Malignant progression of B16 melanoma cells induced in vitro by growth factors produced by highly malignant cells.

Four mouse B16 melanoma subclones (G3.15, G3.5, G3.12 and G3.26) exhibit progressively greater growth capacity in vitro and in vivo. Previously, non-metastatic G3.15 cells were sequentially converted, in monolayer cultures, to the moderately-metastatic G3.5 cells, and then to a highly-metastatic G3.5* phenotype. Both conversions were induced by hypoxia followed by confluence, and also occurred in tumors. G3.5* cells were comparable with, yet distinguishable from, G3.12 cells in being growth-autonomous in culture. In this study, the presumption that rapidly-growing G3.26 cells represented the ultimate progression step in this clonal system was examined. Both G3.12 and G3.5* cells converted in vitro to the G3.26 phenotype during growth in serum-free medium conditioned by G3.26 cell growth. By selective filtration of conditioned medium and characterization of the stability of growth- and conversion-promoting activities, three distinct activities were found to promote a two-step G3.12 to G3.26 phenotype conversion: (1) a < 10 kDa filtrate stimulated slight attachment and proliferation of G3.12 cells, effects that were reversible, partly attributable to accumulated lactate, and fully mimicked by medium acidification to pH 6.5; (2) medium acidification, together with a heat- and acid-stable but partially trypsin-sensitive > 10 kDa activity, induced G3.12-->G3.5* conversion that resulted in acquisition of growth autonomy; and (3) a heat-, acid- and trypsin-sensitive > 10 kDa activity induced G3.5*-->G3.26 conversion, characterized by anchorage-independent growth in soft agar, and potent lung colonization following intravenous injection. Phenotype analysis of G3.12 tumors and lung metastases revealed that G3.5*-like cells were regularly present in tumors and metastases, whereas G3.26-like cells occurred almost exclusively in large lung metastases. While G3.12 cells might convert to G3.5* cells in order to disseminate, G3.26 cells are apparently not involved in metastatic spread but probably account for the rapid growth of established metastases.

Acquisition of in vitro growth autonomy during B16 melanoma malignant progression is associated with autocrine stimulation by transferrin and fibronectin.

Four mouse B16 melanoma subclones representing distinct stages in the benign-to-malignant progression of that tumor (G3.15, G3.5, G3.12, and G3.26), and three phenotype conversion variants with enhanced malignancy (G3.15*, G3.5*, and G3.12*), were comparatively examined for exogenous mitogen and growth factor requirements and for responsiveness to exogenous and endogenous growth modulators in monolayer culture. Growth behavior in serum-free medium with or without mitogen or growth factor supplements, and in supplemented quiescent serum-containing medium, confirmed previous indications that the G3.5 and G3.15* phenotypes were identical, as were the G3.26 and G3.12* phenotypes. However, G3.12 differed from the closest conversion equivalent, G3.5*, and probably represents an aberrant phenotype within this sequence. There was a direct relationship between degree of malignancy (G3.15-->G3.5-->G3.5*-->G3.26), growth capacity in serum-free medium, and responsiveness to transferrin. Only G3.5*, G3.26, and G3.12* cells were growth-autonomous in serum-free medium and also highly responsive to mitogens. The polypeptide growth factors epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, transforming growth factor-alpha, and insulinlike growth factor-1 and -2 were generally stimulatory in quiescent medium, but the degree of growth promotion was unrelated to malignancy level. Transforming growth factor-beta 1 was inhibitory to the more benign populations (G3.15, G3.5, and G3.15*) but stimulated proliferation of other cells. All populations produced autocrine fibronectin, and G3.12, G3.5*, G3.26, and G3.12* cells also produced autocrine transferrin. Only G3.12 cells failed to utilize both of those factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and transcriptional activity of AP-1, CRE, and URE binding proteins in B16 mouse melanoma subclones.

The expression and DNA binding activity of members of the activating protein-1 (AP-1) and activating transcription factor (ATF) families of transcription factors were analyzed in sham and ultraviolet (UV)-irradiated subclones of the B16 mouse melanoma cell system. The four subclones we used represent sequential stages in the development and progression of malignant melanoma and exhibit differences in growth and metastatic potential. Western blot analysis revealed differential expression of some AP-1 (c-jun, jun-B, and jun-D) and ATF (43- and 47-kDa cyclic AMP-responsive element binding protein (CREB) family members) in the different subclones; while c-jun expression was noted in the subclones with the greater malignant potential, jun-D was expressed in those with the lesser malignant potential. Furthermore, a delicate balance between the two forms of CREB was noted; the 47-kDa CREB appeared, when expressed exclusively, in subclones that exhibit a greater malignant potential. Electrophoretic mobility shift assays using AP-1, CRE, and UV-responsive element (URE) consensus sequences indicated that distinct complexes were formed with extracts from each of the four subclones. The complexes were competitively inhibited by each of the target sequences used, suggesting that "cross-talk" occurs between some AP-1 and ATF family members in this cell system. Moreover, a multimer of the URE sequence, cloned upstream of a chloramphenicol acetyltransferase reporter gene, was transcriptionally active and responsive to UV irradiation in two of the four subclones. UV-related transcriptional activation was directly correlated with the expression of a 43-kDa CREB. Together, these observations identify members of AP-1 and CREB families whose expression and activities correlate with the malignant potential of subclones that represent different stages in melanoma development and progression.

Benign-to-malignant B16 melanoma progression induced in two stages in vitro by exposure to hypoxia.

BACKGROUND: Benign tumors apparently become malignant by generating a succession of variants with ever-greater growth potential and autonomy. Such stepwise progression has not been achieved in vitro under conditions likely to occur in developing tumors. PURPOSE: Tumors initiated by clone G3.5 of the mouse B16 melanoma regularly generate stable variants that are more malignant. We investigated the possibility that hypoxia might promote stepwise progression along a benign-to-malignant pathway in monolayer cultures of G3.5 cells. METHODS: Confluent monolayers of metastatic clone G3.5 and the nonmetastatic clone G3.15 were subjected to severe hypoxia (< 50 ppm O2) for up to 72 hours, or to moderate hypoxia (300-1200 ppm O2) for up to 12 days, and were then maintained subconfluent or at confluence for several weeks to permit emergence of progression variants. The relative malignancy of variants was assayed in vivo after subcutaneous injection into mice, by measuring tumor growth rate and counting lung metastases, and after intravenous injection, by counting lung colonies. In vitro assessment of the variants involved growth as monolayers with or without serum, growth in soft agar, and measurement of invasiveness. RESULTS: G3.5 cells were converted to a more malignant variant (G3.5*) by 12-48 hours of severe hypoxia, or longer periods of moderate hypoxia, when followed by maintenance at confluence for 3-5 weeks. Conversions occurred in discrete foci of morphologically-discernible cells (optimum focus formation about one in 1-2 x 10(5) cells) that rapidly expanded to dominate the cultures. The G3.5* phenotype was comparable to the conversion phenotype generated in tumors and included acquisition of growth autonomy in serum-free medium. G3.15 cells were converted to a G3.5-like phenotype by one round of exposure to hypoxia and confluence, and then to the G3.5* phenotype during a second round, at a low frequency (one focus in 5 x 10(6) cells). This behavior was consistent with a failure of all but the largest G3.15 tumors to generate G3.5* conversion cells. CONCLUSIONS: Progression from a relatively benign phenotype, G3.15, to a highly malignant phenotype, G3.5*, can be produced in monolayer culture in two stable stages by sequential rounds of exposure to hypoxia and confluence. The resulting conversions corresponded to phenotypes generated within tumors. Both conversions resulted in populations with enhanced growth capabilities, which could establish dominance within tumors. IMPLICATIONS: The stepwise conversion of B16 melanoma clones provides a unique model for the in vitro investigation of mechanisms underlying acquisition of malignancy during tumor development.

Patterning of B16 melanoma metastasis and colonization generally relates to tumor cell growth-stimulating or growth-inhibiting effects of organs and tissues.

The mouse B16 melanoma metastasizes first to the lungs and secondarily to systemic sites, involving mainly the adrenals, ovaries and pancreas. Systemic colonization effected by intracardiac injection of tumor cells establishes similar patterning, but in addition frequently colonizes the bones. To assess possible systemic site influences on metastasis and colony formation, the capacity of B16 melanoma cells to proliferate in these sites in vivo and in ex vivo explants following intracardiac injection was examined. Effects of cells isolated from these sites, and of organ- or tissue-conditioned medium, on growth of B16 cells in monolayer culture were also studied. Injected fluorochrome-labeled tumor cells initially distributed without site preference, but within 48 h had begun proliferating in the adrenals, ovaries and lungs, while remaining static in the pancreas and bones, and disappearing from the spleen, liver, kidneys, brain, and skeletal muscles. Mitogenic activity releasable in soluble form was associated with all favorable organs and tissues and was the predominant influence of those tissues on cultured tumor cells. In contrast, the overall effects of liver, spleen, kidney, and brain tissues were to inhibit tumor cell growth. Soluble growth-promoting activity enhanced clonogenic growth of isolated tumor cells stimulated by mouse serum, suggesting that metastasis or colony formation might be stimulated in favorable sites by those factors together with blood-borne growth factors. The observed effects of organ- and tissue-derived cells and soluble factors on tumor cells generally reflected the in vivo consequences of tumor cell entrapment in the corresponding sites. However, the failure of metastases to develop in the bones, which are favorable sites for colonization by the same cells, remains puzzling.

B16 melanoma variants selected by one or more cycles of spontaneous metastasis to the same organ fail to exhibit organ specificity.

Metastatic clones of the mouse B16 melanoma spontaneously disseminate from subcutaneous tumors throughout the body in two stages, initially to the lungs and secondarily from established lung metastases to systemic sites. From the heterogeneous 'parent' B16 melanoma cell line and from two representative clones, G3.5 and G3.12, cell populations were selected after one or more cycles of tumor growth or metastasis to a particular site, to determine whether metastatic variants with greater organ preference or specificity could be generated. Variants with enhanced secondary metastatic activity were obtained only from G3.12 tumor-disseminated metastases growing in the lungs or in systemic organs. Regardless of the organ of selection or the number of selection cycles, all variants exhibited an overall increase in secondary metastasis incidence and burden in the brain, adrenals, kidneys and ovaries, but no organ preference or specificity was obtained. Populations that grew especially well in the brain, ovaries or liver following intravascular injection were either non-metastatic or exhibited no organ preference during spontaneous metastasis. The increased secondary metastatic activity of G3.12 variants was apparently not due to either longer host survival or to tumor-disseminated cells bypassing the lungs, but may result from enhanced growth potential or greater secondary dissemination capability imparted during growth as lung metastases.

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.

Hemodynamic considerations in organ and tissue patterning of B16 melanoma systemic metastasis and colonization.

Several populations of the mouse B16 melanoma that are highly metastatic from subcutaneous transplants but differ in growth characteristics were compared with regard to systemic site patterning of visible metastasis, as well as colonization effected by intracardiac injection of tumor cells. In all cases, metastasis proceeded in two stages, initially to the lungs and secondarily from lung metastases to systemic sites. The relative ranking of systemic site involvement by secondary-stage metastasis was basically similar for all tumor cell populations; the overall hierarchy was: kidneys greater than brain greater than adrenals and ovaries greater than pancreas greater than mesentery. Colonization patterns resulting from intracardiac injection were also generally comparable but differed from metastasis patterning in that the kidneys and brain were poorly colonized while the bones were frequent sites of colonization. Enumeration of fluoresceinated tumor cells or microbeads trapped in various sites following intracardiac injection revealed a ranking of initial involvement that differed markedly from colony formation. These results indicate that the hemodynamics of blood flow is not a critical determinant of colonization patterning. Based on the colonizing behavior of microbead-bound tumor cells, the frequent metastatic involvement of the kidneys and brain appears to result from selective trapping of large multicell tumor emboli within arteries in those organs. The occurrence of metastasis in other systemic sites is, like colonization, not readily explained by hemodynamics.

Differences in organization of metastatic and nonmetastatic tumors initiated by the same B16 melanoma clone in mature and young mice.

Subcutaneous transplants of mouse B16 melanoma clone G3.26 grow more slowly, and are markedly more metastatic to the lungs, in mature (greater than 12-month-old) mice than in young (2-month-old) mice. Previous studies suggested that tumors in young mice fail to disseminate viable tumor cells into the hematogenous circulation. To determine if changes in intratumor organization might accompany this altered tumor behavior, G3.26 tumors growing in young and mature mice were examined comparatively at progressive sizes relative to the onset of metastatic dissemination in the older mice. Although the degree of necrosis was comparable in both groups of tumors, vascular density, measured morphometrically in histological sections, was significantly lower in tumors from mature mice at a size when dissemination would be occurring. With the onset of reduced vascular density in tumors in mature mice, there was a substantial increase in the proportion of viable tumor cells that was hypoxic, based on radioresistance and incorporation of the hypoxic cell sensitizer, misonidazole. Quiescent tumor cells, identified by flow cytometry, were also more numerous in tumors from mature mice than in tumors from young mice. Although the importance of these differences in tumor organization to enhanced metastatic behavior is unclear, increased intratumor hypoxia might promote generation of metastatic variants. Alternately, dissemination of tumor cells might be facilitated through a reduced and possibly defective vasculature.

Intrapulmonary spread of established B16 melanoma lung metastases and lung colonies.

Spontaneous metastasis of subcutaneous B16 melanoma transplants proceeds in two distinct stages: initially to the lungs, and secondarily, following tumor removal, from established lung metastases to extrapulmonary systemic sites. Coincident with extrapulmonary metastasis, there is a dramatic amplification of visible lung metastases, with death generally resulting from extensive lung metastasis. The progression of lung metastasis, and lung colonization initiated by intravenous injection of tumor cells, was investigated using B16 melanoma clone G3.12. Analysis of the growth of invisible metastases in organ culture explants of lung revealed that tumors continually disseminated relatively small numbers of lung metastases after reaching a size of about 6 mm in diameter. However, most terminal-stage lung metastases, along with all extrapulmonary metastases, apparently arise from a secondary spread of tumor cells from tumor-derived lung metastases 1-2 mm in size. Individual lung colonies, initiated with G3.12 cells bound to single microbeads, also disseminated large numbers of secondary lung colonies, as well as extrapulmonary colonies, at a 1- to 2-mm size. The mechanism for intrapulmonary spread of secondary metastases and colonies is unclear, but the consequence appears to be a secondary stage of intrapulmonary and extrapulmonary metastasis with selection for tumor cells with rapid growth rates.

Selective fractionation of hypoxic B16 melanoma cells by density gradient centrifugation.

Cells of the mouse B16 melanoma growing in monolayer culture and as tumors were fractionated by isopycnic density centrifugation in a linear-density (1.02-1.20 g/ml) metrizamide gradient. Cultured cells concentrated into one or two distinct bands, with densities of 1.02-1.04 g/ml and 1.06-1.10 g/ml, depending on growth conditions. Cells subjected to extreme hypoxia (less than 0.02% O2) banded predominantly at the lower density, and normally-oxygenated cells banded at the higher density. Fractionated tumor cells concentrated at both densities. Compared with cells at the higher density, lower-density cells incorporated more of the hypoxic cell radiosensitizer [14C]misonidazole and less [3H]thymidine in vivo, were less clonogenic but more resistant to X-irradiation in situ, and labeled to a lesser extent with intravenously-delivered Hoechst 33342 fluorochrome, a marker for cells proximal to tumor blood vessels. Lower-density tumor cells were, therefore, enriched in non-proliferating radioresistant hypoxic cells from tumor regions remote from blood vessels.

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.

B16 melanoma spontaneous brain metastasis: occurrence and development within leptomeninges blood vessels.

Subcutaneous tumors initiated with mouse B16 melanoma clones G3.5 and G3.12 disseminated visible spontaneous brain metastases in 67 per cent and 32 per cent, respectively, of mice with extensive lung metastasis. Most brain metastases appeared as pigmented emboli within blood vessels of the leptomeninges overlying the cerebral cortex. Intravascular metastases consisted of tumor cell aggregates surrounded by fibrous material and generally contained viable cells that proliferated in culture. Some metastatic emboli apparently proliferated intravascularly to such an extent as to cause vessel disruption, permitting tumor invasion into the adjacent cerebral cortex. Cultured cells from G3.12 leptomenings metastases produced tumors that metastasized to a much greater extent than unselected G3.12 tumors, but brain metastasis still occurred only secondarily, after initial dissemination to the lungs. In contrast, G3.5 brain metastasis-derived populations formed tumors that ultimately metastasized to the brain to lesser extents than did unselected G3.5 tumors. One selected variant, G3.12/BM2, reproducibly formed visible and viable brain metastases in more than 80 per cent of tumor-bearing mice, and lethal or potentially lethal brain metastases in 10-15 per cent of mice. This variant may serve as a model for clinical brain metastasis.

Cytotoxic effect of RA233 on hypoxic B16 melanoma cells in vitro.

The pyrimido-pyrimidine derivative RA233 was found to selectively kill cultured mouse B16 melanoma cells after prolonged hypoxia. At the optimum cytotoxic concentration (100 microM), RA233 reduced cell clonogenicity by about 80% when administered during long-term hypoxia of 4 days. Comparable cytotoxicity was also evident when RA233 was present only during re-oxygenation following 4 days of hypoxia. RA233 treatment during both hypoxia and re-oxygenation resulted in the greatest cytotoxicity, with only about 1% of cells surviving such treatment. By contrast, the hypoxic cell sensitizer misonidazole was cytotoxic only when administered during hypoxia. RA233 appears to be a unique hypoxic cell sensitizer that kills long-term hypoxic tumor cells principally during re-oxygenation.

Host immunity to mycoplasma antigens introduced into B16 melanoma cells: effect on tumor growth rate and metastasis.

Immunization of syngeneic C57BL/6 mice with X-irradiated B16 melanoma cells was previously shown to elicit antibodies specific to viral antigens on the melanoma cells. When immunized mice were challenged with viable subcutaneous transplants of B16 melanoma cells that formed non-metastatic tumors in normal mice, tumors failed to develop in some mice, but there was a high incidence of lung metastasis in mice with progressively growing tumors. To determine whether protective immunity and/or enhanced metastasis were the consequences of immune responses specific for inherent tumor-associated viral antigens, non-metastatic B16 melanoma cells were deliberately infected with Mycoplasma arginini. The result was incorporation of perpetuating antigens that elicited, in mycoplasma-immunized mice, humoral and cell-mediated immune responses to infected (B16-M+) but not uninfected (B16-M-) cells. When mycoplasma-immunized mice were challenged with B16-M+ and B-16M- subcutaneous transplants, only B16-M+ tumors were rendered slower-growing and appreciably more metastatic. By contrast, in mice immunized against uninfected B16 melanoma cells, both B16-M+ and B16-M- tumors grew more slowly, and metastasized to a greater extent, than corresponding tumors in unimmunized mice. Enhanced metastasis was not experimentally separable from reduced tumor growth rate and was not simply the consequence of a longer period of tumor growth. Evidence suggests that host immunity does not directly promote metastasis, but that reduced tumor growth rates resulting from protective immunity are more conducive to successful dissemination of metastases.

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.

Syngeneic monoclonal antibodies to B16 melanoma viral antigens.

Four stable IgM monoclonal antibody-producing hybridomas were generated by fusing mouse myeloma cells with spleen lymphocytes from C57BL/6 mice hyperimmunized against the syngeneic B16 melanoma. All four monoclonal antibodies (R31/15, R37/4, R37/6, and R37/7), in common with polyclonal antiserum from immunized mice, recognized antigens on the same complex of related cell surface molecules specified by endogenous AKR-type murine leukemia virus, designated the B16-gp/70/80/85 antigen complex. Reactivity with this antigen complex was demonstrated by radioimmunoprecipitation. Specificity for viral Mr 70,000 glycoprotein-related antigens was indicated by absorption of antibody activity by endogenous AKR virus and by inhibition of antibody binding to B16 melanoma cells by monospecific antiserum to murine leukemia virus Mr 70,000 glycoprotein. Neither polyclonal nor monoclonal antibodies recognized antigens on fish, guinea pig, swine, or human melanoma cell lines. Polyclonal antiserum reacted with several other mouse melanomas and with certain mouse lymphoma lines induced by, or harboring, endogenous murine leukemia viruses, but the monoclonal antibodies were unreactive except for recognition of antigens on Harding-Passey mouse melanoma cells by antibody R37/4 and on RL male 1 mouse lymphoma cells by antibody R37/7. Only monoclonal R37/7 was cytotoxic for cultured B16 melanoma cells in an antibody- and complement-dependent assay with guinea pig complement, although all antibodies were cytotoxic with rabbit complement. In reflecting the predominant humoral immune response to the B16 melanoma detected in syngeneic mice during tumor growth, these monoclonal antibodies will permit experimental amplification of that response to help determine how that immunity influences tumor growth and metastatic dissemination.

Failure of orally administered RA233 to influence B16 melanoma growth or metastasis.

Possible prophylactic antitumor and/or antimetastatic effects of long-term oral administration of a potent inhibitor of platelet aggregation, the pyrimido-pyrimidine derivative RA233, were assessed using four phenotypically distinct clones of the mouse B16 melanoma. The clones tested included: a poorly tumorigenic, very slowly growing and poorly metastatic population (G3.15); a moderately tumorigenic and slowly growing population that frequently metastasizes to the lungs (G3.5); a highly tumorigenic, moderately growing and highly metastatic population (G3.12); and a highly tumorigenic and rapidly growing population that is generally nonmetastatic but can be slightly metastatic when tumors are initiated by very small numbers of cells (G3.26). Addition of 0.5 mg/ml RA233 to the drinking water continuously from the time of subcutaneous injection of cultured tumor cells until death from tumor growth, which resulted in a daily uptake of 80-100 mg/kg of drug per mouse, failed to significantly influence the tumorigenicities, tumor growth rates, metastatic incidences, or metastatic burdens of any of these clones. RA233 at doses equivalent to those delivered daily to experimental animals strongly inhibited ADP-induced aggregation of homologous C57BL/6 mouse platelets and exhibited selective anti-proliferative effects on cultured cells. Although RA233 prolonged bleeding times, pharmacokinetic analysis indicated that clearance of RA233 from mice was so rapid that achievement of sustained circulating levels sufficient to influence tumor cells or platelet-tumor cell interactions by oral administration was unlikely.

Development of host immunity to phenotypically diverse B16 melanoma clones. Implications for tumor growth and metastasis.

A B16 melanoma clone and four derived subclones exhibiting markedly different tumorigenic and metastatic potentials in young C57BL/6 mice were investigated comparatively to determine relative immunogenicities and capacities to induce humoral and cell-mediated immune responses following subcutaneous injection. All five populations stimulated some production of circulating antibody to a cell surface antigen complex (B16-gp70/80/85) specified by endogenous murine leukemia virus, as well as spleen-cell-mediated cytolytic and cytostatic activity apparently directed to the same antigens, but to varying extents. Immunogenicity and relative capacity to induce immunity were inversely related to tumorigenicity and tumor growth rate but were not obviously correlated with metastatic behavior. There were indications that tumor behavior might be influenced by developing or naturally acquired host immunity. The most rapidly growing clone, G3.26, which was poorly immunogenic and nonmetastatic in young mice, grew more slowly and was markedly metastatic in normal-aged mice in which some natural humoral and cellular responses cross-reactive with B16-gp70/80/85 antigens were detected. Furthermore, the highly immunogenic and normally nonmetastatic clone, G3.15, was appreciably metastatic in mice immunosuppressed by T lymphocyte depletion. In other cases, however, tumor behavior in immunosuppressed and immunopotentiated mice did not consistently indicate a critical role for host immunity in determining metastatic or nonmetastatic activity.

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.