Micrometastasis expressing insulin arise in lung and spleen at advanced stage of rip1-tag2 transgenic mice

Abstract

Rip1-Tag2 mice is one overt pancreatic β-cell tumor model, which is widely used for studying pancreas tumor angiogenesis and tumor development. However, tumor metastasis in Rip1-Tag2 mice had rarely been reported, in this present study, we find some micrometastasis in lung and spleen of the Rip1-Tag2 mice at advanced stage, which is important for uncovering metastasis cell characteristics and exploring how to survive in cancer microenvironment. To study the micrometastasis of Rip1-Tag2 mice in advanced pancreatic cancer, we first observed the pathology process of β cell tumor in Rip1-Tag2 mice through HE staining, then we performed immunohistochemistry with insulin antibody, T-antigen antibodies and C-petide antibody on lung and spleen tissues sections from advanced stage, comparing with background wild-type C57BL/6 mice sections. The results indicated that micrometastasis expressing insulin was found in the Rip1-Tag2 mice lung, and spleen. Further evidences demonstrate pathology structure of lung and spleen are damaged. Interestingly and importantly, the expression of T antigen and insulin antibodies are all decreased in advanced stage of primary β cell tumor, which suggest that the at least partly micrometastasis is derived from the early stage or from advanced stage of β cell tumor then return to undifferentiated state like cancer stem cell. The findings contributed to the study of cancer metastasis and cancer stem cell.

Keywords: Rip1-Tag2 transgenic mice; insulin; micrometastasis.

Fig 1

Identification of the gene-type of Rip1-Tag2 mice. T-antigen gene was identified by using PCR assay. The lanes 2, 4, 5 indicated Tag2 were negative (-), but the lanes 3, and 6 show Tag2 were positive (+). Lane 1 was regard as positive control. M was indicated the Marker DL2000.

Fig 2

The process of Pancreatic βcell tumor in Rip1-Tag2 transgenic mice. A. Normal stage, the Islet cell is normal and same as C57BL/6 mice(Fig 2A); B. Hyperplastic islet stage, dysplastic begin to appear, most of cell nuclear/cytoplasm ratio increases(Fig 2B), C. Angiogenic islets stage, angiogenic Islets began to appear(Fig 2C), D. Tumor stage, the vascular network is highly developed, solid tumors formed(Fig 3D). E. Invasion stage. Tumor infiltrate into the surrounding acinar tissue (Fig 2E and 2F).

Fig 3

Micrometastasis appears in lung and spleen in Rip1-Tag2 mice. A. Micrometastasis size was defined as no more than 10 cells per section (10μm). Micrometastasis tumor cell expressing insulin rise in lung and spleen as shown in Fig.3A (magnification: 600×). B. Micrometastasis was further confirmed by IHC assay, C-peptide antibody was used to verify above IHC results, T-antigen antibody was also used in IHC to show that micrometastsis expressed T-antigen as show in Fig 3B (magnification: 1000×). C. Likely, C-peptide was also expression in micrometastasis shown as Fig 3C (magnification: 1000×). The results suggested that micrometastasis was derived from insulinoma. D. Micrometastasis are not find in lung and spleen in C57BL/6 mice but was observed in Rip1-Tag2 mice (P<0.0001, n=3, the number of section is 10μm/slide), shown as Fig.3D. Note: micrometastassis refer to the number of tumor cell was less than 10, and advanced stage was 14 weeks.

Fig 4

Pathological structures altering in lung and spleen in advantage stage. A. Reactive hyperplasia of lymphoid tissue in spleen in Rip-Tag2 mice and C57BL/6 mice (background) as control. B. Thickening and bleeding of the alveolar septa, congestion of capillary, hemorrhagic foci (pink dot) was observed in lung of Rip1-Tag2 mice. Microscope magnification: 400×.

Fig 5

Insulin and T antigen low-expression invasive carcinoma. A. Representative image of Insulin and T-antigen IHC in Hyperplasia (left, 4 weeks) and invasive carcinoma (right, 14 weeks) (magnification: ×1000). B. The results showed insulin and T-antigen down-regulation in hyperplasia comparison with invasive carcinoma (***P<0.01). The mean gray level IOD/Area was used to analysis in 10 fields, the color stained brown was objective to analyze (HRP-DAB).