Angiogenic balance in human melanoma: expression of VEGF, bFGF, IL-8, PDGF and angiostatin in relation to vascular density of xenografts in vivo.

Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis formation, is regulated by pro- and anti-angiogenic factors. We have studied the expression of a panel of angiogenic factors, and of the angiogenesis inhibitor angiostatin, in a panel of human melanoma cell lines giving rise to xenografts with different vascular densities. Angiogenic-factor expression was analyzed in vitro (cell lines) and in vivo (xenografts), both at mRNA (RT-PCR and Northern blot) and at protein level (ELISA and Western blot). In vitro angiostatin generation was assessed by Western-blot analysis. Expression of bFGF and VEGF was clearly correlated with a high degree of vascularization, confirming the importance of these factors for tumor angiogenesis. In addition, there was exclusive or elevated in vitro expression of angiogenic factors IL-8, PDGF-AB, and, to a lesser extent, midkine in cell lines that formed highly vascularized tumors. A similar angiogenic-factor-expression pattern was found in the corresponding xenografts, with the exception of VEGF. In most cell lines, this factor had low expression in vitro which was strongly enhanced in vivo. Although all 8 melanoma cell lines were able to excise the angiostatin fragment from the plasminogen parent molecule in vitro, cell lines BLM and M14 showed the most potent angiostatin generation. In vitro angiostatin generation by cell lysates prepared from melanoma xenografts was comparable in all xenograft types. Thus, in our model system we found no correlation between angiostatin generation and vascular density. Our study has limited the number of pro-angiogenic factors that may be involved in melanoma angiogenesis, and provides evidence for the notion that regulation of tumor angiogenesis is dependent on multiple factors. Inhibition of angiogenesis for therapeutic purposes, therefore, should preferably not concentrate on a single factor.

Basic fibroblast growth factor and receptor expression in human ovarian cancer.

Basic fibroblast growth factor (bFGF) and other members of the FGF family share several biological properties that have the potential to mediate neoplastic cell growth. To test the hypothesis that bFGF may play a role in human ovarian cancer cell growth, three ovarian cancer cell lines, A90, A121(P), and A121(A), were investigated for their ability to respond to bFGF as a mitogen, to express endogenous bFGF protein or message for FGF proteins, and to exhibit FGF receptor or its message. Addition of bFGF to cultures of all three cell lines maintained in chemically defined media resulted in a statistically significant increase in cell number. Cell extracts from A90, A121(P), and A121(A) contained an immunoreactive protein that comigrated with hr-bFGF by Western blot analysis. Several bands of higher molecular weight were also noted. Immunohistochemical staining for bFGF demonstrated a cytoplasmic distribution of bFGF in the three cell lines. Both high- and low-affinity binding sites for human recombinant bFGF (hr-bFGF) were expressed by all three lines. High-affinity sites varied from 2700 sites per cell (Kd = 29 pM) to 13,500 sites per cell (Kd = 71 pM). All three cell lines were screened for mRNA expression for seven FGF proteins and four FGF receptors. In all three lines, mRNA for FGF2 (bFGF) was detected by PCR analysis, and in two lines, mRNA for FGF1 (aFGF) and FGF5 were also found. The FGFR1 receptor subtype (flg) was common to all of the cell lines. Finally, suramin inhibited proliferation of A90 and A121 (P and A) with IC50's of 60 and 210 micrograms/ml, respectively. This is consistent with the A90 cell line having higher levels of endogenous bFGF and flg and therefore being more responsive to suramin inhibition than the A121 cell line. The results indicate that these ovarian cancer cell lines can produce bFGF as well as other members of the FGF family of genes and have the ability to respond to bFGF.

Comparison of cisplatin and carboplatin cytotoxicity in human ovarian cancer cell lines using the MTT assay.

In this study, we compared the cytotoxicity of cisplatin and carboplatin against a panel of human ovarian cancer cell lines using the MTT assay, a rapid colorimetric test that can be used to evaluate the number of residual viable tumor cells following chemotherapy. The established human ovarian cancer cell line OVCAR-3 and the recently isolated and characterized A721, A90, A286, A1, and A121A cell lines were evaluated for chemosensitivity. Each cell line was treated separately with cisplatin and carboplatin at concentrations ranging from 500 to 0.16 micrograms/ml. Various chemotherapeutic exposure periods (1, 4, 24, and 48 hr) were tested to determine maximal efficacy. All cell lines were more susceptible to cisplatin than carboplatin at all drug concentrations and all exposure periods tested (P = 0.005). The overall median 50% inhibitory concentration (ID50) for cisplatin was 107 micrograms/ml compared with 490 micrograms/ml for carboplatin P = 0.005). For both cisplatin and carboplatin a 24-hr exposure was significantly more cytotoxic than a 1-hr exposure (P = 0.003 and P = 0.006, respectively). These in vitro results suggest that cisplatin is significantly more cytotoxic than carboplatin against human ovarian cancer cell lines and that cisplatin should not be replaced by carboplatin in the treatment of advanced epithelial ovarian cancer until randomized trials using maximum dosing of the cisplatin-containing regimen are performed.

Three related near-haploid clones in a primary endometrioid carcinoma of the ovary.

We report the cytogenetic findings in a primary endometrioid carcinoma of the ovary from a 29-year-old woman. Three clones with counts of 30, 29, and 27 chromosomes were observed. The predominant clone had 29 chromosomes and the following karyotype: 29, X, -X, -3, -3, +der(3)ins(3;?) (q21;?), -4, -5, -6, -8, -9, -11, -13, -14, -15, -16, -17, -18, -19, -21, -22, +mar. The clone with 30 chromosomes had an additional marker in the form of a minute chromosome, and the clone with 27 chromosomes was monosomic for chromosomes 12 and 20 also. Interestingly, there was no nullisomy of any of the chromosomes.

Characterization of human ovarian and endometrial carcinoma cell lines established on extracellular matrix.

Six cell lines were established from four patients with advanced carcinoma of the ovary and from one patient with carcinoma of the endometrium. These lines were established from fresh tumor material maintained initially on culture dishes coated with an extracellular matrix (ECM) produced by bovine corneal endothelial cells. Two of the six lines continue to require ECM as a substrate for optimal growth while the remaining four lines will proliferate on ECM or plastic substrate. Four cell lines transplanted into athymic nude mice were tumorigenic and maintained histologic and karyotypic similarities between the patient's original tumor, the cell line, and the transplantable tumor. Furthermore, in vitro degradation of ECM was grossly apparent by those cell lines which formed nude mice xenografts. Tumor cells were characterized by cytology, transmission electron microscopy, karyology, substrate requirements, steroid binding protein analysis, and morphological appearance in culture.

Effective multimodality treatment of epithelial ovarian tumors.

Forty patients with epithelial ovarian tumors underwent cyto-reductive surgery followed by a five drug (Adriamycin, D.D.P., 5FU, M.T.X. and C.T.X.) combination chemo and progestin therapy. They all had an initial complete clinical response and 58% were complete histologic responders. One patient developed reactivated disease six months after a negative second look laparotomy. Two of four fatal outcomes were due to development of mixed mesodermal tumors in patients who originally had serous cystadenocarcinomas. We conclude that this regimen is highly effective, independent of residual tumor size, histologic grade of tumor and prior therapy. Its attendant toxicity is acceptably low.

Borderline malignant serous tumors of the ovary maintained on extracellular matrix: evidence for clonal evolution and invasive potential.

Four cases of advanced stage (II or III) and one case of early stage (IC) borderline malignant serous cystadenocarcinomas of the ovary were maintained on culture dishes coated with an extracellular matrix (ECM) produced by bovine corneal endothelial cells. Cells harvested for chromosomal analysis after 2-3 days showed diploid or near-diploid modalities in all cases. Banded chromosome studies in two cases revealed nonrandom clonal abnormalities with trisomy 2, 7, and 12 in seven of 13 metaphases. No structural abnormalities were noted. These cytogenetic findings differ from those found in malignant serous tumors of the ovary. In addition, borderline tumor cells digested the ECM in all cases and formed a cribiform pattern within a few days of primary culture. This study suggests clonal progression from early to advanced stages of borderline malignant serous tumors; readily distinguishable from overtly malignant serous tumors of the ovary. Ability of tumor cells derived from both primary tumors and metastatic implants to digest the ECM implies the possibility that borderline serous tumors have invasive potential.

Extracellular matrix-coated culture dishes: an in vitro model for primary human gynecologic tumors.

Culture dishes coated with an extracellular matrix (ECM) produced by bovine corneal endothelial cells were utilized to investigate human gynecologic carcinomas of epithelial origin. A high culture success rate was achieved for both solid tumor (83%) and ascitic fluid (75%) derived from specimens from 59 patients. Because of the high percentage of tumor cells attaching to the ECM and actively proliferating, a variety of in vitro studies (karyotypic analysis, morphologic appearance on ECM, and ability to digest the ECM) could be done. A preliminary in vitro profile of the various tumor cell types could be made on the basis of these studies. In addition, five long-term cultures were established utilizing the ECM model system.

Human ovarian carcinoma cells maintained on extracellular matrix versus plastic.

The ability of culture dishes coated with an extracellular matrix (ECM) to act as a suitable substrate for human ovarian carcinoma cells in vitro has been examined. The plating efficiency on ECM was 30 to 80% (dispersed tumor cells from solid tumor tissue and effusions) with active proliferation of tumor cells being observed. Within a few min, ovarian carcinoma cells seeded on an ECM were noted to attach firmly and assume a flattened morphology. In addition, ovarian carcinoma cells maintained on ECM-coated dishes could be released easily via trypsinization or with a cell scraper. This is in marked contradistinction to tumor cells seeded onto plastic dishes without an ECM. Invasion through the ECM by tumor cells from solid tumor tissue was occasionally noted. Nonmalignant cells were removed from dispersed tumor cell preparations by preplating on plastic culture dishes without an ECM. The malignant origin of the tumor cells was confirmed by morphological, histochemical, and cytogenetic criteria. This culture system represents a significant improvement over current methods for routinely culturing human ovarian carcinoma cells. Such a model may be utilized for screening anticancer drugs for their ability to inhibit proliferation of human ovarian carcinoma cells from individual patients. This system also may be useful for elucidating mechanisms of ovarian tumor cell attachment and invasion in the process of metastasis.