Sight and switch off: Nerve density visualization for interventions targeting nerves in prostate cancer

Abstract

Nerve density is associated with prostate cancer (PCa) aggressiveness and prognosis. Thus far, no visualization methods have been developed to assess nerve density of PCa in vivo. We compounded propranolol-conjugated superparamagnetic iron oxide nerve peptide nanoparticles (PSN NPs), which achieved the nerve density visualization of PCa with high sensitivity and high specificity, and facilitated assessment of nerve density and aggressiveness of PCa using magnetic resonance imaging and magnetic particle imaging. Moreover, PSN NPs facilitated targeted therapy for PCa. PSN NPs increased the survival rate of mice with orthotopic PCa to 83.3% and decreased nerve densities and proliferation indexes by more than twofold compared with the control groups. The present study, thus, developed a technology to visualize the nerve density of PCa and facilitate targeted neural drug delivery to tumors to efficiently inhibit PCa progression. Our study provides a potential basis for clinical imaging and therapeutic interventions targeting nerves in PCa.

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Figures

Fig. 1. The nerve-binding peptide, NP41, can target cancer-related nerves of PCa.

(A) Representative in vivo BLI of BALb/c nude male mice 11 weeks after injection of PC-3luc cells into the ventral prostate (1) and image of the orthotopic prostate tumor (2). Representative H&E staining of orthotopic prostate tumor (3). Neural identity was confirmed by immunofluorescence staining of TH (4), VAChT (5), NF-H (6) and NF-L (7). DAPI (4′,6-diamidino-2-phenylindole), blue; TH, VACHT, NF-H, and NF-L, red. Scale bar, 50 μm. (B) Western blot of the expression of neural laminins in prostate tumor tissues and healthy prostate tissues. (C to F) Immunofluorescence staining of TH (C), VAChT (D), NF-H (E), and NF-L (F) colocalizes with NP41 binding, respectively. Scale bars, 100 μm. (G to K) Immunofluorescence staining of laminin-α4 (Lamα4, G), laminin-β2 (Lamβ2, H), laminin-α2 (Lamα2, I), laminin-β1 (Lamβ1, J), and laminin-γ1 (Lamγ1, K) colocalizes with NP41 binding, respectively. Scale bars, 100 μm.

Fig. 2. Preparation and characterization of PSN NPs.

(A) Schematic of the fabrication process. (B) TEM images of PS NPs before labeling with NP41 (left), and PSN NPs after labeling with NP41 (right). Inset shows the appearance of PSN NPs solution. (C) Hydrated particle size of PS NPs and PSN NPs. (D) Zeta potential of PS NPs and PSN NPs. (E) UV-vis absorption spectra of solutions 1 and 2. Solution 1: Peptide solution before PS NPs labeling with peptides. Solution 2: Supernatants isolated after PS NPs labeling with peptides. a.u., arbitrary units. (F) T2-weighted MRI images and (G) T2 relaxation rate of PSN NPs at various concentrations. The red dashed circles show the black MRI images as the PSN NPs concentration increased. (H) MPI images and (I) MPI signal of PSN NPs at various concentrations. (J) Room temperature magnetization curve of PSN NPs. (K) HPLC analysis of in vitro propranolol release profiles from PSN NPs in buffer at pH 6.0 and 7.4.

Fig. 3. Imaging difference of PSN NPs and PS NPs.

(A) T2-weighted MRI images of nerve density of PCa acquired at different time points before and after systemic administration of PSN NPs and PS NPs, respectively. The red dashed circles indicate the nerve density of PCa in situ. (B) MPI images of nerve density of PCa obtained 24 hours following systemic injection of PSN NPs and PS NPs. (C) Quantification of TNR in the PSN NPs and PS NPs groups at corresponding time points to (A). TNR values show notable difference at 24 and 48 hours after injection of two nanoprobes (P = 0.021 and P = 0.046, respectively). (D) Quantification of MPI signal in the PSN NPs and PS NPs groups at corresponding time point to (B). MPI signal values display notable difference at 24 hours after injection of PSN NPs and PS NPs (P < 0.0001). (E) Nuclear fast red and Prussian blue double staining images of major organs (liver, spleen, and kidney) and tumor after intravenous administration of PSN NPs and PS NPs. *P < 0.05, **P < 0.01, and ****P < 0.0001, Student’s t test. Scale bars, 50 μm. Error bars represent SEM.

Fig. 4. PSN NPs distinguish the distribution of high and low nerve density of PCa induced by drugs.

(A and B) MRI images (A) and MPI images (B) of PBS- and 6OHDA-treated mice at 24 hours after systemic administration of PSN NPs. The red dashed circles indicate the nerve density of PCa in situ. (C and D) Quantification of TNR (C) and MPI signals (D) at corresponding time points to (A) and (B), respectively. (E) Nuclear fast red and Prussian blue double staining images of tumor to compare the accumulation of PSN NPs in PBS- and 6OHDA-treated mice. (F) Immunofluorescence images of TH, VAChT, NF-H, and NF-L in PBS- and 6OHDA-treated mice. DAPI, blue; TH, VACHT, NF-H, and NF-L, red. (G) Immunohistochemistry images of Ki-67 in PBS- and 6OHDA-treated mice. (H) Pearson correlation analysis between TNR and TH, VAChT, NF-H, NF-L, and Ki-67 index, respectively. (I) Pearson correlation analysis between MPI signal and TH, VAChT, NF-H, NF-L, and Ki-67 index, respectively. (J) Pearson correlation analysis between nerve density and Ki-67 index. Spearman’s correlation coefficients and P values are shown. Student’s t test. Scale bars, 50 μm. Error bars represent SEM.

Fig. 5. PSN NPs distinguish the distribution of the high and low nerve density of PCa induced by surgery.

(A and B) MRI images (A) and MPI images (B) in sham-operated and surgical HGNx–treated mice at 24 hours after systemic administration of PSN NPs. The red dashed circles indicate the nerve density of PCa in situ. (C and D) Quantification of TNR value (C) and MPI signal (D) at corresponding time points to (A) and (B), respectively. (E) Nuclear fast red and Prussian blue double staining images of tumor to compare the accumulation of PSN NPs in sham-operated and surgical HGNx–treated mice. (F) Immunofluorescence images of TH, VAChT, NF-H, and NF-L in sham-operated and surgical HGNx–treated mice. DAPI, blue; TH, VACHT, NF-H, and NF-L, red. (G) Immunohistochemistry images of Ki-67 in sham-operated and surgical HGNx–treated mice. (H) Pearson correlation analysis between TNR and TH, VAChT, NF-H, NF-L, and Ki-67 index, respectively. (I) Pearson correlation analysis between MPI signal and TH, VAChT, NF-H, NF-L, and Ki-67 index, respectively. (J) Pearson correlation analysis between nerve density and Ki-67 index. Spearman’s correlation coefficients and P values are shown. Student’s t test. Scale bars, 50 μm. Error bars represent SEM.

Fig. 6. PSN NPs inhibit orthotopic PCa development.

PC-3luc cells were injected into the ventral prostate of BALb/c nude male mice to form orthotopic PCa on day 0. After 10 days, mice were divided into different groups: PBS, SN NPs, free propranolol (P), and PSN NPs. Mice received various formulations by systemic administration for six times. (A) In vivo bioluminescence images indicate the orthotopic PCa development on days 14, 23, 31, and 45 after orthotopic PC-3luc cell xenografts. (B) Images of orthotopic prostate tumors (left) and lymph nodes (right) on day 45. (C) H&E staining sections of orthotopic prostate tumors (left) and lymph nodes (right) from indicated groups. (D) TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) of apoptotic cells in tumors from indicated groups. (E) Serial quantification of in vivo bioluminescence intensity of xenografts in the prostate gland. ns, not significant. (F) The mice survival curves in different groups, evaluated by the Kaplan-Meier method and the log-rank test. (G) The changes of mice body weight during treatments. (H) Quantification of apoptotic TUNEL+ cells in the orthotopic prostate tumors from indicated groups at posttreatment. (I) Immunofluorescence images of TH, VAChT, NF-H, and NF-L from indicated groups after treatment. DAPI, blue; TH, VACHT, NF-H, and NF-L, red. (J) Immunohistochemistry images of Ki-67 from indicated groups after treatment. (K) Quantification of nerve density of TH, VAChT, NF-H, and NF-L. (L) Quantification of Ki-67 index. (M) Immunofluorescence images of lamα4, lamβ2, lamα2, lamβ1, and lamγ1 after treatments. DAPI, blue; lamα4, lamβ2, lamα2, lamβ1, and lamγ1, red. (N) Quantification of laminin density of lamα4, lamβ2, lamα2, lamβ1, and lamγ1. Data were obtained from five fields per section from field surface of 0.27 mm2. *P < 0.05, **P < 0.01, and ns, P > 0.05. The statistical analysis was based on nonparametric one-way analysis with Kruskal-Wallis. Scale bars, 50 μm. Error bars represent SEM (n = 7 to 12 per group).

Fig. 7. Illustration of nerve density visualization by MRI and MPI with high sensitivity and specificity and targeted delivery of the nerve blocker propranolol to effectively inhibit PCa progression.