Activity-Based NIR Bioluminescence Probe Enables Discovery of Diet-Induced Modulation of the Tumor Microenvironment via Nitric Oxide.

Nitric oxide (NO) plays a critical role in acute and chronic inflammation. NO's contributions to cancer are of particular interest due to its context-dependent bioactivities. For example, immune cells initially produce cytotoxic quantities of NO in response to the nascent tumor. However, it is believed that this fades over time and reaches a concentration that supports the tumor microenvironment (TME). These complex dynamics are further complicated by other factors, such as diet and oxygenation, making it challenging to establish a complete picture of NO's impact on tumor progression. Although many activity-based sensing (ABS) probes for NO have been developed, only a small fraction have been employed in vivo, and fewer yet are practical in cancer models where the NO concentration is <200 nM. To overcome this outstanding challenge, we have developed BL660-NO, the first ABS probe for NIR bioluminescence imaging of NO in cancer. Owing to the low intrinsic background, high sensitivity, and deep tissue imaging capabilities of our design, BL660-NO was successfully employed to visualize endogenous NO in cellular systems, a human liver metastasis model, and a murine breast cancer model. Importantly, its exceptional performance facilitated two dietary studies which examine the impact of fat intake on NO and the TME. BL660-NO provides the first direct molecular evidence that intratumoral NO becomes elevated in mice fed a high-fat diet, which became obese with larger tumors, compared to control animals on a low-fat diet. These results indicate that an inflammatory diet can increase NO production via recruitment of macrophages and overexpression of inducible nitric oxide synthase which in turn can drive tumor progression.

Activity-based photoacoustic probe for biopsy-free assessment of copper in murine models of Wilson's disease and liver metastasis.

The development of high-performance photoacoustic (PA) probes that can monitor disease biomarkers in deep tissue has the potential to replace invasive medical procedures such as a biopsy. However, such probes must be optimized for in vivo performance and exhibit an exceptional safety profile. In this study, we have developed PACu-1, a PA probe designed for biopsy-free assessment (BFA) of hepatic Cu via photoacoustic imaging. PACu-1 features a Cu(I)-responsive trigger appended to an aza-BODIPY dye platform that has been optimized for ratiometric sensing. Owing to its excellent performance, we were able to detect basal levels of Cu in healthy wild-type mice as well as elevated Cu in a Wilson's disease model and in a liver metastasis model. To showcase the potential impact of PACu-1 for BFA, we conducted two blind studies in which we were able to successfully identify Wilson's disease animals from healthy control mice in each instance.

A General Approach to Convert Hemicyanine Dyes into Highly Optimized Photoacoustic Scaffolds for Analyte Sensing*.

Most photoacoustic (PA) imaging agents are based on the repurposing of existing fluorescent dye platforms that exhibit non-optimal properties for PA applications. Herein, we introduce PA-HD, a new dye scaffold optimized for PA probe development that features a 4.8-fold increase in sensitivity and a red-shift of the λabs from 690 nm to 745 nm to enable ratiometric imaging. Computational modeling was used to elucidate the origin of these enhanced properties. To demonstrate the generalizability of our remodeling efforts, we developed three probes for ß-galactosidase activity (PA-HD-Gal), nitroreductase activity (PA-HD-NTR), and H2 O2 (PA-HD-H2 O2 ). We generated two cancer models to evaluate PA-HD-Gal and PA-HD-NTR. We employed a murine model of Alzheimer's disease to test PA-HD-H2 O2 . There, we observed a PA signal increase at 735 nm of 1.79±0.20-fold relative to background, indicating the presence of oxidative stress. These results were confirmed via ratiometric calibration, which was not possible using the parent HD platform.

Development of NIR-II Photoacoustic Probes Tailored for Deep-Tissue Sensing of Nitric Oxide.

Photoacoustic (PA) imaging has emerged as a reliable in vivo technique for diverse biomedical applications ranging from disease screening to analyte sensing. Most contemporary PA imaging agents employ NIR-I light (650-900 nm) to generate an ultrasound signal; however, there is significant interference from endogenous biomolecules such as hemoglobin that are PA active in this window. Transitioning to longer excitation wavelengths (i.e., NIR-II) reduces the background and facilitates the detection of low abundance targets (e.g., nitric oxide, NO). In this study, we employed a two-phase tuning approach to develop APNO-1080, a NIR-II NO-responsive probe for deep-tissue PA imaging. First, we performed Hammett and Brønsted analyses to identify a highly reactive and selective aniline-based trigger that reacts with NO via N-nitrosation chemistry. Next, we screened a panel of NIR-II platforms to identify chemical structures that have a low propensity to aggregate since this can diminish the PA signal. In a head-to-head comparison with a NIR-I analogue, APNO-1080 was 17.7-fold more sensitive in an in vitro tissue phantom assay. To evaluate the deep-tissue imaging capabilities of APNO-1080 in vivo, we performed PA imaging in an orthotopic breast cancer model and a heterotopic lung cancer model. Relative to control mice not bearing tumors, the normalized turn-on response was 1.3 ± 0.12 and 1.65 ± 0.07, respectively.

Acoustic-based chemical tools for profiling the tumor microenvironment.

Acoustic-based imaging modalities (e.g. ultrasonography and photoacoustic imaging) have emerged as powerful approaches to noninvasively visualize the interior of the body due to their biocompatibility and the ease of sound transmission in tissue. These technologies have recently been augmented with an array of chemical tools that enable the study and modulation of the tumor microenvironment at the molecular level. In addition, the application of ultrasound and ultrasound-responsive materials has been used for drug delivery with high spatiotemporal control. In this review, we highlight recent advances (in the last 2-3 years) in acoustic-based chemical tools and technologies suitable for furthering our understanding of molecular events in complex tumor microenvironments.