Angiogenic Vessels in HepG2-GFP Liver Cancer Mouse Model and In Vivo Fluorescence Imaging

Angiogenesis is the process by which the body builds new blood vessels. The mechanism of angiogenesis is highly complex, involving multiple pathways and steps, and interactions with different growth factors and cells. Whilst blood cell formation is an important physiological process in normal (i.e. non-diseased) tissue, it also plays an important role in several diseased states, such as cancers.

Vascular expansion via angiogenesis occurs to ensure that tissues are being supplied with all of the nutrients and oxygen that they require as they grow. Angiogenesis can occur by one of two mechanisms:

  • Vasculogenesis – the growth of brand new blood vessels
  • Classic angiogenesis – the growth of capillaries from existing vessels

Vasculogenesis plays an important role in the growth and development of embryos, whereas angiogenesis is associated with organ regeneration and growth.

Analytik-Jena’s iBox® Explorer imaging microscope system (Figure 1) can be used for the in vivo fluorescence imaging of angiogenesis. These images can be used to determine and further understand what conditions promote and inhibit angiogenesis in preclinical models and diseased humans.

iBox Explorer imaging microscope

Figure 1. iBox Explorer imaging microscope

The iBox® Explorer possesses a cooled CCD camera. The high resolution of the camera allows images with low fluorescence to be captured, allowing the angiogenic process to be imaged in living animals across a wide wavelength range.

In this article, the system is used to observe angiogenesis in rodents with liver cancer.

Materials and Methods

Vector, Cell Line and Animal

A plasmid containing genes for GFP was used to transfect Hepatocellular Carcinoma cells, which were then cultured, with a selection of GFP-expressing cells. Six-week-old Male nude mice (BALB/c-nu/nu), from Beijing HFK Bio-Technology, China were acclimated for a week. Following acclimation, the mice were injected with the HepG2-GFP cells (1 × 107 cells in 0.1 mL phosphate buffered saline).

Orthotropic Transplantation Tumor Model

Three weeks after the HepG2-GFP inoculation mice with cancer growths underneath underwent orthotropic transplantation. Their tumor tissues were gathered and aseptically sliced into sections. These sections were then orthotopically transplanted into the mice’s liver lobes and the mice were then grown in specific pathogen free (SPF) conditions.

Fluorescent Microscopy

A month after tumor implantation the mice underwent anesthesia and images were taken using the iBox Explore. The system was set up as follows:

  • BioLite™ - Automated MultiSpectral Light Source
  • OptiChemi 610  - 3.2 MP camera
  • Motorized platform with built-in warming plate at 37 °C
  • GFP excitation and emission filter sets
  • VisionWorks® LS – Software for Acquisition and Analysis

The systems magnification optics can range between magnifications of 0.17x and 16.5x, allowing for a wide range of different magnification options.

Results and Discussion

The imaging of GFP-fluorescence in the nude mice at several different magnifications was carried out using the iBox Explorer (Figures 2 and 3). These signals were present in both the transplant site and other regions of the mice’s bodies, showing that the tumor cells had spread.

In vivo GFP fluorescent imaging of mouse transplanted with HepG2-GFP driven tumor. The image shows the mouse one month after orthotropic transplanted tumor in liver lobes, with survival and migration of transplanted tumor cells.

Figure 2. In vivo GFP fluorescent imaging of mouse transplanted with HepG2-GFP driven tumor. The image shows the mouse one month after orthotropic transplanted tumor in liver lobes, with survival and migration of transplanted tumor cells.

Images of the angiogenic vessels around the tumor were also taken. Figure 3 (A) shows images of GFP fluorescence at a magnification of 0.5x with a 30 mm2 field of view. Figures 3 (B), 3 (C) and 3 (D) show images of a higher magnification which were taken to view the vessels around the tumors.

(A) GFP-Fluorescent signals were imaged with iBox Explorer at 0.5x magnification corresponds to a field of view of 30 mm2 window. Areas with angiogenic vessels around tumor were marked. Figs. 3 (B, C, D) correspondent areas with angiogenic vessels were imaged at 1.66x or 2.50x magnifications, which correspond to fields of view of 9 mm2 or 6 mm2 respectively. Angiogenic vessels were marked with yellow dashes.

(A) GFP-Fluorescent signals were imaged with iBox Explorer at 0.5x magnification corresponds to a field of view of 30 mm2 window. Areas with angiogenic vessels around tumor were marked. Figs. 3 (B, C, D) correspondent areas with angiogenic vessels were imaged at 1.66x or 2.50x magnifications, which correspond to fields of view of 9 mm2 or 6 mm2 respectively. Angiogenic vessels were marked with yellow dashes.

(A) GFP-Fluorescent signals were imaged with iBox Explorer at 0.5x magnification corresponds to a field of view of 30 mm2 window. Areas with angiogenic vessels around tumor were marked. Figs. 3 (B, C, D) correspondent areas with angiogenic vessels were imaged at 1.66x or 2.50x magnifications, which correspond to fields of view of 9 mm2 or 6 mm2 respectively. Angiogenic vessels were marked with yellow dashes.

(A) GFP-Fluorescent signals were imaged with iBox Explorer at 0.5x magnification corresponds to a field of view of 30 mm2 window. Areas with angiogenic vessels around tumor were marked. Figs. 3 (B, C, D) correspondent areas with angiogenic vessels were imaged at 1.66x or 2.50x magnifications, which correspond to fields of view of 9 mm2 or 6 mm2 respectively. Angiogenic vessels were marked with yellow dashes.

Figure 3. (A) GFP-Fluorescent signals were imaged with iBox Explorer at 0.5x magnification corresponds to a field of view of 30 mm2 window. Areas with angiogenic vessels around tumor were marked. Figs. 3 (B, C, D) correspondent areas with angiogenic vessels were imaged at 1.66x or 2.50x magnifications, which correspond to fields of view of 9 mm2 or 6 mm2 respectively. Angiogenic vessels were marked with yellow dashes.

Vessels were viewed at magnifications of 2.5x and 1.66x with respective field views of 6 mm2 and 9 mm2. In these images, angiogenic vessels are indicated with yellow dashes.

Conclusion

A month after implantation with the tumor, images of strong fluorescence in the mice were taken. These images showed that the tumor cells were not only located in the liver lobes, but had also spread to other bodily tissues.

High resolution, magnified imaging was used to observe the angiogenic response of vessels in the proximity of the tumor. As part of this observation, the length and number of angiogenic vessels were recorded. This research demonstrates that using a murine model for HepG2-GFP liver cancer is an accurate and precise method of studying angiogenesis.

Analytik-Jena’s iBox® Explorer imaging microscope is a key tool for the preclinical imaging of fluorescence for in vivo research into angiogenesis. The system can be used to provide high-quality images for the observation of changes in vascular structure.

The imaging method detailed can be used for both qualitative and quantitative analysis of angiogenesis and the testing of anti-angiogenic treatments. This could involve, for example, the investigation of pathways that regulate angiogenesis and the angiogenic response to different potential stimulators and inhibitors.

The iBox Explorer, from Analytik-Jena, is an easy-to-use, rapid and accurate method of vessel imaging in mice populations.

References

  • Imaging tumour angiogenesis, Cancer Imaging. 2005; 5(1): 131–138.
  • Challenges for imaging angiogenesis, British Journal of Radiology (2001) 74, 886-890

This information has been sourced, reviewed and adapted from materials provided by Analytik Jena US.

For more information on this source, please visit Analytik Jena US.

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