Smart Sorting of Tumor Phenotype with Versatile Fluorescent Ag Nanoclusters by Sensing Specific Reactive Oxygen Species

Abstract

Reactive oxygen species (ROS) play a crucial role in cancer formation and development, especially cancer metastasis. However, lack of a precise tool, which could accurately distinguish specific types of ROS, restricts an in-depth study of ROS in cancer development and progression. Herein, we designed smart and versatile fluorescent Ag nanoclusters (AgNCs) for sensitive and selective detection of different species of ROS in cells and tissues. Methods: Firstly, dual-emission fluorescent AgNCs was synthesized by using bovine serum albumin (BSA) to sense different types of ROS (H₂O₂, O2•-, •OH). The responsiveness of the AgNCs to different species of ROS was explored by fluorescence spectrum, hydrodynamic diameter, and so on. Furthermore, dual-emission fluorescent AgNCs was used to sense ROS in tumor with different degrees of differentiation. Finally, the relationship between specific types of ROS and tumor cell invasion was explored by cell migration ability and the expression of cell adhesion and EMT markers. Results: This dual-emission fluorescent AgNCs possessed an excellent ability to sensitively and selectively distinguish highly reactive oxygen species (hROS, including O₂•^-and •OH) from moderate reactive oxygen species (the form of H₂O₂), and exhibited no fluoresence and green fluorescence, respectively. The emission of AgNCs is effective in detecting cellular and tissular ROS. When cultured with AgNCs, malignant tumor cells exhibit non-fluorescence, while the benign tumor emits green and reduced red light and the normal cells appear in weak green and bright red fluorescence. We further verified that not just H₂O₂ but specific species of ROS (O₂•^-and •OH) were involved in cell invasion and malignant transformation. Our study warrants further research on the role of ROS in physiological and pathophysiological processes. Conclusion: Taken together, AgNCs would be a promising approach for sensing ROS, and offer an intelligent tool to detect different kinds of ROS in tumors.

Keywords: Ag Nanoclusters; ROS; Tumor Phenotype.

Figure 1

Schematic presentation of the synthesis of AgNCs for selective detection of various species of ROS in cells.

Figure 2

Characterization of AgNCs. (A) Fluorescence spectrum of AgNCs. The black line represents the excitation spectrum of AgNCs; blue and green lines represent the emission spectrum under excitation at 400 and 500 nm, respectively. The inset photograph was pictured under UV light. (B) UV-Vis spectrum of AgNCs. The inset photograph is enlargement of 240 to 340 nm absorption wavelength. (C) TEM and HRTEM images of AgNCs. (D) Hydrodynamic diameter of AgNCs detected by DLS. (E) Mass peaks of BSA and AgNCs measured by MALDI-TOF-MS. Blue and red lines show the mass peaks of BSA and AgNCs, respectively. (F) XPS spectrum of AgNCs. The original and composite spectra are in black and red, respectively. The pink line represents Ag (0) binding energy.

Figure 3

Responsiveness of AgNCs to various species of ROS. (A) FL emission changes of AgNCs excited at 400 nm with an increased amount of H₂O₂. (B) FL emission changes of AgNCs excited at 400 nm with an increased amount of •OH. (C) Dose-response of H₂O₂ on AgNCs FL emission. The F500 and F650 are fluorescence emission intensities at 500 and 650 nm. (D) Dose-response of •OH on AgNCs FL emission. The F and F0 are fluorescence emission intensities in the absence and presence of •OH, respectively.

Figure 4

Mechanism of AgNC responsiveness to various species of ROS. Emission spectra and photographs under UV light (inset) for AgNCs (A), AgNCs with H₂O₂ (B), and AgNCs with O₂•^- (C). Hydrodynamic diameter and HRTEM image (inset) for AgNCs (D), AgNCs with H₂O₂ (E), and AgNCs with O₂•^- (F). XPS spectrum of AgNCs (G), AgNCs with H₂O₂ (H), and AgNCs with O₂•^- (I).

Figure 5

Time-stability and photo-stability of AgNCs. Emission spectrum of AgNCs excited at 400 nm under lights (A) and away from lights (B). Emission spectrum of AgNCs excited at 500 nm under lights (C) and away from lights (D). UV-vis absorption spectra of AgNCs under lights (E) and away from lights (F).

Figure 6

Biological toxicity of AgNCs. (A) Cell viability of DC2.4 cells after treatment with 10 mg/mL AgNCs for specific times (2 h, 4 h, 12 h, 24 h). (B) Cell viability of DC2.4 cells incubated with 0-15 mg/mL AgNCs for 2 h.

Figure 7

Live cell images of cellular ROS with AgNCs. (A) Confocal images of cellular ROS with AgNCs in dendritic cells (DC2.4) and thyroid cancer cell lines (FTC-133, B-CPAP, OCUT-2, TPC-1). (B) Average red fluorescence intensity of dendritic cells (DC2.4) and thyroid cancer cell lines (FTC-133, B-CPAP, OCUT-2, TPC-1).

Figure 8

FlowSight cellular ROS with AgNCs. (A) FlowSight image of cellular ROS with AgNCs in dendritic cells (DC2.4) and thyroid cancer cell lines (FTC-133, B-CPAP, OCUT-2, TPC-1). The ch07 and ch08 can check blue (435-505 nm) and green (505-560 nm) fluorescent signal under ultraviolet light (405 nm). The cho9 is in charge of bright field. (B) ROS levels in dendritic cells (DC2.4) and thyroid cancer cell lines (FTC-133, B-CPAP, OCUT-2, TPC-1) measured by FlowSight cytometry with AgNCs.

Figure 9

ROS-blocking imaging with AgNCs. Malignant thyroid carcinoma cells (OCUT-2) cultured in RPMI-1640 with various ROS-blocking agents (CAT, DPI, NAC and MLT). Cellular ROS was detected by confocal imaging with AgNCs.

Figure 10

Measurement of cellular ROS by commercial reagents. Cellular ROS of dendritic cells (DC2.4) and thyroid cancer cell lines (FTC-133, B-CPAP, OCUT-2, TPC-1) are detected by commercial reagents DCHF-DA, DHE, and APF for the detection of H₂O₂, O₂•^-, •OH, respectively.

Figure 11

Tissular ROS imaging with AgNCs. Frozen sections of the liver, kidney, thyroid gland, and anaplastic thyroid cancer tissues were used to sense tissular ROS with AgNCs.

Figure 12

Migration ability of dendritic cells and thyroid cancer cells with various grades of differentiation. (A) Cell wound scratch assay of dendritic cells (DC2.4) and thyroid cancer cells with various grades of differentiation (FTC-133, B-CPAP, OCUT-2, TPC-1). (B) Migration speed of dendritic cells (DC2.4) and thyroid cancer cells with various grades of differentiation (FTC-133, B-CPAP, OCUT-2, TPC-1). (C) percent migrated distance of the five cell lines (DC 2.4, FTC-133, B-CPAP, OCUT-2, TPC-1) at 24 h.

Figure 13

Effect of ROS on the migration ability of OCUT-2 anaplastic thyroid tumor cells. (A) Cell wound scratch assay of OCUT-2 in RPMI-1640 with various ROS-blocking agents (CAT, NAC, and MLT). (B) Migration of OCUT-2 in the presence of various ROS-blocking agents (CAT, NAC, and MLT).

Figure 14

Genes related to malignant phenotype (E-cadherin and MMP-9) and ROS production (NOX4) in thyroid cancer cells. (A) mRNA levels of E-cadherin, MMP-9, and NOX4 in thyroid cancer cells with various grades of differentiation (FTC-133, B-CPAP, OCUT-2, TPC-1) detected by RT-PCR. (B) Protein levels of E-cadherin, MMP-9, and NOX4 in thyroid cancer cells with various grades of differentiation (FTC-133, B-CPAP, OCUT-2, TPC-1) measured by immunofluorescence.