Experimental Study On MRI Evaluation Of Breast Cancer Lymphangiogenesis And Podoplanin Expression

Objective(1) To observe pathological features and lymphangiogenesis of rat breast cancer growth and differentiation; (2) synthesis podoplanin targeted MRI (magnetic resonance imaging) contrast agent, investigate the physical and chemical characteristics of the nano-scale contrast agent in vitro; (3) podoplanin targeted MR imaging in vivo to evaluatebreast cancer lymphangiogenesis. Methods(1) Goldmag was applied to synthesis molecular probe by its superparamagnetic characteristic and the feature of binding biomolecule by colloidal gold. Goldmag was used to bind with anti-podoplanin antibody to synthesis podoplanin targeted MRI contrast agent (PodAb-GoldMag), and bind with non-specific IgG to synthesis IgG-GoldMag as contrast. Measured before and after chelation of two antibodies absorbance at 280nm, and calculated the surface of the antibody coupling efficiency in GoldMag. ELISA double antibody sandwich method was performed to detect the immunoreactivity of molecular probe. (2) Laser particle size analyzer was used to detect the Zeta electric potential of ultra-small superparamagnetic iron oxide (USPIO), GoldMag, IgG-GoldMag, PodoAb- GoldMag. Transmission electron microscopy (TEM) was applied to observe the size and morphology of USPIO, GoldMag and PodoAb-GoldMag. (3) USPIO and GoldMag was diluted to different concentration (0.5～1000μg/ml) by PBS. And PBS was taken as contrast, so totally got of 48 concentrations. Each sample was performed FSE T1WI, FSE T2WI and GRE T2*WI MR imaging to investigate signal intensity variation of different concentration of USPIO and GoldMag. And calculated the signal intensity ratio, rendered concentration-intensity curve. (4) 80 Wister female rats were administrated intragastrically with DMBA to induce breast tumor. 75 rats were successfully induced breast tumor, and randomly divided into 4 groups, included A (PodoAb-GoldMag, 30 rats), B (IgG-GoldMag, 15 rats), C (GoldMag, 15 rats) and D (Gd-DTPA, 15 rats). 1.5 T MR imaging system was applied to perform FSE T1WI, FSE T2WI, pressure grease sequence and diffusion-weighted imaging. Group A, B and C was taken GRE T2*WI enhanced scanning the time before and after (0, 5, 15, 30min, 1h and 2h) injection of contrast agent. Group D was taken FSE T1WI enhanced scanning in the same time points. Detected signal intensity and rendered dynamic enhanced scan time-signal intensity curve. (5) After MRI examination and perfusion-fixation procedure, the same tissue of breast tumor was drawn in the central and around area. Breast tumor was performed HE stain and podoplanin related antigen immunohistochemisty, immunofluorescence and prussian blue iron stain to observe the pathological type and lymphangiogenesis of breast tumors. (6) HE stain, immunohistochemisty stain, LVD results and MRI images were contrast to analysis pathological basic of breast tumor MRI perform and investigate the value of podoplanin targeted MRI evaluation of breast cancer lymphangiogenesis.Results(1) Anti-podoplanin antibody was successfully bound with GoldMag to synthesis PodAb-GoldMag, and the mean binding rate was 64.81%, immunoreactivity of PodAb-GoldMag was 43% of original. (2) The Zeta electric potential of USPIO, GoldMag, IgG-GoldMag and PodAb-GoldMag was 12.0±18.9mv, 25.2±95.2mv, 18.2±1.6mv, 25.5±20.8 mv respectively. And the particle size distribution of USPIO, GoldMag and PodAb-GoldMag was 15~30 nm, 40~80 nm, 80~100 nm respectively. (3) MRI signal intensity variation of USPIO and GoldMag was similar. As the concentration increased, FSE T1WI signal intensity stepped-up, FSE T2WI signal intensity dropped a little, and GRE T2*WI signal intensity dropped quickly. Took two contrast agent signal intensity to compare, the signal intensity had significant differences under FSE T1WI when the concentration above 30μg/ml (P<0.05), the signal intensity had significant differences under FSE T2WI when the concentration between 40~90μg/ml (P<0.05), the signal intensity was similar under GRE T2*WI (P>0.05). (4) 75 rats breast tumor were successfully induced, 21 were breast proliferative disease, 2 were inflammatory granuloma, and 52 were breast cancer. The total achievement ratio of induced breast lump was 93.8%, and the achievement ratio of induced breast cancer was 69.3%. (5) Breast benign and malignant disease showed hypointensity or isointensity under T1WI; benign mainly showed hyperintensity and isointensity under T2WI, 42 malignant showed hyperintensity and isointensity and 10 malignant showed hypointensity under T2WI; benign and malignant both showed hyperintensity under pressure grease sequence. (6) After injection of PodAb-GoldMag, signal intensity of group A, B and C breast cancer dropped down immediately (P<0.01), and gradually recovered over time. 1h after injection, the signal strength was stable, then signal intensity of group A was lower than before injection, the difference was significant (P <0.05); signal intensity of group B, C was back to levels before injection, no significant difference (P> 0.05). Signal intensity in different histological types of breast cancer before and after contrast agent injection was no significant difference (P> 0.05), the signal intensity of different degree of differentiation of breast cancer in the immediate injection of contrast agent to within 5min significant difference (P <0.05), other time points no significant difference. (7) LVD with the decrease of the degree of differentiation of breast cancer was significantly higher (P <0.05), LVD and group A signal change were significantly negatively correlated (r =-0.814,P = 0.000), and no significant difference in correlation with group B and C. LVD and group D signal intensity was significantly correlated (r = 0.972,P = 0.000).Conclusion(1) GoldMag was a core-shell superparamagnetic composite particles, and the core of GoldMag was USPIO. USPIO and GoldMag MRI signal characteristics were similar, as concentration increased, FSE T1WI signal intensity tended to increase, FSE T2WI signal intensity decreased slightly, GRE T2 * WI signal intensity decreased significantly. GRE T2*WI was the most appropriate MRI imaging sequences for USPIO and GoldMag.(2) PodAb-GoldMag nanos-scale contrast agent was successfully synthesized. And the mean binding rate was 64.81%. Immunoreactivity of PodAb-GoldMag was 43% of original. The Zeta electric potential of PodAb-GoldMag was 25.5±20.8 mv. Particle size distribution of PodAb-GoldMag was 80~100 nm.(3) MRI T1WI scan of rat induced breast cancer showed hypointensity or isointensity; T2WI showed hyperintensity and isointensity; pressure grease sequence T2WI scan appeared hyperintensity, and similar to human breast cancer MRI performance.(4) Proliferative breast disease can be seen only a little lymphangiogenesis and a bit larger, more mature lymphatic vessels. Lymphangiogenesis always showed in stroma around the edges of tumor nests. LVD with the decrease of the degree of differentiation of breast cancer increased.(5) PodAb-GoldMag enhanced breast cancer MRI signal intensity reduction in surrounding area was more obvious than central area. Different differentiation and histological types of breast cancer, PodAb-GoldMag enhanced MRI signal intensity changes were similar.(6) PodAb-GoldMag targeted enhanced MRI correlated with tumor lymphatic vessel density. Rat breast cancer PodAb-GoldMag enhanced scan can help to initially identify the lymphangiogenesis of breast cancer in vivo.