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

Abstract

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.

Figure 1

(a) Schematic representation of the BL₆₆₀-NO reaction with NO and wild-type luciferase to produce an NIR bioluminescent signal. (b) Synthesis of BL₆₆₀-NO.

Figure 2

(a) Spectra of luciferin and BL₆₆₀ in the presence of recombinant luciferase. (b) In vitro assay demonstrating that the probe, NO, and luciferase must all be present to generate a signal: BL₆₆₀-NO (5 μM), DEA NONOate (250 μM), and luciferase (0.05 mg/mL). (c) Bioluminescent signal as a function of BL₆₆₀-NO concentration (0, 1.25, 2.5, 5 μM). (d) Bioluminescent signal as a function of DEA NONOate concentration (0, 62.5, 125, 250, 500 μM). (e) Selectivity assay against a panel of biologically relevant analytes. All analytes were present in 75-fold excess relative to BL₆₆₀-NO (5 μM). All data is reported as the mean ± standard deviation (n = 3).

Figure 3

(a) Representative images of BL signals from A549-Luc2 cells upon treatment with vehicle (DMSO), BL₆₆₀-NO (10 μM), or pretreatment with inhibitor L-NMMA (1 mM) for 30 min followed by BL₆₆₀-NO (10 μM). (b) Quantified data from panel a. (c) Representative images of BL signals from 4T1-Luc upon treatment with vehicle (DMSO), BL₆₆₀-NO (10 μM), or pretreatment with inhibitor L-NMMA (1 mM) for 30 min followed by BL₆₆₀-NO (10 μM). (d) Quantified data from panel c. All data is reported as the mean ± standard deviation (n = 3). Color scales represent luminescence counts. The exposure time, 60 s; emission, open; binning factor, 8; and f number, 1. Dotted white circles represent ROIs used for quantification. A statistical analysis was performed using Student’s t-test (α = 0.05), **: p < 0.01.

Figure 4

(a) Representative BL images of mice pretreated with a vehicle control (saline) or L-NMMA (35 mM, 50 μL). (b) Normalized data from panel a. Vehicle treatment data is reported as the mean ± standard deviation (n = 3), and L-NMMA treatment data is reported as the mean ± standard deviation (n = 5). Exposure time, 25 s; emission, 660 nm; binning factor, 8; and f number, 1. Dotted white ovals represent ROIs used for quantification. A statistical analysis was performed using Student’s t-test (α = 0.05). No statistical significance was observed.

Figure 5

(a) Representative BL images of mice pretreated with a vehicle control (saline) or L-NMMA (35 mM, 50 μL). (b) Normalized data from panel a. All data is reported as the mean ± standard deviation (n = 3). Exposure time, 60 s; emission, open; binning factor, 8; and f number, 1. Dotted white ovals represent ROIs used for quantification. A statistical analysis was performed using Student’s t-test (α = 0.05), *: p < 0.05.

Figure 6

(a) Schematic representing the workflow for the generation of mouse models to study the effect of diet on tumorigenesis and NO production by BL imaging. (b) Representative BL images of mice on low-fat and high-fat diets for 24 weeks, respectively, upon treatment with BL₆₆₀-NO. (c) Quantified data from panel b. All data is reported as the mean ± standard deviation (n = 4). Exposure time, 60 s; emission, open; binning factor, 8; and f number, 1. Dotted white ovals represent ROIs used for quantification. A statistical analysis was performed using Student’s t-test (α = 0.05), *: p < 0.05. Representative images of tumors excised from mice fed a (d) low-fat diet and (e) high-fat diet for 24 weeks with CD68 staining. Scale bar = 25 μm. Representative images of tumors excised from mice fed a (f) low-fat diet and (g) high-fat diet for 24 weeks with iNOS staining. Scale bar = 25 μm.