Abstract
Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. 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. 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). Multimarker analysis can identify potential targets for immune therapy against solid tumors. 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. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.
Highlights
Analysis of cancer cell diversity and immune contexture is of high relevance for tumor subclassification and the development of novel targeted immunotherapies
Besides inherent obstacles presented by the tissue microenvironment that can hamper T cell infiltration, there is a lack of cell surface target molecules that are suitable for CAR T cells, i.e. high coverage of tumor cells and low on-target/off-tumor toxicity
Applied to the screening of glioblastoma multiforme (GBM), high-grade serous ovarian carcinoma (HGSOC), and pancreatic ductal adenocarcinoma (PDAC), MICS-based screening reveals marker combinations with a preferable on-target/on-tumor vs. on-target/off-tumor profile suitable for CAR T cell development
Summary
Analysis of cancer cell diversity and immune contexture is of high relevance for tumor subclassification and the development of novel targeted immunotherapies. 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. Applied to the screening of glioblastoma multiforme (GBM), high-grade serous ovarian carcinoma (HGSOC), and pancreatic ductal adenocarcinoma (PDAC), MICS-based screening reveals marker combinations with a preferable on-target/on-tumor vs on-target/off-tumor profile suitable for CAR T cell development. We validate this screening approach using Adapter CAR T cells for an AND gated combinatorial targeting of cancer cells co-expressing EPCAM and THY1
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