Establishment of NOD/SCID mouse models of human hepatocellular carcinoma via subcutaneous transplantation of histologically intact tumor tissue

Abstract

Hepatocellular carcinoma (HCC) is one of the most deadly human cancers, but it is very difficult to establish an animal model by using surgical specimens. In the present experiment, histologically intact fresh surgical specimens of HCC were subcutaneously transplanted in non-obese diabetic/severe combined immunodeficienccy (NOD/SCID) mice. The biological characteristics of the original and the corresponding transplanted tumors and cell lines were investigated. The results showed that 5 new animal models and 2 primary cell lines were successfully established from surgical specimens. Hematoxylin-eosin staining showed that xenografts retained major histological features of the original surgical specimens. The two new cell lines had been cultivated for 3 years and successively passaged for more than 100 passages in vitro. The morphological characteristics and biologic features of the two cell lines were genetically similar to the original tumor. The subcutaneous transplant animal models with histologically intact tumor tissue and primary cell lines could be useful for in vivo and in vitro testing of anti-cancer drugs and be ideal models to study various biologic features of HCC.

Keywords: Animal model; hepatocellular carcinoma; subcutaneous transplantation; surgical specimens.

Figure 1

The in vivo growth of subcutaneous transplantation of human HCC surgical specimens in NOD/SCID mice. The cancerous tissues were cut into small pieces of approximately 2 mm³ in size, and four or five pieces of tissue were transplanted subcutaneously into the right flanks of NOD/SCID mice (n=8). Tumor growth was monitored once per week and tumor diameters were measured using vernier calipers. The tumor volume was expressed as x̄±s. A. The animal weight and tumor volume of human HCC patient No.5; B. The animal weight and tumor volume of HCC patient No.10; C. H&E staining of surgical tumor tissue and their orthotopic transplant tumor. The magnification of photograph was 200×

Figure 2

The identification of human HCC cell lines by immunocytochemistry. AFP, Albumin, CK 18, CK 19, Hep Par1, Fibroblast, Oct4, Notch1, CD133, CD90, DLK1 and CD44 were detected by immunocytochemistry, respectively. A. The liver cell associated proteins; B. The liver cancer stem cells associated proteins. The magnification of photographs was 200×

Figure 3

Biological characteristics of the primary cell lines. A. Morphological appearance of each of the established HCC cell lines in culture medium; B. The in vitro growth curves of HCC-LY5 and HCC-LY10 cells. 1×10⁴ cells were seeded in 96-well plates, and cell proliferation was measured by a hemocytometer every 24 h for 7 consecutive days. The results are expressed as x̄±s; C. Transwell migration assay in HCC-LY5 and HCC-LY10 cells. 1×10⁵ cells were seeded into Transwell plates and cultured for 12 and 24 h at 37 °C; D. Transwell Matrigel invasion assay in HCC-LY5 and HCC-LY10 cells. 1×10⁵ cells were seeded into Matrigel-coated Transwell plates and cultured for 24 and 36 h at 37 °C. The cells that had invaded or migrated to the underside of the inserts were then stained with crystal violet. The magnification of photograph was 200×. The results are expressed as the x̄±s. E. The in vivo growth of primary HCC cell line. 2×10⁶ cells were injected subcutaneously into the right flank of nude mice (n=8), tumor growth was monitored twice per week, and tumor diameters were measured using vernier calipers. Tumor volume=1/2 (length × width × height)

Figure 4

Detection of HCC molecular markers by flow cytometry in HCC-LY5 and HCC-LY10 cells. CD133, EpCAM, CD44 and CD24 were detected by flow cytometry. CD45 was detected as a control