Electron Microscopy of the Amphibian Model Systems Xenopus laevis and Ambystoma mexicanum

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy

Differential localization of PD-L1 and Akt-1 involvement in radioresistant and radiosensitive cell lines of head and neck squamous cell carcinoma

Immunotherapy by blockade of the PD-1/PD-L1 checkpoint demonstrated amazing tumor response in advanced cancer patients including head and neck squamous cell carcinoma (HNSCC). However, the majority of HNSCC patients still show little improvement or even hyperprogression. Irradiation is currently investigated as synergistic treatment modality to immunotherapy as it increases the number of T-cells thereby enhancing efficacy of immunotherapy. Apart from this immunogenic context a growing amount of data indicates that PD-L1 also plays an intrinsic role in cancer cells by regulating different cellular functions like cell proliferation or migration. Here, we demonstrate opposing membrane localization of PD-L1 in vital and apoptotic cell populations of radioresistant (RR) and radiosensitive (RS) HNSCC cell lines up to 72 h after irradiation using flow cytometry. Moreover, strong PD-L1 expression was found in nuclear and cytoplasmic cell fractions of RR. After irradiation PD-L1 decreased in nuclear fractions and increased in cytoplasmic fractions of RR cells. In contrast, RS cell lines did not express PD-L1, neither in the nucleus nor in cytoplasmic fractions. Additionally, overexpression of PD-L1 in RS cells led to a proportional increase of vital PD-L1 positive cells after irradiation. Moreover, co-immunoprecipitation experiments revealed an interaction between Akt-1 and PD-L1, mostly in irradiated RR cells compared to RS cells suggesting a differential influence of PD-L1 on cell signaling. In summary, our data imply the need for different therapeutic strategies dependent on the molecular context in which PD-L1 is embedded