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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 μm 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 × 10⁵ cells were implanted orthotopically, by 16 days the cell mass had expanded and had invaded the cortex, whereas when 1 × 10⁵ cells were used, tumor masses were much smaller. We therefore subsequently used 3 × 10⁵ 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 × 10⁵ 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.

Keywords

Adrenal gland Orthotopic Cortex Medulla Tumor Neuroblastoma

Introduction

Cell transplantation has been very valuable in studies of adrenal cell function (6, 7). In immunodeficient mice, transplantation of adrenocortical cells has been ectopic, with formation of tissue structures under the capsule of the kidney or in subcutaneous sites (15, 20, 21, 25). However, an orthotopic cell transplantation model would be valuable for investigation of the biology of both the cortex and the medulla and the interactions between these two components of the gland (3). The site of transplantation, orthotopic versus ectopic, is an important consideration both for studies of normal cell function following transplantation as well as a for a complete understanding of tumor biology (12, 13, 16, 22, 24). In the case of the adrenal gland, the use of immunodeficient mice as the host animal for orthotopic intra-adrenal growth of xenografts presents a particular challenge because of the small size of this organ. Prior studies with neuroblastoma cells have suggested that orthotopic intra-adrenal transplantation is feasible (8, 9). However, in our preliminary studies, we observed that it was very difficult to ensure that cells were confined within the adrenal gland (2). Earlier studies used injection of cells into the retroperitoneal space as a substitute for true intra-adrenal injection (10). It was pointed out in a previous study that, because the adrenal gland is only ~2 mm in diameter in the mouse, leakage of cells during injection is very likely (5). These authors attempted to address this issue by comparing injection into the gland with the results of cells deposited next to the adrenal gland, but they did not solve the problem of confining the cells to the gland. In another study, 2 × 10⁶ neuroblastoma cells were injected through the left adrenal fat pad into the adrenal gland, but tumor growth began in the fat pad and later invaded the adrenal gland (11).

It is therefore evident that previously used methods for orthotopic intra-adrenal injection are unreliable in successfully confining injected cells within the adrenal gland. In the present experiments, we used fibrin clot formation to ensure that leakage of cells from the injection site was minimized during intra-adrenal injection in the mouse. The technique of immobilizing cells with a fibrin clot was first introduced for subrenal capsule cell transplantation (4). An additional benefit of placing cells within a fibrin matrix during transplantation is that it may aid in cell survival and growth. Fibrin and fibrinogen have been shown to have roles in muscle regeneration, wound healing and recovery from peripheral nerve injury (1).

In this study, we demonstrate the optimization of intra-adrenal orthotopic transplantation using both tumorigenic adrenocortical cells and neuroblastoma cells injected in a fibrinogen/thrombin mixture. We optimized the number of cells transplanted so as to produce substantial tumor growth within 15–30 days.

Materials and Methods

Cell Culture

A previously described line of tumorigenic bovine adrenocortical cells (18) was used. Cells were derived by coinfection with two retroviruses (one encoding Ras, SV40 large T antigen and GFP and a second encoding hTERT). Cells were grown as previously described. All cells prior to the transplantation were positive for expression of both SV40 T antigen and GFP (18). The human neuroblastoma cell line SK-N-AS, originally derived from an adrenal neuroblastoma (17), was obtained from the American Type Culture Collection. Cells were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM) and 10% cosmic calf serum (Hyclone Labs). Both types of cells were released from the culture dish by digestion with trypsin/EDTA, pelleted in culture medium, and kept on ice until transplantation. Immediately prior to transplantation, the pellet was embedded in thrombin and fibrinogen as described below. For each animal 1–3 × 10⁵ cells were transplanted in a total volume of no more than 7 μl.

Fibrin Clot Formation

We used fibrin clots to retain cells within the adrenal gland during orthotopic transplantation. They were also used together with fluorescent microspheres in experiments to investigate retention of injected material within the gland. The injected mixture comprised cells or microspheres together with 4 mg/ml bovine fibrinogen and 4 U/ml bovine thrombin. The injection was performed immediately after mixing.

Intra-adrenal Orthotopic Cell Transplantation

RAG2^-/-, γc^-/- mice originally purchased from Taconic (Germantown, NY) were maintained in an animal barrier facility as a breeding colony. Female animals aged over 6 weeks (approx. 20 g body weight) were used in the experiments. Procedures were approved by the Institutional Animal Care Committee and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Under tribromoethanol anesthesia, a longitudinal incision was made with fine scissors in the dorsal skin of the retrocostal area. A 1-cm incision in the lateral body wall was made to open the retroperitoneal space. The left adrenal gland was exteriorized. Immediately following mixing of cells with fibrinogen and thrombin, they were injected into the adrenal gland using a custom-made 50-μl glass syringe with a cemented 1.3-cm 30-gauge blunt needle (Hamilton Co., Reno, NV). Following transplantation of the cells, the adrenal gland was returned to the retroperitoneal space. The body wall was closed with 6–0 nylon sutures and the skin closed with surgical staples. Animals were maintained at 35°C ambient temperature until recovery from the anesthetic. Postoperative care for the animals was previously described (23). Animals were sacrificed at 8, 16, or 26 days.

Fluorescent Microspheres

We used fluorescent polystyrene microspheres (10 μm diameter, excitation 468 nm, emission 508 nm; Thermo Scientific) in order to test retention of injected materials within the adrenal gland. Microspheres (3 × 10⁵) were injected into the adrenal gland as described above for cells. Alternatively, they were suspended in PBS to a total volume of <8 μl and were also injected into the adrenal gland. Immediately after injection of the microspheres, the adrenal gland was photographed in situ under illumination with a 470 nm light source (Lightools Inc).

Histology and Immunohistochemistry

The adrenal gland and neighboring tissues excised from the animals were fixed overnight in 4% paraformaldehyde and treated as described previously (23). Tissue sections (6 μm) were deparaffinized and rehydrated using graded alcohol concentrations. Antigen retrieval was performed by heating to 105°C for 20 min in citrate buffer, followed by cooling to room temperature for 20 min in the same buffer. The sections were then incubated with the first antibody at 4°C for 16 h, after blocking nonspecific binding with horse serum for 15 min. The following primary mouse monoclonal antibodies were used: anti-PCNA (proliferating cell nuclear antigen, clone PC10, DakoCytomation), 1:500 dilution; anti-Simian virus large T antigen (SV40 TAg, clone PAb416, Calbiochem) 1:25 dilution; MAP-2a,b (antimicrotubule associated protein antigen 2a,b, clone AP20, Thermo Scientific), 1:100 dilution. Unbound antibody was removed by washing in PBS at room temperature. A secondary antibody conjugated with biotin (Vector Laboratories, Burlingame, CA) was added to the sections for 15 min. Positive cells were visualized by an avidin-biotin-peroxidase complex (Vector Laboratories) under the light microscope as recommended by the manufacturer. The sections were counterstained with hematoxylin.

Results

Optimizing Orthotopic Cell Transplantation

Despite prior publications on orthotopic cell transplantation in the adrenal gland, our preliminary observations indicated that injection of loose cells in suspension into the adrenal gland resulted in extensive inadvertent loss of cells from the gland during injection. We hypothesized that this resulted from the absence of factors that positively retain the cells within the gland. To address this, we developed cell transplantation using injection of the cells within a mixture of fibrinogen and thrombin, which within about a minute forms a semisolid clot. In order to test the retention of the clot within the adrenal gland, we used brightly fluorescent microspheres that could be readily detected macroscopically under appropriate illumination. We mimicked orthotopic transplantation of cells by injecting 3 × 10⁵ fluorescent microspheres, either as a loose suspension in PBS, or in a fibrinogen/thrombin mixture. Figure 1 shows that, when orthotopic transplantion was modeled using fluorescent microspheres, the use of fibrinogen/thrombin resulted in retention of the fluorescent material entirely within the gland, whereas the use of a suspension had the result that a large fraction of the fluorescent material was extruded to the outside of the gland.

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.

Orthotopic Transplantation of Tumorigenic Adrenocortical Cells

These observations obtained with fluorescent microspheres enabled us to optimize a method for intra-adrenal cell transplantation that did not result in extensive loss of cells during the injection and also assisted us in defining a suitable cell number for orthotopic transplantation. We next considered it to be important to define a minimal number of tumorigenic cells that would initiate tumor development following intra-adrenal transplantation. Using a fibrinogen/thrombin mixture as described above, we compared the results of orthotopic transplantion of either 1 × 10⁵ or 3 × 10⁵ tumorigenic bovine adrenocortical cells expressing SV40 TAg (18) in female immunodeficient (RAG2^-/-, γc^-/-) mice. In preliminary studies, we found that implanted cells were localized at the boundary of the cortex and medulla. When 3 × 10⁵ cells were injected, by 16 days the implanted cells had begun to invade the neighboring medullary tissue (Fig. 2b), whereas this was not observed with 1 × 10⁵ cells (Fig. 2a). The expression of SV40 TAg by the transplanted cells greatly assisted their localization within the adrenal gland. We therefore performed all experiments using 3 × 10⁵ cells. Preliminary studies using 5 × 10⁵ cells showed that it was more difficult to confine the transplanted cells entirely within the adrenal gland (2).

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. Immunopositive cells are indicated in the photographs by a dashed white line. (a, a′) 1 × 10⁵ cells; (b, b′) 3 × 10⁵ cells. Scale bars: 500 μm.

Using 3 × 10⁵ cells for orthotopic transplantation, we then investigated the time course of tumor development within the mouse adrenal gland (Fig. 3). We used both SV40 TAg and PCNA as markers to localize the tumorigenic cells. While PCNA is not specific for the tumorigenic cells, the much greater level of PCNA expressed in the transplanted cells in comparison to the host mouse tissues enabled us to use PCNA as a second marker. At 8 days after transplantation the cells were localized to the border of the cortex and medulla of the gland. At 16 days cells were observed to be invading neighboring tissues, and at 26 days cells had more or less completely replaced the medulla. To varying extents, cells had also invaded the cortex, in some cases almost completely replacing the cortex (Fig. 3).

Figure 3.

Time course of intra-adrenal tumor development of orthotopically transplanted bovine adrenocortical cells expressing SV40 large T antigen. Cells (3 × 10⁵) 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. Scale bars: 500 μm.

Orthotopic Transplantation of Neuroblastoma Cells

We then compared orthotopic growth of neuroblastoma cells with the results we had obtained using tumorigenic adrenocortical cells. SK-N-AS cells are an adrenal neuroblastoma cell line derived from an 8-year-old female patient (17). We transplanted 3 × 10⁵ SK-N-AS cells orthotopically in fibrinogen/thrombin in the left adrenal of female RAG2^-/-, γc^-/- mice. Animals were sacrificed at 16 and 26 days following transplantation. The adrenal gland and surrounding tissues were evaluated macroscopically (Fig. 4). At 16 days the gland was enlarged, while by 26 days the tumor had grown well beyond the gland itself and was surrounding the anterior pole of the kidney. This indicated that the rate of growth of orthotopically transplanted neuroblastoma cells was greater than that of the tumorigenic adrenocortical cell line. We evaluated the pattern of tumor growth within the mouse adrenal gland using immunohistochemistry against a general marker of cells of neuroendocrine origin, MAP-2 (14). At day 16 the transplanted cells had already increased in number and had partly replaced the medulla and begun to invade the cortex (Fig. 5). At 26 days they had completely replaced the cortex and medulla or had expanded beyond the adrenal gland into the periadrenal fat. The remaining cortex was distorted or compressed. At this time point, metastases were also observed in the liver of two of five mice (data not shown), as also previously observed (5).

Figure 4.

Orthotopic transplantation of neuroblastoma cells. Human neuroblastoma cells (SK-N-AS) (3 × 10⁵) 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 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. SK-N-AS cells (3 × 10⁵) 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). Scale bars: 500 μm.

Discussion

In xenograft cancer models in immunodeficient mice, orthotopic cell transplantation is considered to be highly desirable for proper modeling of the behavior of the tumor cells. The microenvironment into which the cells are transplanted may cause the behavior of the cells to differ considerably from that in ectopic sites.

In the case of the adrenal gland, orthotopic transplantation would be valuable for studying the biology of neoplastic cells that arise from either component of the gland, the cortex, and the medulla. Prior observations on orthotopic transplantation of cells into the mouse adrenal gland have shown that there are substantial difficulties in confining the transplanted cells to the interior of the gland. When cells are deliberately or inadvertently placed in the vicinity of the gland, they grow into an extra-adrenal mass (e.g., in the periadrenal fat pad) and then invade the gland itself. However, this pattern of growth may not resemble the pattern of primary tumor growth within the gland. Thus, a method is needed in which tumor growth is reliably initiated within the adrenal gland. Here we show that transplantation of cells in a fibrinogen/thrombin mixture, which clots immediately after injection, confines cells within the gland. We tested two lines of neoplastic adrenal cells: an experimentally produced bovine adrenocortical tumor cell line (18) and a human neuroblastoma cell line. Neuroblastoma is thought to arise from neoplastic transformation of cells of the neural crest, which give rise to the adrenal medulla, among other tissues (19). Both cell types formed expanding invasive tumor masses when small numbers of cells were implanted orthotopically in the mouse adrenal gland.

Prior studies and our own preliminary investigations showed two principal problems for intra-adrenal orthotopic cell transplantation. First, when cells are injected through the capsule of the gland into the center, it is impossible to avoid an extrusion of cells out of the gland via the needle track, either during the injection itself or immediately following injection. Here we demonstrated this problem by using brightly fluorescent microspheres that can be readily detected in small numbers. Second, prior studies have used a minimum of 6 × 10⁵ cells for intra-adrenal injections. Our preliminary studies showed that this exceeds the capacity of the adrenal gland to contain the injected cells. Here we showed that the use of a smaller number of cells, 3 × 10⁵, combined with the use of a fibrin clot to retain the injected cells within the gland, provides a true orthotopic model in which tumor formation originates from within the adrenal gland. A further reduced cell number (1 × 10⁵) also resulted in intra-adrenal tumor formation but with a more extended time course. For practical studies of orthotopic tumor formation in <30 days, 3 × 10⁵ cells is optimal. This was shown for both the adrenocortical tumor cell line and neuroblastoma cells.

In the present experiments, we did not extend our studies to functional assessment of the transplanted cells. In prior studies of ectopic adrenal cell transplantation, we showed that the transplanted cells are capable of secretion of appropriate hormones into the bloodstream of the host animal (20, 21). It would be of interest to examine whether hormone secretion from orthotopic transplants differs from that of ectopic transplants. However, a significant difficulty is that the host animals' adrenal glands may be completely removed in the ectopic transplant experiments, thus eliminating endogenous sources of adrenocortical hormones, whereas in the orthotopic transplants, host adrenal tissue remains in situ. If these issues can be solved, studies of hormone secretion from orthotopic adrenal cell transplantation should be very valuable.

In summary, the present results demonstrate the feasibility of an orthotopic cell transplantation method for the mouse adrenal gland. In the future, these studies can be extended to the study of the behavior of nonneoplastic cells of cortical or medullary origin implanted within the gland, or to study various aspects of the interaction between cells of the cortex and medulla (3).

Footnotes

Acknowledgments

This work was supported by grants from the Wilhelm Sander-Stiftung, Germany (to S.R.B.), and grants from the National Institute on Aging, Owens Medical Foundation, Shelby Rae Tengg Foundation, and Glenn Foundation for Medical Research (to P.J.H.).

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Optimizing Orthotopic Cell Transplantation in the Mouse Adrenal Gland

Abstract

Keywords

Introduction

Materials and Methods

Cell Culture

Fibrin Clot Formation

Intra-adrenal Orthotopic Cell Transplantation

Fluorescent Microspheres

Histology and Immunohistochemistry

Results

Optimizing Orthotopic Cell Transplantation

Orthotopic Transplantation of Tumorigenic Adrenocortical Cells

Orthotopic Transplantation of Neuroblastoma Cells

Discussion

Footnotes

Acknowledgments

References