Human Renal Cell Carcinoma in the “Nude” Mouse : Long-term Observations

Introduction

Renal cell carcinoma differs from other malignancies in many respects. The number of affected male individuals is three fold that of women (Bennington, 1973). The tumor grows slowly and in some cases, metastases regress after resection of the primary tumor (Goodwin et al., 1967; Garfield and Kennedy, 1972). Women, in whom a renal cell carcinoma has been diagnosed and removed during pregnancy, seem to survive longer (Fetter and Koppel, 1963; Grabstald, 1964; Anderson and Atkinson, 1973). Whether these characteristics would manifest themselves also under experimental conditions, was largely unknown. Therefore, 17 human renal cell carcinomas were implanted into thymus-dysgenetic male and female “nude” mice. This animal has been found to be a suitable model for study of malignant tumors under defined experimental conditions. The morphological findings in both the primary tumors and in the successive transplants are reported.

Materials and Methods

Animals: Nude (nu/nu) mice with the genetic background Balb/c were bred under SPF-conditions and transfered, when 4-6 weeks old, to a conventional animal room for the transplantation experiments.

Transplantations

Small fragments (approx. 1 X 1 X 5 mm) of the renal cell carcinomas were washed in phosphate-buffered saline containing 0.5% (w/v) Minocyclin® and implanted subcutaneously into the scapular region of 3-8 male and female “nude” mice. The animals were killed when the implants regressed or grew. The growing tumors were transfered to additional mice.

Morphology

Both the primary tumors and the transplants were studied by light- and electron microscopy. For light microscopy, the tissues were fixed in Bouin's solution, embedded in paraffine and stained with either hematoxilin and eosin or Azan or PAS. For electron microscopy, small fragments of the tumors were prefixed in 2.5% glutaral-dehyde in 0.1 M cacodylate buffer (pH 7.2) and postfixed in 1% osmium tetroxide (buffered to pH 7.2 with 0.1 M S-collidine). After dehydratation in an ethanol series, the material was embedded in Epon. Section contrast was enhanced by uranyl acetate and lead citrate.

In vitro studies

Fragments of the 10th nude mouse-passage of the tumor H 1077 were minced under aseptic conditions in HAM-medium containing 15% calf serum and trypsinized. Cell suspensions were washed and dispersed in 30 ml Falcon tissue culture flasks or 30 mm Petri dishes. After 1-3 in vitro-passages, the cells were either processed for light- and electron microscopy or re-injected into nude mice.

Results

Nine implanted renal cell carcinomas (4 clear cell-type, 2 granular-cell type, and 3 mixed-cell type, see Table I) were found to regress in the nude mice after variable periods of time. The subcutaneous residual nodules consisted of a dense collagenous connective tissue, surrounded by a few granulocytes, macrophages and - in some cases - by small groups of lymphocytes. No tumor cells were found in these nodules.

Seven tumor implants (4 clear-cell type, 1 granular-cell type and 2 mixed-cell type) were found to persist in the animals for up to 112 days after inoculation (Table I). Light- and electron microscopically, the nodules contained small groups of tumor cells, the morphology of which was very similar to that of the primary tumors (Fig. 1 and 2). In the transplants, however, the tumor cells lacked often a well defined basal lamina (Fig. 2 a). Many of the capillaries of the surrounding connective tissue were lined by a fenestrated endothelium (Fig. 2 b).

One mixed-type renal cell carcinoma, which was characterized by very pleomorphic cells (Fig. 3) and by a high mitotic activity, was found to proliferate rapidly in the subcutis of the nude mice. The tumor could be serially transplanted for more than 24 mouse passages (998 days). Throughout the passages, the transplants grew well in both male and virgin female mice. Metastases of tumor cells were never detected in lymph nodes or other organs. In general, the transplants maintained the morphological characteristics of the primary tumor. However, they contained a higher number of clear cells (Fig. 5 b) as compared to the primary implants, and the granular cells displayed more lysosomes and micropinocytotic vesicles (Fig. 6 a and c). The microvilli at the apical cell surface were often found to form a brush border (Fig. 6 a). In contrast, a regression or even disappearance of the tumor was noted in female nude mice which had become pregnant shortly before or after transplantation. In these animals, a dense connective tissue scar with a few mononuclear cells was usually the only remnant of the transplanted tumor at the inoculation site.

Cells obtained by trypsination of tumor fragments from the 10th nu/nu mouse passage formed, in vitro, monolayers which consisted of epitheloid and fibroblastoid elements (Fig. 9). The cultures were subcultivated for up to 3 passages and their cells re-injected into nude mice. Within a few weeks, the animals developed typical tumor nodules, the morphology of which was identical to those of the “conventional” transplants.

Discussion

In the experiments reported, in which 17 human renal cell carcinomas were implanted subcutaneously into thymusdysgenetic nude mice with the genetic background Balb/C, 9 tumors were found to disappear completely, and to be replaced by connective tissue. In 7 other cases, groups of morphologically intact tumor cells could be observed as long as 112 days after implantation. In these nodules, no tumor cell mitoses were found. Only one very pleomorphic, mixed-cell type renal carcinoma displayed rapid growth and could be serially transplanted over 24 nude mouse-passages. These observations are in contrast to those of Katsuoka et al. (1976), who reported the successful transplantation of 5 out of 9 human renal cell carcinomas in nude mice. Whether this difference in tumor takes reflects differences in the genetic background of the nude mice used, or is due to the breeding or maintenance conditions of the animals, is at present unknown. Although still other factors cannot be excluded, it is interesting to note that the only proliferating tumor in our series was a poorly differentiated renal cell carcinoma with a high mitotic activity. This is in accordance with findings reported by Merenda et al. (1975). These authors observed that only undifferentiated endometrial carcinomas would grow in the nude mouse, whereas differentiated ones persisted in the animals without proliferation.

Our observations that tumor fragments regularly regressed and eventually disappeared when transplanted in female nude mice which became pregnant during the transplantation experiments need further investigation. Whether this elimination is caused by changes in the endocrine and/or immune system of the animals during pregnancy, is presently unknown. In this context, it is interesting to note that women, in whom a renal cell carcinoma was removed during pregnancy, seem to display a prolonged survival (Fetter and Koppel, 1963; Grabstald, 1964; Anderson and Atkinson, 1973).