Reactivity of Tumor Cells in Malignant Effusions with a Panel of Monoclonal and Polyclonal Antibodies

14123 Background: Tumor progression and metastasis formation are often associated with enhanced angiogenesis and with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and a mediator of vascular permeability. We here describe that VEGF is produced and secreted by neoplastic cells in various solid tumors and its production mediated through mTOR. Methods and Results: As assessed by ELISA, the VEGF protein was detected in supernatants of cell lines derived from breast cancer (MDA-MB231), pancreatic carcinoma (BxPC-3), lung cancer (A-427), colon carcinoma (HCT8), and cholangiocellular carcinoma (EGI-1). In addition, VEGF was detected in supernatants of primary tumor cells obtained from malignant effusions in various malignancies (breast cancer, n=4; pancreatic cancer, n=1; ovarial cancer, n=1; parotic carcinoma, n=1; oesophageal carcinoma, n=1). In each case, VEGF protein was detectable in neoplastic cells by immunocytochemistry, and was found to accumulate in supernatants of cultured tumor cells over time, suggesting constant production and secretion. Correspondingly, as assessed by RT-PCR, primary tumor cells as well as the cell lines tested were found to express VEGF mRNA in a constitutive manner. Since mTOR is a well known regulator of VEGF synthesis, we applied rapamycin on primary neoplastic cells and on tumor cell lines. Rapamycin (20–200 nM) was found to counteract the production and secretion of VEGF in all tumor cells tested (VEGF in supernatants in cultures supplemented with rapamycin at 100 nM compared to control=100% on day 6: MDA-MB231: 11.8±0.2%; BxPC-3: 23.6±18.8%; A-427: 30.1±3.4%; HCT8 17.2±0.5%; EGI-1 28.4±1.1%; p<0.05). By contrast, neither rapamycin nor VEGF were found to modulate growth of primary tumor cells or the growth of the tumor cell lines tested. Conclusions: Various human tumor cells express and secrete VEGF. VEGF production is mediated through mTOR. These observations may have implications for the design of new treatment approaches attempting to counteract VEGF production/secretion and thus VEGF-dependent angiogenesis and effusion- formation in solid tumors. No significant financial relationships to disclose.

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Greater specificity of p40 compared with p63 in distinguishing squamous cell carcinoma from adenocarcinoma in effusion cellblocks

CytoJournal ◽

10.25259/cytojournal_78_2019 ◽

2020 ◽

Vol 17 ◽

pp. 13

Author(s):

Nah Ihm Kim ◽

Ji Shin Lee

Keyword(s):

Squamous Cell Carcinoma ◽

Positive Predictive Value ◽

Cell Carcinoma ◽

Negative Predictive Value ◽

Tumor Cells ◽

Squamous Cell ◽

Predictive Value ◽

Immunocytochemical Staining ◽

Nuclear Staining ◽

Malignant Effusions

Objective: Squamous cell carcinoma (SCC) rarely causes malignant effusions. Distinguishing between SCC and adenocarcinoma in effusion cytology can be a challenge. p63 and p40 have been frequently used to support squamous cell differentiation in both histological and cytological specimens. However, similar results in cytological preparations of effusion fluids have been rarely reported. This study was designed to assess the diagnostic value of p63 and p40 immunoreactivity for the differentiation of SCC from adenocarcinoma in malignant effusions. Materials and Methods: Immunocytochemical staining of p63 and p40 was performed on thirty cellblock specimens, including ten malignant effusions carrying SCC and twenty malignant effusions showing adenocarcinoma. Any degree of nuclear staining was considered positive. Results: Of the ten SCC cases, 100% tested positive for both p63 and p40, and most cases showed diffuse staining (>25% of tumor cells). The expression of p63 and p40 was detected in 4 (20%) and 2 (10%) of twenty adenocarcinoma cases, and the extent of staining was all focal (≤25% of tumor cells). The p63 reactivity showed 100% sensitivity, 80% specificity, 71% positive predictive value, and 100% negative predictive value for the differentiation of SCC from adenocarcinoma in malignant effusions. The sensitivity of p40 for SCC was 100%, the specificity was 90%, the positive predictive value was 83%, and the negative predictive value was 100%. Conclusion: Although p63 and p40 are both useful markers for the diagnosis of SCC in malignant effusions, p40 is more specific than p63 in distinguishing SCC from adenocarcinoma.

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Interlaboratory Comparison of In Vitro Cloning of Fresh Human Tumor Cells from Malignant Effusions

Predictive Drug Testing on Human Tumor Cells - Recent Results in Cancer Research ◽

10.1007/978-3-642-82295-7_20 ◽

1984 ◽

pp. 197-201 ◽

Cited By ~ 1

Author(s):

V. Hofmann ◽

E. E. Holdener ◽

M. Müller ◽

U. Früh

Keyword(s):

Tumor Cells ◽

Interlaboratory Comparison ◽

Human Tumor ◽

Human Tumor Cells ◽

Malignant Effusions

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Biologically Active Tissue Factor-Bearing Larger Ectosome-Like Extracellular Vesicles in Malignant Effusions from Ovarian Cancer Patients: Correlation with Incidence of Thrombosis

International Journal of Molecular Sciences ◽

10.3390/ijms22020790 ◽

2021 ◽

Vol 22 (2) ◽

pp. 790

Author(s):

Corinna Steidel ◽

Fanny Ender ◽

Achim Rody ◽

Nikolas von Bubnoff ◽

Frank Gieseler

Keyword(s):

Ovarian Cancer ◽

Ovarian Carcinoma ◽

Cancer Patients ◽

Tissue Factor ◽

Tumor Cells ◽

Extracellular Vesicles ◽

Thromboembolic Events ◽

Advanced Ovarian Carcinoma ◽

Venous Thromboembolic Events ◽

Malignant Effusions

The development of malignant effusions such as ascites reflects a massive progression of a malignant disease. In patients with ovarian carcinoma, a high amount of ascites (>500 mL) is an independent negative prognostic marker. The composition and constituents of ascites reflect the inflammatory environment of the underlying tumor. Increased cellular resistance of ascites-derived tumor cells and the development of venous thromboembolic events (VTE) are major risks for these patients, especially in patients with advanced ovarian carcinoma. In this study, we discuss the release of tissue factor-bearing extracellular vesicles (TF+ EVs) from tumor cells into the environment (ascites fluid) and their systemic spreading as a possible causal explanation of the pathologic coagulation status in these patients. We obtained ascites from patients with advanced ovarian carcinoma, collected during surgery or therapeutic paracentesis (n = 20). Larger ectosome-like EVs were isolated using sequential centrifugation, quantified by high-resolution flow cytometry and analyzed using nanoparticle tracking analysis. Furthermore, the pro-coagulant properties (TF activity) of EVs were determined. Compared to published TF activities of EVs from healthy persons, TF activities of EVs derived from ascites of patients with ovarian cancer were very high, with a median of 80 pg/mL. The rate of VTE, as reported in the patient files, was high as well (35%, 7 out of 20). Furthermore, all but one patient with VTE had EV concentrations above the median within their ascetic fluid (p < 0.02). Since VTE continues to be a frequent cause of death in cancer patients, prophylactic antithrombotic treatment might be worth considering in these patients. However, given the risk of bleeding, more clinical data are warranted. Although the study is too small to enable reaching a conclusion on direct clinical implementation, it can well serve as a proof of principle and a rationale to initiate a prospective clinical study with different patient subgroups. We also show ex vivo that these larger ectosome-like EVs induce intracellular ERK phosphorylation and tumor cell migration, which is not directly related to their pro-coagulative potency, but might help to understand why cancer patients with thromboembolic events have a poorer prognosis.

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Applications of post-embedding immunogold labelling in the study of human tumors

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156365 ◽

1989 ◽

Vol 47 ◽

pp. 876-877

Author(s):

Stephen Hearn ◽

Theodosios Kontozoglou ◽

Harvey Cramer

Keyword(s):

Cell Carcinoma ◽

Tumor Cells ◽

Polyclonal Antibodies ◽

Protein A ◽

Immunogold Labelling ◽

Needle Aspiration ◽

Peripheral Blood Cell ◽

Specimen Preparation ◽

Thin Sections ◽

Molecular Features

Post-embedding immunogold labelling (IEM) can provide an exact correlation between the ultrastructure of tumor cells and the location of specific antigens. This technology links the molecular features of tumor cells derived from immunohistochemistry with our knowledge of tumor ultrastructure from electron microscopy. Criteria for the use of IEM in the study of human tissues include: the ability to localize many different antigens with commercially available antibodies; specimen preparation that is rapid, standardized and effective for different biopsies and tissue culture; results should compare with immunoperoxidase labelling on formalin-fixed, paraffin-embedded tissue.The tumors examined in this study included: squamous carcinomas, melanomas, multiple myelomas, schwannomas, medullary carcinomas of the thyroid, adenocarcinomas, leiomyomas, germ-cell carcinoma, myeloid leukemia, lymphomas, merkel cell carcinoma, and carcinoid tumors. Fine-needle aspiration biopsies (FNAB), core biopsies of bone marrow, peripheral blood, cell culture monolayers and surgically removed tissues were fixed in 1.6% glutaraldehyde. Samples were dehydrated, infiltrated in Lowicryl K4M and UV polymerized (total preparation time: 3 days). Technique for FNAB samples used mesh filtration to purify tumor cells which were then entrapped in albumin prior to embedding to prevent loss of cells. Glass coverslips (4x2mm) bearing cell monolayers were fixed in situ, infiltrated, placed in gelatin capsules and the cells separated from the coverslips after polymerization for en face sectioning. Thin sections were immunolabelled with readily available monoclonal and polyclonal antibodies using the protein A-gold technique. Samples of normal tissues were labelled for each antigen as positive controls and the negative controls used had appropriate low levels of non-specific labelling.

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