Ultrasound Molecular Imaging as a Potential Non-invasive Diagnosis to Detect the Margin of Hepatocarcinoma via CSF-1R Targeting

Abstract

Though radiofrequency ablation (RFA) is considered to be an effective treatment for hepatocellular carcinoma (HCC), but more than 30% of patients may suffer insufficient RFA (IRFA), which can promote more aggressive of the residual tumor. One possible method to counter this is to accurately identify the margin of the HCC. Colony-stimulating factor 1 receptor (CSF-1R) has been found to be restrictively expressed by tumor associated macrophages (TAMs) and monocytes which more prefer to locate at the boundary of HCC. Using biotinylation method, we developed a CSF-1R-conjugated nanobubble CSF-1R (NB_CSF-1R) using a thin-film hydration method for margin detection of HCC. CSF-1R expression was higher in macrophages than in HCC cell lines. Furthermore, immunofluorescence showed that CSF-1R were largely located in the margin of xenograft tumor and IFRA models. In vitro, NB_CSF-1R was stable and provided a clear ultrasound image even after being stored for 6 months. In co-culture, NB_CSF-1R adhered to macrophages significantly better than HCC cells (p = 0.05). In in vivo contrast-enhanced ultrasound imaging, the washout half-time of the NB_CSF-1R was significantly greater than that of NB_CTRL and Sonovue^® (p = 0.05). The signal intensity of the tumor periphery was higher than the tumor center or non-tumor region after NB_CSF-1R injection. Taken together, NB_CSF-1R may potentially be used as a non-invasive diagnostic modality in the margin detection of HCC, thereby improving the efficiency of RFA. This platform may also serve as a complement method to detect residual HCC after RFA; and may also be used for targeted delivery of therapeutic drugs or genes.

Keywords: CSF-1R targeting; HCC tumor margin; macrophage; non-invasive tumor margin detection; ultrasound imaging.

SCHEME 1

After radiofrequency ablation (RFA), macrophages are highly condensed at the tumor margin, highly expressing CSF-1R. By using nanobubbles targeting CSF-1R (NB_CSF1R), we developed an ultrasound-guided CSF-1R specific targeted real-time monitoring of tumor margin. NB_CSF1R could penetrate through the vascular and adhered to the macrophages, which can provide ultrasound molecular imaging to reveal the accurate margin of HCC.

FIGURE 1

(A) qRT-PCR was used to determine the expression of colony-stimulating factor 1 receptor (CSF-1R) in different cell lines. (B) Western blot was used to access the protein level of CSF-1R in macrophages, THP-1, and SMMC-7721. (C) Showed the CSF-1R expression intensity in macrophages, THP-1, and SMMC-7721, macrophages showed the highest intensity than THP-1 and SMMC-7721, (***indicates p < 0.01). (D) The expression level of CSF-1R in macrophages and SMMC-7721 were detected by fluorescence-activated cell sorting (FACS) blue indicated the control group and, red indiacted the experiment group. (E) IHC verified the expression distribution of CSF-1R in human liver cancer tissues (the red line indicated the boundary of hepatocellular carcinoma (HCC). (P, peritumoral, T, tumor). (F) Semi-quantitatively analyzed the expression of CSF-1R by image-pro plus (IPP) software (***indicates p < 0.01).

FIGURE 2

(A) The process of synthesis of nanobubble CSF-1R (NB_CSF–1R) through the streptavidin/biotin chemical effect, CSF-1R_mAb was biotinylated and then conjunction to the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀)-Biotin. (B) Western blot showed that the NB_CSF–1R band was at approximately the same position as CSF-1R_mAb, and the NB_CTRL showed no expression of CSF-1R. (C) Showed the Western blot intensity of NB_CSF–1R, CSF-1R _mAb, NB_CTRL (**indicates p < 0.05, ***indicates p < 0.001).

FIGURE 3

(A) Showed the structure of the NB_CTRL and NB_CSF–1R, (B) scanning electron microscopy (SEM) image of NB_CTRL with a scale bar of 500 nm (C) SEM image of NB_CSF–1R with a scale bar of 500 nm. (D) Summary of the average size, and zeta potential of NB_CTRL and NB_CSF–1R as measured by dynamic laser scattering. Concentration of NBs were measured by Coulter counter. Data represent mean ± SD (n = 5).

FIGURE 4

Fluorescence microscopy image of target NBs. (A) CSF-1R_mAb (FITC showed green fluorescence), (B) DiI-dyed (nanobubbles showed red fluorescence), and (C) their co-localization (merge) under fluorescence microscope, with a sale bar of 200 μm.

FIGURE 5

(A) Cell viability test for NB_CSF–1R determined through CCK-8. In vitro cytotoxicity assays using macrophage and (high CSF-1 expression) and SMMC-7721 cells (low CSF-1 expression) incubated with NB_CSF–1R for 24 h; there was no significant difference in the viability of macrophages and SMMC-7721. (B) In vitro ultrasound images of NB_CSF–1R stored for 0, 30, 90, 120, 180 days, and the Sonovue^® stored for 0 days as a control. Ultrasound frequency, 20 MHz.

FIGURE 6

(A) In vitro ultrasound images of various concentrations of NB_CSF–1R dilute for different times, imaging was still viable even when diluted 2000 times. Ultrasound frequency, 20 MHz. (B) In vitro, the same quantity of NB_CSF–1R and NB_CTRL were added to SMMC-7721 and macrophages and then observed using confocal laser scanning microscopy (CLSM). There were few NB_CTRL adhered to SMMC-7721 and macrophages, and also few NB_CTRL bounded to macrophages. There were a lot NB_CSF–1R bounded to macrophages, and showed the specificity of targeting. With a scale bar of 100 μm.

FIGURE 7

(A) In vivo tumor imaging, images were taken at the indicated time points (0, immediate, 5, 15, and 30 min) after NB_CSF–1R, NB_CTRL, and Sonovue^® were injected into tail vein of the transplanted tumors. (B) In vivo tumor imaging, images were taken at the indicated time points (0, immediate, 5, 15, and 30 min) after NB_CSF–1R, NB_CTRL, and Sonovue^® were injected into tail vein of insufficient radiofrequency ablation (IRFA) models. Perfusion imaging was also taken to show the nanobubble diffusion. (C) The peak intensity of NB_CS–F1R, NB_CTRL, and Sonovue^® was showed and there was no significant difference in each group. (D) The washout half-time in six group was show, and the NB_CSF–1R can provide longer imaging time in the xenograft tumor and IRFA models (**indicates p < 0.05, ***indicates p < 0.001).

FIGURE 8

(A) The echo intensity fitting curve of the same area around or in the center of transplant tumor with NB_CSF–1R, NB_CTRL, and Sonovue^®. The intensity of the peritumoral echo signal of the NB_CSF–1R was significantly higher than that of the central tissue at the peak time, 5 and 15 min (p = 0.05). Data was shown in panel (B).

FIGURE 9

The distribution of CSF-1R in xenograft tumor observed by fluorescence immunoassay, higher fluorescence intensity was observed at the peritumor, lower fluorescence intensity was observed at the tumor center. [(A) boundary of the tumor, (B) central of tumor, (C) boundary of the residual tumor after IRFA, and (D) residual tumor after IRFA. With a scale bar of 200 μm. The yellow line showed the tumor margin].