Overall: PROJECT SUMMARY Cancer stem cells (CSCs) play a critical role in fostering tumor resistance to therapies and relapse after treatment. This presents a crucial barrier to the development of successful anti-cancer therapeutics. Transcriptional reprogramming and plasticity play a critical role in and out of the CSC state, which in turn are interdependent on the regulatory function of the three-dimensional (3D) structure of chromatin, epigenetic states, and other molecular events. Our understanding of fundamental CSC biology has been hampered by the need for cellular nanoscale imaging technologies that provide both highly detailed structural information regarding 3D chromatin organization and highly multiplexed molecular imaging of the many molecular regulators and events involved in CSC processes. We propose to establish the Northwestern University Center for Chromatin Nanoimaging in Cancer (NU-CCNIC) to address this fundamental technology gap in cellular nanoscale imaging and deploy the new technologies to address the fundamental knowledge gap in CSC biology. The Center converges experts in cellular nanoscale imaging, computational imaging, molecular modeling, computational genomics, CSC biology, and oncology. The Center will develop, test, validate, iterate, and deploy an integrated and co-registered Multi-scale Chromatin Nanoimaging Platform that will comprise three “nested-doll” imaging techniques: chromatin scanning transmission electron microscopy, optical spectroscopic super-resolution nanoscopy, and optical spectroscopic nanosensing. The Nanoimaging Platform will enable quantitative imaging of chromatin structure and highly multiplexed molecular and gene-specific localization, at the most fundamental length-scale approaching 1 nm resolution, including the imaging of statistically significant cell populations and live cells with high temporal resolution over prolonged temporal follow-up times. The Nanoimaging Platform will be bridged to computational genomics, epigenomics, genome mapping, and predictive transcriptional modeling datasets. These technologies will be deployed to answer several long-standing open questions in CSC biology. We will elucidate whether CSCs can originate from non-CSCs via transcriptional reprogramming, test the role of chromatin structure in fostering transcriptional plasticity in CSC processes, and explore the possibility of transcriptionally reprogramming CSCs to exit the stem-state as a new therapeutic strategy. All aspects of the technology development will be guided by the needs of the CSC biology testbed through a series of research feedback loops. In the long term, such single-cell nanoimaging technologies will help comprehensive understanding of the complex interplay between structural, physico-chemical, and molecular genomic events. We anticipate that these convergence studies will provide new insights into CSC biology, which are impossible to reveal with the use of any single method, and open new opportunities for identifying therapeutic strategies.

U54 CENTER NARRATIVE Despite rapid advances in new anti-cancer therapeutics, for many solid cancers complete remission is still rare. In many cases, tumors eventually develop resistance to treatments. One of the major barriers to effective therapeutics is likely to be potentiated by highly transcriptionally plastic cancer stem cells. In this U54 Northwestern University Center for Chromatin Nanoimaging in Cancer, we will develop, optimize, test, and deploy new nanoimaging technologies, integrated with state-of-the-art molecular and computational methods, to address some of the long-standing questions related to the origin of cancer stem cells and their ability to adapt to chemotherapies. Addressing these fundamental knowledge and technology gaps may help identify new therapeutic strategies that prevent tumor resistance to therapeutics and relapse after treatment.