Purification and some properties of a lung-derived growth factor that differentially stimulates the growth of tumor cells metastatic to the lung.

The ability of malignant cells to respond to growth factor(s) present in or secreted by a distant target organ may be important in tumor metastasis. We used metastatic cell lines and clones of the rat 13762NF mammary adenocarcinoma that show reproducible spontaneous metastatic behavior from the mammary fat pad to regional lymph nodes and lung sites. Whereas poorly lung metastatic MTPa and MTC cells did not grow in response to lung-conditioned medium, highly lung-metastatic MTLn3 cells responded and grew rapidly in lung-conditioned medium. The major growth-promoting factor for MTLn3 cells from porcine and rat lung-conditioned media was purified by using hydroxylapatite affinity and anion exchange chromatography, chromatofocusing, size exclusion chromatography, and preparative native gel electrophoresis. The activity in each of the purification fractions was measured by determining their ability to increase the number of MTLn3 cells in serum-deprived culture. The major component that differentially stimulated the growth of highly metastatic MTLn3 cells was a glycoprotein of Mr approximately 66,000. Under reducing conditions, its apparent Mr was approximately 72,000. This lung-derived mitogen was stable at pH 4.0-9.0, possessed a pI of 6.9-7.0, and preferentially promoted the growth of lung-metastasizing tumor lines over their poorly lung-metastasizing counterparts in rat 13762NF mammary adenocarcinoma and murine B16 melanoma tumor systems. The activity of porcine lung-derived growth factor was not affected by pretreatment with antisera to porcine insulin, human granulocyte-macrophage colony-stimulating factor, human platelet-derived growth factor, or murine epidermal growth factor. It was inactivated by reduction with dithiothreitol or exposure to high temperature (95 degrees C). The results suggest that specific organ-derived growth factors are important in metastatic colonization and organ growth of particular malignant cells.

Purification and identification of mouse lung microvessel endothelial cell-derived chemoattractant for lung-metastasizing murine RAW117 large-cell lymphoma cells: identification as mouse monocyte chemotactic protein 1.

We reported recently that medium conditioned with mouse lung microvessel endothelial cells possessed chemotactic activity for a highly lung-metastatic variant (L17) of the RAW117 murine large-cell lymphoma cell line but not for the poorly metastatic parental cells (P) or a liver-metastasizing variant (H10). The chemotactic factor was purified to homogeneity by a five-step procedure involving hydrophobic interaction, Cibacron blue F3GA affinity, metal affinity, anion exchange, and reversed phase chromatography, followed by preparative gel electrophoresis. The purified material appeared as a single broad band when analyzed by SDS-PAGE, with an average molecular weight of 26,000. The factor was cleaved by cyanogen bromide treatment, and a partial amino acid sequence of one of the cleaved polypeptides proved identical to mouse monocyte chemotactic protein 1 (mMCP-1/JE). The amino acid composition of the factor also indicated similarity to mMCP-1/JE. Separately purified mMCP-1/JE significantly stimulated the chemotactic migration of RAW117 cells (L17 >> H10, P). When recombinant human monocyte chemotactic protein 1 was compared to the purified endothelial cell chemotactic factor as a chemoattractant, similar migratory responses were observed in the RAW117 sublines. The chemotactic activity for L17 cells was significantly reduced from lung microvessel endothelial cell-conditioned medium after treatment with anti-mouse MCP-1 antibody. In contrast, the migration-stimulating activity of liver sinusoidal endothelial cell-conditioned medium to H10 cells was not affected by anti-mouse MCP-1. A major function of mMCP-1/JE is to recruit monocytes to inflammatory sites, and our results suggest that mMCP-1/JE also facilitates lymphoma lung invasion and metastasis.

Differential expression of a Mr approximately 90,000 cell surface transferrin receptor-related glycoprotein on murine B16 metastatic melanoma sublines selected for enhanced brain or ovary colonization.

A cell surface Mr approximately 90,000 glycoprotein (gp90) was found in higher amounts on brain-colonizing than on lung-colonizing murine B16 melanoma sublines. The possible role of gp90 in determining the brain-associated metastatic properties of B16 cells was examined by purifying the glycoprotein and studying the effects of anti-gp90 on the growth, adhesion, and organ colonization properties of B16 cells. The specificity of the anti-gp90 was demonstrated in immunoprecipitation studies where a cell surface- or metabolically labeled Mr approximately 90,000 glycoprotein of pI approximately 4 was exclusively found upon two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Immunoprecipitation analysis, enzyme-linked immunosorbent assays, and the lectin-binding properties of gp90 on lectin affinity columns indicated that it is a Mr approximately 180,000 disulfide-linked dimer, probably related to the transferrin receptor. B16 sublines selected for various organ colonization properties differentially expressed gp90, bound 125I-labeled transferrin, and responded differently to purified transferrin in proliferation assays in relation to their metastatic properties (B15b greater than O13 greater than F10 greater than F1). Anti-gp90 immunoglobulin G affected the growth of brain-colonizing B16-B15b more than B16-F1 cells, but had no effect on the adhesion of B16-B15b or -F1 cells to microvessel endothelial cells in vitro, and anti-gp90 immunoglobulin G F(ab')2 had little effect on the brain colonization properties of B16-B15b cells in syngeneic mice. In blocking assays, anti-gp90 inhibited the binding of 125I-labeled transferrin to B16-B15b cells in a dose-dependent manner. The results suggest that the differential growth-stimulating effects of transferrin on highly metastatic B16 melanoma cells may be due to their differential expression of a Mr approximately 90,000 glycoprotein that is related to the transferrin receptor. In organ sites, such as the brain, differential expression of a transferrin-like receptor may allow metastatic cells to respond to low concentrations of growth factors known to be present in certain organs.

A paracrine migration-stimulating factor for metastatic tumor cells secreted by mouse hepatic sinusoidal endothelial cells: identification as complement component C3b.

Selective malignant cell invasion at secondary sites mediated by organ-specific (paracrine) motility factors may be of importance in preferential organ colonization of metastatic cells. In this study we isolated and examined a migration-stimulating activity present in mouse hepatic sinusoidal endothelial cell-conditioned medium (HSE-CM). HSE-CM contains migration-stimulating activity for highly liver-metastatic (RAW117-H10) and highly lung- and liver-metastatic (RAW117-L17) mouse large cell lymphoma sublines but not for the poorly metastatic parental line (RAW117-P). A migration-stimulating factor for H10 cells was purified from HSE-CM by hydroxylapatite affinity and DEAE anion exchange chromatography, Sephacryl S-200 gel filtration, and preparative native gel electrophoresis. The activity in each of the purification fractions was measured in a Transwell chamber assay using 3-microns diameter pore filters. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the component migrated as a single component of M(r) > 200,000 (nonreducing conditions) or as two components or M(r) approximately 110,000 and approximately 67,000 (reducing conditions). The factor was bound to Concanavalin A-Sepharose but not to heparin- or gelatin-Sepharose affinity columns, induced mainly H10 chemotactic cell activity and some chemokinetic activity, and preferentially stimulated the chemotaxis of liver-colonizing RAW117 sublines (H10 > L17 > P). NH2-terminal amino acid sequence analysis of each subunit indicated that the HSE-CM-derived migration-stimulating factor was a proteolytic fragment of complement component C3. HSE-derived migration-stimulating factors may be important in determining the ability of RAW117 tumor cells to invade and colonize the liver.

Differential stimulation of the growth of lung-metastasizing tumor cells by lung (paracrine) growth factors: identification of transferrin-like mitogens in lung tissue-conditioned medium.

Certain metastatic tumor cells successfully form metastases at particular organ sites, and their organ colonization properties cannot be explained by mechanical or anatomic factors. These tumor cells possess the ability to colonize such sites through preferential adhesion to organ microvessel endothelial cells, preferential organ invasion by expression of particular degradative enzymes and response to organ motility factors, and preferential organ growth by response to growth factors present at relatively higher concentrations in the target organ. The likelihood that target organ-associated growth factors exist and are important in metastatic colonization has been approached by studying the mitogenic effects of target organ extracts, fragments, or conditioned media on poorly and highly metastatic tumor cells that show organ preference of metastasis. We previously described the isolation of a major organ-derived (paracrine) growth factor from lung tissue-conditioned medium. Characterization of this mitogen has demonstrated that it is a transferrin or a transferrin-like glycoprotein, and antibodies to transferrin can remove significant growth activity from lung tissue-conditioned medium. Further demonstration of the existence and characterization of metastasis-associated organ (paracrine) growth factors and their receptors will be helpful in understanding the organ preference of metastasis.

Partial purification of a liver-derived tumor cell growth inhibitor that differentially inhibits poorly-liver metastasizing cell lines: identification as an active subunit of arginase.

Organ specific tumor metastasis is thought in part to require the ability of metastatic cells to respond to target-organ-associated growth factors or to avoid the effects of target organ associated growth inhibitors. We previously found that murine and rat liver-conditioned media inhibited the growth of the poorly-liver metastasizing murine RAW117-P large-cell lymphoma cells more than their highly liver-metastasizing RAW117-H10 counterparts. Using a six step chromatographic procedure, the major RAW117-P cell proliferation inhibitor from a rat liver extract was purified. The factor displayed a Mr of approximately 35,000 and an isoelectric point > 8.5. This material inhibited the growth of many cells at high concentration; however, in dose-response studies it displayed a higher IC50 for highly-liver metastatic murine RAW117-H10 lymphoma and human KM12SM colon carcinoma cells than for their poorly-liver metastatic counterparts. Attempts to identify the growth inhibitor led to the supplementation of tissue culture inhibitor assays with various components, including excess amino acids, and this was found to completely abrogate the factor's activity. Specifically, the addition of excess arginine resulted in the complete cellular recovery from inhibitor exposure. This tentatively identified the liver growth inhibitor as the enzyme arginase, a Mr approximately 10,000 multisubunit protein. A microtiter plate-based assay for arginase was developed and the purification repeated using human liver as a source of activity and the human KM12C colon carcinoma line as a target. The growth inhibitory and arginase activities were found to co-purify, identifying the factor as arginase. Highly-metastatic cells displayed no ability to preferentially inactivate or inhibit the activity of arginase, but they did they display slightly greater amounts of intracellular arginine. The liver is a major site of arginase localization as the enzyme is required for the functioning of the urea cycle. The results indicate that certain liver-colonizing tumor cells can escape, to a degree, the proliferation-damping effects of arginine depletion.

Differential motility stimulation but not growth stimulation or adhesion of metastatic human colorectal carcinoma cells by target organ-derived liver sinusoidal endothelial cells.

Liver is the most common distant metastatic site for colorectal cancers and when blood-borne colorectal cancer cells reach the liver, they first encounter hepatic capillary and sinusoidal endothelial cells. Thus we studied differences between highly (HT-29LMM) and poorly (HT-29P) liver-metastatic sublines of human colorectal cancer cells by examining the interactions between tumor cells and liver microvessel endothelial cells. Using hepatic sinusoidal endothelial (HSE) and lung microvessel endothelial (MLE) cell-conditioned medium we measured the growth and motility stimulating activities released from these endothelial cells and adhesion of these cancer cells to the endothelial cells. Differences in the ability of HSE-conditioned medium (HSE-CM) or MLE-conditioned medium (MLE-CM) to stimulate HT-29 cell growth were not observed. There was a small but significant increase in the rate of adhesion of highly metastatic HT-29LMM cells to HSE cell monolayers than poorly metastatic HT-29P cells, but there was no difference in adhesion to MLE cell monolayers. HSE-CM stimulated the motility of highly metastatic colorectal cancer cells to a greater extent than the poorly metastatic cells. Motility-stimulating activity for the colorectal cancer cell lines was not detected in MLE-CM. The HSE-CM motility-stimulating activity for human HT-29 cells was not removed using antibodies against hepatocyte growth factor (HGF/SF), complement component C3 or laminin, indicating that it is not related to these known liver-derived motility factors. The results suggest that the ability of highly metastatic HT-29LMM colorectal cancer cells to colonize the liver is related to their ability to respond to liver sinusoidal endothelial cell-derived motility factors and to a lesser degree to adhere to liver sinusoidal endothelial cells.

Involvement of a cathepsin B-like cysteine proteinase in platelet aggregation induced by tumor cells and their shed membrane vesicles.

Murine 15091A mammary adenocarcinoma cells and membrane vesicles spontaneously shed from these tumor cells in culture can induce aggregation of washed human platelets. A spectrum of proteinase inhibitors was tested for their ability to inhibit 15091A induced platelet aggregation. Of the inhibitors tested the most effective were those selective for cysteine proteinases. The effect of the spectrum of proteinase inhibitors on 15091A induced platelet aggregation was compared to the effect on cathepsin B-like cysteine proteinase activity in homogenates of 15091A tumor cells and their spontaneously shed vesicles. The results suggest that there is a correlation between activity of a cathepsin B-like proteinase in 15091A cells and vesicles and the ability of these cells and vesicles to induce aggregation of washed human platelets.

Purification and characterization of platelet aggregating activity from tumor cells: copurification with procoagulant activity.

The platelet aggregating component from murine 15091A mammary adenocarcinoma cells was purified by solubilization of activity with CHAPS (3-[(3-cholidamidopropyl)-dimethylammonio]-1-propane sulfonate), fractionation with ammonium sulfate, ion exchange chromatography on DEAE cellulose, and hydrophobic interaction chromatography on dodecyl agarose. A purification of 90-100 fold over the initial cell homogenate was achieved. SDS-PAGE of the purified material resulted in a single major band with a molecular weight of 51,000 +/- 2,000. Procoagulant activity was found to copurify with platelet aggregating activity. Reconstitution with phospholipids was necessary to obtain platelet aggregating activity and procoagulant activity. Trypsin abolished both platelet aggregating and procoagulant activities. The irreversible proteinase inhibitors phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethyl ketone, iodoacetamide or phenanthroline had no effect on platelet aggregating or procoagulant activities. Platelet aggregation induced by this material was inhibited by low concentrations of the specific irreversible thrombin inhibitors, dansylarginine N-(3-ethyl-1, 5-pentanediyl) amide and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone. This is the first report of copurification of tumor cell platelet aggregating and coagulating activities.

Purification and characterization of a Mr approximately 66,000 lung-derived (paracrine) growth factor that preferentially stimulates the in vitro proliferation of lung-metastasizing tumor cells.

In medium containing low concentrations of serum, rat 13762NF mammary adenocarcinoma cell lines and clones (MTPa and MTC; isolated from the locally growing tumor) of low metastatic potential to lung did not exhibit a growth response to lung-conditioned medium, whereas a highly metastatic cell clone isolated from a spontaneous lung metastasis (MTLn3) did. The major growth-promoting factor for MTLn3 cells from porcine and rat lung-conditioned media was isolated by using a five-step procedure (anion exchange chromatography, Affi-gel blue affinity chromatography, chromatofocusing, size exclusion chromatography, and preparative native gel electrophoresis). The lung-derived factor that stimulated the growth of highly metastatic MTLn3 cells was a glycoprotein of Mr approximately 66,000 (non-reduced) or Mr approximately 72,000 (reduced) and possessed a pI of 6.9-7.0. It preferentially promoted the growth of lung-metastasizing tumor lines over their poorly lung-metastasizing counterparts in three tumor systems: rat 13762NF mammary adenocarcinoma, murine B16 melanoma, and murine RAW117 large-cell lymphoma. The factor's growth-stimulatory affect was inactivated by reduction or exposure to high temperature (95 degrees C). Although the growth factor appears to be glycosylated, its molecular weight was not altered by treatment with the protein-deglycosylating agent, trifluoromethane sulfonic acid. Cleavage of the protein by cyanogen bromide resulted in the formation of five fragments. Malignant cell response to this lung-derived paracrine growth factor may be important in the successful formation of lung metastases.

Lung-derived growth factor that stimulates the growth of lung-metastasizing tumor cells: identification as transferrin.

We have previously shown that culture medium conditioned by lung fragments contains mitogenic activity for lung-metastasizing tumor cells but not for their non-metastatic counterparts. The growth-promoting component from media conditioned by rat and porcine lungs has been purified and partially characterized as a Mr approximately 66,000 (unreduced) or Mr approximately 72,000 (reduced) glycoprotein [Cancer Res 49:3928, 1989; J Cell Biochem 43:127, 1990]. Here we report that this factor is the iron transport protein transferrin. Migration distances in sodium dodecyl sulfate and native gel polyacrylamide electrophoresis systems were similar, as were the specific activities and spectrum of mitogenic activities of the lung-derived growth factor and transferrin. Electrophoretically separated holo-rat transferrin and rat lung-derived growth factor displayed similar positive stains for iron. A polyclonal antibody generated against the lung-derived growth factor cross-reacted with human and rat transferrin in Western blots, and anti-human transferrin cross-reacted with rat lung-derived growth factor. All of the mitogenic activity contained in crude lung conditioned media could be removed by antibody-mediated transferrin depletion. The putative cell receptor molecular weights for the lung-derived growth factor and transferrin were similar as were the molecular weights of polypeptides produced by partial trypsin cleavage of the two. Finally, the amino acid sequence of certain regions of rat lung-derived growth factor demonstrated a high degree of homology to human transferrin. The physical and biochemical properties, antigenicity, and mitogenic activity of a previously unidentified lung-derived growth factor for lung-metastasizing tumor cells indicate that it is transferrin.

Selection of highly metastatic rat MTLn2 mammary adenocarcinoma cell variants using in vitro growth response to transferrin.

We previously found that the proliferative response to transferrin and the expression of transferrin receptors (TfR) on the cell surface of various rat 13762NF mammary adenocarcinoma cell sublines correlated with their spontaneous metastatic capability. To further assess the involvement of transferrin and TfR in metastasis, transferrin-responsive cells were selected from the poorly-metastatic, low-transfferin responsive 13762NF MTLn2 subline. When maintained in low serum (0.3%) conditions, MTLn2 cells failed to survive. However, if like medium was supplemented with 0.5 microgram/ml rat transferrin, some colonies emerged, presumably due to their ability to proliferate in response to the added transferrin. The surviving cells were expanded and exposed to ten or 20 similar cycles of transferrin growth selection to obtain the sublines MTLn2-Tf10 and MTLn2-Tf20, respectively. The MTLn2-Tf20 cells proliferated in response to transferrin at a rate similar to that of the high metastatic 13762NF sublines. Using immunofluorescent staining, Scatchard analysis, and affinity isolation of TfR, we discovered that the MTLn2-Tf20 cells had 5 to 6 times more TfR than did the parental MTLn2 line. When injected into the mammary fat pads of rats, the MTLn2-Tf20 line metastasized to the axillary lymph node in seven out of ten animals and to the lungs in six out of ten (median number = 13). No metastases were seen in the MTLn2 parental line. The MTLn2-Tf10 cells showed intermediate properties compared with the MTLn2 and MTLn2-Tf20 cells. The results indicate that variant cells with a high response to transferrin may be more metastatic than the bulk cells in a poorly metastatic population. The selection of cells with high levels of TfR and a higher proliferative response to transferrin results in sublines with greater potentials for spontaneous metastasis.

Responses to paracrine chemotactic and autocrine chemokinetic factors and lung metastatic capability of mouse RAW117 large-cell lymphoma cells.

We studied the cell migration properties of poorly metastatic murine RAW117-P large-cell lymphoma cells, a highly lung metastatic subline (RAW117-L17) and a highly liver metastatic subline (RAW117-H10). L17 cells responded to the serum-free conditioned medium (CM) of mouse lung microvessel endothelial cells (MLEs) and mouse lung fibroblasts (MLFs). The migration of L17 cells was also stimulated by its own CM and, to a lesser extent, by the CM of parental (P) and H10 cells. RAW117-P and -H10 cells responded poorly to all of the CM tested. Chequerboard analyses revealed that the migration-stimulating activities of MLE CM and MLF CM were mainly chemotactic, whereas those of L17, P and H10 CM were chemokinetic. We also analysed the effect of MLE CM and MLF CM in combination with L17, P or H10 CM on cell migration of the RAW117 sublines. The migration of lung metastatic subline L17 cells to MLE or MLF CM was enhanced when L17 CM was also present. This enhancement effect was not seen when P or H10 cells were exposed to MLE or MLF CM plus the CM from P or H10 cells respectively. Thus we found that the chemotactic response of lung metastatic large-cell lymphoma cells to paracrine migration stimulation factors from lung endothelial cells and fibroblasts in concert with an autocrine chemokinetic factor may be involved in RAW117 lung-specific invasion and metastasis.

Role of the coagulation system in tumor-cell-induced platelet aggregation and metastasis.

The ability of tumor cells to initiate coagulation and subsequent platelet aggregation is believed to facilitate the metastatic process. The mechanism by which tumor cells initiate thrombotic alterations is unclear. We have purified a plasma membrane protein platelet aggregating activity/procoagulant activity (PAA/PCA) from several rodent tumors which initiates the coagulation of homologous plasma and aggregation of homologous platelets by a mechanism independent of factor VII. This protein does not possess any proteinase activity; however, its activity is dependent upon the presence of factor X. In addition, PAA/PCA requires reconstitution with phospholipid for expression of activity. These results suggest that tumor cells express a unique protein which possesses procoagulant activity resulting in thrombin generation. Thrombin is responsible for subsequent tumor-cell-induced platelet aggregation.

Separable growth and migration factors for large-cell lymphoma cells secreted by microvascular endothelial cells derived from target organs for metastasis.

Metastatic variant sublines of the murine large-cell lymphoma cell line RAW117 were tested for their growth and migration properties in vitro in medium conditioned by soluble factors released from syngeneic mouse liver-, lung-, and brain-derived microvessel endothelial cells. Medium conditioned with hepatic sinusoidal endothelial cells stimulated the growth of highly liver-colonising (RAW117-H10) and highly liver- and lung-colonising (RAW117-L17) sublines at higher rates than the poorly metastatic parental line (RAW117-P) (H10 greater than L17 greater than P). Medium conditioned with lung microvessel endothelial cells selectively stimulated the growth of the lung-colonising RAW117-L17 subline. Medium conditioned with brain microvessel endothelial cells showed no growth selectivity, and equivalently stimulated the growth of various RAW117 cell sublines. Medium conditioned with hepatic sinusoidal endothelial cells preferentially promoted the migration of the liver-colonising H10 and L17 sublines, and medium conditioned with lung endothelial cells differentially stimulated the migration of the lung-colonising L17 subline; whereas medium conditioned with brain endothelial cells only slightly stimulated the migration of L17, but not H10 or P cells. Fractionation of medium conditioned with hepatic sinusoidal endothelial cells by DEAE Sephacel anion exchange chromatography revealed that the growth-stimulating activities were clearly separable from migration-stimulating activities. The growth- and migration-stimulating activities released from organ microvessel endothelial cells may be important in determining the ability of RAW117 cells to selectively form metastatic colonies in particular organs.

Preferential growth stimulation of metastatic rat mammary adenocarcinoma cells by organ-derived syngeneic fibroblasts in vitro.

To determine whether organ-derived fibroblasts differentially affect the growth of cells from tumors that preferentially metastasize to specific organs, we investigated the effect of medium conditioned with primary cultured rat fibroblasts from various organs on the in vitro growth of metastatic cell lines and clones of the rat 13762NF mammary adenocarcinoma. The conditioned medium from fibroblasts derived from rat mammary fat pad differentially stimulated tumor cell growth in monolayer culture and clonogenic growth in soft agarose of the highly metastatic clone MTLn3 in a dose-dependent manner. Conditioned medium from fibroblasts derived from the lung and liver also stimulated the growth of clone MTLn3 cells but to a lesser extent than did mammary fat pad fibroblasts. In contrast, poorly metastatic cell clones (MTC, MTPa) did not respond to the growth stimulatory factor(s) from the fibroblast-conditioned medium. The factor(s) responsible for the growth stimulation were inactivated by heat and trypsin treatment and inhibited by low pH and cycloheximide. The result suggest that fibroblasts in different organs have different effects on tumor cell growth, and they may determine, in part, the organ specificity of tumor development and metastasis.

Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials.

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain-colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF-7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf.

Transferrin receptor overexpression enhances transferrin responsiveness and the metastatic growth of a rat mammary adenocarcinoma cell line.

We previously found that breast cancer cell transferrin receptor expression and proliferative response to transferrin often correlated with metastatic capability. To further explore this, we transfected mammary tumor cells with a cDNA coding for the transferrin receptor and examined the effects of its overexpression on various cellular properties. A human transferrin receptor expression plasmid was made by excising the cDNA for the receptor from pcDTR1 and ligating it into the multiple cloning site of pcDNAINeo. The resulting construct was transfected into the poorly metastatic rat MTLn2 line that expresses low endogenous levels of rat transferrin receptor, and transfection-induced receptor expression was ascertained using antibodies specific for the human protein. Approximately 50% of the initial geneticin-resistant transfected MTLn2 cells overexpressed human transferrin receptor protein. High expressors were further isolated by four sequential FACS sorts. The final cell population expressed approximately 3-7 times more cell surface transferrin receptor than did vector transfected controls. Both lines proliferated at the same rate in normal (medium plus 5% FBS) culture conditions. However, in serum-free conditions, the transferrin receptor overexpressor cells displayed a pronounced proliferative response to transferrin whereas the control line did not. When injected into the mammary fat pads of female nude mice, cells from both lines formed micrometastases to the lung that were specifically visualized by immunohistochemical staining of rat cytokeratin 17. This revealed that the transferrin receptor transfected line formed larger lesions of this nature than did cells from the vector transfected controls. When injected into the tail vein of female nude mice, the transferrin receptor overexpressors likewise formed gross lung metastases of remarkably greater size than did the vector only transfectants. Overexpression of cell surface human transferrin receptor on MTLn2 cells appeared to affect their in vitro growth response to transferrin and their ability to grow at a secondary site in vivo.

Adhesive, invasive, and growth properties of selected metastatic variants of a murine large-cell lymphoma.

The adhesive, invasive, and growth properties of parental murine large-cell lymphoma cells of low metastatic potential (RAW117-P) were compared to in-vivo-selected sublines of high metastatic potential to liver (RAW117-H10) or lung (RAW117-L17). Using small (approximately 0.5 mm3) pieces of syngeneic organ tissue (lung, liver, kidney) we found that RAW117-L17 cells selectively attached to and invaded lung tissue, whereas RAW117-H10 cells preferentially attached to and invaded liver tissue. We measured adhesion to microvessel endothelial cells established from syngeneic lung and liver and found that the RAW117-L17 cells bound to lung microvessel endothelial cells at significantly higher rates than the other lines, and RAW117-H10 and -L17 cells attached to hepatic sinusoidal endothelial cells at significantly faster rates than RAW117-P cells. Such organ specificity of adhesion was not found at the level of the subendothelial matrix, and the rates of adhesion of RAW117 cells to subendothelial matrix were lower than to endothelial cells. RAW117 cells of low or high metastatic potential bound to immobilized extracellular matrix components, such as fibronectin, at high rats, but adhesion to laminin or collagen IV was minimal. Previous studies indicated that RAW117 lines could proliferate in vitro in certain organ-conditioned media under limiting serum conditions. We therefore examined the ability of a purified paracrine lung growth factor (LDGF-1) to stimulate growth of RAW117 cells in limiting serum-containing medium. The high lung-colonizing L17 line was stimulated to proliferate by LDGF-1 at faster rates than the other lines. The data support Paget's hypothesis that the organ specificity of tumor metastasis is determined by specific tumor cell and host properties.

Malignant melanoma metastasis to brain: role of degradative enzymes and responses to paracrine growth factors.

Mouse and human melanoma cells metastatic to the brain express degradative enzyme activities that are used for invasion of brain basement membrane and parenchyma. Compared to poorly metastatic or lung- or ovary-metastatic murine melanoma lines, the brain-metastatic sublines secreted higher levels of a variety of degradative enzymes. Brain-metastatic murine and human melanoma cells also degraded subendothelial basement membrane and reconstituted basement membrane at rates higher than other metastatic melanoma cells. In some cases these degradative activities in mouse and human melanoma cells can be induced by paracrine factors known to be present in the brain parenchyma, such as nerve growth factor (NGF). NGF stimulates the expression of degradative enzymes, such as the endo-beta-glucuronidase heparanase, that are important in basement membrane penetration but this factor does not stimulate melanoma cell growth. The growth of brain-metastasizing melanoma cells appears to be stimulated by other paracrine growth factors, such as paracrine transferrin. Melanoma cells metastatic to brain express higher numbers of transferrin receptors and respond and proliferate at lower concentrations of transferrin than do melanoma cells metastatic to other sites or poorly metastatic melanoma cells. The results suggest that degradation and invasion of brain basement membrane and responses to paracrine neurotrophins and paracrine transferrins are important properties in brain metastasis of murine and human malignant melanoma cells.