Translational 3D spatiomolecular imaging in health and disease

<p dir="ltr">Volumetric microscopic imaging, also known as three-dimensional (3D) imaging, is an advanced approach that enables spatial visualization of molecular components such as proteins and RNAs within intact tissues. It provides an unbiased, panoramic view for investigating spatial heterogeneity and molecular organization in complex biological systems. To achieve deep imaging of large samples, tissue-clearing methods have been established to make biological specimens optically transparent, thereby greatly advancing the capability of 3D imaging. However, current methods remain limited in their ability to visualize RNA and to support multimodal molecular imaging. To date, most published 3D imaging studies have focused primarily on proteins-either through endogenous fluorescent reporters (e.g., CUBIC) or antibody-based labeling for spatial profiling of target proteins (e.g., iDISCO+) in whole tissues. This thesis aims to further develop and apply 3D imaging methods to translational research in health and disease.</p><p dir="ltr">In Papers I and II, we developed and presented a whole-organ RNA detection method termed TRISCO (Tris buffer-mediated retention of in situ hybridization chain reaction signal in cleared organs). This method effectively preserves RNA integrity, enables uniform staining, and addresses transparency challenges associated with combining in situ hybridization with tissue clearing. Using TRISCO, we monitored the expression of immediate early genes in mouse brains following administration of the anti-obesity drug semaglutide. This application illustrates the potential of TRISCO for studying drug-induced gene expression changes at the spatial transcriptional level.</p><p dir="ltr">In Paper III, the iDISCO+ method was applied to investigate microglia-myelin interactions in the mouse spinal cords. We found that myelin degeneration was particularly prominent in the dorsal column (DC), accompanied by molecular and functional alterations in DC microglia and an upregulation of TGF-ß signaling. Disruption of TGF-B signaling in microglia led to excessive microglial activation, loss of MBP+ myelin, and aggravated neurological deficits.</p><p dir="ltr">In Paper IV, we applied 3D imaging to clinical upper tract urothelial carcinoma (UTUC) biopsies, enabling histopathological assessment without sectioning. We found that phosphorylated ribosomal protein S6 (pS6) served as a spatial biomarker for improving diagnosis and prognosis.</p><p dir="ltr">In Paper V, we integrated and optimized the co-detection of RNA and protein method and named as ARO-DIIFCO (accelerated robust and operator-friendly technique for simultaneous 3D mRNA and protein detection). This method enables comprehensive dual-molecular mapping-of both mRNA and protein-at single-cell resolution, while reducing processing time by one-third compared to the previous protocol. Applying ARO-DIIFCO to a cohort of breast cancer patients, we demonstrate its potential for identifying personalized risk-associated features linked to disease progression.</p><p dir="ltr">In summary, the 3D imaging strategies developed and applied in this thesis provide a translational framework for spatially resolving molecular landscapes, bridging fundamental biology with clinical applications.</p><h3>List of scientific papers</h3><p dir="ltr">I. Whole-Brain Spatial Transcriptional Analysis at Cellular Resolution. Shigeaki Kanatani, Judith C. Kreutzmann*, <b>Yue Li</b>*, Zoe West*, Lea Lydolph Larsen, Danai Vougesi Nikou, Ilse Eidhof, Abigail Walton, Songbai Zhang, Leslie Rubio Rodríguez-Kirby, Jacob Lercke Skytte, Casper Gravesen Salinas, Kimiharu Takamatsu, Xiaofei Li, Daisuke H. Tanaka, Dagmara Kaczynska, Keishiro Fukumoto, Razieh Karamzadeh, Yujiao Xiang, Naofumi Uesaka, Tsutomu Tanabe, Mikael Adner, Johan Hartman, Ayako Miyakawa, Erik Sundström, Gonçalo Castelo-Branco, Urmas Roostalu, Jacob Hecksher- Sørensen, and Per Uhlén. Science 2024. <a href="https://doi.org/10.1126/science.adn9947" rel="noreferrer" target="_blank">doi.org/10.1126/science.adn9947</a></p><p dir="ltr">II. Whole-organ spatial transcriptional analysis at cellular resolution using TRISCO.<b> </b><b>Yue Li</b>*, Abigail Walton*, Judith C. Kreutzmann, Lea Lydolph Larsen, Urmas Roostalu, Jacob Hecksher-Sørensen, Shigeaki Kanatani, Per Uhlén. Nature Protocols under revision 2025 [Accepted]</p><p dir="ltr">III. TGF-B signaling mediates microglial resilience to spatiotemporally 1 restricted myelin degeneration. Keying Zhu, Yun Liu, Jin-Hong Min, Vijay Joshua, Jianing Lin, <b>Yue Li</b>, Judith C. Kreutzmann, Yuxi Guo, Wenlong Xia, Elyas Mohammadi, Melanie Pieber, Valerie Suerth, Yiming Xia, Zaneta Andrusivova, Jean-Philippe Hugnot, Shigeaki Kanatani, Per Uhlén, Joakim Lundeberg, Xiaofei Li, Stephen P.J. Fancy, Heela Sarlus, Robert A. Harris, Harald Lund. [Manuscript]</p><p dir="ltr">IV. Three-Dimensional Imaging of Upper Tract Urothelial Carcinoma Improves Diagnostic Yield and Accuracy. Keishiro Fukumoto*, Shigeaki Kanatani*, Georg Jaremko, Zoe West, <b>Yue Li</b>, Kimiharu Takamatsu, Ibrahim Al Rayyes, Shuji Mikami, Naoya Niwa, Tomas Andri Axelsson, Nobuyuki Tanaka, Mototsugu Oya, Ayako Miyakawa, Marianne Brehmer, Per Uhlen. JCI Insight. 2024. <a href="https://doi.org/10.1172/jci.insight.175751" rel="noreferrer" target="_blank">//doi.org/10.1172/jci.insight.175751</a></p><p dir="ltr">V. ARO-DIIFCO: Simultaneous 3D Mapping of mRNA and Protein to Aid Personalized Medicine in Breast Cancer.<b> </b><b>Yue Li</b>, Shigeaki Kanatani, Ceren Boyaci, Stephanie Robertson, Wenchao Shao, Zoe West, Judith C. Kreutzmann, Songbai Zhang, Johan Hartman, Per Uhlén [Manuscript]</p><p dir="ltr">*: These authors contributed equally to this study.</p>