TY - THES ID - 138245523 TI - Validation of Multiple Iteractive Labelling by Antibody Neodeposition (MILAN) for Characterization of Immunological Components in Alveolar Soft-part Sarcoma AU - Vermeulen, Eline AU - Thomas More Kempen. More Life Sciences & Chemistry PY - 2019 PB - Geel : Thomas More Kempen DB - UniCat UR - https://www.unicat.be/uniCat?func=search&query=sysid:138245523 AB - Soft tissue sarcoma (STS) is a group of rare and heterogeneous malignancies that can occur in various types of connective tissue (Vodanovich & Choong, 2018). It accounts for about 70 clinically relevant subtypes of STS based on their morphology and tissue origin (Tuna, Ju, Amos, & Mills, 2012). A proper diagnosis and treatment of patients with STS is difficult due to the rarity and heterogeneity of this disease (Schöffski, Cornillie, Wozniak, Li, & Hompes, 2014). Complete resection is the main therapeutic option for operable STS and can be supported by adjuvant therapy such as chemotherapy and radiotherapy. However, many patients develop resistance to chemotherapy, which means there is no effective treatment for inoperable STSs. Many studies are in progress for developing new drugs based on the understanding of subtype-specific cancer biology. One of the rarest subtypes of STS is alveolar soft-part sarcoma (ASPS). It harbours specific chromosome translocations which directly/indirectly lead to tumorigenesis (Folpe & Deyrup, 2006). The better understanding of disease biology can foster the development of therapeutic agents and leads to better disease management for inoperable STSs. ASPS might be the most sensitive subtype of STS for immunotherapy (Groisberg, et al., 2017). The human immune system can be restored to eliminate tumour cells by using immune checkpoint inhibitors to block immune proteins (Lewin, et al., 2018). This has prompted us to characterize immunological components to have a better understanding of immunologic components in ASPS. In the Laboratory of Experimental Oncology (LEO), tissue microarrays (TMAs) have been constructed to study STS. TMA, which assembles small representative tissue samples from different patients in a single tissue block, allows to perform simultaneous staining for markers of relevance (Singh & Sau, 2010). Ultimately microarrays help to have a better understanding of the biology of ASPS. In this project, TMA is used for further experiments with multiple iteractive labelling by antibody neodeposition (MILAN). MILAN is a novel multiplex immunofluorescence assay which is currently established and validated. Multiplexing means in situ labelling for multiple immunostainings in the same tissue section or even the same cell of interest (Cattoretti, Bosisio, Marcelis, & Bolognesi, 2018). This technique is based on applying the combination of primary and fluorophore conjugated secondary antibodies. And it is used to determine the status, localization and interaction of multiple immunological components and to amplify the use of precious material. Results from the single-colour immunofluorescence staining proved the applicability of the antibodies and the optimization of the MILAN protocol. Microscopic evaluation was used to determine the expression of targeted molecules by the presence and localisation of a fluorescent signal. The experiment for dual-colour immunofluorescence staining proved that there is no interaction between primary antibodies and non-specific staining when using antibody mixture. The results of the multiple rounds of immunofluorescence staining proved that the stripped slides can be applied with other antibodies and that the cycling is applicable for multiple cycles. The MILAN protocol and the MILAN setting were validated and established. After the validation of MILAN, it was applied on TMAs for characterization of immunological components in ASPS. ER -