Protonation and Trapping of a Small pH-Sensitive Near-Infrared Fluorescent Molecule in the Acidic Tumor Environment Delineate Diverse Tumors in Vivo

Abstract

Enhanced glycolysis and poor perfusion in most solid malignant tumors create an acidic extracellular environment, which enhances tumor growth, invasion, and metastasis. Complex molecular systems have been explored for imaging and treating these tumors. Here, we report the development of a small molecule, LS662, that emits near-infrared (NIR) fluorescence upon protonation by the extracellular acidic pH environment of diverse solid tumors. Protonation of LS662 induces selective internalization into tumor cells and retention in the tumor microenvironment. Noninvasive NIR imaging demonstrates selective retention of the pH sensor in diverse tumors, and two-photon microscopy of ex vivo tumors reveals significant retention of LS662 in tumor cells and the acid tumor microenvironment. Passive and active internalization processes combine to enhance NIR fluorescence in tumors over time. The low background fluorescence allows tumors to be detected with high sensitivity, as well as dead or dying cells to be delineated from healthy cells. In addition to demonstrating the feasibility of using small molecule pH sensors to image multiple aggressive solid tumor types via a protonation-induced internalization and retention pathway, the study reveals the potential of using LS662 to monitor treatment response and tumor-targeted drug delivery.

Keywords: cancer imaging; extracellular pH; fluorescence; pH-sensitive probe; small animal; tumor model.

Figure 1

(A) LS662 is a pH activated probe that becomes highly fluorescent in acidic solution. (B) In vivo, LS662 is activated in tumors and the inactive probe clears from uninvolved tissue, simultaneously increasing contrast and decreasing the dose of LS662 to nontumor regions. (C) LS662 selectively activates in infiltrating tumor. (D) Once LS662 is activated by the protonation of the indolium ring nitrogen, it acts as an amphiphilic small molecules that can penetrate into tumor cells by destabilization of the cell membrane. Once, inside the cell, it can be seen activated in the acidic lysosomes.

Figure 2

(A) Synthesis route of LS662. (B) Chemical structure of cypate. (C) Absorption and emission spectra of pH titrated LS662 in water from pH 2–10. Absorption in basic solution primarily occurs in the visible range, while absorption in acid solution occurs in the NIR. In acidic solution, excitation at 720 nm results in fluorescence at 758 nm. LS662 has a pK_a of 5.2. (D) Pseudocolor images of a solution of LS662 at pH 6.0, 6.5, 7.0 and 7.5 (see methods section for solution preparation).

Figure 3

(A) Images of cellular uptake of LS662 in A431 cells for 2 or 8 h at normal physiological pH 7.4 (neutral) or acidic pH 6.4 (acid) media. Sodium azide (NaN3+) was added to each condition to determine whether the internalization of LS662 is energy dependent. (B) Quantification of the cellular uptake, described in A, in A431 and 4T1/luc cells with LS662 and cypate, *p-value < 0.05. (C) Cellular uptake of LS662 and cypate in A431 cells treated in three conditions: media, PBS, and 50% ethanol, to probe healthy, dying and dead cells, respectively. The images show LS662 fluorescence; cypate images not shown. (D) Healthy, dying and dead cells were coincubated with LS662 (red) lysotracker (green), to investigate the subcellular localization of LS662. Scale bar = 10 μm.

Figure 4

(A, B, C) LS662 in 4T1/luc murine breast cancer, A, A431, human epidermal cancer, B, and PyMT spontaneous breast cancer C. (i) Fluorescence images of tumor bearing mouse at indicated times post-injection. Red arrows indicate the location of the tumor. Pre-injection image show the bright field image of the mouse. (ii) Plot of the average fluorescence values of the tumor region compared to normal tissue control region obtained from the in vivo images. The values are normalized to the post injection fluorescence intensity of each region, respectively. (iii) Fluorescence intensity in organs of interest after the mouse was sacrificed and the organs were excised. All values are normalized to the muscle signal. (D) Cypate, in 4T1/luc murine breast cancer model (n = 3). Error bars are shown for cases of n ≥ 3.

Figure 5

(A) 2-photon tomographic reconstruction of 4T1/luc tumor, excised immediately after the animal was sacrificed, 24 h post initial injection of LS662, (Green-elastin, blue- collagen, Red-LS662.) Matching fluorescence sectioned histology showing LS662 fluorescence and H&E (B,C, D) Fluorescence (red) and H&E stained images, from a 4T1/luc, A431, and PyMT tumor, respectively. (E) Lymph node infiltrated with tumor cells shows LS662 fluorescence. (F) Muscle samples from 4T1/luc (top), A431 (middle), and PyMT (bottom) bearing mice, show no LS662 fluorescence.