sepharose and Ovarian-Neoplasms

Epithelial ovarian cancers are among the most aggressive forms of gynecological malignancies. Despite the advent of poly adenosine diphosphate-ribose polymerase (PARP) and checkpoint inhibitors, improvement to patient survival has been modest. Limited in part by clinical translation, beneficial therapeutic strategies remain elusive in ovarian cancers. Although elevated levels of extracellular proteins, including collagens, proteoglycans, and glycoproteins, have been linked to chemoresistance, they are often missing from the processes of drug- development and screening. Biophysical and biochemical signaling from the extracellular matrix (ECM) determine cellular phenotype and affect both tumor progression and therapeutic response. However, many state-of-the-art tumor models fail to mimic the complexities of the tumor microenvironment (TME) and omit key signaling components. In this article, two interpenetrating network (IPN) hydrogel scaffold platforms, comprising of alginate-collagen or agarose-collagen, have been characterized for use as 3D in vitro models of epithelial ovarian cancer ECM. These highly tunable, injection mold compatible, and inexpensive IPNs replicate the critical governing physical and chemical signaling present within the ovarian TME. Additionally, an effective and cell-friendly live-cell retrieval method has been established to recover cells post-encapsulation. Lastly, functional mechanotransduction in ovarian cancers was demonstrated by increasing scaffold stiffness within the 3D in vitro ECM models. With these features, the agarose-collagen and alginate-collagen hydrogels provide a robust TME for the study of mechanobiology in epithelial cancers. STATEMENT OF SIGNIFICANCE: Ovarian cancer is the most lethal gynecologic cancer afflicting women today. Here we present the development, characterization, and validation of 3D interpenetrating platforms to shift the paradigm in standard in vitro modeling. These models help elucidate the roles of biophysical and biochemical cues in ovarian cancer progression. The agarose-collagen and alginate-collagen interpenetrating network (IPN) hydrogels are simple to fabricate, inexpensive, and can be modified to create custom mechanical stiffnesses and concentrations of bio-adhesive motifs. Given that investigations into the roles of biophysical characteristics in ovarian cancers have provided incongruent results, we believe that the IPN platforms will be critically important to uncovering molecular dri

Topics: Alginates; Biophysics; Carcinoma, Ovarian Epithelial; Collagen; Extracellular Matrix; Female; Humans; Hydrogels; Mechanotransduction, Cellular; Ovarian Neoplasms; Sepharose; Tumor Microenvironment

Topics: Aged; Ascitic Fluid; Carcinoma; Female; Histocytological Preparation Techniques; Humans; Immunohistochemistry; Ovarian Neoplasms; Sepharose

Ten ovarian and 2 cervical tumour cell lines were analysed for the production of pregnancy-associated proteins. Pregnancy-associated plasma protein-A (PAPP-A) was detected by radioimmunoassay in culture media of 2 out of 4 (50%) tumour granulosa cell lines (mean = 104 microIU/10(5) cells/24 h) but not in any ovarian (n = 6) or cervical (n = 2) tumour cell lines. By contrast, human chorionic gonadotrophin (hCG), pregnancy specific beta 1-glycoprotein and alpha-fetoprotein (AFP) were not detected in any of the PAPP-A positive media. Only two cell lines produced hCG (58.5 and 25.5 mIU/10(5) cells/24 h). No AFP was produced by any of these 12 cell lines, whereas placental protein 5 was positive in 7. None of these proteins were detected in the culture media of 4 cell lines. In vitro derived PAPP-A was immunologically indistinguishable from either pregnancy or ovarian follicular PAPP-A. All PAPP-A species interacted reversibly with immobilised heparin and were determined by molecular sieve chromatography to have an apparent molecular weight of 820,000 daltons. Cultured tumour granulosa cells specifically synthesised and secreted a large protein which was immunologically and physicochemically indistinguishable from in vivo (pregnancy and ovarian follicular) derived PAPP-A.

Topics: Female; Granulosa Cell Tumor; Heparin; Humans; In Vitro Techniques; Molecular Weight; Ovarian Neoplasms; Pregnancy Proteins; Pregnancy-Associated Plasma Protein-A; Protein Binding; Sepharose

The in vitro antiproliferative activity of human recombinant interferon-gamma (IFN-gamma) was tested against human tumor cells in vitro in combination with doxorubicin, cisplatin, or vinblastine. Using a human tumor clonogenic assay (HTCA), IFN-gamma alone showed dose-dependent inhibition of colony growth in six or seven human tumor cell lines as well as in each of nine fresh ovarian tumor specimens. The combination of IFN-gamma and either doxorubicin or cisplatin showed additive antiproliferative effects against all the cell lines with the exception of an IFN-gamma-resistant endometrial cancer cell line (HEC-1A). In combination with vinblastine, IFN-gamma rarely had an additive effect. Inclusion of macrophages from malignant effusions in the HTCA potentiated the antiproliferative effect of IFN-gamma alone as well as the combination of IFN-gamma and doxorubicin; however, the efficacy of the two agents was never more than additive. The results show that combinations of IFN-gamma with doxorubicin or cisplatin are additive and warrant further investigation. The antitumor effect of IFN-gamma alone or in combination with cytotoxic drugs may be significantly enhanced by tumor-associated macrophages.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line; Cisplatin; Colony-Forming Units Assay; Doxorubicin; Female; Humans; Interferon-gamma; Macrophages; Ovarian Neoplasms; Sepharose; Tumor Stem Cell Assay; Vinblastine

We used affinity chromatography on concanavalin A Sepharose to study the serum alpha-fetoprotein of 10 patients with histologically proven germ-cell tumors and 12 patients with primary liver cancer. Less than 50% of the fetoprotein from germ-cell tumors bound to concanavalin A, as compared with more than 80% of the alpha-fetoprotein from primary liver cancers.

Topics: Adult; Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Female; Humans; Liver Neoplasms; Male; Mesonephroma; Middle Aged; Ovarian Neoplasms; Sepharose; Testicular Neoplasms

Human ovarian tumor-associated antigen (TAA) has been purified from ovarian tumor tissue by affinity chromatography on concanavallin A-Sepharose and three different gamma globulin-Sepharose columns. The resulting ovarian TAA appears to be contaminated by one normal antigen or family of antigens. Rabbit antiserum prepared against this purified ovarian TAA (antiserum 404) was coupled to CNBr-activated Sepharose 4B. This coupled Sepharose was added to fractionated serum from ovarian cancer patients with Stage III and IV malignancy. Bound protein was eluted with 0.2M glycine buffer and tested against antiserum 404. The bound protein contained TAA identical to the TAA isolated from ovarian tumor tissue.

Topics: Animals; Antigens, Neoplasm; Chromatography, Affinity; Concanavalin A; Cyanogen Bromide; Female; gamma-Globulins; Humans; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Mannose; Ovarian Neoplasms; Ovary; Rabbits; Sepharose