We describe a novel imaging system for fully automated cyclic immunofluorescence analysis including a mechanism for most gentle erasure of signal with the potential to apply hundreds of binders to a single specimen. A number of cell surface markers are currently being investigated for CAR T cell therapy in solid tumors 11 and strategies have been outlined to combine different markers to circumvent tumor escape mechanisms (“OR” gated combinatorial CARs) or to reduce off-tumor toxicity (“AND” gated CARs) 12. high coverage of tumor cells and low on-target/off-tumor toxicity. Besides inherent obstacles presented by the tissue microenvironment that can hamper T cell infiltration, there is also a lack of cell surface target molecules that are suitable for CAR T cells, i.e. Antibody-oligonucleotide conjugates used for cyclic immunofluorescence analysis need to be carefully selected, limiting these technologies to roughly 60 analytes 9.ĬAR T cell–based therapies have resulted in a remarkable success in the treatment of hematopoietic malignancies 10, but have not yet led to a breakthrough in solid tumors. Transition element isotopes chelated to antibodies as used in mass spectrometric readouts are confined to about 40 unique labels 3. However, there are still limitations in spatial resolution, degree of binder-based multiplexing, and tissue integrity. The analysis can be improved by new multiplexing technologies using either optical or mass spectrometric readouts such as multi-epitope-ligand cartography (MELC) 1, ChipCytometry 2, mass cytometry 3, multiplexed ion beam imaging (MIBI) 4, cyclic immunofluorescence (CycIF) 5, multiplex immunohistochemistry 6, co-detection by indexing (CODEX) 7, or InSituPlex 8. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.Īnalysis of cancer cell diversity and immune contexture is of high relevance for tumor subclassification and the development of novel targeted immunotherapies. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Multimarker analysis can identify potential targets for immune therapy against solid tumors. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level.
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