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.

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.

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.

Contribution of oxygen-derived free radicals to experimental necrotizing enterocolitis.

Oxygen-derived free radicals, particularly superoxide anion, are considered important mediators of intestinal injury induced by ischemia/reperfusion based on the protective effects of superoxide dismutase and allopurinol. A role for free radicals was investigated in a model of necrotizing enterocolitis (NEC) which was initiated by a luminal, as opposed to a vascular, insult. Intestinal loops of weanling rabbits received either saline (control loops) or a solution of 10 mg/ml casein and 50 mg/ml calcium gluconate acidified to pH 4 with proprionic acid (treated loops). When the animals were sacrificed 3 hours later, severe damage was noted in the treated loops, which included blunting of villi and edema, with all animals surviving. At 16 hours only 5 of 8 rabbits survived, and 3 had hemorrhagic necrosis. Control loops were normal in each case. Intravenous infusion of superoxide dismutase (4 mg/kg/hr), commencing 15 minutes after NEC induction, totally prevented intestinal injury. On the other hand, pretreatment with allopurinol, an inhibitor of xanthine oxidase, for 2 days (30 and 60 mg/kg by mouth) was not protective against intestinal damage. A cellular infiltration in treated loops was not histologically evident in the majority of animals at 3 hours after treatment, a finding confirmed by the minimal accumulation of 111In-labeled leukocytes in damaged and intact intestinal tissue. These results suggest that superoxide generated locally from sources other than xanthine oxidase play a critical and early role in experimental NEC and that superoxide dismutase may prove to be an effective therapy in this devastating neonatal disease.

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.

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.

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.

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.

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.