Generation of phenotypic diversity in the B16 mouse melanoma relative to spontaneous metastasis

Serial s.c. transplantation of the B16 melanoma in syngeneic mice for nearly 30 monthly generations effected gradual changes in the incidence of spontaneous pulmonary metastasis. Cell lines derived from s.c. tumors and from secondary tumor growths in the lungs were comparably variable in metastatic predilection and also differed in ability to produce tumor colonies in the lungs following i.v. injection of cultured cells. Some lines metastasized to the lungs from s.c. tumors and also colonized the lungs; others were metastatic but noncolonizing, colonizing and nonmetastatic, or nonmetastatic and noncolonizing ("null"). Metastatic and colonizing activities of all cell lines except the most potent colonizers were unstable in culture and during s.c. growth. Over 150 clones and subclones were obtained from B16 melanoma cell lines, and four distinct categories were defined on the basis of dissemination-related phenotypic characteristics: slow-growing null (Ns); rapid-growing null; metastatic; and colonizing. No single cell belonged to more than one category at the same time, but interconversions occurred rapidly and consistently during growth in vitro and in vivo. Progenitor Ns cells generated metastatic cells in culture and in tumors and became rapid-growing null cells and colonizers solely within tumors. Metastatic activity was transient, with cells reverting back to an Ns phenotype in culture and s.c. or converting to rapid-growing null cells and colonizers in vivo. Only potent colonizers were stable, an apparent end result of phenotypic diversification, but formation or proliferation of these cells within tumors was somehow regulated. Comparable heterogeneity was generated within lung metastases, except that reversion of metastatic cells to Ns cells and regeneration of metastatic activity were not demonstrated; the result was a progressive loss of metastatic cells within developing metastases.     

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.     

DNA amplification and metastasis of the human melanoma cell line MeWo

A positive correlation was found between the presence of amplified DNA in the form of homogeneously staining regions (HSRs), and the formation of spontaneous metastases by the human melanoma cell line MeWo. HSR+ and HSR- MeWo sublines and clones were injected s.c. into BALB/c nude mice. All MeWo lines produced primary tumors that were allowed to grow to a similar size before the animals were sacrificed and examined for the presence of metastatic nodules in the lungs and abdominal cavity. HSR+ lines gave extensive metastases (greater than 100 nodules) in the lungs and/or liver and abdomen in 18 of 19 animals. The HSR- MeWo lines were effectively nonmetastatic, producing metastases in 3 of 20 animals, two of which had only a single metastatic lung nodule each. Evidence for the presence of HSRs in the primary tumors and metastatic nodules was obtained by DNA dot-blot hybridization to a sequence (D15Z1) amplified in the HSRs, flow cytofluorometry for cellular DNA content, and quinacrine staining of metaphase spreads. The HSR+ clones also colonized the lung to a much greater extent than HSR- clones following i.v. injection. In addition, the HSR+ clone had a selective advantage in lung colonization, since i.v. injection of a 50:50 mixture of HSR+ and HSR- clones resulted in extensive metastases populated exclusively by HSR+ cells. The results suggest that DNA sequences amplified in the HSRs of human melanoma MeWo cells may confer enhanced metastatic properties to these cells.     

Colonization of human lung grafts in SCID-hu mice by human colon carcinoma cells

Human colon carcinoma cell lines were examined in a colonization assay using SCID-hu mice engrafted with human fetal lung (HFL) tissues. Cell lines SW620 and COLO 320DM, derived from metastatic tumors, colonized HFL grafts after i.v. injection into SCID-hu mice. Cell lines SW480 and T34 initiated from primary colon tumors were unable to colonize HFL grafts. The ability to colonize HFL grafts but not mouse lungs of SCID-hu-L mice correctly reflects the clinical origin of these human colon carcinoma cell lines. Expression of a number of cell adhesion molecules was examined on SW480, SW620 and in vivo selected highly aggressive variants of SW620. NCAM and integrin α3 expressed on the surface of SW480 cells were lost from metastatic cells, while carbohydrate ligands sialyl Lewis x and a, previously shown to be upregulated in metastatic colorectal tumors, were expressed at higher levels on colonizing cells. Unlike SW480, SW620 and its in vivo selected variants expressed RNA for calcium binding protein calbindin-D28K, a neuroendocrine marker. Acquisition of neuroendocrine features might be of potential importance in the development of the metastatic phenotype. © 1996 Wiley-Liss, Inc.     

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.     

Follistatin suppresses the production of experimental multiple-organ metastasis by small cell lung cancer cells in natural killer cell-depleted SCID mice.

PURPOSE: Follistatin (FST), an inhibitor of activin, regulates a variety of biological functions, including cell proliferation, differentiation, and apoptosis. However, the role of FST in cancer metastasis is still unknown. Previous research established a multiple-organ metastasis model of human small cell lung cancer in natural killer cell-depleted SCID mice. In this model, i.v. inoculated tumor cells produced metastatic colonies in multiple organs including the lung, liver, and bone. The purpose of this study is to determine the role of FST in multiple-organ metastasis using this model. EXPERIMENTAL DESIGN: A human FST gene was transfected into the small cell lung cancer cell lines SBC-3 and SBC-5 and established transfectants secreting biologically active FST. The metastatic potential of the transfectants was evaluated using the metastasis model. RESULTS: FST-gene transfection did not affect the cell proliferation, motility, invasion, or adhesion to endothelial cells in vitro. I.v. inoculated SBC-3 or SBC-5 cells produced metastatic colonies into multiple organs, including the lung, liver, and bone in the natural killer cell-depleted SCID mice. FST transfectants produced significantly fewer metastatic colonies in these organs when compared with their parental cells or vector control clones. Immunohistochemical analyses of the liver metastases revealed that the number of proliferating tumor cells and the tumor-associated microvessel density were significantly less in the lesions produced by FST transfectants. CONCLUSIONS: These results suggest that FST plays a critical role in the production of multiple-organ metastasis, predominantly by inhibiting the angiogenesis. This is the first report to show the role of FST in metastases.     

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.     

Correlation of frequency of induced mutation and metastatic potential in tumor cell lines from a single mouse mammary tumor

Spontaneous mutation rates were determined in mouse mammary tumor subpopulation lines that differ in metastatic phenotype. Although there was almost a 9-fold difference in spontaneous rates to ouabain resistance among the three lines tested, the difference did not correlate with ability to metastasize. Similarly a 10-fold difference in spontaneous rates to 6-thioguanine resistance did not correlate with metastatic ability. In contrast, the frequency of ethyl methanesulfonate-induced mutations was associated with metastatic potential. Thus, ethyl methanesulfonate only induced significant numbers of 6-thioguanine resistant colonies in 66 and 410.4 cells, the only 2 of 5 lines tested that spontaneously metastasize at high frequency, and of ouabain resistant colonies in 66, 410.4, and 168 cells, the only lines tested that produce experimental lung metastases after i.v. injection. Differential sensitivity to induced mutation was not correlated with differences in plating efficiency, wild type sensitivity to ethyl methanesulfonate, 6-thioguanine, or ouabain toxicity, ploidy, cell shape, cell size, or ability to engage in metabolic cooperation.     

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.     

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.     

Malignant potential of cells isolated from lymph node or brain metastases of melanoma patients and implications for prognosis

We studied the correlation between the formation of brain metastasis and the malignant growth potential of seven human melanoma cell lines, isolated from lymph node metastases (A375-SM, TXM-1, DM-4) or from brain metastases (TXM-13, TXM-18, TXM-34, TXM-40), and the potential of three variants of the mouse K-1735 melanoma. Growth rates in different concentrations of fetal bovine serum and colony-forming efficiency in semisolid agarose were measured, and the tumorigenicity and metastatic ability were determined in nude mice (for the human melanoma cell lines) or in C3H/HeN mice (for the K-1735 variants). The ability to form brain metastasis was tested by injection of cells into the carotid artery. A high colony-forming efficiency in agarose, especially at concentrations of agarose greater than 0.6%, corresponded with high tumor take rates, rapid tumor growth rates, and metastatic colonization of the lungs of the recipient mice. For the human melanomas, the lymph node metastasis-derived cells were more tumorigenic and metastatic than the brain metastasis-derived cells. In the K-1735 mouse melanoma, the tumorigenic and metastatic behavior of the cells after i.v. and s.c. injection corresponded with growth in agarose cultures. However, for growth in the brain after intracarotid injection, the different melanoma cell lines showed similar frequencies of tumor take, regardless of tumorigenicity in other sites of the recipient mice, although mice given injections of brain metastasis-derived cells survived longer than mice given injections of lymph node metastasis (human melanoma) or lung metastasis (K-1735 M-2)-derived cell lines. The results from the human and mouse melanoma cell lines show that the brain metastasis-derived cell lines were not more malignant than the lymph node or lung metastasis-derived cells. These data imply that the production of brain metastasis is not always the final stage of a metastatic cascade.     

Metastatic growth of a murine tumor: evidence of dissemination to the lungs in the absence of subcutaneous growth

Growth of MCA-38/B colon adenocarcinoma was detectable 30-33 days after subcutaneous (s.c.) tumor cell inoculation in mice. Seventy percent of the mice receiving 10(7) tumor cells, 50% of those receiving 10(6), and 15% of the mice given 10(5) cells developed s.c. tumors (mean of 4 experiments, total of 80 mice per group). Metastases in the presence of a primary tumor were observed in 11% of 10(7) and in 10% of 10(6) tumor-cell injected animals. Lung metastases were detected in the absence of tumor growth at the site of s.c. cell injection in 19% of 10(7), in 8% of 10(6) and in 5% of 10(5) and 10(4) tumor-cell inoculated mice. In parallel experiments an intravenous (i.v.) inoculum of tumor cells produced lung colonies in 40% of 10(6) and in 14% of 10(5) tumor-cell injected animals. Smaller inocula did not give rise to lung colonies, thus making it unlikely that accidental i.v. inoculations of tumor cells during the s.c. injections caused the observed metastatic dissemination to the lungs.     

Promotion of pulmonary metastasis in mice by bleomycin-induced endothelial injury

The passage of circulating tumor cells across the vascular wall is an important step in the evolution of cancer metastases. Since tumor cells attach preferentially to subendothelial matrix at sites of endothelial injury and retraction in vitro, we have used an established in vivo model of pulmonary endothelial damage to examine the effects of endothelial injury on the localization and metastasis of circulating tumor cells in vivo. C57BL/6 mice were given a single i.v. dose of bleomycin (120 mg/kg) or multiple i.p. injections (10 mg/kg, twice weekly for 6 wk). Five days after the single injection or 4 days after the last i.p. injection, 2 X 10(5) [131I] iododeoxyuridine-labeled fibrosarcoma cells or unlabeled cells were injected i.v. Two to 8 times as many labeled cells were found in the lungs of bleomycin-treated animals after 24 h. Two and 3 wk after injecting unlabeled fibrosarcoma cells, 1.4 to 5 times more metastatic lung colonies were counted in bleomycin-treated animals than in controls. Morphometric analysis of histological sections demonstrated that the percentage of lung area occupied by tumor in bleomycin-treated animals was between 4 and 16 times that of controls. Analysis of bronchoalveolar lavage fluids demonstrated 5-fold increases of total protein content and leakage of previously injected 125l-labeled albumin, indicating increased endothelial permeability. Electron microscopic examination of lungs of bleomycin-treated mice demonstrated endothelial retraction with exposure of the underlying basement membrane. Electron microscopy of [3H]thymidine-labeled tumor cells, located by autoradiography, demonstrated their attachment to exposed basal lamina. Data from these experiments in vivo support the hypothesis that endothelial damage can facilitate the metastasis of circulating tumor cells.     

Metastatic phenotype of murine tumor cells expressing different cooperating oncogenes.

Four murine cellular tumor models expressing various combinations of oncogenes (SV40 large T and v-Ha-ras, SV40 large T and v-src, SV40 large T and neu, adenovirus EIA and v-Ha-ras) induce sarcoma when they are inoculated s.c. into the DBA/2 syngenic mice. The metastatic patterns, distribution and fate of these tumor cells transplanted by two different routes into syngenic DBA/2 mice have been studied. All the tumor cell lines except EIA-ras, induce massive overt artificial metastases principally in the lung after i.v. injection. In s.c. tumor-bearing mice, a few resting cells colonize the lung as micrometastases. When removed from this tissue context and injected s.c. these cells regain their proliferative potential and grow as local tumors which again give rise to occult pulmonary micrometastases.     

A murine model of experimental metastasis to bone and bone marrow

Bone is a common site of metastasis in human cancer. A major impediment to understanding the pathogenesis of bone metastasis has been the lack of an appropriate animal model. In this paper, we describe an animal model in which B16 melanoma cells injected in the left cardiac ventricle reproducibly colonize specific sites of the skeletal system of mice. Injection of 10(5) cells resulted in melanotic tumor colonies in most organs, including the skeletal system. Injection of 10(4) or fewer cells resulted in experimental metastasis almost entirely restricted to the skeletal system and ovary. In contrast, i.v. injection of 10(5) cells resulted in tumor colonies in the lung only. Left cardiac injection of 10(2) cells caused bone colonization, but the same number of cells injected i.v. did not colonize the lung. The number of bones with tumor colonies increased with increasing number of cells injected. Melanotic tumor colonies in the bone were characteristically distributed in the metaphysis of long bones and in the periphery of flat bones. Most animals developed paraplegia due to spinal cord compression by bony metastasis to the spine. Tumor colonization of bone occurred only in regions of bone containing hematopoietic bone marrow. This suggests that the injected tumor cells lodge, survive in the hematopoietic bone marrow environment, and grow to destroy adjacent bone. This experimental model of metastasis to bone will facilitate future studies of the pathophysiology and treatment of bone and bone marrow metastasis.     

99mTcOAADT]-(CH2)2-NEt2: a potential small-molecule single-photon emission computed tomography probe for imaging metastatic melanoma

Evaluation of [99mTc]oxotechnetium(V) complexes of the amine-amide-dithiol (AADT) chelates containing tertiary amine substituents as small-molecule probes for the diagnostic imaging of metastatic melanoma has shown that technetium-99m-labeled AADT-(CH2)2-NEt2 (99mTc-1) has the highest tumor uptake and other favorable biological properties. We have, therefore, assessed this agent in a more realistic metastatic melanoma model in which, after i.v. tail injection, a highly invasive melanoma cell line, B16F10, forms pulmonary tumor nodules in normal C57BL6 mice. Small melanotic lesions develop in the lungs and, on histologic examination, appear as small black melanoma colonies, increasing in size and number with time after tumor cell injection. Groups of mice received tumor cell inocula of 2 x 10(5), 4 x 10(5), or 8 x 10(5) B16F10 cells; 14 days later, 2 hours after 99mTc-1 administration, lung uptake of 2.83 +/- 0.21%, 3.63 +/- 1.07%, and 4.92 +/- 1.61% injected dose per gram of tissue (% ID/g), respectively, was observed, compared with normal lung uptake of 2.13 +/- 0.2% ID/g (P < 0.05). Additionally, a higher level of 99mTc-1 accumulation was seen 17 days after tumor cell inoculation as the lung lesions grew. These in vivo studies coupled with additional in vitro and ex vivo assessment show that 99mTc-1 has high and specific uptake in melanoma metastases in lungs and can potentially follow the temporal growth of these tumors.     

Metastatic heterogeneity of cells from Lewis lung carcinoma

To allow investigations of the role of tumor cell proteases in invasion and metastasis, an attempt was made to obtain well-defined homogeneous populations of Lewis Lung carcinoma cells differing widely in their metastatic potential. From a single Lewis lung carcinoma, a parental line of cells was established and subsequently cloned so as to provide 18 clonal tumor cell lines. These clones differed in their ability to produce spontaneous, macroscopically visible metastases in the lung after i.m. inoculation into syngeneic C57BL/6 mice. Several of them were less metastatic than the parental line. The parental line expressed a metastatic behavior close to that of the high-metastatic cell subpopulations that it contained. There was, within certain limits, a good correlation between the potential for spontaneous lung metastases arising from a primary tumor and that for "artificial" lung colonies obtained after i.v. injection of the Lewis lung carcinoma cells. Although positively correlated with the growth rate of the tumor cells, the metastatic ability of the clones could not be considered as a mere reflection of the proliferation rates of the cells constituting the primary tumors. Differences in metastatic behavior observed among clones persisted in several cases after the cells had been maintained in culture for prolonged periods. However, this stability of the clones in vitro was not absolute. Indeed, some subclones isolated from the low-metastatic clone H122 displayed metastatic abilities which were lower than that of the parent clone. Furthermore, a significant increase in metastatic potential was once observed after a prolonged culture period of that same clone, H122. Thus, new metastatic phenotypes can emerged under in vitro culture conditions. However, the relative rarity of this event suggests that some metastatic heterogeneity already preexisted in vivo among the tumor cells.     

Metastatic potential of four human melanoma xenografts in young athymic mice following tail vein inoculation

We have investigated the lung colonizing ability of four newly established human metastatic melanoma xenografts, designated CRML1, CRML2, CRML3, and CRML4 following i.v. tail vein inoculation into 3- to 4-week-old gnotobiotic CD-1 athymic mice. The experimental metastatic potential of the tumors was assessed from the primary tumor samples through eight progressively growing s.c. passages. CRML1 and 2 were investigated in detail; five sublines (two from CRML1 and three from CRML2) were established from these tumors with various growth rates and lung colonizing abilities. The histopathologies of the patients' biopsies and the s.c. passaged parental lines were compared with these i.v.-derived sublines as one measure of tumor heterogeneity, in conjunction with the kinetics of lung tumor formation. The frequency and distribution of extrapulmonary tumor growth was also investigated after i.v. inoculation. In general, it reflected the clinical distribution of metastases, although their frequency of appearance was reduced. While CRML3 was the most aggressive disease clinically, it did not demonstrate the reproducible experimental metastasis of the other lines. CRML4 produced lung colonies routinely, but with latency periods of 20 weeks or more. On the other hand, the most rapidly growing sublines of CRML1 and CRML2 essentially replaced the normal lung tissue within 4 to 6 weeks following inoculation of 10(5) cells. The two sublines of CRML1 with higher effective metastatic potential were maximally able to colonize the lungs within only two i.v. cycles in one case, while the other line required six i.v. to i.v. passages to reach its maximum ability. In CRML2, two sublines were identified that rapidly increased in their lung colonizing ability, while another line remained effectively equal to the parental s.c. line over four cycles of reinoculation i.v. These results demonstrated that the athymic mouse can serve as a model for experimental metastasis of human tumors, and that aspects of the metastatic heterogeneity of these tumors can be investigated by using this system.     

Effects of the immunomodulator LS 2616 on growth and metastasis of the murine B16-F10 melanoma

The carboxamide-quinoline LS 2616 is a novel immunomodulator augmenting natural killer (NK) cell activity and T-lymphocyte related effector functions. To investigate the possible usefulness of LS 2616 in immunotherapy of tumors, the effect of the substance on growth and metastasis of the B16-F10 melanoma in syngeneic C57BL/6 mice was investigated. Treatment with LS 2616 from the time of s.c. inoculation of B16-F10 cells significantly reduced tumor take. Continuous treatment of mice with LS 2616 initiated 4 days prior to i.v. injection of tumor cells reduced the number of pulmonary metastases by 85%. When treatment with LS 2616 was started 4 days after i.v. injection of tumor cells, a time when established tumor foci were readily detectable in the lungs, a significant reduction in the number of pulmonary metastases resulted. LS 2616 significantly reduced the number of spontaneous pulmonary metastases developing from a B16-F10 tumor growing in the footpad. When treatment with LS 2616 was initiated after the establishment of grossly visible spontaneous pulmonary metastases, no significant effect on the number of metastases was found after 2 weeks of treatment. However, combined treatment with a dose of cyclophosphamide which in itself was ineffective resulted in a statistically significant 70% reduction in the number of remaining pulmonary metastases. Injection of antibodies to asialomonoganglioside which strongly reduce NK cell activity in various organs was used as a probe for the involvement of NK cells in the effects of LS 2616 on the B16-F10 tumor. The therapeutic efficiency of LS 2616 on tumor take when given from the time of s.c. inoculation, on the number of i.v. induced pulmonary metastases when treatment was started before tumor cell injection, as well as the spontaneous development of pulmonary metastases during exposure to the substance was abrogated by simultaneous injection with antibodies to asialomonoganglioside. In contrast, the beneficial effects of LS 2616 on already established i.v. produced or spontaneous pulmonary metastases were unaltered in mice made NK cell deficient by injection of anti-asialomonoganglioside antibodies. In conclusion, LS 2616 has potent antitumor activities mediated by NK cells as well as non-NK cell related defense mechanisms.