Project Summary The identity and spatial context of biomolecules (e.g., protein and RNA) in cancer cells are crucial components of their pathology. Therefore, a scalable and multiplexed imaging platform that can simultaneously map the protein and RNA landscapes tissue would be a vital tool towards deeply profiling and mapping cancer cell types in their spatial context. Extensive efforts have been made toward this end to reveal unprecedented details at both cellular level and molecular level. However, these methods generally lack high multiplexity or suffer low sensitivity as the target abundance decreases. In addition, highly multiplexed methods for co-imaging of protein and RNA at the whole tissue level still lag behind. We here aim to address these limitations by developing a versatile imaging platform for highly multiplexed, rapid, scalable proteomic and transcriptomic mapping of cell line and mammalian tissue samples with high-plex signal amplification. We propose to (Aim 1) develop a simple, highly controllable polymerase mediated iterative in situ DNA extension (ISE) and concatenation. We will demonstrate the application of ISE on the imaging of protein and RNA targets in both tissue and cell sample with high signal-to-noise and signal specificity. We will also (Aim 2) validate the scalability of ISE imaging in cell and tissue samples. We will optimize the technology to achieve spatial mapping of 50 to 100-plex protein and RNA targets in mammalian tissue samples. Finally, we will (Aim 3) validate ISE imaging in both FFPE and thick tissue, both normal and cancerous sample types. We will co-detect both RNA and protein tumor biomarkers in clinical FFPE samples and finally, we will integrate the ISE imaging methods with existing tissue clearing methods (iDISCO, CLARITY, etc.) to enable high-throughput and highly multiplexed tissue imaging from cellular level to molecular level for both normal and cancerous brain tissue. We will establish a platform for 50-plex imaging in hundreds micrometer to millimeter thick mammalian tissue specimens to unveil unprecedented detail in the tissue. The proposed work will deliver a comprehensive imaging toolset including a low-cost, simple design of orthogonal DNA probes for multiplexing imaging on protein and RNA molecules, and a scalable signal amplification method for multiplexed fluorescence imaging in different types of tissues. We envision that our technologies will be widely accessible and seamlessly incorporated into the pipelines, workflows, and coordinate frameworks of the mission for clinical researchers, pathologists, as well as the wider bio-imaging community to facilitate cancer research and clinical practice.