Shift of syndecan-1 expression from epithelial to stromal cells during progression of solid tumours.

Syndecan-1 (SDC-1), a protein found on cells and in the extracellular matrix, participates in cell proliferation, cell migration and cell-matrix interactions. SDC-1 expression correlates with the maintenance of epithelial morphology and inhibition of invasiveness. In the present study, a second SDC-1 mRNA isoform was identified and the expression of both transcripts was investigated in various normal and malignant tissues. Both transcripts were coexpressed at equal levels in all tissues and organs analysed. Cancer-profiling array (CPA) analysis of 241 non-enriched tumour and normal cDNAs revealed stronger upregulation of SDC-1 in tumour tissues as compared with oligonucleotide array-based expression analysis of SDC-1 in microdissected breast, prostate, lung, and colon carcinoma cells. With in situ hybridisation and immunohistochemistry it was demonstrated that this difference in SDC-1 expression originates from stromal cells present in tumour connective tissue. But only the cells in connective tissue surrounding breast, lung, colon and bladder carcinoma showed upregulation of SDC-1. These stromal cells were characterised as spindle cells with myofibroblastic differentiation and they may contribute to the dedifferentiation of tumour cells and the development of metastasis.

Subcellular distribution of epothilones in human tumor cells.

Epothilones are a new class of natural and potent antineoplastic agents that stabilize microtubules. Although 12,13-epoxide derivatives are potent antiproliferative agents, the activities of the corresponding 12,13-olefin analogs are significantly decreased. These data were confirmed for two new analogs, 6-propyl-EpoB (pEB) and 6-propyl-EpoD (pED), in comparison with the natural compounds EpoB/EpoD, by using human A431, MCF7, and MDR1-overexpressing NCI/Adr cells. By using tritiated pEB/pED, compound uptake, release, and nuclear accumulation were investigated in A431 and NCI/Adr cells. In these cells, epothilones can principally be recognized and exported by Verapamil-sensitive efflux pumps, which are not identical to MDR1. The degree of export depends on the structure, olefin vs. epoxide-analog, and also on the intracellular drug concentration. The accumulation of pED used at 3.5 or 70 nM, respectively, was increased in the presence of 10 microM Verapamil in both cell lines 2- to 8-fold. In contrast, the intracellular levels of pEB were affected by Verapamil only at 3.5 nM pEB in NCI/Adr (2-fold) and not in A431 cells. In addition, strong nuclear accumulation was observed for pEB (40-50%) but not paclitaxel or pED (5-15%) in both cell lines. Our study suggests that differences in growth inhibitory efficacy between epoxide and olefin analogs may be based on different mechanisms of drug accumulation and subcellular distribution.

Signaling-inactive epidermal growth factor receptor/ligand complexes in intact carcinoma cells by quinazoline tyrosine kinase inhibitors.

Several inhibitors of EGF receptor (EGFR) tyrosine kinase activity have been developed that compete with ATP at its binding site such as the quinazolines PD 153035 and ZD 1839 or the 4,5-dianilino-phthalimides DAPH1 and DAPH2. When tested on human A431 cells, the quinazolines completely blocked EGF-induced receptor phosphorylation at 100 nM, whereas it was inhibited by DAPH1 and DAPH2 by only 20% at 3 microM. Quinazoline-treated A431 as well as tumor cells expressing less EGFR (A549, MDA MB 231, and T47D) bound 3- to 6-fold more (125)I-labeled EGF than untreated intact control cells. Scatchard analysis revealed the disappearance of low- and high-affinity EGFR on A431 cells upon PD 153035 treatment. A single receptor class of intermediate ligand binding affinity emerged and its number corresponded to the sum of the two classes. DAPH1 and DAPH2 did not change ligand binding properties of EGFR. PD 153035 exerted the most potent effects on EGF binding to A431 or on inhibiting EGF-stimulated growth of rat MTLn3 cells at low ligand concentrations. Cross-linking of EGFR on PD 153035-treated A431 cells indicated the formation of inactive dimers that further increased upon addition of EGF. Chemical cross-linking of (125)I-labeled EGF to PD 153035-treated A431 cells revealed increased binding to monomeric and dimeric EGFR. Thus, the quinazolines sequestered EGFR plus the ligand into inactive receptor/ligand complexes. This novel mode of action of quinazoline tyrosine kinase inhibitors may be the basis for their extraordinary potency especially in conditions when the ligand is present in limiting amounts.

Selective increase of alpha2-integrin sub-unit expression on human carcinoma cells upon EGF-receptor activation.

The effects of chronic EGF exposure on expression of the alpha2beta1 collagen and alpha5beta1 fibronectin receptor in a pair of human carcinoma cell lines (A431 and A549) with differential responses to EGF in a short-term ECM-cell adhesion assay were investigated. Treatment with EGF at 10 ng/ml for 24 hr increased on both cell lines the expression of the alpha2- but not the beta1- or alpha5-integrin sub-units, and concomitantly cellular adhesion was increased on collagen IV but not on fibronectin. Increased collagen adhesion of A549 cells could be blocked by alpha2- and beta1-integrin-sub-unit antibodies down to control levels, while it was blocked by alpha2-integrin-sub-unit antibody only by 60% and completely by the beta1-integrin-sub-unit antibody on A431 cells. EGF induced disparate shifts in cell morphologies (dome-like structures, A431, vs. spindle-like fibroblastoid, A549) with concomitant opposite changes in the expression/localization of E-cadherin in cell-cell contacts. This could be taken as an indication for cell-type-specific differential changes in the ratio of cell-ECM vs. cell-cell contacts. The EGF-induced up-regulation of the alpha2beta1 integrin was instrumental in increasing collagen adhesion of A549 but only partly in the case of A431 cells, in which cells the alpha2beta1 integrin may have additional functions besides serving as cell-ECM receptor.

Differential cross-talk of estrogen and growth factor receptors in two human mammary tumor cell lines.

Cultured human mammary MCF7 and T47D tumor cell lines were used to test the interference of the partial antiestrogen 4'-hydroxytamoxifen (4-OH-TAM) and the pure antiestrogen ZM 182780 with growth factor (IGF-I, heregulin) signaling pathways. Growth of both cell lines was stimulated by IGF-I (20 ng/ml) or heregulin (3 nM). ZM 182780 effectively blocked growth factor induced as well as basal proliferation of MCF7 cells while the compound was ineffective in interfering with growth factor mitogenic activity in T47D cells. On both cell lines the IGF-I or heregulin- induced proliferation was enhanced further by 4-OH-TAM. This synergism could be inhibited dose-dependently by ZM 182780. When cells were grown in the presence of estradiol plus growth factors, the antiestrogenic potencies of both compounds and the efficacy of ZM 182780 were unaffected, while the efficacy of 4-OH-TAM was reduced. Our data show cell type specific cross-talk between the receptor for estrogen and that for IGF-I or heregulin, which is different in MCF7 and T47D cells, respectively. In MCF7 cells with demonstrable cross-talk, a clear superiority exists for a pure antiestrogen over a partial agonist in interfering with growth factor mitogenic activity.

Suppression of ruffling by the EGF receptor in chemotactic cells.

To clarify the relationship between ruffling and lamellipod extension in growth factor-stimulated chemotactic responses, we utilized cell lines derived from the rat 13762 NF mammary adenocarcinoma. Nonmetastatic MTC cells expressing the human EGF receptor (termed MTC HER cells) demonstrated chemotactic responses to TGF-alpha, an EGF receptor ligand typically present in mammary tissue. In microchemotaxis chambers, peak chemotactic responses occurred in response to 5 nM TGF-alpha. MTC HER cells showed dramatic ruffling edges in the absence of external stimuli, and addition of 5 nM TGF-alpha led to a transient reduction in ruffling concomitant with lamellipod extension. Lamellipod extension correlated with an overall increase in actin polymerization. These responses were blocked by the PI 3 kinase inhibitor wortmannin but not by the MAP kinase inhibitors PD98059 and SB203580. We conclude that the initial chemotactic response to TGF-alpha involves lamellipod extension and that ruffling reflects a dynamic turnover of lamellipodia that is arrested during lamellipod extension. By regulating the dissolution of ruffles and extension of lamellipods, a chemotactic response can be achieved, which may contribute to the metastatic process.

Tumor metastasis inhibition with the prostacyclin analogue cicaprost depends on discontinuous plasma peak levels.

Stable prostacyclin analogues exert a strong inhibitory effect on lymphogenous as well as haematogenous tumor metastasis in a series of tumor lines. The strong inhibition of metastasis was achieved by repeated once-daily i.g. applications. The mechanism of antimetastatic action is related to the expression of functional IP-receptors (PGI-receptors). As cellular assay systems indicated that the IP-receptor mediated signalling is down-regulated upon continuous exposure to prostacyclin or stable derivatives, it has been questioned whether a mode of drug application with constant plasma drug levels may potentially result in a decrease of the antimetastatic effect. We addressed this question using the stable prostacyclin analogue cicaprost in a disease model by comparing i.g. applications given once daily with a continuous administration of equivalent doses via drinking water. Very similar to our previous investigations in the 13762NF MTLn3 rat mammary carcinoma model, cicaprost administered by i.g. application strongly reduced lung and lymph node metastasis. In contrast, administration of equivalent doses via drinking water leading to lower but constant steady-state plasma levels failed to exert inhibitory effects. Plasma and urine levels of cicaprost were measured with a sensitive radioimmunoassay on the last treatment day. Pharmacokinetic evaluation demonstrated a similar bioavailability of cicaprost in both groups. This result first demonstrates a treatment failure of a prostacyclin derivative in a chronic disease model in association with a continuous drug administration leading to constant plasma levels. A desensitization of receptor signalling by constant plasma levels may be a possible mechanism for treatment failure.

Negative cooperativity between alpha 3 beta 1 and alpha 2 beta 1 integrins in human mammary carcinoma MDA MB 231 cells.

The alpha 3 beta 1 integrin has been implicated as a receptor for several matrix components, including collagen, fibronectin, and laminins. The function of alpha 3 beta 1 seems to be very versatile involving cell adhesion to or migration on ECM, establishment of cell-cell contacts in aggregates, as well as linkage to intracellular tyrosine phosphorylation cascades. Here we report a strong induction of attachment of alpha 3 beta 1 integrin expressing human breast carcinoma cell line MDA MB 231 to matrix proteins by two alpha 3 integrin subunit function-blocking monoclonal antibodies (P1B5 and ASC-1). In contrast, stimulation of adhesion to ECM by inhibitory alpha 3 integrin-specific antibodies was not observed in the alpha 3 beta 1 integrin-expressing nonmalignant human mammary epithelial cell line MCF-10A or the human breast carcinoma cell line MDA MB 468 that expressed relatively low amounts of alpha 3 beta 1 integrin at the cell surface. This increase was specific for collagens and not observed on fibronectin or laminin. Physiological concentrations of bivalent cations were not required. MAb P1B5 did not induce homotypic aggregation of MDA MB 231 cells. The P1B5-induced increase in cell attachment to collagens could be prevented but not reduced below control levels by blocking mAb to the alpha 2 integrin subunit. Function blocking anti-alpha 5 integrin subunit mAb was without effect while anti-beta 1-mAb completely abolished adhesion. Our data indicate that negative cooperativity between integrins results in transdominant inhibition of alpha 2 beta 1 function by alpha 3 beta 1 in human MDA MB 231 but not MDA MB 468 tumor cells or nonmalignant MCF-10A cells.

Prevention of EGF-modulated adhesion of tumor cells to matrix proteins by specific EGF receptor inhibition.

The adhesion of tumor cells to various extracellular matrix (ECM) proteins is influenced by epidermal growth factor (EGF). Maximal effects are obtained at low EGF concentrations, at which mostly the cytoskeleton-associated high-affinity EGF receptors (EGFRs) are saturated. Tumor cells expressing EGFR either endogenously (MDA MB 231, MTLn3) or, for the human EGFR, ectopically (MTC HER1/1) in intermediate amounts exhibited, upon EGF addition, increased cellular adhesion to various ECM proteins, such as fibronectin, collagens and vitronectin. In contrast, human A431 and MDA MB 468 cells, over-expressing EGFR, demonstrated reduced attachment in similar experimental conditions. Both increased as well as reduced EGF-dependent adhesion could be blocked using either ligand-blocking monoclonal antibody 14E1 or the potent EGFR tyrosine kinase inhibitor PD 153035. Our data indicate that signals downstream of EGFR activation are responsible for the opposing effects of EGF on cellular adhesion since both can be prevented by EGFR inhibition. Thus, the integration of EGFR- and integrin-dependent signals can be different in carcinoma cell lines and might be influenced by EGFR numbers.

Induction of apoptosis by EGF receptor in rat mammary adenocarcinoma cells coincides with enhanced spontaneous tumour metastasis.

The low metastatic MTC (13762NF) rat mammary adenocarcinoma cell line is devoid of epidermal growth factor receptor (EGFR). To test for a link between expression of EGFR and the ability of tumour cells to metastasise from their orthotopic site (spontaneous metastasis), stable subclones of this line (S+) that had been retrovirally transduced to express an ectopic full length HER1 were established and characterised. Proliferation, survival, and response to TGF-alpha were investigated and related to the tumorigenic growth and metastatic properties of the cells. S+ clones responded in vitro to ligand stimulation by growth inhibition and apoptosis. Upon orthotopic inoculation into the mammary fat pad of nude (nu/nu) mice, S+ clones showed retarded growth and apoptosed in situ, while MTC cells or neoR control cells showed no signs of apoptosis. Yet, S+ cells exhibited more spontaneous metastasis than the MTC parental cells or neoR control cells. Spontaneous metastasis requires cellular detachment (primary site) as well as attachment (secondary site) and growth in target organs. Neither the HER1 mediated increased ECM adhesion nor its negative effect on growth potential explains the observed effect. This is the first direct demonstration of the potential of EGFR to promote spontaneous metastasis of mammary adenocarcinoma cells from their orthotopic site.

Signaling by epidermal growth factor differentially affects integrin-mediated adhesion of tumor cells to extracellular matrix proteins.

The adhesion of different epidermal growth factor (EGF) receptor (EGFR) expressing cell lines to various extracellular matrix (ECM) proteins is influenced by EGF. To investigate a putative receptor crosstalk between EGFR and integrins we chose two cell lines for a more detailed analysis: the highly metastatic rat mammary carcinoma clone MTLn3 that showed increased adhesion to a panel of ECM proteins in the presence of 10 ng/ml EGF and the nonmetastatic human vulva carcinoma cell line A431 which showed a decreased adhesion under the same conditions. These EGF-mediated stimulatory or inhibitory effects on adhesion were observed within a few minutes. On human A431 cells the inhibitory effect was blocked by an EGFR specific antibody that interferes with ligand binding. In cell adhesion assays performed in the presence of divalent cations MTLn3 and A431 cells exhibited the typical behavior described for integrin-dependent matrix adhesion: Mn2+ enhanced binding to collagen IV and fibronectin whereas Ca2+ inhibited adhesion to collagen IV but not to fibronectin. Adhesion-inhibition assays with anti-human integrin antibodies revealed that A431 cells adhere to collagen via alpha 1 beta 1 and alpha 2 beta 1, and that adhesion to fibronectin is mediated predominantly through alpha 5 beta 1. The interaction of MTLn3 cells with fibronectin was in part RGD dependent, indicating the involvement of either alpha 3 beta 1 or alpha 5 beta 1. Addition of EGF in these assays showed that affecting the integrin extracellular domains by addition of either bivalent cations, RGD peptides, or function-blocking integrin antibodies did not prevent the effects mediated by EGF. We conclude that signals downstream of EGFR can modulate integrin-mediated adhesion to ECM proteins in both an inhibitory and a stimulatory manner.

Antimetastatic action of the prostacyclin analogue cicaprost in experimental mammary tumors.

In breast cancer, the survival rate strongly depends on the number of lymph nodes involved. A drug with a specific inhibitory activity on lymph node and organ metastases would therefore be a candidate for adjuvant therapy after surgery. Prostacyclin and its stable analogues have been shown to interfere with certain steps of the metastatic cascade and to inhibit the number of lung colonies after i.v.-inoculation of various tumor cell lines. Our data reveal that cicaprost, a metabolically stable and orally active analogue of prostacyclin, has pronounced antimetastatic effects in a series of spontaneously metastasizing rodent tumors. In the SMT 2a and 13762 MTLn3 mammary carcinomas of the rat, cicaprost given daily from the day of tumor implantation strongly inhibits the number of lung metastases as well as lymph node weights without exerting an effect on the primary tumor. Even starting treatment when palpable primary tumors are present gives a pronounced antimetastatic activity. To demonstrate that cicaprost has an effect on metastases already settled in the respective organ, treatment was started after surgical removal of the primary tumor. In the SMT 2a tumor, a strong inhibition of the number of metastases was shown. Interestingly, a perioperative treatment schedule was also effective in both models used. As primary tumor growth in vivo or proliferation in vitro remained unchanged by cicaprost, its mode of action seems to be related to one or more mechanisms of the metastatic process. In tumor cell lines expressing a functional prostacyclin receptor, stimulated tumor cell migration is inhibited and changes of differentiation status are obvious. In conclusion, cicaprost strongly inhibits lymph node and organ metastases of spontaneously metastasizing rodent mammary tumors with a mode of action different from cytostatic or antihormonal drugs.

Ligand mediated activation of ectopic EGF receptor promotes matrix protein adhesion and lung colonization of rat mammary adenocarcinoma cells.

Increased expression of EGF receptor (EGFR) in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive causative link, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of functional receptors. A full length cDNA of the human EGFR (HER) was introduced by infection with a retroviral vector into MTC cells. Expression of HER was stable and receptors were functional with respect to surface expression, ligand binding and EGF-stimulated phosphorylation. Independent clones of the transfectants were isolated and characterized. Ligand stimulation of MTC HER cells and derived clones led to enhanced adhesion of cells to extracellular matrix proteins. Implantation of cells intravenously into female nu/nu mice revealed ligand-dependent enhancement of lung colonizing potential of EGFR-expressing cells.

EGF enhances attachment of metastatic rat mammary adenocarcinoma cell clone MTLn3 to fibronectin and collagen.

Attachment of highly metastatic rat mammary adenocarcinoma MTLn3 cells to matrix proteins and its modulation by EGF was examined. Plastic plates were coated with varying amounts of collagen or fibronectin. MTLn3 cells exhibited a dose-dependent adhesion to both matrix proteins, however, they attached more efficiently to collagen than to fibronectin. When EGF or TGF alpha were added at 0.3 to 10 ng/ml for 30 min, a dose-dependent increase in adhesion to both matrix proteins was observed. Maximal stimulation (2-fold) was seen with 10 ng/ml of either growth factor. However, EGF was more potent at lower concentrations (0.3-3 ng/ml) than TGF alpha. The ability of growth factors to stimulate adhesion was also dependent on the amount of matrix the cells were exposed to. While EGF increased rapid attachment of MTLn3 cells to both matrix proteins similarly, subsequent cell spreading and formation of lamellar extensions was faster in cells plated on collagen. These results are suggestive of a functional link between EGF receptor and specific integrin activities.

The stable prostacyclin analogue Cicaprost inhibits metastasis to lungs and lymph nodes in the 13762NF MTLn3 rat mammary carcinoma.

Prostacyclin and its stable analogues have been shown to interfere specifically with certain steps of the metastatic cascade. The antimetastatic activity of the stable prostacyclin analogue Cicaprost (Schering AG) on haematogenous metastasis in a series of tumours in rats and mice has been well established. In order to test the effect of Cicaprost on lymphogenous metastasis we chose the metastatic cell clone MTLn3 derived from the 13762NF rat mammary carcinoma. The effect of Cicaprost on prevention of lung metastasis, lymph node metastasis and primary tumour growth was investigated. Cicaprost given in daily doses of 0.01, 0.03 and 0.1 mg/kg orally, reduced the number of lung metastases in a dose-dependent manner. Whereas the median number of lung metastases in the controls was greater than 1000, Cicaprost at a dose of 0.1 mg/kg reduced the number of lung metastases to between 11 and 100. The weight of the ipsilateral axillary lymph nodes was diminished by Cicaprost to 30-50% of controls. Moreover, metastasis to the contralateral axillary lymph node was completely inhibited by Cicaprost at all three doses tested. Cicaprost did not influence the growth rate of the MTLn3 cell clone implanted into the mammary fat pad or the weight of the primary tumour at the end of treatment. In conclusion, in addition to its dose-dependent effect on haematogenous metastasis, Cicaprost strongly inhibits lymph node metastasis.

EGF receptor in neoplasia and metastasis.

EGFR is a member of the tyrosine kinase family of cell surface receptors with a wide range of expression throughout development and in a variety of different cell types. The receptor can transmit signals to cells: i) upon interaction with ligands such as EGF, TGF alpha, amphiregulin or heparin binding EGF, ii) upon truncation or mutation of extracellular and/or intracellular domains, iii) upon amplification of a basal receptor activity (in the absence of ligand) through cooperation with other cellular signaling pathways or nuclear events (e.g. expression of v-erbA). The activated EGFR can exert pleiotropic functions on cells, depending on their tissue origin and state of differentiation. Under certain conditions it can also contribute to neoplasia and development of metastases. Such conditions can exist upon aberrant receptor/ligand expression and activation (e.g. in the wrong cell; at the wrong time; in the wrong amounts). Aberrant signalling can also occur through constitutive EGFR activation. Oncogenic potential of EGFR has been demonstrated in a wide range of experimental animals. EGFR is also implicated in human cancer, where it may contribute both to the initiation (glioblastoma) and progression (epithelial tumors) of the disease. EGFR may influence key steps in the processes of tumor invasion and dissemination. Involvement of EGFR in tumor spread may indicate a potential use of this receptor as a target for antimetastatic therapy.

A link between ras and metastatic behavior of tumor cells: ras induces CD44 promoter activity and leads to low-level expression of metastasis-specific variants of CD44 in CREF cells.

The activated oncogene c-Ha-ras induces expression of the surface glycoprotein CD44 in cloned rat embryonic fibroblasts (CREF). Induction is transcriptional as shown by transient cotransfections of c-Ha-ras expression constructs and CD44 promoter reporter gene constructs and depends on the presence of an AP-1 binding site at position -110. Increased transcript levels for the standard isoform of CD44 (CD44s) are accompanied by the appearance of alternatively spliced RNAs and the synthesis of variants of CD44 (CD44v). These CD44v molecules differ from the standard type by the addition of sequences in the extracellular portion of the molecules. The occurrence of CD44v molecules in CREF cells upon induction of the CD44 promoter is probably due to leakiness of the splice control in these cells since stable transfection with c-Ha-ras does not alter the CD44v/total CD44 ratio. Upon ras overexpression, however, using an inducible mouse mammary tumor virus-ras construct, a transient increase of CD44v/total CD44 ratio of 3-4 has been determined suggesting that a burst of ras expression, in the genetic background of CREF cells, influences both promoter activity and splice control or accuracy. The expression of CD44v proteins is responsible for the metastatic potential in a variety of tumors (U. Günthert et al., Cell, 65: 13-24, 1991). Also in CREF cells expression of CD44v correlates with metastatic behavior, ras-transfected CREF cells are not only fully transformed but also give rise to metastatic spread as measured in the spontaneous metastasis assay. The adenoviral oncogene E1A counteracts ras-induced promoter function and, consequently, inhibits metastatic behavior without extinguishing transformation.

Rapid effects of EGF on cytoskeletal structures and adhesive properties of highly metastatic rat mammary adenocarcinoma cells.

In the highly metastatic rat mammary adenocarcinoma cell clone MTLn3, EGF induced increased adhesion to fibronectin while in the human epidermoid carcinoma cell line A431 EGF induced diminished adhesive properties. Flattening of cells with extensive formation of filopodia was observed in MTLn3 cells within 5 min of EGF addition, while in A431 cells EGF induced rounding up and only occasional formation of filopodia. Immunofluorescent analysis revealed extension of microtubules (MT) into the filopodia and Western blot analysis demonstrated an EGF-induced 2- to 3-fold increase in the amount of assembled tubulin in MTLn3 but not in A431 cells. In MTLn3, but only marginally in A431 cells, EGF treatment resulted in phosphorylation of a 280 kD cytoskeleton-associated protein, which was rapid and dose-dependent. These results suggest differential signal transduction pathway of cytoskeleton-associated EGFRs in highly metastatic MTLn3 as compared with A431 cells.

Transforming growth factor-beta production and induction of cellular responses in 13762NF rat mammary adenocarcinoma cell clones.

We characterized three clones of different metastatic capacity (MTC, MTLn2 and MTLn3) derived from the 13762NF rat mammary adenocarcinoma for their production and response to TGF-beta. All three clones expressed comparable amounts of TGF-beta 1 mRNA and secreted 100-300 pg/10(6) cells/24 h in a soluble latent form. TGF-beta was found in extracellular matrices produced by all three tumor clones. Addition of exogenous TGF-beta induced different responses. While the low metastatic clone MTC was highly sensitive to the growth inhibitory effect of TGF-beta (ID50 approximately 50 pg/ml), a 6-fold higher dose was necessary for the high metastatic clone MTLn3 (ID50 approximately 300 pg/ml). The clone with intermediate metastatic potential MTLn2 was unresponsive to TGF-beta (1 pg/ml to 3 ng/ml). Our data suggest that tumor cells can modulate their biological properties in an autocrine and/or paracrine fashion by virtue of expression of TGF-beta.