Inhibition and promotion of tumor growth by BCG: evidence for stimulation of humoral enhancing factors by BCG

Effects of pretreatment with BCG, strain Japan, on tumor growth were studied using a transplatable methylcholanthrene (MCA)-induced fibrosarcoma in C3H/He mice. Injection of BCG7 weeks before tumor inoculation at a site distant from the tumor caused a slight inhibition of tumor growth. A low dose of tumor cells did not grow at the BCG-primed site when BCG was injected 7 and 11 weeks before the tumor. When a high dose was inoculated into the BCG-primed site, inhibition of the primary tumor occurred in mice which had received BCG 7 weeks previously, but the number of distant metastases in the popliteal lymph node and the lungs was increased in mice pretreated with BCG at any time. Furthermore, post treatment with BCG at a site distant from the tumor caused promotion of tumor growth. Enhanced antibody formation and suppression of delayed type hypersensitivity (DTH) occurred in tumor-bearing mice. BCG treatment of such mice caused a vigorously enhanced antibody formation and a marked suppression of DTH. The sera from tumor-bearing mice enhanced tumor growth. Tumor growth was suppressed in splenectomized mice. These findings suggested that antibodies against tumor-specific antigens enhanced tumor growth in this system and that BCG treatment of tumor-bearing mice stimulated formation of antibodies probably acting as blocking factors.     

The role of blood platelets in experimental metastases

After the intravenous injection of Walker 256 tumour cells into rats the platelet count decreased rapidly and remained low during the following period of observation. The platelet decrease was closely related to the number of cells injected. Intra-arterial tumour cell injections required a considerably higher tumour cell count to produce a comparable thrombocytopenia. Non-viable tumour cells and tumour cell fragments induced a similar decrease of circulating platelets. Neither viable tumour cells nor tumour cell fragments aggregated rat platelets in vitro. The presence of fibrin monomers in tumour cell injected animals suggested intravascular fibrin deposition; the plasma fibrinogen level, however, did not decrease significantly. Isotope studies using ⁵¹Cr labelled platelets revealed a rapid disappearance of the platelets from the circulation and their trapping in the lung—the primary site of tumour cell lodgement. Dipyridamole and ancrod pretreatment did not influence the decrease of platelets and their accumulation in the lung after tumour cell injection. In contrast, heparin completely prevented the thrombocytopenia and the platelet trapping in the lung. From the present experiments it is concluded that embolic tumour cells lead to early endothelial damage, resulting in local thrombin formation with subsequent irreversible platelet aggregation.     

The effects of N-methylformamide on artificial and spontaneous metastases from a murine hepatocarcinoma

The effects of the differentiation-inducing polar solvent N-methylformamide (NMF) on artificially induced and spontaneous metastases from a murine hepatocarcinoma (HCA-1) in C3Hf/Kam mice were investigated. Exposure of HCA-1 cells in vitro for 6 days to 1.0% or 1.25% NMF resulted in an increase in the number of lung nodules formed in mice when these cells were injected into their tail veins. This in vitro NMF exposure increased cell volume and induced only a slight amount of cytotoxicity. Administration of NMF to mice 1 day before i.v. tumour cell inoculation resulted in a dose-dependent increase in the number of lung nodules formed, beginning at an NMF dose of 600 mg kg-1. NMF caused a similar magnitude of metastasis enhancement in immunosuppressed mice. However, when the maximum dose tested (1,800 mg kg-1) was administered as 6 daily fractions of 300 mg kg-1 each, no increase in artificial metastases was detected. Administration of NMF to mice one day after i.v. tumour cell injection resulted in a dose-dependent decrease in the number of lung nodules. In mice bearing 5-6 mm HCA-1 leg tumours, treatment with 6 daily fractions of NMF (300 mg kg-1 each) significantly reduced the number of spontaneous pulmonary metastases, yet had very little effect on the growth of the primary tumour. These data suggest that, in a clinically relevant treatment setting, NMF can reduce metastasis formation.     

Antitumour responses induced by short-term pretreatment with tumour cells.

The injection (s.c. or i.p.) of 10(6) live or lethally irradiated methylcholanthrene-induced fibrosarcoma cells into CBA/Ca mice one or 2 days before i.v. challenge with the same tumour inhibited the formation of artificial lung tumour metastases. In addition, it also frequently enhanced the cytostatic effect of peritoneal exudate cells on monolayers of the same tumour. The effects on lung tumour metastasis were not noted if X-irradiated tumour was injected i.v., or if s.c. administration was delayed until one day after i.v. challenge. Similar effects on tumour growth were also observed in C3Hf/Bu mice and (CBA/Ca x A/HeJ) F1 hybrids which were pretreated (s.c.) with tumour shortly before i.v. challenge with the same tumour. Further studies in CBA/Ca mice suggested that the protective effect was tumour-specific, for the growth of i.v. injected tumour was not significantly inhibited by pretreatement with a number of other MC-induced or spontaneous tumours from the same and different strains.     

A randomised phase II study of weekly paclitaxel or vinorelbine in combination with cisplatin against inoperable non-small-cell lung cancer previously untreated

Cyclooxygenase-2 (COX-2) expression is increased in breast cancer and surgery has been shown to increase the growth of metastatic tumours. We investigated the effect of selective COX-2 inhibition on the growth of metastases in either an experimental metastasis model or following excision of a murine primary breast tumour. 50,000 4T1 mammary carcinoma cells were injected into the mammary fat pad of female BALB/c mice. When the mean TD reached 8+/-0.4 mm, tumours were excised and the mice were randomised into two groups (n=12 per group) to receive daily intraperitoneal injections of the selective COX-2 inhibitor, SC-236 or drug vehicle for 14 days. Alternatively, experimental metastases were established by tail-vein injection of 50,000 4T1 cells. Mice received either the selective COX-2 inhibitor, SC-236 or drug vehicle for 14 days (n=12 per group). SC-236 treatment significantly reduced tumour burden, the number and size of spontaneous metastases following primary tumour excision. SC-236 treatment also reduced tumour burden, the number and size of experimental metastases. Immunohistochemical staining demonstrated that COX-2 inhibition reduced microvessel density and increased apoptosis within both spontaneous and experimental metastases. These data clearly demonstrate that the selective COX-2 inhibitor, SC-236, has potent antimetastatic activity against both spontaneous metastases arising following primary tumour excision and experimental metastases.     

Effect of Corynebacterium parvum on peripheral blood platelets

The level of peripheral blood platelets was determined after i.v. injection of Corynebacterium parvum in normal C57BL mice and in those bearing the Lewis lung carcinoma. Twenty minutes after injection of a formalin-killed active strain (CN6134, (CN6134, which inhibited tumour metastases) or a killed inactive strain (CN 5888, which did not inhibit metastases) the number of circulating blood platelets was reduced by 50%. The level of platelets returned to control values by 8 h after the active, and by approximately 3 days after the inactive strain. The active strain alone caused a second and prolonged fall in platelet numbers, from approximately 16 h to 21 days after injection. Heparin given 3 X weekly to these mice restored the platelet count to normal values by 10 days after injection of active-strain C. parvum. The level of platelets in tumour-bearing mice was essentially similar to that in normal mice. Possible causes of the thrombocytopenia and the significance of platelets in metastasis are discussed.     

Carcinoembryonic antigen: enhancement of liver colonisation through retention of human colorectal carcinoma cells.

Carcinoembryonic antigen (CEA) is an oncofetal antigen whose function in the progression of colorectal carcinoma remains unclear although recent studies suggest it participates in homotypic cellular adhesion. We have previously shown that 40 micrograms of CEA injected intravenously into athymic nude mice enhances experimental metastasis in liver and lung by two human colorectal carcinoma cell lines that are injected intrasplenically 30 min later. The metastatic potential of another three moderately to highly metastatic colorectal carcinoma cell lines and of one weakly metastatic line has now been analysed in this model. CEA pretreatment only enhanced colony formation by cell lines that were weakly metastatic in untreated nude mice; it did not affect experimental metastasis by highly metastatic lines. CEA pretreatment enhanced the retention of 125I Idudr-labelled weakly metastatic tumour cells within the liver and lungs 4 h after intrasplenic injection but not the retention of highly metastatic tumour cells or inert latex beads. A significant correlation existed between the formation of experimental metastases and the early retention of tumour cells within the liver after intrasplenic injection. Aggregation did not appear to be important for retention in liver because CEA did not aggregate colorectal carcinoma cells in vitro. Also CEA did not alter natural host effector cell function in a cytolysis assay in vitro. We suggest that CEA facilitates liver colonisation by three of eight human colorectal carcinomas in athymic nude mice by increasing the hepatic retention of tumour cells. The potential mechanisms by which CEA may increase the retention of tumour cells in the liver are discussed.     

Adenovirus-mediated interferon alpha gene transfer induces regional direct cytotoxicity and possible systemic immunity against pancreatic cancer

We previously demonstrated a characteristically high sensitivity of pancreatic cancer cells to interferon alpha (IFN-alpha) gene transfer, which induced a more prominent growth suppression and cell death in pancreatic cancer cells than in other types of cancers and normal cells. The IFN-alpha protein can exhibit both direct cytotoxicity and indirect immunological antitumour activity. Here, we dissected and examined the two mechanisms, taking advantage of the fact that IFN-alpha did not show any cross-species activity in its in vivo effect. When a human IFN-alpha adenovirus was injected into subcutaneous xenografts of human pancreatic cancer cells in nude mice, tumour growth was significantly suppressed due to cell death in an adenoviral dose-dependent manner. The IFN-alpha protein concentration was markedly increased in the injected subcutaneous tumour, but leakage of the potent cytokine into the systemic blood circulation was minimal. When a mouse IFN-alpha adenovirus was injected into the same subcutaneous tumour system, all mice showed significant tumour inhibition, an effect that was dependent on the indirect antitumour activities of IFN-alpha, notably a stimulation of natural killer cells. Moreover, in this case, tumour regression was observed not only for the injected subcutaneous tumours but also for the untreated tumours at distant sites. This study suggested that a local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its dual mechanisms of antitumour activities and lack of significant toxicity.     

Intrabronchial implantation of the Lewis lung tumor cell does not favor tumorigenicity and metastasis

The purpose of these studies was to determine whether the orthotopic implantation of the Lewis lung tumor favors tumorigenicity and production of metastasis as compared with implantation at an ectopic site. Different numbers of viable cells were implanted subcutaneously (footpad), intrabronchially, or intrathoracically into groups of syngeneic mice. Tumorigenicity, production of distant metastases and survival were recorded. Tumor cells implanted subcutaneously produced fast-growing local tumors and numerous spontaneous lung metastases. Tumor cells implanted intrabronchially produced slow-growing tumors that did not produce distant metastasis. Since radiolabeled cells implanted into different sites exhibited similar patterns of survival, the differences in behavior of 3LL cells in the skin or lung were due to other factors. We conclude that for the Lewis lung carcinoma, orthotopic implantation does not favor increased tumorigenicity and metastatic potential.     

Evidence for effects of heparin on cell surfaces influencing experimental metastases

The effect of heparin on tumour cells and on intravenously induced metastases was studied in a syngeneic tumour-host system, MCG1-SS in CBA mice. In vitro heparin induced significant changes in the volumes of cells, but did not affect their aggregability or vitality. In vivo heparin did not affect the subcutaneous transplantability of tumour cells. Metastasis formation from intravenously injected cells was differently affected when heparin was given as intravenous pretreatment to animals, than when the same amount was given in the cell suspensions. Thus, pretreatment of animals with heparin affected pulmonary metastases only to a small degree, but gave an increased number of tumour takes in extrapulmonary organs. These changes were ascribed to systemic effects of heparin, above all on blood coagulability. When given in the cell suspensions, heparin did not affect the number of extrapulmonary “takes”, but induced significant changes in pulmonary metastases. The average volume of such metastases became consistently smaller, while their number, if anything, rose. These effects were interpreted as being due to direct actions of heparin on the surfaces of tumour cells, probably related to its polyanion character. It was pointed out that in previous experiments with animals given large heparin doses, a direct cell effect might also prevail. Another polyanion, chondroitin sulphate, caused similar although less pronounced changes than heparin in vitro and in vivo. The polycation protamine had roughly opposite effects.     

Chemocoagulation of Metastatic Lymph Nodes Using a Suspension of Mitomycin C—Adsorbing Activated Carbon Particles in 80% Ethanol

Chemocoagulation therapy was evaluated in an experimental model of metastasis of marine lymph nodes following injection of a suspension of mitomycin C-containing activated carbon particles in 80% ethanol (MMC-CH-ET) into the popliteal lymph node. Lymph node metastasis was induced in the left popliteal and the lumbar lymph nodes 8 days after injection of mouse leukemia P388 cells into the footpad of the left hindleg of BDFI mice. When MMC-CH-ET was injected into the left popliteal lymph node, it immediately left this site and entered the lumbar lymph node via lymphatic vessels. When compared with tissue concentrations of mitomycin C following injection of an aqueous solution of this drug, the mitomycin C concentration of MMC-CH-ET was maintained at significantly higher levels for 2 hr following injection both at the site of injection and at secondary lymph nodes. Furthermore, coagulative necrosis was identified histologically throughout the injected lymph node and the secondary lymph node, including the metastatic site. The mortality of mice treated with MMC-CH-ET injection was significantly reduced and lymph node metastasis was controlled with MMC-CH-ET when compared with the results for mice treated with an aqueous solution of mitomycin C or treated by surgical lymph node dissection. In this report, we suggest that the use of MMC-CH-ET as a therapeutic agent may be useful in targeting lymph node metastasis.     

In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases

The role of host cells in tumor progression and metastasis is critical. Intrasplenic injection of tumor cells has long been known as an effective method of developing liver metastases in nude mice, whereas portal vein (PV) injection of tumor cells can result in rapid death of the tumor cells. Host cells were thought to play a role in these phenomena. We report here that after splenic injection of tumor cells, splenocytes cotraffic with the tumor cells to the liver and facilitate metastatic colony formation. Human colon cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP) were injected in either the PV or spleen of nude mice and imaged at the subcellular level in vivo. Extensive clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours after PV injection and essentially all the cancer cells died. In contrast, splenic injection of these tumor cells resulted in the aggressive formation of liver and distant metastasis. GFP spleen cells were found in the liver metastases that resulted from intrasplenic injection of the tumor cells in transgenic nude mice ubiquitously expressing GFP. When GFP spleen cells and the RFP cancer cells were coinjected in the PV, liver metastasis resulted that contained GFP spleen cells. These results suggest a novel tumor-host interaction that enables efficient formation of liver metastasis via intrasplenic injection.     

Increased experimental pulmonary metastasis in pregnant mice

The effect of pregnancy on experimental pulmonary metastasis was studied. Compared to the incidence of pulmonary metastasis induced by G6 cells in non-pregnant mice, the incidence of such metastasis was found to be greatly enhanced when the cells were injected i.v. in the latter half of pregnancy. The maximum enhancement was seen on the 15th day of pregnancy. The incidence of pulmonary metastasis returned to the level observed in non-pregnant mice when the cells were injected 4 days after parturition. Pregnancy also significantly increased the incidence of pulmonary metastasis of 2 other cell lines (3LL and Colon 26). Injection of G6 cells after hysterectomy performed on the 15th day of pregnancy resulted in decreased lung colonization, similar to that seen after parturition. Quantificative analysis of the arrest of G6 cells labeled with [¹²⁵1]-5-iodo-2′-deoxyuridine in the lungs showed that the tumor-cell clearance from the lungs during the 24–72 hr after tumor-cell injection was much slower in pregnant than in non-pregnant mice. The continuous administration of β-estradiol and/or progesterone, which maintained serum levels of the hormones equivalent to those prevailing on the 15th day of pregnancy, did not affect the lung colonization of G6 cells. Tumor-cell-platelet aggregation was more extensive with platelets obtained from mice at the 15th day of pregnancy than with those from non-pregnant mice. When platelets isolated from pregnant mice were injected into normal mice 5 min before G6 injection, lung metastasis was also enhanced. These findings suggest that a pregnant host is handicapped with regard to pulmonary metastasis, this being partly due to increased platelet-aggregating activity in response to tumor cells. © 1995 Wiley-Liss Inc.     

诱导小鼠肺泡巨噬细胞产生一氧化氮抑制肿瘤实验性肺转移

本文报道卡介苗(BCG)致敏小鼠雾化吸入基因重组白细胞介素-2(rIL-2)抑制小鼠乳腺癌实验性肺转移。结果显示,小鼠经治疗后肺表面转移瘤结节数显著减少,肺泡灌洗液一氧化氮(NO)含量显著增高。单独雾化吸入rIL-2对部分种瘤小鼠也有一定的抑瘤作用。用NO合成阻断剂(N-单甲基-L-精氨酸,NMA)能几乎完全阻断rIL-2的抑瘤作用。结果表明,雾化吸入rlL-2加BCG处理是治疗小鼠乳腺癌肺转移的一种有效方法,其机制主要是通过诱导活化肺泡巨噬细胞(Mφ)产生NO发挥抑瘤作用。     

The growth and metastasis of an allografted lymphoma in normal, deprived and reconstituted mice

The growth of the EL⁴ lymphoma has been studied in thymus-deprived, reconstituted and normal CBA/Lac mice. In normal mice the lymphoma grew slowly and progressively after intradermal injection, killing the host between 39 and 64 days later, depending upon the number of cells injected: no metastases were seen. In deprived mice the tumour grew more rapidly and killed all mice between 17 and 20 days: 50% had distant metastases. Reconstituted mice resembled normal mice. Intravenous injection of lymphoma cells was also followed by early death, within 16 days, of deprived mice: all had widespread tumour growth in the viscera. Normal and reconstituted mice survived considerably longer, some being free of tumour at over 200 days after injection; death was associated with localized tumour growth in soft tissues. Studies of the clearance of ₁₂₅IUDR-labelled tumour cells and an examination of early histological changes showed that this difference in the distribution of tumour growth was not due to differences in rate of clearance of cells from the blood or lungs. It appeared likely that the differences between thymus-deprived and reconstituted mice were due to the survival of rather small numbers of tumour cells in a variety of tissues.     

In vivo electroporation of plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas combined with electrochemotherapy induces long-term antitumour immunity

When cancer cells, including melanoma cells, are genetically altered to secrete cytokines, irradiated and injected into subjects, long-term antitumour immunity is induced. Optimally, existing melanomas induced to produce cytokines in vivo could stimulate this same immune response. Although in vivo electroporation enhances plasmid expression, electroporation of plasmids encoding granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-2 (IL2) into B16 mouse melanomas did not significantly alter tumour growth at the concentration tested. Electrochemotherapy, which causes short-term, complete regressions of treated tumour but no resistance to challenge, was combined with plasmid delivery. The combination treatment resulted in the induction of long-term immunity to recurrence and resistance to challenge in up to 25% of mice.     

Development of new spontaneous metastatic heterotopic model of lewis lung carcinoma imaged by GFP expression

Many studies have demonstrated the importance of spontaneous metastases in cancer research. Until now, we still had only a few spontaneous metastatic models with high occurrence rate of metastasis in distant lymph and visceral tissues. We report a syngeneic heterotopic metastatic model using the Lewis lung cancer cell line with high metastatic ratio in C57BL/6 mice after transplantation by injection of cancer cells and without surgical intervention. Metastatic process was declared for each mouse in two groups ?sacrificed 3 or 5 weeks after subcutaneous (s.c.) injection of the tumor cells into the dorsal side of the tail. The total number of metastases was counted as the sum of observed macrometastases. Our model produced produced a 100% rate of spontaneous lymphatic and visceral metastases after a simple injection transplantation into the heterotopic site. In mice with large primary tumors which are non-lethal, visceral and lymph macrometastases were observed. Tumor volume correlated linearly not only with the tumor growth time, but also with the number of metastases in lymph nodes and organs. This new metastatic model could be useful for studying the metastasis mechanism and for developing therapy for lymph and visceral metastases.     

Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice

There are few reports describing experimental models of the growth and metastasis of human breast carcinomas. This article discusses the tumorigenic and metastatic properties of two estrogen receptor-negative breast carcinomas injected into nude mice. Tumor growth in the mammary fatpad (m.f.p.) and the subcutis was compared in female nude mice. The injection of 10(5) viable cells of two human breast carcinoma cell lines (MDA-MB-231 and MDA-MB-435) gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c. injections produced tumors and these occurred several weeks after the appearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is a favorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant metastases in 80% to 100% of recipients. The most common sites for metastasis were the lymph nodes and lungs, with a lower incidence of metastases in muscle (chest wall and thigh), heart, and brain. New variant cell lines were isolated from metastases in the lungs, brain, and heart. All the cell lines were tumorigenic in the m.f.p., and the lung- and heart-derived metastasis lines produced slightly more lung metastases than the original cell line. However, the brain metastasis variant produced significantly fewer lung metastases. Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimental lung metastases showed enhanced lung colonization potential when reinjected i.v. Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable tool for analysis of the cellular and molecular basis of the metastasis of advanced breast cancer.     

Functional role of platelets in experimental metastasis studied with cloned murine fibrosarcoma cell variants

The involvement of platelets in experimental metastasis was studied with cloned cell lines derived from PAK 17, a recently induced methylcholanthrene-induced C57BL/6 mouse fibrosarcoma. Tumor cell-induced platelet aggregation and lung colonization assays were used to distinguish three major stable phenotypes among the clones: a low metastatic-low platelet aggregating type, e.g., clone PAK 17.12; a low metastatic-high platelet aggregating type, e.g., clone PAK 17.14; and a high metastatic-high platelet aggregating phenotype, e.g., clone PAK 17.15. Clones with high metastatic but low platelet aggregating potential were not observed in the study. Intravenously injected PAK 17.14 and PAK 17.15 cells, but not PAK 17.12 cells, induced greater than 50% reductions in circulating platelet levels in C57BL/6 mice. Since highly metastatic clone PAK 17.15 cells consistently induced high levels of tumor cell-induced platelet aggregation regardless of the platelet donor, it was selected to study the relationship between its tumor cell-induced platelet aggregation and lung colonizing abilities. (a) A 93% decrease in lung colony number resulted in mice injected with 100 micrograms of prostacyclin immediately before injection of clone PAK 17.15 cells. Prostacyclin was also able to inhibit, in a dose dependent fashion (0-5 ng), platelet aggregation induced by clone PAK 17.15 cells in vitro. (b) A 92% reduction in lung colony number occurred in mice showing marked thrombocytopenia following injection of 100 micrograms of rabbit anti-mouse platelet antibody 24 h before tumor cell injection. (c) A greater than 80% reduction in clone PAK 17.15 lung colony number was observed in mice rendered thrombocytopenic by i.v. injection of 0.038 units of neuraminidase 24 h before i.v. injection of 10(5) tumor cells. These results suggest that platelets are required for successful lung colonization by clone PAK 17.15 cells. However, the presence in this fibrosarcoma of high platelet aggregating-poorly metastatic cells, such as clone PAK 17.14, demonstrates that while the ability to aggregate platelets is necessary for successful metastasis by some tumor cells, it is insufficient if tumor cells lack other critical properties required for completion of the metastatic cascade.     

Growth and metastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice

The purpose of this study was to determine whether the methods for isolating tumor cells from a human renal cell carcinoma (HRCC) influence the biological behavior of the cancer cells. Renal cell carcinoma obtained from a surgical specimen was dissociated by enzymatic treatment and cells were plated into culture dishes or injected s.c. into the kidney of BALB/c nude mice. The resultant kidney tumor produced liver metastasis and ascites. All tumors growing in nude mice (s.c., kidney, liver, ascites) were also established in culture. The human origin of all five lines was ascertained by karyotypic and isoenzyme analyses. Cells from all lines were injected, s.c., i.p., i.v., intrasplenically, and beneath the renal capsule of nude mice. All the lines were tumorigenic after s.c. or renal subcapsule injection, although the rate of tumor growth varied among the five lines. The metastatic behavior of the HRCC cells was influenced by both the nature of the tumor cells and the route of injection into nude mice. In general, cells derived from the liver metastasis produced more metastases in nude mice than other lines. The lines established in culture from the primary HRCC and the ascites were poorly metastatic. Even with highly metastatic cells, i.v. injection did not yield significant metastasis, but the injection of cells into the renal subcapsule resulted in extensive metastasis to the lungs and in all peritoneal organs. These results indicate that nude mice can be used for the isolation of populations of HRCC cells with different growth and metastatic potential and that, of the organ sites tested, the renal subcapsule is the most advantageous site for implantation of HRCC cells.