Noninvasive Mapping of Extracellular Potassium in Breast Tumors via Multi-Wavelength Photoacoustic Imaging

Abstract

Elevated extracellular potassium (K⁺) in the tumor microenvironment (TME) of breast and other cancers is increasingly recognized as a critical factor influencing tumor progression and immune suppression. Current methods for noninvasive mapping of the potassium distribution in tumors are limited. Here, we employed photoacoustic chemical imaging (PACI) with a solvatochromic dye-based, potassium-sensitive nanoprobe (SDKNP) to quantitatively visualize extracellular potassium levels in an orthotopic metaplastic breast cancer mouse model, Ccn6-KO. Tumors of three distinct sizes (5 mm, 10 mm, and 20 mm) were imaged using multi-wavelength photoacoustic imaging at five laser wavelengths (560, 576, 584, 605, and 625 nm). Potassium concentration maps derived from spectral unmixing of the photoacoustic images at the five laser wavelengths revealed significantly increased potassium levels in larger tumors, confirmed independently by inductively coupled plasma mass spectrometry (ICP-MS). The PACI results matched ICP-MS measurements, validating PACI as a robust, noninvasive imaging modality for potassium mapping in tumors in vivo. This work establishes PACI as a promising tool for studying the chemical properties of the TME and provides a foundation for future studies evaluating the immunotherapy response through ionic biomarker imaging.

Keywords: CCN6; breast cancer; chemical imaging; metaplastic breast carcinoma; nanoparticle; photoacoustic; potassium.

Figure 1

Illustration of photoacoustic K⁺ imaging with solvatochromic dye-based K⁺-sensing nanoparticles (SDKNP). The SDKNP potassium probe has a characteristic absorption change depending on the K⁺ levels. The K⁺ image was obtained using a multi-wavelength ratiometric photoacoustic imaging method.

Figure 2

Optical properties of SDKNP. (A) Spectroscopic optical absorption of SDKNP at different K⁺ levels. (B) Absorption ratios between each wavelength relative to 560 nm, which is the isosbestic point of the absorption changes.

Figure 3

Calibration and characterization of SDKNP. (A) A ratiometric calibration of SDKNPs’ optical absorption changes against various ions. Ratios of the absorption peaks at 520 nm over 600 nm were plotted against ion concentrations. All measurements were taken at a constant ionic strength using LiCl to maintain constant tonicity. (B) Dynamic light scattering measurements of SDKNP size at both 0 and 100 mM K⁺, again using LiCl to maintain constant tonicity. (C) Measurement and distribution of zeta potential for the SDKNPs.

Figure 4

Results of photoacoustic K⁺ imaging with SDKNP. (A) Representative K⁺ maps acquired by the photoacoustic and ultrasound dual-modality imaging system. Color-coded K⁺ concentration maps were superimposed on grayscale ultrasound images for anatomical localization. (B) K⁺ levels in tumors of three different size groups (Group 1 [5 mm]: n = 4; Group 2 [10 mm]: n = 3; Group 3 [20 mm]: n = 4) Group-wise mean potassium levels ± standard deviations are shown. Statistical comparisons were performed using two-sample t-tests. * p < 0.01. Scale bar = 5 mm.

Figure 5

Quantitative analysis of Ccn6-KO tumor K⁺ levels using ICP-MS. K⁺ levels in tumors of three different size groups (Group 1 [5 mm]: n = 4; Group 2 [10 mm]: n = 3; Group 3 [20 mm]: n = 4) were quantified using inductively coupled plasma mass spectrometry (ICP-MS). Bar graphs show mean values ± standard deviations for potassium levels calculated by background-subtracted signals in the three groups. Statistical comparisons were conducted using two-sample t-tests. * p < 0.05.