Optimizing orthotopic cell transplantation in the mouse adrenal gland

Abstract

Orthotopic cell transplantation models are important for a complete understanding of cell-cell interactions as well as tumor biology. In published studies of orthotopic transplantation in the mouse adrenal gland, human neuroblastoma cells have been shown to invade and occupy the adrenal, but in these investigations a true orthotopic model was not established. Here we show an orthotopic model in which transplanted cells are retained within the adrenal gland by formation of a fibrin clot. To establish an appropriate technique, we used brightly fluorescent 10 microm polystyrene microspheres injected into the mouse adrenal gland. In the absence of fibrinogen/thrombin for clot formation, much of the injected material was extruded to the outside of the gland. When the microspheres were injected in a fibrinogen/thrombin mixture, fluorescence was confined to the adrenal gland. As a model neoplastic cell originating from the cortex of the gland, we used a tumorigenic bovine adrenocortical cell line. When 3 x 10(5) cells were implanted orthotopically, by 16 days the cell mass had expanded and had invaded the cortex, whereas when 1 x 10(5) cells were used, tumor masses were much smaller. We therefore subsequently used 3 x 10(5) cells. When mice were sacrificed at different time points, we found that tumor growth resulting was progressive and that by 26 days cells there was extensive invasion into the cortex or almost complete replacement of the cortex with tumor cells. As a model neoplastic cell of neural crest origin, we used SK-N-AS human neuroblastoma cells. Orthotopic transplantation of 3 x 10(5) cells resulted in extensive invasion and destruction of the gland by 26 days. In summary, the present orthotopic model for intra-adrenal cell transplantation is valuable for investigation of growth of neoplastic cells of both cortical and medullary origin and should be useful for future studies of cortex-medulla interactions.

Figure 1

Testing intra-adrenal orthotopic cell transplantation using fluorescent microspheres. Successful orthotopic transplantation was tested using fluorescent microspheres as a surrogate for mammalian cells. The left mouse adrenal gland was exposed and 3 × 10⁵ green fluorescent microspheres (10 µm diameter) were injected into the gland either as a suspension in PBS or in a thrombin/fibrinogen mixture, which clots immediately after transplantation. Under illumination with a blue light source (470 nm) fluorescent microspheres could be observed either within the gland or externally in the vicinity of the gland. (a, b) Microsphere suspension; (c, d) microspheres injected in thrombin/fibrinogen mixture.

Figure 2

Optimization of cell number for intra-adrenal cell transplantation. Either 1×10⁵ or 3×10⁵ tumorigenic bovine adrenocortical cells expressing SV40 large T antigen were transplanted orthotopically in the left adrenal gland of female RAG2^−/−, γc^−/− mice. The animals were sacrificed at 16 days following transplantation. Tissues were fixed, embedded in paraffin and sectioned. The transplanted cells were localized by immunohistochemistry using anti-SV40 T antigen and a peroxidase-conjugated secondary antibody. Sections were lightly counterstained with hematoxylin. Immuno-positive cells are indicated in the photographs by a dashed white line. (a, b) 1×10⁵ cells; (c, d) 3×10⁵ cells. Bars = 500 µm.

Figure 3

Time-course of intra-adrenal tumor development of orthotopically transplanted bovine adrenocortical cells expressing SV40 large T antigen. 3×10⁵ cells were transplanted in the left adrenal gland of female RAG2^−/−, γc^−/− mice. The animals were sacrificed at 8, 16 and 26 days following surgery. The tissues were fixed, embedded in paraffin and sectioned for histological and immunohistological analyses in order to evaluate the behavior of the transplanted cells over time. Sections were stained with hematoxylin and eosin (H&E), and transplanted cells were visualized with anti-SV40 T antigen and additionally with anti-PCNA (proliferating cell nuclear antigen). Examples of transplants at 8 and 16 days are shown, and two examples at 26 days. Bars = 500 µm.

Figure 4

Orthotopic transplantation of neuroblastoma cells. 3×10⁵ human neuroblastoma cells (SK-N-AS) were injected in a thrombin/fibrinogen mixture into the left adrenal gland of female RAG2^−/−, γc^−/− mice. In order to evaluate the rate of growth of the tumors formed from the transplanted cells, animals were sacrificed at 16 and 26 days following surgery. The macroscopic appearance of the kidney and adrenal gland is shown. A clear expansion of the tumor formed from the neuroblastoma cells is observed as an enlargement of the the adrenal gland a 16 days (a) and as an expansion outside of the gland at 26 days (b).

Figure 5

Time-course of intra-adrenal tumor development of orthotopically transplanted neuroblastoma cells. 3×10⁵ SK-N-AS cells were transplanted in the left adrenal gland of female RAG2^−/−, γc^−/− mice. The animals were sacrificed 16 and 26 days following surgery. The tissues were fixed, embedded in paraffin and sectioned for histological and immunohistological analyses in order to evaluate the behavior of the transplanted cells over time. Sections were stained with hematoxylin and eosin (H&E), and transplanted neuroblastoma cells were visualized using an antibody against microtubule-associated protein 2 (MAP-2). Bars = 500 µm.