B16 melanoma metastasis to an "artificial organ" implant.

The mouse B16 melanoma metastasizes in two stages, first to the lungs and then from lung metastases to systemic organs. Despite widespread dissemination, visible metastases generally occur only in the brain, adrenals, kidneys, ovaries, pancreas, and mesentery. As a novel approach to investigate the basis of metastatic patterning in this system, the possibility was explored that an implantable "artificial organ" could serve as a site for the occurrence and experimental modulation of secondary-stage metastasis. Each implant consisted of a cellulose disc 4 mm in diameter, with a central 1-mm polymer pellet to effect local sustained release of angiogenic or growth factors in a s.c. environment. During the secondary spread of tumors initiated with the B16 melanoma clone G3.12 and with the more metastatic variant G3.12/BM2, metastatic involvement of implants containing angiogenic factors was mainly as invisible micrometastases demonstrable by bioassay; visible metastases were rare and were located in implant blood vessels. Metastasis occurred in about 30% (G3.12) and 50% (G3.12/BM2) of implants with vasculature induced by ethylene-vinyl acetate copolymer alone. Endothelial cell growth factor and heparin promoted greater vascularization but did not significantly alter metastatic involvement of implants. Release of tumor cell mitogenic activity from pellets containing a crude extract of mouse lungs increased the incidence of G3.12/BM2 metastasis in implants to over 70% and stimulated growth of visible metastases within the cellulose matrix. In contrast, liver extract inhibited metastasis growth. Colonization of implants following intracardiac injection of G3.12/BM2 cells was generally similar to metastasis, but visible colonies formed more readily and were less dependent on the influence of lung extract. These results indicate that metastasis and colonization can occur regularly in implants and that the relative favorability of the implant environment for secondary tumor growth can be altered by incorporation of tumor cell growth modulators.     

Metastatic dissemination of B16 melanoma: pattern and sequence of metastasis

The progressive metastatic spread from subcutaneous transplants of two subpopulations of the mouse B16 melanoma, slow-growing clone G3.5 and fast-growing clone G3.12, was examined during tumor growth in C57BL/6 mice and after surgical excision of tumors of various sizes. In addition to enumeration of visible and lethal or potentially lethal ("clinically relevant") metastases, the occurrence of visibly undetectable proliferating (occult) or nonproliferating (dormant) micrometastases was assessed by implanting lymph nodes and organs subcutaneously into normal mice and monitoring for resulting tumor growth. Occult or dormant metastases were disseminated initially to the lungs from G3.5 tumors of 3-4 mm in mean geometric diameter (MGD) and G3.12 tumors of 6-7 mm in MGD. The ipsilateral axillary lymph node (IALN), the regional draining lymph node for these tumors, received metastases after the lungs, initially from 10 to 12-mm tumors. Subsequently, occult or dormant and visible metastases first appeared in systemic organs and lymph nodes (kidneys, adrenal glands, ovaries, and contralateral axillary lymph node) at tumor sizes of about 26 mm in MGD. Systemic metastases occurred only in mice with large and numerous lung metastases and did not depend on the continuing presence of the subcutaneous tumor or on the presence of IALN metastases, which indicated that established lung metastases were a generalizing site from which systemic metastatic spread initiated. After tumor excision, death generally resulted from extensive lung metastasis. Occasional lethal or clinically relevant metastases were also observed in the IALN, kidneys, adrenal glands, ovaries, brain, eyes, and urinary bladder; liver involvement was evident exclusively as occult or dormant micrometastases. Terminal metastatic patterns of these B16 melanoma transplants were as widespread and indiscriminate as those of malignant melanoma in humans.     

Metastatic dissemination of B16 melanoma: evidence that metastases can result from nonspecific trapping of disseminated tumor cells

Spontaneous metastasis from tumor transplants of two representative mouse B16 melanoma clones, G3.5 and G3.12, was examined experimentally to determine whether initial dissemination to the lungs, or secondary systemic spread from established lung metastases, resulted from organ-specific tropism or from nonspecific trapping of circulating tumor cells in capillary beds. In parabiosed mice, subcutaneous tumors metastasized extensively within hosts, but guests remained metastasis-free except following the rare involvement of the parabiotic junction during secondary spread. Intrasplenic tumor transplants metastasized to the liver, whereas intrarenal transplants metastasized to the lungs, reflecting patterns of venous drainage. Subcutaneous implants of neonatal lung and kidney in the flank opposite from the site of tumor initiation acquired metastases only during secondary systemic spread, and there was no evidence of organ selectivity. Metastases from various organs, and derived cell lines, when transplanted subcutaneously grew into tumors that initially metastasized exclusively to the lungs. These results indicate that both initial and secondary metastases of these B16 melanoma transplants occurred by nonspecific trapping of tumor cells in the first capillary bed encountered. In contrast, organ colonization following intravenous injection of tumor cells frequently proceeded beyond the first capillary bed.     

DT-5461, a new synthetic lipid A analogue, inhibits lung and liver metastasis of tumor in mice.

We have investigated the antimetastatic effect of a new synthetic lipid A analogue, of low endotoxicity, DT-5461, against two highly metastatic tumor cell lines, L5178Y-ML25 T-lymphoma and B16-BL6 melanoma cells in mice. Four intermittent i.v. administrations of DT-5461 at intervals of 4 days resulted in a significant inhibition of liver metastasis caused by i.v. injection of L5178Y-ML25 cells and lung metastasis of B16-BL6 cells in the experimental metastasis models. Intraperitoneal and intranasal administrations as well as i.v. administration of DT-5461 were also effective in preventing lung metastasis of the melanoma cells. Multiple administrations of DT-5461 before the surgical excision of primary tumors significantly reduced the number of lung colonies of melanoma cells and primary tumor size. Similarly, this treatment modality after the surgical excision of primary tumors showed a greater reduction of lung tumor colonies as compared with lipopolysaccharide, a synthetic lipid A (No. 506) and its analogue as well as untreated control in the spontaneous lung metastasis model. Furthermore, the group that received DT-5461 after the inoculation of lymphoma or melanoma cells showed significantly enhanced survival rate compared with the untreated control. These results suggested that DT-5461 may be therapeutically useful for the inhibition of tumor metastasis.     

Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in mice

This study is designed to establish a pulmonary tumour model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma (LLC) cell suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 s per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph node metastasis was observed in all mice that formed a lung nodule after intrapulmonary implantation. The size of tumour nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21+/-2 days (range 19-24 days). Histopathological analysis revealed that no metastatic tumour was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumour cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of cis-diamine dichloro platinum on day 1 after tumour implantation tended to suppress the primary tumour nodule and significantly inhibited lymph node metastasis. Thus, a solitary pulmonary tumour nodule model with lymph node metastasis approximates clinical lung cancer and may provide a useful basis for lung cancer research.     

Targeting mitogen-activated protein kinase/extracellular signal-regulated kinase kinase in the mutant (V600E) B-Raf signaling cascade effectively inhibits melanoma lung metastases

Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by (V600E)B-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)-mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant (V600E)B-Raf signaling cascade to inhibit melanoma metastases.     

The role of intratumor environment in determining spontaneous metastatic activity of a B16 melanoma clone

B16 melanoma-derived cell lines and clones that initiate rapid-growing and nonmetastatic tumors in normal young (2-month-old) mice were previously shown to form slower-growing and highly metastatic tumors in normal mature or aged (greater than 10-month-old) mice. Similarly, slower tumor growth and enhanced metastasis occurred in young mice hyperimmunized against tumor-associated antigens. The metastatic characteristics of subcutaneous tumors initiated by one B16 melanoma clone, G3.26, were examined in normal young mice, normal mature mice, young mice immunized against G3.26 cells, and young mice maintained on a diet of 50% less food than usual. In normal young mice, tumors rarely disseminated viable lung metastases, even at very large sizes, and viable tumor cells were not detected in blood obtained by whole-body vascular perfusion. In contrast, tumors in mature, in immunized, and in calorie-restricted mice gave rise to visible lung metastases in 60-90% of mice, with dissemination beginning at relatively small tumor sizes. These tumors grew 27-78% slower than tumors in normal young mice, but in no case was expression of metastatic activity dependent on longer host survival. In all three experimental hosts, metastatic activity was transient and not expressed during subsequent growth of metastases in young mice. Different host mechanisms operating in mature, immune, and calorie-restricted mice were probably responsible for suppressing tumor growth. However, the consistent generation of metastatic activity under such diverse conditions suggests a common basis for promotion of metastasis, possibly related to intratumor environment alterations resulting from slower tumor growth.     

Phenotypic interconversion of B16 melanoma clonal cell populations: relationship between metastasis and tumor growth rate

Three distinct dissemination-related phenotypes have been recognized in clones of the mouse B16 melanoma based on in vivo behavior: metastatic (spontaneously disseminating to the lungs from solid tumors), colonizing (capable of forming tumor colonies in the lungs following intravenous injection), and null (tumorigenic but non-metastatic and non-colonizing). From a progenitor null clone, G3, subclones that became phenotypically diversified in vitro (metastatic G3.5 and null G3.15) and in vivo (metastatic G3.12 and colonizing G3.26) were derived. During long-term culturing, G3 cells became metastatic and then lost that activity, G3.5 and G3.12 cells gradually lost metastatic activity, and G3.26 cells became slightly metastatic and non-colonizing. Subclone G3.15 became highly metastatic after a single subcutaneous (s.c.) tumor passage. In aged mice, and in young mice injected with incompletely-tumorigenic cell doses, G3 and G3.26 s.c. tumors were metastatic, but cells cultured from those tumors or metastases were non-metastatic when tested in young mice at standard highly-tumorigenic cell doses. The behavior of G3.5 and G3.12 tumors was not altered in aged mice or when tumors were initiated with small cell inocula. Analysis of growth characteristics associated with these phenotypic interconversions indicated that lung-colonizing potential was directly related to the ability of the cells to grow as multicell colonies in 0.3% agar, and that metastatic activity was expressed by tumors that grew at moderate rates. In young mice receiving standard cell doses, G3.5 and G3.12 tumors inherently grew at that rate, whereas G3 and G3.26 tumors grew more rapidly and G3.15 tumors grew more slowly. Regardless of inherent phenotype, all clones were capable of expressing metastatic activity, at least transiently, as tumor growth was altered to moderate rates. Expression of metastatic behavior might, therefore, be regulated to some extent by tumor growth characteristics.     

Helix pomatia agglutinin binding in human tumour cell lines: correlation with pulmonary metastases in nude mice.

The extent of lectin binding by three human melanoma (LOX, FEMX-1 and SESX) and two sarcoma lines (MHMX and OHSX) was related to their potential for experimental metastasis formation in athymic nude mice. The Helix pomatia agglutinin (HPA), which recognises the N-acetyl-D-galactosamine ligand, showed differential binding to the cell lines in a manner that correlated with their ability to give lung colonies after i.v. injection in the mice (P < 0.005). The degree of HPA binding and lung colony formation of the cell lines studied was ranked in the following order, LOX > MHMX > OHSX > SESX > FEMX-I. Similar patterns were not observed with the other lectins used in this study (WGA, Con A, PNA and UEA-I). The high HPA reacting LOX melanoma line shows extensive pulmonary metastatic formation with no extrapulmonary colonies, whereas the low HPA reacting FEMX-I cells give only extrapulmonary metastases with no detectable colonies in the lungs. Precoating of tumour cells with HPA prior to injection did not reduce the ability of cells to give pulmonary metastases, suggesting that the HPA epitope was not functionally associated with the pulmonary metastatic potential observed in nude mice. These findings support recent human studies of a correlation between HPA binding and incidence of metastasis, however, our data indicate that there is no causal relationship. Further analyses are required to identify the specific HPA-binding glycoconjugates that may be involved.     

Expression of matrix metalloproteinases in the microenvironment of spontaneous and experimental melanoma metastases reflects the requirements for tumor formation

Expression of matrix metalloproteinases (MMPs) and their activation in tumor cells, as well as tumor surrounding stromal cells have been implicated in tumor cell invasion and metastasis. By means of a syngeneic tumor model for either experimental or spontaneous metastases, the differential expression of MMPs and tissue inhibitors of MMPs (TIMPs) in relation to the microenvironment and the way of metastasis induction was characterized. In vitro characterization revealed that increased levels of secreted MMP-2, MMP-9, and TIMP-1 were only detectable in the most aggressive cell line, B16G3.12BM2. Remarkably, active MMP-2 was restricted to this cell line, whereas TIMP-2 and membrane type (MT) 1-MMP expression was comparable in all three of the spontaneously metastasizing melanoma cell lines investigated. In vivo analysis demonstrated that MMP-2, MMP-9, and MT1-MMP were predominantly expressed at the tumor-stroma border of s.c. tumors. Furthermore, functional active MMP-2 was restricted to this invasive front. In spontaneous lymph node or lung metastases, however, MMP-9 was expressed both in the center and the periphery of tumors; these areas were largely negative for MMP-2 and MT1-MMP. Notably, tumor cells of experimental lung metastases did not express MMP-9 at all. These results indicate that expression of MMPs in melanoma metastases is not only influenced by their localization but also the nature of tumor induction, suggesting that individual MMPs serve specific roles during the different stages of metastasis formation.     

Comparison of 'spontaneous' and 'experimental' metastasis using rat 13762 mammary adenocarcinoma metastatic cell clones

Rat 13762NF mammary adenocarcinoma cloned cell lines were assayed at different in vitro passage numbers and compared for their abilities to form 'spontaneous' metastases by subcutaneous injection of cells and 'experimental' metastases by intravenous injection of cells. Tumor cell clones were established from locally growing tumor and spontaneous lung metastases, and these clones were found to possess heterogeneous metastatic potentials in both metastasis assays. The rank order of clonal metastatic potentials based on either the average number of lung tumor colonies or the average total lung tumor volume was generally equivalent for 'spontaneous' and 'experimental' metastases, but some differences were noted. Ranking of 'spontaneous' metastasis by average total lung tumor volumes more closely resembled the rank order of 'experimental' metastasis than by the average number of spontaneous metastases. The results demonstrated that in the 13762NF mammary adenocarcinoma system (i) there is heterogeneity in tumor cell clonal metastatic potential using either 'spontaneous' or 'experimental' assays; (ii) these two assay methods yield generally the same rank order of metastatic potential; (iii) the metastatic potential of each of the tumor cell clones drifts with time (passage number) in cell culture, and (iv) ranking by average tumor burden calculated from total lung tumor volumes may yield a better estimate of metastatic potential than ranking by the average number of lung tumor colonies.     

Augmentation of B16 melanoma lung colony formation in C57BL/6 mice having marked granulocytosis

The effect that polymorphonuclear leukocytes (PMN) may have on the metastatic colonization of tumor cells is controversial: some laboratories have reported that PMN can inhibit metastasis whereas others have shown an augmentation effect. We have exploited our finding that a particular fibrosarcoma (BMT-11) transplanted subcutaneously into syngeneic C57BL/6 mice induces a progressive increase in the number of circulating PMN, to re-examine this question. We used such "granulocytosis-positive" mice as recipients of B16 melanoma cells to examine the influence of significant granulocytosis on the level of lung tumor colonies. The number of B16 melanoma lung colonies detected after intravenous (i.v.) injection was significantly higher in BMT-11 tumor-bearing mice with granulocytosis than in control (non-tumor-bearing) mice. Retention of 125IUdR-labelled B16 cells 24 hr after the i.v. injection was 3 to 10 times greater in mice with granulocytosis than in controls. Either simultaneous injection, or preinjection of PMN with B16 cells, increased the lung-colonizing capacity of B16 melanoma cells. These results suggest that abnormally increased numbers of PMN in the peripheral blood, particularly in the lung circulation, can enhance the ability of tumor cells to colonize or metastasize.     

Inhibition of the metastatic spread and growth of B16-BL6 murine melanoma by a synthetic matrix metalloproteinase inhibitor

The synthetic matrix metalloproteinase inhibitor batimastat was tested for its ability to inhibit growth and metastatic spread of the B16-BL6 murine melanoma in syngeneic C57BL/6N mice. Intraperitoneal administration of batimastat resulted in a significant inhibition in the number of lung colonies produced by B16-BL6 cells injected i.v. The effect of batimastat on spontaneous metastases was examined in mice inoculated in the hind footpad with B16-BL6 melanoma. The primary tumor was removed surgically after 26-28 days. Batimastat was administered twice a day from day 14 to day 28 (pre-surgery) or from day 26 to day 44 (post-surgery). With both protocols, the median number of lung metastases was not significantly affected, but there was a significant reduction in the weight of the metastases. Finally, the effect of batimastat was examined on s.c. growth of B16-BL6 melanoma. Batimastat administered daily, starting at day of tumor transplantation, resulted in a significant growth delay, whereas treatment starting at advanced stage tumor only reduced tumor growth marginally. Our results indicate that a matrix metalloproteinase inhibitor can not only prevent the colonization of secondary organs by B16-BL6 cells but also limit the growth of solid tumors.     

9-Nitrocamptothecin liposome aerosol treatment of melanoma and osteosarcoma lung metastases in mice.

The response rates of relapsed osteosarcoma and melanoma pulmonary metastases to traditional i.v. chemotherapeutic regimens have been disappointing. Direct drug delivery of chemotherapy to the lungs could increase the drug concentration in the tumor area and may offer a new therapeutic approach for these patients. Previous studies demonstrated that drugs delivered to the respiratory tract in liposomal formulation resulted in high pulmonary drug concentration, reduced systemic toxicity, and reduced dosage requirements compared with parenteral and oral administration. To determine whether this approach has utility against pulmonary metastases, the efficacy of aerosol therapy with liposome-encapsulated 9-nitrocamptothecin (L-9NC) was determined using two different experimental lung metastasis models. C57BL/6 mice were treated the day after the i.v. injection of B16 melanoma cells with aerosol L-9NC for 1 h (153 microg 9-nitrocamptothecin/kg) for 5 days per week for up to 3 weeks. Aerosol L-9NC treatment resulted in a reduction in lung weights (P = 0.005) and number of tumor foci (P < 0.001). Visible tumor nodules were fewer and smaller in the 9-nitrocamptothecin-treated group than in untreated control mice (P < 0.001). Using a newly developed human osteosarcoma experimental metastasis model in nude mice, we demonstrated that aerosol L-9NC was also effective against established lung metastases. Aerosol therapy initiated on the ninth week after i.v. tumor injection and continued for 8 or 10 weeks produced highly significant reductions in the number of animals with both visible and microscopic disease (P < 0.02), the total number of tumor foci in the lungs (P < 0.005), and the size of the individual tumor nodules (P < 0.02). These data suggest that L-9NC aerosol therapy may offer significant advantage over existing methods in the treatment of melanoma and osteosarcoma pulmonary metastases.     

DT-5461, a New Synthetic Lipid A Analogue, Inhibits Lung and Liver Metastasis of Tumor in Mice

We have investigated the antimetastatic effect of a new synthetic lipid A analogue, of low endo-toxicity, DT-5461, against two highly metastatic tumor cell lines, L5178Y-ML25 T-lymphoma and B16-BL6 melanoma cells in mice. Four intermittent i.v. administrations of DT-5461 at intervals of 4 days resulted in a significant inhibition of liver metastasis caused by i.v. injection of L5178Y-ML25 cells and lung metastasis of B16-BL6 cells in the experimental metastasis models. Intraperitoneal and intranasal administrations as well as i.v. administration of DT-5461 were also effective in preventing lung metastasis of the melanoma cells. Multiple administrations of DT-5461 before the surgical excision of primary tumors significantly reduced the number of lung colonies of melanoma cells and primary tumor size. Similarly, this treatment modality after the surgical excision of primary tumors showed a greater reduction of lung tumor colonies as compared with lipopolysaccharide, a synthetic lipid A (No. 506) and its analogue as well as untreated control in the spontaneous lung metastasis model. Furthermore, the group that received DT-5461 after the inoculation of lymphoma or melanoma cells showed significantly enhanced survival rate compared with the untreated control. These results suggested that DT-5461 may he therapeutically useful for the inhibition of tumor metastasis.     

Characterization of two highly metastatic variants of Lewis lung carcinoma with different organ specificities

The biological properties and metastasis of two sublines of the Lewis lung carcinoma (3LL) which have maintained a stable pattern of organ-selective metastasis have been studied. Subline M-3LL, a lung-specific variant which originated from a lung metastasis of the parent line, metastasized only to the lung following injection of 10(4)-10(6) tumor cells i.v. or s.c. Lymphatic metastases of this tumor were rarely detected. Subline H-3LL which was developed from a rare, spontaneous hepatic metastasis of the parent line metastasized primarily to the liver, but pulmonary metastases have also been observed. While it grew at local s.c. sites, this tumor metastasized to the regional lymph nodes draining the tumor site, as determined by histology and by bioassay of the lymph nodes following their grafting into new recipient animals. Histologically, the two lines were indistinguishable with the exception of a higher incidence of giant cells detected in tissue sections and culture monolayers of the liver-colonizing variant H-3LL. Ten clones derived from each of the variant lines were expanded in vitro and inoculated i.v. While none of the ten clones derived from line M-3LL gave rise to extrapulmonary metastasis, nine of ten clones derived from Tumor H-3LL gave rise to hepatic metastasis. Highly metastatic clones selected from each tumor were subsequently used to study the patterns of distribution and arrest of radiolabeled tumor cells following their inoculation i.v. No correlation could be found between the initial distribution of the radiolabeled tumor cells and the organ selectivity eventually noted in the site of the metastases.     

The primary tumor is the primary source of metastasis in a human melanoma/SCID model. Implications for the direct autocrine and paracrine epigenetic regulation of the metastatic process

Although previous autopsy and experimental studies had indicated that metastases can metastasize, the question of whether metastases from metastases increasingly contribute to the overall metastatic burden is crucial to the basic question of whether the metastatic process is more directly regulated by genetic or by epigenetic mechanisms. The highly metastatic human C8161 melanoma was transfected with either pSV2neo or pSV2hygro and clones of neo-C8161 and hyg-C8161 were injected intravenously and subcutaneously in SCID mice. In combination experiments, both the timing and the size of inoculum of tumor cells were titrated to ensure that the hematogenously injected cells disseminated almost exclusively to the lungs and that the overall pulmonary burden was equal to the primary tumor. In s.c. injection experiments, no spontaneous metastases ever developed when the primary tumor was extirpated before it had grown to more than 0.5 cm in diameter. When the primary tumor approached 1 cm in diameter, widely-disseminated metastases developed within lungs, liver, subcutaneous sites and other internal viscera. In the combination-injection experiments, while large numbers of both hematogenously and spontaneously metastatic clones were recovered from the lungs, a vast excess of only the latter clones was recovered from extrapulmonary sites. Both hematogenously and spontaneously metastatic pulmonary clones recovered showed similar levels of Matrigel invasion and collagenases by substrate gel electrophoresis, but significantly decreased levels when compared to the cell line. Primary tumor clones, in contrast, demonstrated increased invasion and increased collagenases. Our findings argue for the importance of paracrine (orthotopic) and autocrine (size) epigenetic mechanisms in the regulation of metastasis. © 1996 Wiley-Liss, Inc.     

GROWTH OF A LOCAL TUMOR EXERTS A SPECIFIC INHIBITORY EFFECT ON PROGRESSION OF LUNG METASTASES

The effects of local tumor growth on the progression of lung metastasis of two mouse tumors of C57BL origin—the 3LL Lewis lung carcinoma and the B-16 melanoma—were studied. Excision of primary footpad grafts of 3LL or B-16 resulted in a significant increase in incidence and size of lung metastases. The incidence, but not the size, of lung metastases was found to be a function of size of the local tumor at time of excision. Reinoculation of tumor cells in the left footpad following excision of tumor from the right footpad suppressed the acceleration of growth of lung metastases and their increased incidence. Thus, the progression of the local tumor exerts an inhibitory effect on lung metastasis. The extent of inhibition of accelerated metastatic development by reinoculated tumor cells is a function of size of the cell inoculum or, in fact, of mass of the local tumor. The inhibitory effect of the local tumor on lung metastasis seems to be tumor-specific. 3LL metastases were not inhibited by local B-16 or EL4, neither was B-16 metastasis inhibited by 3LL. The specificity of interactions between the local tumor growth and its metastases may suggest the participation of immunological mechanisms in the control of lung metastasis. The involvement of the lymphoid system in the control of metastatic progression is also supported by the observation that total-body X-irradiation was associated with an increase in lung metastasis and that splenomegaly was observed in mice bearing the footpad tumor. Its excision resulted in the prevention of splenomegaly but reinoculation of the tumor, leading to metastatic suppression, was again associated with splenomegaly.     

Novel metastasis model of human lung cancer in SCID mice depleted of NK cells

Metastasis is a critical problem in the treatment of human lung cancer. Thus, a suitable animal model of metastasis of human lung cancer is required for in vivo biological and preclinical studies. In this study, we tried to establish a suitable model for this, using SCID mice. Neither human SCLC H69/VP cells (5 × 10⁶) nor squamous-cell carcinoma RERF-LC-AI cells (1 × 10⁶), injected through a tail vein, formed metastases in untreated SCID mice. Pre-treatment of SCID mice with anti-asialo GM₁ serum resulted in only a few metastases of H69/VP cells, but pre-treatment with anti-mouse IL-2 receptor β chain Ab (TM-β1) resulted in numerous lymph-node metastases 56 days after tumor inoculation. H69/VP-M cells, an in vivo-selected variant line, formed significant numbers of lymph-node metastases even in SCID mice pre-treated with anti-asialo GM₁ serum. SCID mice depleted of NK cells by treatment with TM-β1 showed different patterns of metastasis when inoculated intravenously with the 2 different human lung cancer cell lines (H69/VP and RERF-LC-AI cells): H69/VP cells formed metastases mainly in systemic lymph nodes and the liver, whereas RERF-LC-AI cells formed metastases mainly in the liver and kidneys, with only a few in lymph nodes. A histopathological study showed that the metastatic colonies consisted of cancer cells. The numbers of metastatic colonies formed by the 2 cell lines increased with the number of cells inoculated. TM-β1 treatment of SCID mice efficiently removed NK cells from peripheral blood for at least 6 weeks, whereas, after treatment of the mice with anti-asialo GM₁ serum, NK cells were recovered within 9 days. These findings suggest that NK-cell-depleted SCID mice may be useful as a model in biological and pre-clinical studies on metastasis of human lung cancer. © 1996 Wiley-Liss, Inc.     

Growth characteristics of clonal cell populations constituting a B16 melanoma metastasis model system

Three distinct dissemination-related phenotypes have been distinguished among cell subpopulations of the mouse B16 melanoma: tumorigenicity, spontaneous metastasis from subcutaneous tumors, and organ colonization following intravenous injection of cells. From a progenitor clone (G3) of tumorigenic but nonmetastatic and noncolonizing (null) cells that underwent phenotypic diversification in vitro and in vivo, 4 subclones were obtained: G3.5 (culture-generated metastatic), G3.12 (tumor-generated metastatic), G3.15 (culture-generated null), and G3.26 (tumor-generated colonizing). The growth potentials of the parent clone and derived subclones were investigated comparatively in in vivo assays (tumorigenicity, tumor growth rate, and lung colonization potential), monolayer culture assays (generation time, saturation density, clonogenicity, and rate of detachment by trypsin), and in soft agar. In overall growth potential, G3.26 greater than G3.12 greater than G3, G3.5 greater than G3.15. These results indicate that metastatic populations of the B16 melanoma are not the most rapidly and effectively growing cells obtainable from that tumor.