Young Nude Mice Research Articles

Nutritional and supranutritional levels of selenate differentially suppress prostate tumor growth in adult but not young nude mice

The inhibitory effect of oral methylseleninic acid or methylselenocysteine administration on cancer cell xenograft development in nude mice is well characterized; however, less is known about the efficacy of selenate and age on selenium chemoprevention. In this study, we tested whether selenate and duration on diets would regulate prostate cancer xenograft in nude mice. Thirty-nine homozygous NU/J nude mice were fed a selenium-deficient, Torula yeast basal diet alone (Se−) or supplemented with 0.15 (Se) or 1.0 (Se+) mg selenium/kg (as Na2SeO4) for 6 months in Experiment 1 and for 4 weeks in Experiment 2, followed by a 47-day PC-3 prostate cancer cell xenograft on the designated diet. In Experiment 1, the Se− diet enhanced the initial tumor development on days 11–17, whereas the Se+ diet suppressed tumor growth on days 35–47 in adult nude mice. Tumors grown in Se− mice were loosely packed and showed increased necrosis and inflammation as compared to those in Se and Se+ mice. In Experiment 2, dietary selenium did not affect tumor development or histopathology throughout the time course. In both experiments, postmortem plasma selenium concentrations in Se and Se+ mice were comparable and were twofold greater than those in Se− mice. Taken together, dietary selenate at nutritional and supranutritional levels differentially inhibit tumor development in adult, but not young, nude mice engrafted with PC-3 prostate cancer cells.

Effect of dietary selenium on T cell immunity and cancer xenograft in nude mice

Selenium is known to regulate carcinogenesis and immunity at nutritional and supranutritional levels. Because the immune system provides one of the main body defenses against cancer, we asked whether T cell immunity can modulate selenium chemoprevention. Twenty-four homozygous NU/J nude mice were fed a selenium-deficient, Torula yeast basal diet alone or supplemented with 0.15 or 1 mg Se/kg (as Na2SeO4) for 8 months in Experiment 1 and 11 weeks in Experiment 2. Mice were injected with PC-3 prostate cancer cells (8–20 × 105 cells/site, 2 sites/mouse), followed by a 7-week tumor development. The athymic, immune-deficient nude mice are known to gradually develop CD8 killer, but not CD4 helper, T cells. Here we show that, prior to xenograft, CD4 and CD8 T cell titers are greater in 28-week old nude mice fed a high selenium diet than the other two diets. Tumor development in adult nude mice (Experiment 1) was suppressed whereas in young nude mice (Experiment 2) was promoted by feeding a high selenium diet. Dietary selenium deficiency does not affect tumor weight. After xenograft, dietary selenium status does not affect levels of CD4 and CD8 T cells in adult nude mice in Experiment 1, while high selenium resulted in significant decrease in CD4 T cells in young nude mice in Experiment 2. Taken together, there is an opposing role of excessive selenium on tumor xenograft development in adult and young nude mice carrying differential T cell profiles. Grant Funding Source: UMCP

Realtime photoacoustic microscopy of murine cardiovascular dynamics.

Non-invasive visualization of cardiovascular dynamics in small animals is challenging due to their rapid heart-rates. We present a realtime photoacoustic imaging system consisting of a 30-MHz ultrasound array transducer, receive electronics, a high-repetition-rate laser, and a multicore-computer, and demonstrate its ability to image optically-absorbing structures of the beating hearts of young athymic nude mice at rates of approximately 50 frames per second with 100 microm x 25 microm spatial resolution. To our knowledge this is the first report of realtime photoacoustic imaging of physiological dynamics.

Breast cancer metastasis to bone: evaluation of bioluminescent imaging and microSPECT/CT for detecting bone metastasis in immunodeficient mice

This study sought to determine if weekly X-ray exposure affected breast cancer cell metastasis to bone and to also evaluate the use of bioluminescent imaging (BLI) and microSPECT for detection of metastatic bone lesions. Five week old nude mice were randomly assigned to the CT exposed (n = 7) and no CT exposure (n = 6) treatment groups. Mice received an intracardiac injection of MDA-MB-435 human breast cancer cells transduced with luciferase, or a sham injection (saline). The CT exposed group of mice received CT irradiation once a week for 5 weeks. All mice underwent weekly BLI and select mice received Tc-99m-MDP followed by microSPECT imaging after 5 weeks. Pathological evaluation and histomorphometry were used to assess the affect of CT X-rays on bone metastasis and to evaluate BLI. BLI results found no significant difference in metastasis between animals that received CT and those that did not (P > 0.05); however, histomorphometry of the knee joints revealed a significant increase (P = 0.029) in tumor area of the leg bones in mice that received CT exposure (60% +/- 7%) compared to animals that did not receive CT scans (33% +/- 8%). Compared to histological analysis, BLI of the leg and spine was determined to have excellent sensitivity (100%), good specificity (80-90%) and accuracy (90-96%), a positive predictive value of 81-93% and a 100% negative predictive value. Thus, multi-modality imaging techniques can be very useful for monitoring bone metastasis, however microCT X-rays should be used judiciously in order to limit irradiation that may stimulate increased metastasis to specific regions of the skeleton. MicroSPECT imaging did not detect metastatic lesions in the legs of these young nude mice.

Virus-Induced Demyelination in Nude Mice Is Mediated by γδ T Cells

Infection of mice with mouse hepatitis virus (MHV), strain JHM, results in acute and chronic demyelination with many similarities to the human disease multiple sclerosis. This pathological process is primarily T cell-mediated and MHV infection of mice lacking B and T cells does not result in demyelination. In apparent contradiction to these results, robust demyelination is detected in MHV-infected young nude (athymic) mice. Herein, we show that demyelination in nude mice was mediated by gamma delta T cells. These cells, but not conventional CD4 or CD8 alpha beta T cells, were detected in the central nervous system of MHV-infected nude mice and their depletion with neutralizing antibody resulted in an 80% reduction in demyelination. These results show, for the first time, that gamma delta T cells can substitute for alpha beta T cells in a virus model of demyelination and further support a pathological role for gamma delta T cells in patients with multiple sclerosis.

Actions of bisphosphonate on bone metastasis in animal models of breast carcinoma.

Bone, which abundantly stores a variety of growth factors, provides a fertile soil for cancer cells to develop metastases by supplying these growth factors as a consequence of osteoclastic bone resorption. Accordingly, suppression of osteoclast activity is a primary approach to inhibit bone metastasis, and bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used for the treatment of bone metastases in cancer patients. To obtain further insights into the therapeutic usefulness of BP, the authors studied the effects of BP on bone and visceral metastases in animal models of metastasis. The authors used two animal models of breast carcinoma metastasis that they had developed in their laboratory over the last several years. One model uses female young nude mice in which inoculation of the MDA-MB-231 or MCF-7 human breast carcinoma cells into the left cardiac ventricle selectively develops osteolytic or osteosclerotic bone metastases, respectively. Another model uses syngeneic female mice (Balb/c) in which orthotopic inoculation of the 4T1 murine mammary carcinoma cells develops metastases in bone and visceral organs including lung, liver, and kidney. BP inhibited the development and progression of osteolytic bone metastases of MDA-MB-231 breast carcinoma through increased apoptosis in osteoclasts and breast carcinoma cells colonized in bone. In a preventative administration, however, BP alone increased the metastases to visceral organs with profound inhibition of bone metastases. However, combination of BP with anticancer agents such as uracil and tegafur or doxorubicin suppressed the metastases not only in bone but also visceral organs and prolonged the survival in 4T1 mammary tumor-bearing animals. Of interest, inhibition of early osteolysis by BP inhibited the subsequent development of osteosclerotic bone metastases of MCF-7 breast carcinoma. These results suggest that BP has beneficial effects on bone metastasis of breast carcinoma and is more effective when combined with anticancer agents. They also suggest that the animal models of bone metastasis described here allow us to design optimized regimen of BP administration for the treatment of breast carcinoma patients with bone and visceral metastases.

Differential influence of a thymic extract on alpha- and beta-adrenoceptors of mouse brain cortex.

Experimental findings support the hypothesis that the thymus plays an important role in maintaining the efficient network of interactions among immune, endocrine, and nervous functions.14 One of the numerous targets of thymic action is the adrenergic system. We previously found a restoring influence of a thymus graft on the alterations of some P-adrenergic responsiveness in young athymic nude and old normal mice.5 Both the alterations and the thymus-induced restoring actions involve modifications of P-adrenergic receptors PARS).^.' Thymus control was also demonstrated as effective in the brain cortex of old mice where it exerts selective action on PIARs altered during aging, whereas it does not alter P2AR density which does not show age-related

Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation.

Mouse gut intraepithelial lymphocytes (IEL) consist mainly (90%) of two populations of CD8+ T cells. One bears heterodimeric alpha/beta CD8 chains (Lyt-2+, Lyt-3+), a T cell receptor (TCR) made of alpha/beta chains, and is Thy-1+; it represents the progeny of T blasts elicited in Peyer's patches by antigenic stimulation. The other bears homodimeric alpha/alpha CD8+ chains, contains no beta chain mRNA, and is mostly Thy-1- and TCR-gamma/delta + or -alpha/beta +; it is thymo-independent and does not require antigenic stimulation, as shown by its presence: (a) in nude and scid mice; (b) in irradiated and thymectomized mice repopulated by T-depleted bone marrow cells bearing an identifiable marker; (c) in thymectomized mice treated by injections of monoclonal anti-CD8 antibody, which lead to total depletion of peripheral CD8+ T lymphocytes; and (d) in germ-free mice and in suckling mice. In young nude mice, alpha/alpha CD8 chains, CD3-TCR complexes, and TCR mRNAs (first gamma/delta) are found on IEL, while they are not detectable on or in peripheral or circulating lymphocytes or bone marrow cells. IEL, in contrast to mature T cells, contain mRNA for the RAG protein, which is required for the rearrangement of TCR and Ig genes. We propose that the gut epithelium (an endoderm derivative, as the thymic epithelium) has an inductive property, attracting progenitors of bone marrow origin, and triggering their TCR rearrangement and alpha/alpha CD8 chains expression, thus giving rise to a T cell population that appears to belong to the same lineage as gamma/delta thymocytes and to recognize an antigenic repertoire different from that of alpha/beta CD8+ IEL.

Characterization of Vβ‐bearing cells in athymic (nu/nu) mice suggests an extrathymic pathway for T cell differentiation

In the present article, the expression of the T cell receptor (TcR) beta chain and other T cell molecules was evaluated in surface immunoglobulin-negative spleen cell populations of young and old BALB/c and C57BL/6 nude mice, using a panel of monoclonal antibodies. The results obtained show that in young nude mice, most Thy-1high cells do not express other T cell markers. These mice have, however, a sizable population of Thy-1low cells with the same phenotype of alpha/beta+, CD4-CD8- thymocytes or MRL/lpr peripheral T cells, expressing predominantly genes of the V beta 8 family. The evolution of alpha/beta+ cells in aging nudes is strongly suggestive of an extrathymic pathway of differentiation of these cells since (a) the acquisition of high density TcR and CD3, as well as Thy-1 or CD4CD8 antigens at the cell surface of nude V beta+ T cells is not simultaneous; (b) alpha/beta+ cells in nude mice co-express other T cell markers at random and, even in old mice, they never completely resemble to the predominant high Thy-1+ CD3+ TcR alpha/beta+, CD4+CD8+ cells of euthymic controls; and (c) BALB/c nude T cells express V beta 11 genes, that are deleted in euthymic BALB/c mice. This latter finding may also indicate differences in the mechanisms of selection of T cells specificities in the thymus vs. the peripheral pools.

The nude mouse as a model for the study of human pancreatic cancer

The purpose of this study was to characterize an in vivo model of human pancreatic cancer suitable for chemotherapy and immunotherapy studies. In this study we report a 2-year experience in growing the MIA PaCa-2 (CRL 1420) human pancreatic cancer cell line in 92 adult (8 weeks old) and 256 young (3–6 weeks old) nude mice. Ten million tumor cells were transplanted into orthotopic (duodenal lobe of the pancreas) and/or heterotopic positions (hepatic and subcutaneous) and data on operative mortality, effect of total body irradiation (TBI), tumor growth kinetics, and survival are presented comparing the two age groups. Operative mortality was due to anesthetic intolerance which was higher in the young mouse population (13.4% versus 5.7%). Adult mice withstood TBI (500 rad) without mortality but young mice were highly sensitive to radiation damage and their maximum tolerated dose (LD 50) was 425–450 rad. Subcutaneous tumors grew significantly more often in young compared to adult animals (97.9% versus 69%) and this finding was not affected by TBI (96.9% versus 75%), though tumors did appear more quickly after TBI. An average of 14.7 ± 2.8 days was required for the subcutaneous tumors to become macroscopically apparent in the adult population compared with 3.1 ± 0.8 days in the young mice. The largest subcutaneous tumor diameter 28 days following tumor implant averaged 9.3 ± 0.6 mm in the young animals and 5.5 ± 1.7 mm in the adult population ( P < 0.01). Treatment of young mice with human recombinant interleukin-2 (IL-2) (10,000 Units twice a day for 28 days) produced a 27% decrease in tumor growth. This effect was abolished by prior irradiation of the young mice with 375 rad TBI. Pancreatic tumor growth also occurred more consistently in young than in adult animals (91.2% versus 64.3%) and irradiation did not affect pancreatic tumor take in either group. Occasionally intrapancreatic tumor growth was associated with liver metastases in animals that were killed after 28 days (17.8% in young and 22.2% in adult animals). However, when more than 45 days elapsed before sacrificing the animals, the incidence of hepatic metastases increased to 57.1%. This was slightly less than the incidence of hepatic lesions found after direct injection of cancer cells into the liver by portal vein injection (71.4%). Direct extension of tumor into surrounding tissues was common with frequent involvement of the duodenum (83.7%), kidneys (30.6%), and other intraabdominal organs (43.9%). Survival was significantly longer in adult compared to young mice. All young mice that grew intrapancreatic tumors died within 3 months from the injection of tumor cells, while about 50% of the adult animals with documented pancreatic tumors survived up to 5 months. The model is an efficient means for the in vivo study of human pancreatic cancer and the autologous anti-tumor response can be controlled by the use of young and irradiated nude mice by methods described in this paper.

Evidence for functional heterogeneity in vesicular stomatitis virus (VSV)-induced helper T cells

Syngeneic memory cells can be stimulated to yield a secondary immune response after their transfer into irradiated euthymic recipients as well as into young thymusless nude mice. It is shown that nude mice older than twelve weeks of age are not permissive towards memory cell activation as it is found in non-irradiated euthymic animals. This barrier to isogeneic or congeneic cells seems to be caused by a pool of cyclophosphamide-sensitive cells. Since young nude mice could be rendered as unpermissive as older nude mice by pretreatment with either PNA-agglutinable thymus cells or nylon-wool passed spleen cells, it is suggested that an increased number of precursor T cells in older nude mice might induce this effect. Further experiments with monoclonal antibodies against the Lyt-1, Lyt-2, and L3T4 marker on T cells indicate that T-helper/inducer activity might be required to establish the ≪isogeneic barrier≫ in nude mice.

Thymus-dependent and thymus-independent developmental pathways for peripheral T cell receptor-gamma delta-bearing lymphocytes.

To elucidate the developmental pathways of T cells that bear TCR gamma delta, we have analyzed the kinetics of expression and biochemical characteristics of gamma delta receptors in the thymus and spleen of normal and athymic (nude) mice, as well as nude mice engrafted with neonatal thymuses. TCR gamma delta-bearing thymocytes and splenocytes have a CD4-8- phenotype, and both populations express products of the C gamma 1 locus. TCR gamma delta-bearing cells develop in the thymus before their appearance in the spleen. Young nude mice have no detectable TCR gamma delta-bearing cells in their spleens. When young nude mice are given thymus grafts, TCR gamma delta-bearing cells of host origin first develop in the engrafted thymus, followed by their appearance in the spleen. In the absence of a thymus graft, the spleens of old nude mice eventually develop small numbers of TCR gamma delta + cells, as well as TCR alpha beta + cells. These results demonstrate that there is a major thymic-dependent pathway for TCR gamma delta expression, as well as a minor thymic-independent pathway seen in older nude mice. The development of TCR gamma delta + cells in the thymus before their appearance in the spleen, both in normal ontogeny as well as in the thymus-engrafted nude mouse model, suggests that thymic TCR gamma delta + cells are precursors of the thymus-dependent population of peripheral TCR gamma delta + cells.

The Transplantation Barrier of Nude Mice

Syngeneic memory cells can be stimulated to yield a secondary immune response after their transfer into irradiated euthymic recipients as well as into young thymusless nude mice. It is shown that nude mice older than twelve weeks of age are not permissive towards memory cell activation as it is found in non-irradiated euthymic animals. This barrier to isogeneic or congeneic cells seems to be caused by a pool of cyclophosphamide-sensitive cells. Since young nude mice could be rendered as unpermissive as older nude mice by pretreatment with either PNA-agglutinable thymus cells or nylon-wool passed spleen cells, it is suggested that an increased number of precursor T cells in older nude mice might induce this effect. Further experiments with monoclonal antibodies against the Lyt-1, Lyt-2, and L3T4 marker on T cells indicate that T-helper/inducer activity might be required to establish the ≪isogeneic barrier≫ in nude mice.

Functional alpha and beta T cell chain receptor messages can be detected in old but not in young athymic mice.

The expression and sequences of T cell antigen receptor (TcR) alpha and beta-chain genes were investigated in the spleens from congenitally athymic (nude) mice. A small number of Thy-1+ cells (approximately 7.0%) was found in nylon wool-enriched spleen cells from young (8 weeks old) nude mice but no L3T4 or Lyt-2 surface antigens were detected. These nude mice expressed only a low level of 1.0-kb beta-chain mRNA and little or no alpha-gene transcripts. On the other hand the number of Thy-1+ spleen cells increased slightly (to 24%) in old (20 weeks old) nude mice and a small number of L3T4+ (10%) and/or Lyt-2+ (5%) cells were detectable. "Full length" 1.7-kb alpha-chain and 1.3-kb beta-chain messages could be found in nylon wool-enriched spleen cells from old nude mice. Sequence analysis of the cDNA revealed that no functional alpha and beta-chain genes can be detected in the spleen cells of young athymic mice while some of the old mice with nu/nu genotype are composed of completely rearranged V-(D)-J-C-gene segments which encode potentially functional proteins. Interestingly, two of three independent cDNA clones encoding the alpha chain used the same V alpha and J alpha-gene segments. These results suggest that extrathymic TcR alpha and beta-chain gene rearrangements do occur, though slowly, to some extent, in nude mice and may be responsible for the limited antigen and/or H-2-related T cell functions in these old athymic mice. The data further suggests that the TcR repertoire in athymic mice may also be limited.

T cell involvement in the decrease of antigen-responsive B cells in aged mice.

The role of T cells in the reduced frequency of splenic B cells specific for several antigens in aged mice was studied by assessing B cell responsiveness in (a) aged nude mice and (b) irradiated young mice repopulated with splenic B cells or with Ig- bone marrow cells from young mice and T cells from aged vs. young mice. Using the fragment culture technique to assess B cells specific for 2,4-dinitrophenyl (DNP) and for (4-hydroxy-3,5-dinitrophenyl) acetyl, we found that the frequency of responsive splenic B cells in aged BALB/c nude mice was very similar to that of young nude mice. In addition, we found that in chimeric mice constructed with either bone marrow or splenic B cells from young mice and T cells from aged mice the frequency of DNP-specific splenic B cells was significantly lower than that in control chimeras constructed with T cells from young mice. These results indicate that T cells from aged mice can down regulate B cell responsiveness and that a mature, naive B cell may be its possible target. The results of both experimental approaches are consistent with a role for T cells in the regulation of responsive B cells in aging.

L-Ascorbic acid-induced DNA strand breaks and cross links in human neuroblastoma cells

The effect of high concentrations of l-ascorbic acid on the in vivo and in vitro growth of human neuroblastoma has been investigated. Directly implemented into cell culture it decreased the DNA, RNA and protein synthesis, and mitosis of neuroblastoma cells, without affecting normal neuronal cells. In vivo treatment of young nude mice bearing human neuroblastoma with 500 mg/kg l-ascorbic acid for the first 10 days markedly inhibited the growth of tumor mass. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in tumor cells treated with 1 × 10 −4 M l-ascorbic acid for 2 h. DNA-DNA and DNA-protein cross links in cells treated with l-ascorbic acid were revealed by the proteinase potassium assay. The results indicated that l-ascorbic acid can be a very effective and selective agent against human neuroblastoma.

Factors influencing phenotypic diversity of human prostate carcinoma cells metastasizing in athymic nude mice.

Three related human prostate carcinoma cell lines, PC-3, 1-LN-PC-3-1A (1-LN), and 1-LN-PC-3-1A clone 4 (clone 4) were compared in terms of relative metastatic capacity in adult and young male nude mice. Only 1-LN produced lung metastases after intravenous injection into both 6- to 8-week-old and 4-week-old nude mice, as well as in mice injected intraperitoneally. The extent of the phenotypic diversity exhibited by these human prostate tumor lines was influenced by inherent dissemination ability, age of the host, and route of injection. These lines provide a useful system for the analysis of the biology of human tumor metastasis in nude mice.

Frequency and specificity of precursors of interleukin 2-producing cells in nude mice.

The frequency and specificity of precursors of interleukin 2-producing cells (IL 2-P) in congenitally athymic (nude) N:NIH(s)II mice was investigated. IL 2-P were detected and quantitated in a sensitive limiting dilution microassay in which Lyt-2-depleted lymphoid cell populations were first cultured for 12 days with irradiated allogeneic (DBA/2) stimulating cells and a source of IL 2 and then washed and restimulated with irradiated T cell-depleted stimulating cells for an additional 24 hr. Supernatants from restimulated cultures were assayed for IL 2 activity on CTLL indicator cells, and IL 2-P frequencies were calculated. The results indicated that IL 2-P were undetectable in young (6-wk-old) nude mice, but increased in frequency with age to eventually reach levels five to 10-fold lower than their euthymic (nu/+) littermates. In specificity studies, microcultures established originally with limiting numbers of nude or nu/+ responding cells and DBA/2 stimulating cells were split into three aliquots and restimulated with T cell-depleted stimulating cells of DBA/2, BALB/c, or C57BL/6 origin. Analysis of IL 2 production in these restimulated microcultures clearly demonstrated different patterns of cross-reactivity in individual nude mice that were not seen in nu/+ controls. These results are discussed in the context of a model proposing that the T cell repertoire in athymic mice is oligoclonal in nature.

Human monoclonal antibody to tumor-associated ganglioside GD2: Suppressed growth of human melanoma in nude mice

Human IgM κ monoclonal antibody (anti-GD2) to a tumor antigen, ganglioside GD2, has been produced by Epstein-Barr virus-transformed human B lymphoblasts. In the present study, we demonstrated the anti-tumor effects of passively administered anti-GD2 against an ascites-form human melanoma cell line, M14-A, transplanted into athymic nude mice. M14-A expresses GD2 in vivo. Cells (5 × 10 5) were inoculated intraperitoneally (i.p.) or subcutaneously (s.c.) into CD-1 nude mice that had received i.p. injections of 200 μg anti-GD2 and rabbit complement. Significant tumor-free intervals were observed in the treated mice ( P < 0.005) for i.p. tumors and P < 0.025 for s.c. tumors). M14-A formed well-vascularized s.c. tumors if injected into young nude mice. Three-wk-old CD-1 nude mice bearing 2–3 mm M14-A s.c. tumor nodules were treated i.p. with anti-GD2 and rabbit complement. Tumor growth was delayed for 25 days. On day 15, treated tumors were 20% the size of control tumors. Because most biopsied or autopsied melanomas express GD2, and because patients with melanoma produce autoantibodies to GD2, the results in this study may provide important information for future passive immunization with human monoclonal antibody and for active specific immunization with GD2 antigen.

Use of Young Nude Mice for Selection of Subpopulations of Cells With Increased Metastatic Potential From Nonsyngeneic Neoplasms&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN4"&gt;4&lt;/xref&gt;

The purpose of these studies was to investigate whether young (3-wk-old) nude mice, which lack functional T-lymphocytes and demonstrate low natural killer cell activity, could serve as in vivo models for the selection of metastatic subpopulations of cells from heterogeneous allogeneic melanomas. Three-week-old BALB/cAnN or N:NIH(S) nude mice received iv injections of single cells harvested from either the B16 or K-1735 melanoma. Individual pulmonary metastases were harvested 4 weeks later and implanted sc into nude mice to expand the population. Cells from each of these metastases colonized in the lungs of 3-week-old nude mice and 6-week-old normal syngeneic mice with significantly higher efficiency than did cells from the parent tumors. It was concluded that, in addition to being a useful in vivo model for ascertaining the metastatic potential of neoplasms, the healthy young nude mouse could be used for selecting and maintaining tumor cell variants of high metastatic potential room heterogeneous allogeneic tumors.