Mast cell modulation of tumour cell proliferation in rat mammary adenocarcinoma 13762NF

Mast cells were shown to accumulate around the periphery of the invasive and metastatic rat mammary adenocarcinoma (MTLn3), and histological evidence of mast cell degranulation was observed during the later stages of this model. To assess the physiological role of mast cells in vivo we have used the mast cell-stabilising compound FPL 55618 applied i.p. daily at 1 mg kg-1 for 23 days. Using groups of 12 rats we have found that this compound inhibited tumour growth at the primary site by as much as 70% in most of the treated animals compared with the control group which received equivalent volumes of saline. When the drug treatment was stopped after 23 days, tumour growth of the test group accelerated over the next 7 days and reached a similar tumour size to that of control animals. Histological studies of the tumour and contiguous host tissue at day 24 of the experiment revealed numerous extra-tumoural mast cells often showing signs of degranulation at several sites around the tumour periphery in the control animals. Such observations were not seen in those animals receiving FPL 55618 where, in contrast to controls, numerous intact mast cells were often seen within the tumour mass. Following cessation of the MC-stabilising treatment progressive mast cell activation was evident within 2-4 days, primarily at the tumour periphery. In vitro studies have shown that drug concentrations equivalent to five times the in vivo dose had no effect on the proliferative rate or viability of the MTLn3 cells. Moreover, the proliferative rate of these cells in culture was significantly increased when exposed to soluble mast cell products. Thus our data indicate that a mast cell-stabilising compound has significant benefits in reducing tumour growth in vivo, an observation which supports the concept that mast cell:tumour cell interactions are important for the growth and invasive properties demonstrated by this model of breast carcinoma.     

Candidate metastasis-associated genes of the rat 13762NF mammary adenocarcinoma

Summary Differential hybridization was used to isolate genes potentially involved in the process of metastasis. Ten complementary DNAs (cDNAs) that were differentially expressed between a highly metastatic (MTLn3) and a nonmetastatic (MTC.4) line of the rat 13762NF mammary adenocarcinoma were isolated and sequenced. Examination of the EMBL/GenBank database revealed that one of the genes had a high degree of homology (98.8%) to annexin I (also known as calpactin II). Quantitative analysis of Northern blot hybridizations showed that the annexin I-like sequence was expressed 4- to 7-fold higher in MTLn3 than in MTC.4 cells. Steady state mRNA levels were also low in MTLn2, a cell line of low metastatic potential closely related to MTLn3, but were not related to metastatic potential in colon adenocarcinoma or melanoma cells. Two of the cDNAs (designated 8.11 and 10.14) were found to be novel. The expression of 10.14 mRNA (3.2 kb) was 4-fold higher in MTLn3 than in MTC.4 cells. Sequencing of the 10.14 cDNA (2.2 kb) revealed a putative open reading frame of 583 amino acids that was also novel. Expression of 8.11 mRNA (>7 kb) inversely correlated with metastatic potential. Another differentially transcribed gene was highly homologous to ERK2 (extracellular signal related kinase 2), a mitogen-activated protein kinase (MAPK). Northern analysis of ERK2 expression revealed 3-fold higher amounts of a 1.3 kb mRNA in MTLn3 than in MTC.4 cells. Higher levels of ERK2 mRNA were generally seen in the more metastatic human colon but not in melanoma cell lines. We also corroborated the work of Taniguchi (Nucl Acids Res 19:6949, 1991) by independently identifying EF-1 as a putative metastasis-associated gene.Supported by an educational grant from CIBA-GEIGY Corp., at the San Antonio Breast Cancer Symposium, Dec. 8, 1992.     

Effect of host immune status on the spontaneous metastasis of cloned cell lines of the 13762NF rat mammary adenocarcinoma

The importance of host immune status on the spontaneous metastasis of cloned cell lines of the 13762NF rat mammary adenocarcinoma was examined. Cell lines MTLn3 (high metastatic potential), MTF7 and MTLn2 (intermediate metastatic potential) and MTC (low metastatic potential) were subjected to a series of in vivo assays designed to assess how manipulation of the immune system in the syngeneic F344 host would affect the ability of these cells to metastasise. Treatment of tumour bearing rats with the immunosuppressive agents cyclosporin A or cyclophosphamide had little influence on metastasis in this system. Growth of tumours in congenitally athymic nude rats resulted in reduction of observed metastases. In addition, humoral immune response was not detectable during a 23-day period of tumour growth in F344 rats. Excision of the tumour growing in situ reduced the number of metastases when the tumours were resected early (less than 10 days), but at later times tumour resection did not influence the incidence of metastasis. The importance of initial lymphatic rather than haematogenous routes of dissemination was confirmed in experiments where the draining inguinal and axillary lymph nodes were removed at different times either before, or after, subcutaneous mammary fat pad injection of metastatic tumour cells.     

Organization of cytoskeletal structures in 13762NF rat mammary adenocarcinoma cell lines and clones of different metastatic potentials

The correlation between metastatic potential and the organization of microtubules and microfilaments in nine cell lines and clones derived from the 13762NF rat mammary adenocarcinoma was investigated using immunofluorescence techniques. Cell sizes and morphologies of cell clones isolated from the locally growing 13762NF tumor were heterogeneous; those derived from spontaneous metastases were more homogeneous. The organization of microtubules was similar in cells of various 13762NF lines and clones, and differences could not be detected with the techniques employed. The organization of microfilaments was very heterogeneous within some clones but homogeneous within others. Correlations between microfilament organization and origin of a cell line or clone or its metastatic potential were not found in this tumor system.     

Tumor-cell-platelet aggregation does not correlate with metastatic potential of rat 13762NF mammary adenocarcinoma tumor cell clones

The ability of tumor cells to induce platelet aggregation has been correlated with their capacities to colonize the lungs of experimental animals. We tested this hypothesis by studying the ability of cloned, low-passage metastatic tumor cell lines derived from rat 13762NF mammary adenocarcinoma to aggregate rat platelets in vitro and in vivo, and we then compared this activity to metastatic potential by determining the incidence of lung metastasis after subcutaneous or intravenous inoculation of the tumor cell clones into syngeneic rats. Our results failed to show a correlation between in vitro platelet-aggregating activity and metastatic potential. In this system we could not detect platelet-aggregation activity with the most metastatic tumor clone, while the least metastatic clone clearly possessed high platelet-aggregating activity. In addition, by measuring changes in blood platelet and fibrinogen concentrations at various intervals following intravenous injection of tumor cell clones, we were able to confirm in vivo the observed in vitro differences in their plateletaggregating activities. Thus, platelet aggregating activity is heterogeneously expressed among 13762NF cell clones and appears unrelated to spontaneous or experimental metastasis in this tumor system.     

Heterogeneity in the sensitivities of the 13762NF rat mammary adenocarcinoma cell clones to cytolysis mediated by extra- and intratumoral macrophages

The susceptibility of cloned cell lines of the 13762NF rat mammary adenocarcinoma to macrophage-mediated cytolysis was investigated using both intra- and extratumoral macrophages. The percentage of Fc receptor-positive cells in tumors growing s.c. in syngeneic F344 rats ranged from 8 to 20%, but we could not demonstrate a significant correlation between the number of Fc receptor-positive cells within tumors and their spontaneous metastatic potentials. In macrophage-mediated cytolysis assays, cloned 13762NF cell lines of differing metastatic potential, established from tissue culture lines, fresh tumor explants, or short-term cultures (one passage in vitro), were used as targets. Effector cells were thioglycolate-elicited peritoneal macrophages (activated in vitro with bacterial lipopolysaccharide) or intratumoral macrophages (activated in vitro with lipopolysaccharide). When the effector cells were peritoneal macrophages, established cloned 13762NF cell lines showed little correlation in their susceptibility to macrophage-mediated cytolysis and metastatic potential, while this was not observed when fresh tumor explants were used. Highly metastatic MTLn3 cells were the least sensitive, less metastatic MTF7 and MTLn2 cells were more susceptible, and the low metastatic parental MTPa cells were the most sensitive in 72-h cytolysis assays. When the effector cells were intratumoral macrophages, all 13762NF cell lines showed less sensitivity in cytolysis assays than similar assays using thioglycolate-elicited peritoneal macrophages. With the exception of line MTLn2, short-term cultures (one passage in vitro) did not differ substantially in susceptibility to intratumoral macrophages compared to fresh explants. In this system, the sensitivity of 13762NF cells to macrophage-mediated cytolysis is a function of effector as well as target cell source.     

Increased immunogenicity of a spontaneous variant clone of the 13762A rat mammary adenocarcinoma

The 13762A rat mammary adenocarcinoma is weakly immunogenic and spontaneously metastasizes to regional lymph nodes and lungs. A clone (18A) was isolated from the parental tumor, which grew for 3 weeks in normal F344 rats, forming tumors up to 2-3 cm and some nodal metastases, and then completely regressed. Pretreatment of recipient rats with 450 rad permitted progressive growth and death due to metastases. The behavior of 18A has been stable during a period of 120 days in continuous culture or for 6 in vivo passages in irradiated rats. Regression of 18A was associated with intense tumor mononuclear leukocytic infiltration, whereas parental tumors of the same size recruited few leukocytes. Regressions occurred when 18A cells were placed intradermally, sc, or im, but iv injections were not rejected. Parental tumors grew progressively at all sites. Regressor rats were specifically immune to challenge with both 18A and parental tumor but not to an unrelated mammary carcinoma (R3230AC). Irradiated 18A tumor cell vaccines protected recipients against challenge with parental tumor, but similar vaccines of irradiated parental tumor cells were ineffective. The systemic adoptive transfer of immune lymphocytes more strongly inhibited the growth of established (7 days) 18A than parental tumor. It was concluded that the parental 13762A tumor contained stable variants that were significantly more immunogenic and more susceptible to immune attack than the parental tumor. Such variant tumor lines may be useful in the study of the host response to metastasis.     

Conditions for effective Bacillus Calmette-Guérin immunotherapy of postsurgical metastases of 13762A rat mammary adenocarcinoma.

We evaluated critical variables in Bacillus Calmette-Guérin (BCG) immunotherapy of residual 13762A rat mammary adenocarcinoma. BCG was given intratumorally on Day 7 of tumor growth and followed by primary tumor excisions on Day 20. Untreated animals died on about Day 40 with axillary nodal and pulmonary parenchymal metastases. BCG-treated animals experienced prolonged survival, and some were cured. The highest dose (5.0 X 10(7) colony-forming units) of BCG was more effective than the lowest (0.5 X 10(7) colony-forming units), but 1,500 micrograms Corynebacterium parvum were more effective than even the highest BCG dose. Previous sensitization to BCG did not improve the effects of BCG treatment. BCG treatment was effective when given on Day 7 and sometimes as late as Day 12 or 17, but C. parvum was ineffective if given after Day 7. Repeated injections of BCG or C. parvum were not more effective than single injections were. Rats cured of residual 13762A tumor by BCG treatment were strongly and specifically immune to rechallenge. We concluded that a high dose (5.0 X 10(7) colony-forming units) of BCG given early (7 days) was the most effective presurgical treatment of 13762A metastases. Repeated injections or host presensitization to BCG did not improve the benefits.     

Loss of myoepithelial cell characteristics in metastasizing rat mammary tumors relative to their nonmetastasizing counterparts

A series of WF rat mammary tumors comprising one transplantable nonmetastasizing line (MT-W9), two predominantly lymphatic (SMT-2A and SMT-077) and one lymphatic and hematogenous (MT-450) metastasizing transplantable lines, and 7,12-dimethylbenz[a]anthracene (DMBA)-induced nonmetastasizing primary tumors was examined for the presence of epithelial and myoepithelial cell characteristics with the use of immunocytochemical techniques. Tumor cells staining for myosin were only occasionally observed in a basal orientation in glandular structures in sections of DMBA-induced and MT-W9 tumors; anti-laminin serum stained the peripheries of the glandular structures in the DMBA-induced and MT-W9 tumors but failed to stain the SMT-2A and SMT-077 tumor cells. In the nonmetastasizing tumors immunologically detectable keratin occurred mainly in the outer cellular layer of glandlike structures, whereas milk fat globule membrane immunoreactivity occurred primarily in the luminal cells. Both these types of immunoreactivity were observed in MT-450 tumor cells, but the pattern of keratin staining was random. No such immunoreactivity occurred in SMT-2A or SMT-077 tumors. No tumor cell-associated staining for fibronectin was seen in any of the tumors examined, although host stromal components stained intensely. The nonmetastasizing tumors contained cuboidal epithelial cells with lumen formation, surface microvilli, and intercellular junctional complexes, together with a relatively undifferentiated elongated cell component. Other elongated cells showed hemidesmosomes, pinocytotic vesicles, tonofilaments, and small bundles of cytoplasmic filaments, suggesting gradations toward a myoepithelial phenotype. The MT-450 tumors were ultrastructurally similar to the nonmetastasizing tumors, but no features of myoepithelial cells were seen, although some cuboidal epithelial cells exhibited prominent tonofilaments. The SMT-2A and SMT-077 tumors consisted of nests of cuboidal-like cells with highly pleomorphic nuclei and much intercellular collagen. The results indicate a progressive loss of cellular differentiation characteristics, particularly those of the myoepithelial cell with increasing malignancy in this system.     

Role of T-cell subsets in the destruction of established metastases of 13762A rat mammary adenocarcinoma

The 13762A rat mammary adenocarcinoma metastasizes with high frequency to regional lymph nodes and lungs. The intratumoral injection of Corynebacterium parvum on day 7 followed by primary tumor excision on day 20 significantly prolonged survival and cured 10-40% of syngeneic F344 rats. Established metastases were destroyed by the treatment, and strong and specific tumor rejection immunity was induced. The purpose of the present study was to determine if T-cells were required for the C. parvum treatment to be effective and to identify the subsets of T-lymphocytes that might participate in the response. The results indicated that rats depleted by either neonatal thymectomy or a combination of adult thymectomy, 900 rad, and bone marrow reconstitution did not inhibit tumor growth after C. parvum treatment. Restoration of depleted rats with lymph node cells permitted effective treatment. The lymph node cells that were responsible for restoration expressed both W3/13 (pan-T-cell) and W3/25 (helper T-cell) membrane-associated differentiation antigens. T-cells that bore the MRC OX8 (cytotoxic-suppressor T-cell) antigen did not restore the response to C. parvum treatment. The effect of lymph node restoration was markedly potentiated by simultaneous administration of thymocytes, a T-cell population that expresses both W3/25 and MRC OX8 antigens. In conclusion, the cytotoxic-suppressor T-cells were ineffective in the restoration of T-cell-depleted, tumor-bearing rats to benefit from C. parvum but helper T-cells were highly effective, and their activity was strongly potentiated by administration of thymocyte amplifier cells.     

Systemic adoptive transfer of immunity and low-dose irradiation eradicate metastases of 13762A rat mammary adenocarcinoma

Rats cured of poorly immunogenic 13762A tumor by a combination of surgery and cyclophosphamide (CY) treatments produced peritoneal exudate cells (PEC) that prevented tumor growth when transferred to naive recipients, but they were ineffective against established tumor. A highly immunogenic 13762A clone (18A) induced PEC lymphocytes that completely reversed the growth of established primary tumor and of lymph-node metastases. 18A-immune PEC alone strongly inhibited tumors of 7 days' duration, but only moderately suppressed 14-day tumors, and had no effect on 21-day tumors. Irradiation (450 R) of rats prior to tumor transplantation improved the effectiveness of the PEC given at 7 days, but the benefit had gone by 14 days. Long-term T-cell depletion prior to tumor challenge allowed PEC inhibition of 7- and 14-day tumors, but not 21-day tumors. The most potent strategy was the administration of 450 R followed immediately by immune PEC. When rats with 21-day tumors were so treated, the metastases grew temporarily to a maximum diameter of 2-5 cm and then completely regressed. We concluded that a combination of immune T cells and 450 R can cure established, massive metastases, probably through a combination of an increase in the numbers of T-cell effectors and elimination of suppressor cells.     

Metastasis of ocular and flank implants of the 13762NF mammary adenocarcinoma in syngeneic rats

Solid fragments of a syngeneic tumor of low immunogenicity were implanted on the anterior surface of the iris or posterior surface of the cornea in young Fischer 344 rats. Growth and metastasis of these implants were examined and compared with those occurring following sc tumor implantation in the flank. Corneal implants did not grow well or metastasize. Iridial implants grew well, and tumor typically filled the eye within a few weeks. If tumor-filled eyes were left intact, all rats died or became moribund 7-9 weeks later. Metastasis was seen in the lungs (11/11), cervical lymph nodes (7/11), body wall (5/11), heart (4/11), kidney (4/11), and liver (3/11). Removal (enucleation) of tumor-filled eyes prior to spread to the orbit prevented the development of overt metastatic disease for 1-2 years (11/11). When healthy enucleated rats were necropsied 0 days to 26 weeks post enucleation, small nodules were seen in the lung (9/13), liver (3/13), or spleen (1/13). Nodules were not seen in rats that had been housed in our animal facility for 1-2 years (16/16), had eye surgery with tumor implanted on the posterior surface of the cornea (6/6), or had been subjected to the surgical procedures of corneal implantation and enucleation (3/3). When tumors were implanted sc, rats died after 6-7 weeks. Metastasis was seen in the lung (12/12), lymph nodes (12/12), spleen (1/12), and liver (1/12). Removal of very small flank tumors failed to prevent animals from dying 7-8 weeks later with pulmonary and lymph node metastasis (10/10). In summary, iridial implantation resulted in an altered pattern of metastasis. Animals that had iridial implants and enucleation failed to develop overt metastatic disease and had small nodules in the lung, liver, or spleen.     

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.     

Induction of apoptosis by iron depletion in the human breast cancer MCF-7 cell line and the 13762NF rat mammary adenocarcinoma in vivo

It is known that the interruption of normal iron metabolism with chelators of iron, toxic metals, toxic metals bound to transferrin, or anti-transferrin receptor antibodies leads to significant inhibition of tumor cell growth in cell culture systems and animal models. In the present study, we found that iron depletion was produced by the iron chelator deferoxamine mesylate, the free toxic metals gallium or indium, and the toxic metals gallium or indium bound to transferrin in the MCF-7 human breast cancer cell line, and this induced the condensation and fragmentation of chromatin, and the formation of DNA fragments characteristic of apoptosis. The induction of apoptosis was quantitated with acridine orange and ethidium bromide staining of apoptotic cells, separation of fragmented DNA from radiolabeled cells, and in situ terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assays. The apoptosis, caused by deferoxamine mesylate, and gallium or indium bound to transferrin in the MCF-7 cells, can be completely inhibited by excess ferric chloride or equimolar iron-loaded transferrin. Gallium-transferrin and indium-transferrin complexes induced more apoptosis than their respective salts in the MCF-7 cells. Deferoxamine mesylate induced a small increase in the endogenous expression of both the bcl-2 and bax genes in the MCF-7 cells and this can be prevented by ferric chloride. In the 13762NF rat mammary adenocarcinoma model, in situ TUNEL assays showed that the iron-deficiency following a low iron diet or intravenous injection of deferoxamine mesylate produced 5.32 +/- 3.90% and 6.46 +/- 3.58% of apoptotic cells, respectively, compared to 2.01 +/- 1.20% of apoptotic cells in the control rats maintained on a normal diet (p < 0.05 and p < 0.01, respectively, Student's t-test). This is the first report of iron depletion caused by a low iron diet or deferoxamine mesylate treatment inducing apoptosis in rats bearing the 13762NF marnmary adenocarcinoma.     

Characterization of cytokeratins expressed in metastatic rat mammary adenocarcinoma cells

The expression of cytokeratins (CKs) was investigated in cell lines and clones established from the rat 13762NF mammary adenocarcinoma tumor and its spontaneous lymph node and lung metastases. Two-dimensional polyacrylamide gel electrophoresis of intermediate filament-enriched protein fractions from cultured cells revealed that clones established from spontaneous metastases contained three CKs (Mr approximately 54,000, approximately 52,000, and approximately 40,000) characteristic of simple epithelia and two CKs (Mr approximately 51,000 and approximately 47,000) characteristic of stratified epithelia. CK expression varied qualitatively and quantitatively between the different metastasis-derived cell clones. In contrast, cell clones established from the original mammary fat pad tumor expressed low or undetectable levels of CKs. Western blot analyses with a panel of anti-CK antibodies with defined specificities confirmed the observations. One-dimensional polyacrylamide gel electrophoresis of whole-cell lysates and intermediate filament-enriched extracts were transferred and probed with the panel of antibodies. The relative expression of individual CKs varied according to the cell line or clone examined and environmental conditions (low versus high passage and in vitro versus in vivo growth), whereas the amount of total CKs expressed relative to total cell protein varied according to cell line or clone and growth conditions.     

Lectin-binding patterns in transplantable mouse mammary tumors and their metastases.

Lectin binding was assessed in a transplantable pregnancy-dependent mouse mammary tumor line (TPDMT-4), its autonomous sublines (T4-0196 and T4-01165) and their artificial metastases (lung colonies), using the avidin-biotin-peroxidase technique. Soybean agglutinin (SBA) and peanut agglitinin (PNA) bound to the luminal surfaces of TPDMT-4 tumor cells, while dolicos biflorus agglutinin (DBA) showed no binding. In T4-0196 and T4-01165 tumors as well as their lung metastases, SBA and PNA binding was mixed and both positive and negative cells were detected, indicating that these lectins were not associated with the metastatic phenotype. Although the T4-0196 and T4-01165 sublines had a mixture of DBA-positive and DBA-negative cells, all the metastatic T4-0196 subclones contained only DBA-positive cells and all the metastatic T4-01165 subclones had DBA-negative cells. Thus DBA-positive, and DBA-negative subclones had respectively metastasized to the lungs from these autonomous sublines, implying that the carbohydrate moieties detected by DBA were not associated with metastatic potential but that the lung metastases were clonal in origin.     

The role of polymorphonuclear leukocytes (PMN) on the growth and metastatic potential of 13762NF mammary adenocarcinoma cells

Circulating neutrophil (PMN) levels can increase in rats bearing subcutaneously growing clones of the 13762NF mammary adenocarcinoma and the level of increase correlates with the metastatic potential of the clone. In rats with poorly metastatic MTC tumors, numbers of circulating PMN did not rise, whereas PMN levels rose 50-fold in rats bearing highly metastatic MTLn3, 12-fold in rats with weakly metastatic MTLn2, and 14-fold in those with moderately metastatic MTF7 tumors. Neutrophilia was caused partly by tumor size, but metastatic potential was a stronger determinant, suggesting that PMNs may play a role in the metastatic process. To determine whether circulating PMNs indeed contribute to cellular metastatic potential, we examined effects of PMN on various aspects of the metastatic process. Experimental metastasis assays involving i.v. co-injections of PMNs yielded a dose-dependent increase in extrapulmonary metastases for MTLn3, but no change in lung colonization potential for any of the clones examined. The change in the metastatic profile was not due to any modification in in vivo distribution of i.v. injected tumor cells or in adhesion to endothelial monolayers in vitro. PMNs also had no effect on in vitro DNA, RNA or protein synthesis and were not cytolytic (E:T 100:1). However, PMNs collected from high-passage MTLn3 tumor-bearing rats had a 50% increase in heparanase and type-IV collagenolytic activity as compared to unstimulated PMNs isolated from normal rats. These results indicate that polymorphonuclear cells may contribute to the metastatic potential of highly metastatic clones from the 13762NF mammary adenocarcinoma cells by assisting in the degradation of basement membrane during extravasation.     

Direct effects of the pyrimido-pyrimidine derivative RA 233 (Rapenton) on rat 13762NF mammary tumor cell clones in vitro

Studies with the pyrimido-pyrimidine analogue RA 233 (Rapenton) suggest that its antimetastatic action may not be mediated entirely by inhibition of platelet function. Little is known about its direct effects on tumor cells. We investigated the in vitro effects of RA 233 on clones MTLn3 and MTC of differing metastatic potentials, isolated from the 13762NF rat mammary adenocarcinoma. The results indicated that RA 233 is cytostatic (EC50 of approximately 140 microM and approximately 180 microM for MTLn3 and MTC cells, respectively) rather than cytotoxic by determining changes in viable cell number, thymidine uptake, and incorporation of thymidine and methionine. In both clones RA 233 inhibited cAMP-dependent phosphodiesterase activity and affected cAMP accumulation in intact cells. In contrast, clonal heterogeneity in drug-induced morphological changes, such as vacuole formation and altered organization of cytoskeletal structures, as well as increased tumor cell growth at 50 microM RA 233 was observed between clones MTLn3 and MTC. These data could explain the conflicting results obtained with RA 233 when evaluated as an antimetastatic agent.     

Metastasizing potentials of mouse mammary tumors and their metastases

This in vivo investigation determined the relative potentials of 7 C3H/He and C3Hf/He mammary tumors and their metastases to metastasize spontaneously from intramammary implants. The purpose of the studies was to examine the hypothesis that metastases derive from distinct sub-populations of cells in primary tumors which have specific inheritable characteristics that predispose the cells to form metastases. By serially transplanting, in parallel tests, tissue from autochthonous metastases and tissue from autochthonous primary tumors, the distinction of tumor-cell populations with different metastatic potentials was anticipated. However, the results of the experiments showed that primary tumors and metastases in autochthonous hosts had similar potentials for spontaneous metastasis. Moreover, the primary tumors and the metastases, transplanted as parallel lines through consecutive generations, maintained similar average metastasizing potentials. Changes in metastatic potential which occurred in 3 of the 7 tumors during serial passage appeared in nearly equal transplant generations in the parallel lines of the primary tumor and the metastases. The data suggest that the spontaneous metastases were not derived from a sub-population of cells with inheritable high metastasizing potential, but developed through stochastic events from the average tumor cells that entered the circulation.