Biomedical subjects

C W Stackpole

Publications and source records attributed to C W Stackpole.

At least 19 recordsLinked to original sources

PubMed1995

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.

Animals↗

PubMed1995

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)

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1992

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.

Adrenal Gland Neoplasms↗

PubMed1991

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.

Animals↗

PubMed1991

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.

Animals↗

PubMed1990

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.

Animals↗

PubMed1989

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.

Animals↗

PubMed1989

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.

Aging↗

PubMed1988

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.

Animals↗

PubMed1987

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.

Animals↗

PubMed1987

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.

Aging↗

PubMed1985

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.

Animals↗

PubMed1985

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.

Animals↗

PubMed1985

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.

Agar↗

PubMed1984

Multiple antigens related to the major envelope glycoprotein of murine leukemia virus expressed on B16 melanoma cells as targets of host immune response.

Reactivity of B16 melanoma cell surface proteins with antisera to the major envelope glycoprotein, gp70, of murine leukemia viruses was assessed by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface proteins from cultured monolayers of the B16 melanoma and variant lines B16-F1, B16-F1(1r6), B16-F10, and B16-F10(1r6), and from purified B16 melanoma tumor cells, contained three glycosylated components specifically reactive with gp70 antisera, with apparent molecular weights of 70,000, 80,000, and 85,000 (B16-gp70, B16-gp80, and B16-gp85). Antisera raised in syngeneic C57BL/6 mice by immunizing with X-irradiated B16, B16-F10, or B16-F10(1r6) cells immunoprecipitated only solubilized B16-gp70, B16-gp80, and B16-gp85. Absorption of mouse antiserum to B16-gp70/80/85 antigens with purified viruses from various sources indicated that antigens on all three molecules were related to endogenous AKR-type murine leukemia virus antigens. Mice hyperimmunized against melanoma cells were challenged subcutaneously with 4 X 10(4), 10(5), or 2.5 X 10(5) viable B16 or B16-F10 cells, inocula that were lethal and nonmetastatic in unimmunized mice. The lowest cell dose was rejected by 90% of immunized mice. Tumors grew in an average of 58% of immunized mice challenged with 10(5) cells, pulmonary metastases occurring in 61% of those mice. Inocula of 2.5 X 10(5) cells grew in all immunized mice, with a 60% incidence of metastasis. These studies indicate that host immunity to B16-gp70/80/85 antigens can either inhibit or stimulate B16 melanoma tumor progression.

Animals↗

PubMed1983

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.

Animals↗