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The immune status of tumor‐infiltrating lymphocytes (TILs) is essential for the effectiveness of cancer immunotherapies. However, due to the diversity of immune status in TILs, cellular heterogeneity, and the applicability to the clinic, it is still lacking effective strategies to meet clinical needs. We developed a novel immuno‐recognition‐induced method based on rolling circle amplification (RCA), namely immunoRCA, to in situ visualize the immune status of TILs in actual clinical samples. This developed immunoRCA method, in which, feature mRNAs were used as the biomarkers for the immune status of TILs, has a low fluorescence background, high sensitivity, and specificity. The immunoRCA was able to efficiently evaluate the immune status of CD8+ T cells regulated by activating or inhibiting factors, track the T cell type and immune status during in vitro expansion, and in situ visualize the number, location, and immune status of TILs in clinical specimens. [ABSTRACT FROM AUTHOR]

N6‐methyladenosine (m6A) modification—the most prevalent mammalian RNA internal modification—plays key regulatory roles in mRNA metabolism. Current approaches for m6A modified RNA analysis limit at bulk‐population level, resulting in a loss of spatiotemporal and cell‐to‐cell variability information. Here we proposed a m6A‐specific in situ hybridization mediated proximity ligation assay (m6AISH‐PLA) for cellular imaging of m6A RNA, allowing to identify m6A modification at specific location in RNAs and image m6A RNA with single‐cell and single‐molecule resolution. Using m6AISH‐PLA, we investigated the m6A level and subcellular location of HSP70 RNA103‐m6A in response to heat shock stress, and found an increased m6A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6AISH‐PLA can serve in the study of m6A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis. [ABSTRACT FROM AUTHOR]

Titelbild: Single-Cell Imaging of m6A Modified RNA Using m6A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6AISH-PLA) (Angew. In ihrer Zuschrift auf S. 22828 postulieren Jinghong Li et al. eine leistungsstarke In-situ-Imaging-Methode, die als "m SP 6 sp A-specific in situ hybridization mediated proximity ligation assay" (m SP 6 sp AISH-PLA) bezeichnet wird und die Identifizierung von m SP 6 sp A-Modifikationen an spezifischen Stellen in RNA und die Bildgebung von m SP 6 sp A-RNA mit Einzelzell- und Einzelmolekül-Auflösung ermöglicht. In ihrer Zuschrift auf S. 22828 postulieren Jinghong Li et al. eine leistungsstarke In-situ-Imaging-Methode, die als "m SP 6 sp A-specific in situ hybridization mediated proximity ligation assay" (m SP 6 sp AISH-PLA) bezeichnet wird und die Identifizierung von m SP 6 sp A-Modifikationen an spezifischen Stellen in RNA und die Bildgebung von m SP 6 sp A-RNA mit Einzelzell- und Einzelmolekül-Auflösung ermöglicht. [Extracted from the article]

In their Communication on page 22646, Jinghong Li et al. propose a powerful in situ imaging method, termed m SP 6 sp A-specific in situ hybridization mediated proximity ligation assay (m SP 6 sp AISH-PLA), that enables the identification of m SP 6 sp A modifications at a specific location in RNAs and the imaging of m SP 6 sp A RNA with single-cell and single-molecule resolution. Keywords: in situ imaging; m6A; proximity ligation; single-molecule; spatiotemporal information EN in situ imaging m6A proximity ligation single-molecule spatiotemporal information 22589 22589 1 10/06/21 20211011 NES 211011 B N b SP B 6 b sp B -methyladenosine b (m SP 6 sp A)-modified RNA analysis is limited at the bulk-population level, thus not giving access to spatiotemporal and cell-to-cell variability information. Cover Picture: Single-Cell Imaging of m6A Modified RNA Using m6A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6AISH-PLA) (Angew. [Extracted from the article]