Molecular imaging of tumor-infiltrating macrophages in a preclinical mouse model of breast cancer

Abstract

Significant evidence has indicated that tumor-associated macrophages (TAMs) play a critical role in the proliferation, invasion, angiogenesis, and metastasis of a variety of human carcinomas. In this study, we investigated whether near-infrared fluorescence (NIRF) imaging using a macrophage mannose receptor (MMR; CD206)-targeting agent could be used to noninvasively visualize and quantify changes in TAMs in vivo. The CD206-targeting NIRF agent, Dye-anti-CD206, was prepared and characterized in vitro and in vivo. By using NIRF imaging, we were able to noninvasively image tumor-infiltrating macrophages in the 4T1 mouse breast cancer model. Importantly, longitudinal NIRF imaging revealed the depletion of macrophages in response to zoledronic acid (ZA) treatment. However, ZA alone did not lead to the inhibition of 4T1 tumor growth. We therefore combined anti-macrophage ZA therapy and tumor cytotoxic docetaxel (DTX) therapy in the mouse model. The results demonstrated that this combination strategy could significantly inhibit tumor growth as well as tumor metastasis to the lungs. Based on these findings, we concluded that CD206-targeted molecular imaging can sensitively detect the dynamic changes in tumor-infiltrating macrophages, and that the combination of macrophage depletion and cytotoxic therapy is a promising strategy for the effective treatment of solid tumors.

Keywords: CD206; Image-guided therapy.; Macrophage depletion; Optical imaging; Tumor-associated macrophage.

Figure 1

(A) The synthesis scheme of Dye-anti-CD206. (B) SDS-PAGE and NIRF imaging of anti-CD206 antibody and Dye-anti-CD206. (C) CD206 and F4/80 staining of RAW 264.7 murine macrophage cells. (D) Direct fluorescence staining of RAW 264.7 and 4T1 cells using Dye-anti-CD206 or Dye-IgG.

Figure 2

(A) In vivo NIRF imaging of 4T1 tumor-bearing BALB/c normal mice at 2, 8, 24, and 48 h after intravenous injection of Dye-anti-CD206 or Dye-IgG. Tumors are indicated by the dashed circles. (B) Quantification and kinetics of in vivo tumor targeting characteristics of Dye-anti-CD206 or Dye-IgG. (C) Representative images of dissected organs of 4T1 tumor-bearing mice sacrificed at 24 h after intravenous injection of Dye-anti-CD206 or Dye-IgG. (D) Quantified biodistribution of Dye-anti-CD206 and Dye-IgG in 4T1 tumor-bearing mice at 24 h postinjection. *, P <0.05; **, P <0.01.

Figure 3

(A) Overlay immunofluorescence staining of F4/80 and CD206 in 4T1 tumor tissue. (B) Immunofluorescence staining of F4/80 in 4T1 tumor tissues harvested from tumor-bearing mice 24 h postinjection of Dye-anti-CD206 or Dye-IgG. Green color from FITC for murine F4/80, red color from DyLight680 (Dye680) for Dye-anti-CD206, blue color from DAPI for visualization of nuclei.

Figure 4

Longitudinal in vivo NIRF images (A) and quantified tumor uptake (B) of 4T1 tumor-bearing mice at 24 h after Dye-anti-CD206 injection on days 0, 3, 7, and 11 after zoledronic acid (ZA; 150 μg/kg in PBS daily for 7 days) or PBS treatment (control). Tumors are indicated by the dashed circles. (C-D) Immunofluorescence staining (C) and quantified integrated optical density (IOD) expressed in relative percentages (D) of murine CD206 in 4T1 tumor tissues on days 0, 3, 7, and 11 after ZA or PBS treatment. *, P <0.05; **, P <0.01; ***, P <0.001.

Figure 5

(A) In vitro cytotoxic effect of zoledronic acid (ZA), docetaxel (DTX), or ZA plus DTX on 4T1 tumor cells. (B-C) Photography of the 12-well-plate (B) and microscopy examination (C) of the colony formation of 4T1 cells treated with the vehicle control, ZA, DTX, or ZA plus DTX. (D) Tumor growth curves of the 4T1 tumor bearing mice after treatment with the vehicle control, ZA (150 μg/kg daily for 7 days), DTX (5 mg/kg daily for 7 days), or ZA (150 μg/kg daily for 7 days) plus DTX (5 mg/kg daily for 7 days). *, P <0.05; **, P <0.01.

Figure 6

(A-B) Immunofluorescence staining of Ki67 (A) and percent Ki67-positive cells (B) in 4T1 tumor tissues harvested on day 12 from mice treated with vehicle (control), zoledronic acid (ZA), docetaxel (DTX), or ZA plus DTX. (C-E) Photographs of India ink-filled lungs (C), counted average of tumor metastatic lesions in the lungs (D), and the H&E staining of lung slices (E) from 4T1 tumor-bearing mice (on day 12) treated with vehicle (control), ZA, DTX, or ZA plus DTX. Tumor metastases appear as white nodules on the black lung surfaces and are indicated by red arrows. Tumor metastases in the H&E stained lung slices are indicated by the dashed circles. *, P <0.05; ***, P <0.001.