Changes in the tumorigenic and metastatic properties of murine melanoma cells treated with a triazene derivative

Treatment of B16/BL6 murine melanoma cellsin vitro with the xenogenizing agent potassiump-(3-methyl-1-triazeno)benzoate (MM-COOK) had a profound impact on the tumorigenic and metastatic properties of the tumor, an effect that was only detectable in immunologically intact hosts. The treated tumor cells gave rise to a considerably smaller number of experimental and spontaneous pulmonary metastases and displayed an impaired growth ratein vivo, but were highly tumorigenic and metastatic in irradiated recipients. Moreover, the drug-treated cells retained thein vitro growth pattern and plating efficiency of the parent line, and were able actively to immunize intact hosts. Studies aimed at clarifying the mechanisms responsible for the decreased metastatic potential of the cells treated with MM-COOK indicated the involvement of host immune responses largely mediated by cells in the T-dependent compartment with no major contribution of natural immunity effector mechanisms.     

Hyaluronidase expression induces prostate tumor metastasis in an orthotopic mouse model

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.     

Dynamic heterogeneity: isolation of murine tumor cell populations enriched for metastatic variants and quantification of the unstable expression of the phenotype

Recent studies have indicated that KHT fibrosarcoma or B16 melanoma cell variants capable of forming experimental metastases in the lungs of mice after i.v. injection are created stochastically at high rates (10^–5/cell/generation). Expression of this phenotype is unstable and hence expanding populations of tumor cells establish a dynamic equilibrium between a small subpopulation of metastatic variants and a large compartment of nonmetastatic cells. In the present experiments, cell suspensions were prepared from the lungs of mice bearing experimental metastases and the tumor cells contained in them were tested for their metastatic efficiency (ME) using the lung colony assay. The ME of the recovered tumor cell populations was found to be a function of the time of metastatic growth in the animal. Tumor cells isolated soon after the initial i.v. injection, i.e. derived from micrometastases, are highly metastatic while populations recovered from macroscopic nodules are similar to parental lines in their ability to colonize the lung. These results are consistent with the prediction of the above dynamic heterogeneity model that nascent lung metastases should be composed largely of tumor cells expressing the variant metastatic phenotype, but that the proportion of such variants should decline during growth to the equilibrium (parental population) level. Mathematical analysis of the results indicates that the effective rate of reversion of the variant phenotype is 10^–1/cell/generation.     

Prophylaxis and treatment of experimental lung metastases in mice after treatment with liposome-encapsulated 6-O-stearoyl-N-acetylmuramyl-L-α-aminobutyryl-D-isoglutamine

A new lipophilic muramyl dipeptide analog, 6-O-stearoyl-N-acetylmuramyl-L--aminobutyryl-D-isoglutamine, when incorporated in liposomes, was effective in both the prevention and eradication of experimental pulmonary metastases in mice. Multilamellar vesicles composed of synthetic phospholipids (phosphatidylglycerol and phosphatidylcholine) containing saturated myristoyl or unsaturated dioleoyl acyl chains were found to potentiate the antimetastatic activity of this glycopeptide. Prophylactic and therapeutic efficacy was observed against the three murine tumors tested: FSa, an immunogenic fibrosarcoma; NFSa, a nonimmunogenic fibrosarcoma; and B16 melanoma. Neither the administration of empty liposomes or free glycopeptide, nor their coadministration, had a significant antimetastatic effect. This approach is promising for the therapy of cancer metastases in humans, particularly in the prevention of metastatic seeding and in the treatment of micrometastases.This is contribution No. 180 from the Institute of Bio-Organic Chemistry, Syntex Research.     

Differential uptake of systemic fluorochrome Hoechst 33342 in lung and liver metastasis of B16 melanoma

Summary The growth and vascularization patterns of B16 melanoma colonies in the liver and lungs were measured and compared by histological techniques and dye diffusion patterns after injection of the fluorochrome Hoechst 33342. In the liver, the fluorescent pattern of dye diffusion revealed that uninodular tumours measuring up to 146 n in diameter were not functionally vascularized. However, when the nodules fused to give rise to multinodular tumours measuring between 256 and 366 n in diameter, a reticular dye diffusion pattern revealed functional tumour vascularization. In the lungs, subpleural, parenchymal and peritubular (i.e. surrounding blood vessels and airways) tumours were observed. The two former classes were vascularized down to thicknesses and diameters of 49 and 24 m respectively. In contrast, dye diffusion was never seen in peritubular tumour cuffs up to 609 m in thickness. The results indicate differences in vascularization patterns in B16 tumours in the liver and lungs, and differences between tumours growing in different sites within the lungs. If these results are applicable to metastases in these two organs, they indicate potential diffusion-mediated resistance to chemotherapy, and potential hypoxia-mediated resistance to radiotherapy of both metastases and micrometastases.     

Tumorigenicity,invasiveness and metastatic capability of FR3T3 rat cells before and after transfection with bovine papilloma virus type 1 DNA

Fischer rat FR3T3 cells were tested for tumorigenicity, invasive and metastatic capabilities before and after transfection, either with the entire bovine papilloma virus type 1 (BPV-1) genome or with a plasmid (pV69) containing a 69 per cent Bam H1-Hind III fragment of the BPV-1 genome as well as bacterial sequences. Cell lines were grouped as parental, pV69-transfectants, BPV-1 transfectants,in vitro derivatives, andin vivo derivatives. The tumorigenic, invasive and metastatic capabilities of these cell lines were examinedin vivo through s.c., and i.p. injections of cell suspensions and through s.c. implantations of cellular aggregates into syngeneic rats. Invasiveness was testedin vitro through confrontations with embryonic chick heart fragments in organ culture. All cell lines including parental lines, were found to be invasivein vitro and tumorigenicin vivo; all tumors were invasive. It is, therefore, not possible to draw conclusions about the role of BPV-1 gene sequences in the acquisition of the invasive phenotype. Transfection with BPV-1 genes conveyed the metastatic phenotype upon parental FR3T3 cells, which were themselves found to be non-metastatic. With regards to this, no differences were found between BPV-1 transfectants compared with pV69 transfectants. Untransfected cells became metastatic also through passagein vivo as an s.c. tumor. The expression of the metastatic phenotype was not noticeably correlated with alterations of growth characteristics of the cell lines. We concluded that the implication of BPV-1 gene sequences in conveying the metastatic phenotype upon FR3T3, if any, was indirect, presumably through alterations of the host cell genome. Our experiments illustrate the need for long-term observations with parental cell lines before drawing conclusions about the role of oncogenes in the acquisition of the malignant phenotype.     

Low E-cadherin and β-catenin expression correlates with increased spontaneous and artificial lung metastases of murine carcinomas

This study examined the relationship between the expression of E-cadherin or -catenin in murine adenocarcinomas and their hematogenous metastatic propensity, assessed by both spontaneous and artificial lung metastasis. Seven different carcinomas, syngeneic to C3Hf/Kam mice were used: 4 mammary carcinomas (MCa-4, MCa-29, MCa-35, and MCa-K), ovarian carcinoma OCa-I, hepatocarcinoma HCa-I, and adenosquamous carcinoma ACa-SG. These tumors vary widely in their ability to spontaneously metastasize to the lung (from 0 to 100% metastatic incidence), and their cells greatly differ in their ability to form artificial lung nodules when injected i.v. Primary tumors in the leg were assessed for E-cadherin and -catenin expression by western botting. The expression of both proteins showed wide variation among the tumors; however, the expression of E–cadherin correlated well with that of -catenin. There was significant inverse correlation between the expression of E-cadherin, as well as -catenin, and the incidence of both spontaneous and artificial lung metastases from these tumors. Spontaneous metastases of highly metastatic HCa-I and moderately metastatic MCa-35 were significantly lower in E-cadherin and -catenin expression than their corresponding primary tumors were. Thus, the propensity of murine carcinomas for hematogenous spread is highly related to E-cadherin and -catenin levels in primary tumors. The inverse correlation between the expression of these molecules and spontaneous and artificial metastases implies that tumor cells with low E-cadherin and -catenin content have increased ability to enter the vascular circulation at the primary tumor site and to colonize distant tissues.     

EWS/FLI-1 oncoprotein subtypes impose different requirements for transformation and metastatic activity in a murine model

Ewing sarcoma/primitive neuroectodermal tumors (EWS/PNET) are characterized by specific chromosomal translocations most often generating a chimeric EWS/FLI-1 gene. Depending on the number of juxtaposed exons assembled, several fusion types have been described with different incidences and prognoses. To assess the impact of each fusion type on the specific phenotypic, tumorigenic, and metastatic features of EWS/PNET, we developed an amenable system using a murine mesenchymal multipotent C3H10T1/2 cell line. Upon transduction of EWS/FLI-1, cells acquired dramatic morphological changes in vitro, including a smaller size and "neurite-like" membrane elongations. Chimeric fusion proteins conferred oncogenic properties in vitro, including anchorage-independent growth and an increased rate of proliferation. Furthermore, EWS/FLI-1 expression blocked mineralization, with concomitant repression of osteoblastic genes, and induced a dramatic repression of the adipocytic differentiation program. Moreover, EWS/FLI-1 promoted an aberrant neural phenotype by the de novo expression of specific neural genes. The intramuscular injection of transduced cells led to tumor development and the induction of overt osteolytic lesions. Analogously, to what was observed in human tumors, type 2 EWS/FLI-1 cells formed primary tumors in immunodeficient mice with a higher incidence and a lower latency than cells bearing types 1 and 3 fusions. By contrast, cells expressing types 2 and 3 fusions showed specific metastatic activity with a higher number of macroscopic metastases in soft tissues and osteolytic lesions in the limbs as compared to type-1-expressing cells. Therefore, the structure of each oncoprotein strongly influenced its tumorigenicity and metastagenicity. Thus, this model provides a basis for understanding the genetic determinants involved in Ewing tumor development and metastatic activity and represents a cellular system to analyze other oncoproteins involved in human sarcomagenesis.     

Spontaneous but not experimental metastatic activities differentiate primary tumor-derived vs metastasis-derived mouse prostate cancer cell lines

We previously developed an in vivomouse prostate reconstitution (MPR) model of metastatic prostate cancer using p53 knockout mouse urogenital sinus tissue for retroviral transduction of rasand myconcogenes (Thompson et al., Oncogene, 10, 869, 1995). We further demonstrated contrasting responses to transforming growth factor beta-1 (TGF-1) in three matched pairs of early passage cell lines derived from primary prostate tumors and lung metastases generated by this model system (Sehgal et al., Cancer Res, 56, 3359, 1996). In this study we tested these cell lines for growth potential in subcutaneous and orthotopic (dorso-lateral prostate) locations and metastatic activities in both spontaneous and experimental assays. Subcutaneous and orthotopic tumors produced by cell lines derived from metastatic lesions tended to grow less rapidly but demonstrated greater spontaneous metastatic potential than the cell lines derived from primary tumors. In contrast all cell lines produced lung colonies in an experimental metastasis assay (tail vein inoculation) with the primary tumor-derived cell lines yielding higher activities in two of three matched pair analyses. The ability of all cell lines to produce lung metastases in the experimental assay, while only the metastasis-derived cell lines retain the ability to initiate and complete the entire metastatic pathway in the spontaneous assay, suggests that intravasation may be the rate-limiting step in metastasis in this model system.     

B16 melanoma variants selected by one or more cycles of spontaneous metastasis to the same organ fail to exhibit organ specificity

Metastatic clones of the mouse B16 melanoma spontaneously disseminate from subcutaneous tumors throughout the body in two stages, initially to the lungs and secondarily from established lung metastases to systemic sites. From the heterogeneous parent B16 melanoma cell line and from two representative clones, G3.5 and G3.12, cell populations were selected after one or more cycles of tumor growth or metastasis to a particular site, to determine whether metastatic variants with greater organ preference or specificity could be generated. Variants with enhanced secondary metastatic activity were obtained only from G3.12 tumor-disseminated metastases growing in the lungs or in systemic organs. Regardless of the organ of selection or the number of selection cycles, all variants exhibited an overall increase in secondary metastasis incidence and burden in the brain, adrenals, kidneys and ovaries, but no organ preference or specificity was obtained. Populations that grew especially well in the brain, ovaries or liver following intravascular injection were either non-metastatic or exhibited no organ preference during spontaneous metastasis. The increased secondary metastatic activity of G3.12 variants was apparently not due to either longer host survival or to tumor-disseminated cells bypassing the lungs, but may result from enhanced growth potential or greater secondary dissemination capability imparted during growth as lung metastases.     

Enhanced antiproliferative activity by metastatic RAW117 lymphoma cells

The highly malignant/metastatic murine large cell lymphoma cell line RAW117-H10 forms 100–200 times more liver metastatic tumors than its parental counterpart cell line RAW117-P. RAW117-H10 cells, but not the less malignant/metastatic parental cells, significantly inhibited the mitogen-induced proliferation of normal syngeneic Balb/c and allogeneic ICRC mouse spleen cells. Such an inhibition also occurred when mitomycin-C treated metastatic lymphoma cells were added 24 h after initiation of culture, indicating that no competition with mitogen binding sites on the lymphocytes was necssary for inhibition of proliferation. Antiproliferative cell surface molecules were extracted non-cytolytically from the RAW117-H10 cells using butanol. The butanol extracts from the metastatic RAW117-H10 cells also inhibited the mitogen-induced proliferation and natural killer (NK) cell-mediated cytotoxicity of normal spleen cells. Our results indicate that these antiproliferative cell surface molecules of metastatic murine RAW117-H10 lymphoma cells may have important role(s) in tumor-mediated host immunosuppression.     

Thymosin-beta4 regulates motility and metastasis of malignant mouse fibrosarcoma cells

We identified a thymosin-beta4 gene overexpression in malignant mouse fibrosarcoma cells (QRsP-30) that were derived from clonal weakly tumorigenic and nonmetastatic QR-32 cells by using a differential display method. Thymosin-beta4 is known as a 4.9-kd polypeptide that interacts with G-actin and functions as a major actin-sequestering protein in cells. All of the six malignant fibrosarcoma cell lines that have been independently converted from QR-32 cells expressed high levels of thymosin-beta4 mRNA and its expression in tumor cells was correlated with tumorigenicity and metastatic potential. Up-regulation of thymosin-beta4 in QR-32 cells (32-S) transfected with sense thymosin-beta4 cDNA converted the cells to develop tumors and formed numerous lung metastases in syngeneic C57BL/6 mice. In contrast, antisense thymosin-beta4 cDNA-transfected QRsP-30 (30-AS) cells reduced thymosin-beta4 expression, and significantly lost tumor formation and metastases to distant organs. Vector-alone transfected cells (32-V or 30-V cells) behaved like their parental cells. We observed that tumor cell motility, cell shape, and F-actin organization is regulated in proportion to the level of thymosin-beta4 expression. These findings indicate that thymosin-beta4 molecule regulates fibrosarcoma cell tumorigenicity and metastasis through actin-based cytoskeletal organization.     

Stem cell-related markers in primary breast cancers and associated metastatic lesions

It has been reported previously that: (1) normal-breast epithelial cells that are CD24-/44+ express higher levels of stem/progenitor cell-associated genes; (2) cancer cells that have undergone epithelial to mesenchymal transition display CD24-/44+ cell-surface expression, a marker for breast cancer stem cells; (3) loss of E-cadherin is a preliminary step in epithelial to mesenchymal transition; and (4) vimentin is a marker of mesenchymal phenotype. We hypothesized that stem cell subpopulations would be more frequent in metastatic than in primary tumors. Therefore we assessed by immunohistochemical analysis, tissue microarrays containing tissue from primary and associated metastatic breast cancers for expression of CD24, CD44, E-cadherin and vimentin to evaluate candidate cancer-initiating cell populations in breast cancer subtypes and metastatic lesions. The occurrence of CD24-/44+ and CD24+/44- cells did not differ in primary vs matched lymph node or distant and locoregional metastatic lesions; E-cadherin expression was decreased in primary vs lymph node metastases (P=0.018) but not decreased in distant and locoregional metastases relative to primary tumor, whereas vimentin, was more frequently expressed in lymph node and distant and locoregional metastases (P=0.013, P=0.004) than in matched primary cancers. Thus, the frequency of CD24-/44+ cells does not differ in metastases relative to the primary breast cancer but differs by tumor stage and subtype.     

Polyomavirus-transformed FR 3T3 rat cells are able to form metastases in syngeneic rats

A series of polyomavirus-transformed FR 3T3 rat cell lines were tested for their tumorigenic and metastatic properties after subcutaneous inoculation of syngeneic Fisher rats. All of them grew into tumors, which appeared with variable latency periods; the TD50 varied from cell line to cell line. Eight of the 18 transformants that were inoculated gave rise to metastases, always localized in the lung. The capacity to form metastases, though at a low frequency, was also conferred on FR 3T3 cells upon transformation with a recombinant plasmid encoding only the middle-T protein. Fibroblast-like cells were predominantly observed upon histological examination of the metastases. Culture cell lines were derived from independent tumors and metastases induced by two transformants with low and high metastatic potentials, respectively. Metastasis-derived cell lines exhibited metastatic potentials similar to those of the respective original transformants. All the tumor- and metastasis-derived cell lines synthesized the same early viral polypeptides as the respective original transformants; in contrast, the viral DNA integrations evolved during tumor and metastasis formation.     

TNF-α mediated selection of macrophage-resistant gene-regulatory tumor variants

In vitro macrophage-or TNF--mediated selection procedures on 3LL tumor cells have led to the selection of 3LL variants manifesting a highly reduced sensitivity towards the cytotoxic effects of both TNF- and tumoricidal macrophages, while retaining the parental sensitivity to the cytolytic activity of (i) H₂O₂, (ii) macrophage-ADCC reactions and (iii) NK cells. A correlation was observed between the TNF- binding capacity of the 3LL cell lines and their susceptibility towards macrophage-and TNF--mediated cytotoxicity, indicating that macrophage and TNF- sensitivity may partially be regulated at the TNF- receptor level. Further, the selected 3LL variants are gene-regulatory variants rather than cellular mutants, as upregulation of the TNF- receptor by interferon-gamma (IFN-) or 5-azacytidine treatment resulted in an increased vulnerability of the selected 3LL variants to the killing activity of macrophages and TNF-. The resistance of the 3LL variants to macrophage-and TNF-mediated cytotoxicityin vitro was reflected by a higher tumorigenic and metastatic potentialin vivo. Therefore, the generation of TNF--and macrophage-resistant variants through immunoselection may contribute to the basic mechanisms of tumor progression and metastasis.     

Increased levels of α6 integrins are associated with the metastatic phenotype of human breast cancer cells

Integrins play an important role in interactions between cells and the extracellular matrix, and thus have a potential role in metastasis. Expression levels of 6, 1 and 4 integrin sub-units were measured in a panel of human breast cancer cell lines by RT/PCR, immunoprecipitation and flow cytometry. All the lines expressed 6, with the highest levels in the MDA-MB-231 and MDA-MB-435 cells. These grew the most aggressively and were metastastic in nude mice. Low levels of 6 protein were measured in breast cancer cells that were poorly tumorigenic and non-metastatic in nude mice, and there was an inverse relationship between ER and 6 expression. RT/PCR revealed that all lines expressed the 2 isoforms of 6, with the 6A isoform generally more abundant than 6B isoform. Clones of MDA-MB-435 were isolated by sterile sorting for cells with high or low 6 expression, and two variants established from metastases in nude mice were found to differ in 6 expression. When injected into nude mice, the 6-high variants produced significantly more lung metastases than the 6-low variants. 1 was abundant in all lines, while 4 was not detected in MDA-MB-134 cells, and in the MDA-MB-435 cells an alternately spliced variant of 4 was identified. Sequencing of the alternate variant revealed a novel sequence from a splicing event in the cytoplasmic tail of 4. None of the cells with this variant mRNA expressed detectable levels of 4 protein. Our results suggest that high 6 expression in human breast cancer cells is associated with tumorigenicity and metastatic potential.