costimulation of the T cell. Particularly only limited information on the genetic expression programs linked to CD28 costimulation exists (18 -20), whereas to our best knowledge no study to date has assessed the protein expression patterns of both prestimulated and naïve T cells upon CD28 costimulation. To address the complexity of the T cell response directed against a defined, B7-transduced tumor cell vaccine, here we applied proteome-based technologies and identified a variety of differentially expressed proteins in T cells following B7-1 and