Genomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the in-situ higher-order folding of chromatin structure during malignant transformation remains largely unknown. Here, using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncover a gradual decompaction and fragmentation of higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, and prior to tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal functional consequences in enhanced transcription activities and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin disruption to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention.

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Multiple myeloma (MM) is characterized by uncontrolled proliferation of malignant plasma cells in the bone marrow and peripheral blood. Here, we found that CD40 and CD40L co-expressed on XG1 MM cells and the coordinated expression of CD40-CD40L was critical for production and autocrine IL-6 in XG1 cells. Furthermore, TNF-α enhanced the expression of both CD40 and CD40L expression on XG1 cells. We also found that persistent CD40L/CD40 signaling was required for the constitutive activation of NF-κB in the cells.

Use of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology to detect pancreatic cancer is limited, with a high false negative rate mainly due to the relatively fewer number of completely cancerous cells. To improve the accuracy of EUS-FNA cytological diagnosis, we evaluated a novel optical system—spatial-domain low-coherence quantitative phase microscopy (SL-QPM)—to analyze nanoscale nuclear architecture on original cytology samples, especially those diagnosed as indeterminate for malignancy, with the goal of maintaining high specificity and reducing false positive rate. We performed SL-QPM on original cytology samples obtained by EUS-FNA from 40 patients with suspicious pancreatic solid lesions (27 adenocarcinomas, 5 neuroendocrine tumor, 8 chronic pancreatitis), including 13 cases that were cytologically indeterminate. Each diagnosis had been confirmed by follow-up surgical pathology. The SL-QPM-derived nanoscale nuclear architectural parameters distinguished pancreatic cancer from cytologically indeterminate cells. A logistic regression model using nuclear entropy and SD increased the sensitivity of cytology in identifying pancreatic cancer from 72% to 94% while maintaining 100% specificity. The SL-QPM-derived nanoscale nuclear architecture properties show great promise in improving the cytological diagnosis of EUS-FNA for pancreatic cancer and could be used when traditional cytopathology does not get an accurate diagnosis, and can be easily translated into a traditional clinical device.

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SUMMARYAberrant chromatin structure is a hallmark in cancer cells and has long been used for clinical diagnosis of cancer. However, underlying higher-order chromatin folding during malignant transformation remains elusive, due to the lack of molecular scale resolution. Using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncovered a gradual decompaction and fragmented higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, even prior to the tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal the functional consequences in enhanced formation of transcription factories, spatial juxtaposition with relaxed nanosized chromatin domains and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin decompaction to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal the gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention.SIGNIFICANCEGenomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although much progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the higher-order folding of chromatin structure remains largely unknown. Using optimized super-resolution microscopy, we uncover de-compacted and fragmented chromatin folding in tumor initiation and stepwise progression in multiple tumor types, even prior to the presence of tumor cells. This study underlines the significance of unfolding higher-order chromatin structure as an enabling characteristic to promote tumorigenesis, which may facilitate the development and evaluation of new preventive strategies. The potential of imaging higher-order chromatin folding to improve cancer detection and risk stratification is demonstrated by detecting high-risk precursors that cannot be distinguished by conventional pathology.