In Vivo Flow Cytometric Evaluation of Circulating Metastatic Pancreatic Tumor Cells after High-Intensity Focused Ultrasound Therapy

Abstract

We examined our hypothesis that high-intensity focused ultrasound (HIFU) treatment of pancreatic ductal adenocarcinoma (PDAC) in nude mice models may lead to an increased occurrence of hematogenous metastasis. The human PDAC cell line BxPC-3 transfected with mCherry was implanted into nude mice to establish orthotopic and subcutaneous xenograft (OX and SX) tumor models. Mice were exposed to HIFU when tumor sizes reached approximately 200-300 mm³ . The OX and SX tumor models were monitored continuously for tumor growth characteristics and hematogenous metastasis using in vivo flow cytometric (IVFC) detection of circulating tumor cells (CTCs) from the pancreas. We chose an appropriate mouse model to further examine whether or not HIFU increases the potential risk of hematogenous metastasis, using IVFC detection. Our results showed that the CTC number was greater in the OX model than in the SX model. The CTC number in the OX model increased gradually over time, whereas the CTC number in the SX model remained low. Therefore, the OX model was better for studying tumor metastasis by IVFC detection. We found significantly decreased CTC numbers and tumor volume after HIFU ablation. Our results showed the applicability of the PDAC OX tumor model for studying the occurrence of tumor metastasis due to the generation of CTCs. HIFU ablation substantially restricted PDAC hematogenous metastasis and provided effective tumor control locally. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals Inc., on behalf of International Society for Advancement of Cytometry.

Keywords: circulating tumor cells; high-intensity focused ultrasound; in vivo flow cytometry; pancreatic cancer.

Figure 1

Schematic diagram of high‐intensity focused ultrasound (HIFU) ablation and in vivo flow cytometry (IVFC) detection. (A) The schematic illustration of the ultrasound‐guided HIFU ablation on the orthotopic mCherry‐BxPC‐3 tumor‐bearing nude mouse. (B) Red fluorescence‐labeled tumor cells intravasate into the vascular and circulate throughout the body as circulating tumor cells (CTCs). (C) Optical path design of 561 nm excited IVFC. M1: reflection mirrors. CL: cylinder lens. DM1, DM2: dichroic mirrors. MS1, MS2: mechanical slits. F1, F2: filters. AL1, AL2, AL3: achromats lens. PMT: photomultiplier tube. CCD: charge coupled device. (D) Mouse ear blood vessels obtained by light‐emitting diode (LED) transmission illumination. The mCherry‐BxPC‐3 cells passed through the laser slit, then the fluorescence signals from the excited cells were recorded. Scale bar, 50 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

The brightest (top 1%) mCherry‐BxPC‐3 cells were sorted by fluorescence‐activated cell sorting(FACS), then CTCs measurement by in vivo flow cytometry(IVFC) after nude mice implantation. (A) Differential interference contrast image of tumor cells; (B) Fluorescent image of tumor cells. Scale bar, 50 μm; (C) before FACS, the proportion of the brightest cells was 73.52%; (D) After FACS, the brightest cells accounted for 99.38%; (E) Showing off a piece of blank control data. The animals in the blank control group (n = 6) did not undergo tumor implantation; (F) Visualization of digitized IVFC signals of mCherry‐BxPC‐3 cells using software developed on MATLAB platform. A 10 min recorded data on day 12 of the OX tumor model was shown (arrow, a single cell trace). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Growth characters and CTCs dynamics of mCherry‐BxPC‐3 cells in the OX and SX tumor models. (A) The line graph showed mice weight in the OX tumor model, SX tumor model, and normal control group. 10 days after tumor implantation, the OX tumor model weight began to lose, which were significant differences compared to the SX tumor model and normal control group. There was no difference in mice weight between the SX tumor model and the normal control group. (B) The survival rate of the OX model (n = 12) was significantly lower than that of the SX model (n = 7). (C) The column graph showed tumor volume at each point. 10 days after tumor implantation, the tumor volume in the OX tumor model was significantly larger than the SX tumor model. (D) Measured by IVFC for once every 3 days and at least 1 h, there was a difference in CTCs counts between the OX and SX tumor model. (n = 6 for each group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Ultrasound imaging of the OX tumor model before and after HIFU ablation. (A) a representative image of the OX tumor on day 12 acquired by the B‐mode ultrasound. The tumor possesses a long diameter of 10.0 mm and a short diameter of 7.5 mm, total volume almost 281.25 mm³. (B and C) The same section shown by superb microvascular imaging (SMI) mode, circular blood flow around the tumor. (D) Before or (E) after HIFU ablation immediately, image of the tumor (line). (d) Before or (e) after HIFU ablation, measurement of mean tumor gray intensity by ImageJ software. (F) The column graph showed a significant difference in mean gray intensity of before and after HIFU ablation. (n = 6 for each group, *P < 0.05). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

The HIFU ablation restricted hematogenous metastasis of OX tumor model, and provide effective local tumor control. (A) In the OX tumor model, CTCs dynamics after HIFU ablation and the control group. HIFU treatment taken on day 12 after IVFC, monitoring CTCs counts for every 3 days. (B) The volume of tumor in the HIFU ablation group and the control group were shown above. (n = 6 for each group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

Histological observation of the tumor collected from the tumor‐bearing nude mice, the tissue sections were stained with H&E. (A) The SX tumor limited to subcutaneous growth with a clear boundary on day 12. (B) the OX tumor invasive growth in pancreatic tissue on day 12. (C) The OX tumor 24 h after HIFU ablation, the lesions showed significant necrosis with fuzzy cellular structures. Scale bar, 50 μm. [Color figure can be viewed at wileyonlinelibrary.com]