scholarly journals Multi-Marker Immunofluorescent Staining and PD-L1 Detection on Circulating Tumour Cells from Ovarian Cancer Patients

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6225
Author(s):  
Du-Bois Asante ◽  
Michael Morici ◽  
Ganendra R. K. A. Mohan ◽  
Emmanuel Acheampong ◽  
Isaac Spencer ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Vascular Endothelial Cells ◽  
Tumour Cells ◽  
Immunofluorescent Staining ◽  
Vascular Endothelial ◽  
Circulating Tumour Cells ◽  
Antibody Staining ◽  
Epithelial Marker ◽  
Staining Protocol ◽  
Insight Into

Detection of ovarian cancer (OC) circulating tumour cells (CTCs) is primarily based on targeting epithelial markers, thus failing to detect mesenchymal tumour cells. More importantly, the immune checkpoint inhibitor marker PD-L1 has not been demonstrated on CTCs from OC patients. An antibody staining protocol was developed and tested using SKOV-3 and OVCA432 OC cell lines. We targeted epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC-specific (PAX8) markers for detection of CTCs, and CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. CTCs were enriched using the ParsortixTM system from 16 OC patients. Results revealed the presence of CTCs in 10 (63%) cases. CTCs were heterogeneous, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) for both (hybrid). PAX8 was only found in 11/157 (7%) CTCs. In addition, 62/157 (39%) CTCs were positive for PD-L1. Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial (p = 0.007) and mesenchymal (p = 0.0009) expressing CTCs. Characterisation of CTC phenotypes in relation to clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity plays in OC and its relationship with PD-L1.

scholarly journals 599 Circulating tumour cells in breast and ovarian cancer: size-based isolation and ex vivo expansion

2020 ◽  
Author(s):  
Bashir Mohamed ◽  
Mark Ward ◽  
Mark Bates ◽  
Cathy Spillane ◽  
Tanya Kelly ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Tumour Cells ◽  
Circulating Tumour Cells ◽  
Breast And Ovarian Cancer

scholarly journals Altered Functions of Human Blood-Derived Vascular Endothelial Cells by Simulated Microgravity

2020 ◽  
Vol 4 (1) ◽  
pp. 2-16
Author(s):  
Vidhya Ramaswamy ◽  
Allison Goins ◽  
Josephine B. Allen
Keyword(s):  
Endothelial Cells ◽  
Human Blood ◽  
Simulated Microgravity ◽  
Cardiovascular Health ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Altered Gravity ◽  
Adaptation Mechanism ◽  
Insight Into

AbstractRecently, the increase in incidence of cardiovascular degeneration associated with weightlessness has drawn much attention to the detrimental effects of space travel on cardiovascular health. Particularly, the regulatory role of the endothelium in cardiovascular degeneration has been studied extensively. The goal of this study was to understand the effects of simulated microgravity on the proliferative, secretory, and anti-thrombogenic functions of endothelial cells differentiated from human blood-derived progenitor cells. Exposure to simulated microgravity enhanced proliferation, as well as the release of soluble nitric oxide while downregulating the release of pro-inflammatory cytokines, such as interleukin-6 (IL-6). Interestingly, the cells also upregulated gene expression of heat shock protein 70 (hsp70), which may be a potential adaptation mechanism of the cells to altered gravity conditions. However, the secretory and proliferative functions had no effect on the anti-thrombogenic functions of these cells. Their anti-coagulative and anti-thrombogenic abilities, as assessed by both upregulation of tissue plasminogen activator (tPA) and their ability to delay plasma clotting, were impaired on exposure to simulated microgravity. These results collectively provide a useful insight into various mechanisms involved in regulating anti-thrombogenic ability of the endothelium, as well as cardiovascular health in altered gravity conditions.

scholarly journals Localization of factor-VIII-related antigen in human vascular subendothelium

Blood ◽  
1980 ◽  
Vol 55 (5) ◽  
pp. 752-756
Author(s):  
JH Rand ◽  
II Sussman ◽  
RE Gordon ◽  
SV Chu ◽  
V Solomon
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Blood Vessel ◽  
Factor Viii ◽  
Platelet Adhesion ◽  
Vascular Endothelial Cells ◽  
Staining Technique ◽  
Immunofluorescent Staining ◽  
Vascular Endothelial ◽  
Factor Viii Related Antigen

Factor-VIII-related antigen has previously been shown to be synthesized by vascular endothelial cells. Using both an immunofluorescent staining technique and electron microscopy, we have demonstrated the presence of factor-VIII-related antigen in human vascular subendothelium. This finding may have implications in the mechanism of platelet adhesion to deendothelialized blood vessel surfaces.

scholarly journals Localization of factor-VIII-related antigen in human vascular subendothelium

Blood ◽  
1980 ◽  
Vol 55 (5) ◽  
pp. 752-756 ◽  
Author(s):  
JH Rand ◽  
II Sussman ◽  
RE Gordon ◽  
SV Chu ◽  
V Solomon
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Blood Vessel ◽  
Factor Viii ◽  
Platelet Adhesion ◽  
Vascular Endothelial Cells ◽  
Staining Technique ◽  
Immunofluorescent Staining ◽  
Vascular Endothelial ◽  
Factor Viii Related Antigen

Abstract Factor-VIII-related antigen has previously been shown to be synthesized by vascular endothelial cells. Using both an immunofluorescent staining technique and electron microscopy, we have demonstrated the presence of factor-VIII-related antigen in human vascular subendothelium. This finding may have implications in the mechanism of platelet adhesion to deendothelialized blood vessel surfaces.

scholarly journals Identification of a New Way to Induce Differentiation of Dermal Fibroblasts Into Vascular Endothelial Cells

2021 ◽  
Author(s):  
Xiaoling Cui ◽  
Jie Wen ◽  
Xiao Li ◽  
Nan Li ◽  
Xuxiao Hao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Vascular Function ◽  
Vascular Endothelial Cells ◽  
Dermal Fibroblasts ◽  
Angiogenic Factors ◽  
Immunofluorescent Staining ◽  
Vascular Endothelial ◽  
Qrt Pcr ◽  
Small Chemical

Abstract Background: Human dermal fibroblasts (HDFs) have the potential to differentiate into vascular endothelial cells (VECs), but their differentiation rate is low and the mechanism involved is unclear. The small molecule pathway controls the phenotype of fibroblasts by activating cellular signaling pathways, which is a more convenient method in the differentiation strategy of dermal fibroblasts into vascular endothelial cells.Methods: In this study, dermal fibroblasts were treated with the different doses of CPP, and the mRNA level and protein level were detected by qPCR, Western blot and immunofluorescent staining. Matrigel assays also were used to teste the angiogenic ability of vascular endothelial cells derived from dermal fibroblasts.Results: Here, we report that a small chemical molecule, CPP ((E)-4-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl) piperazin-1-yl) styryl)-1-methylpyridin-1-ium iodide), efficiently induces the differentiation of dermal fibroblasts into Vascular endothelial cells. First, we observed that the morphology of CPP-treated dermal fibroblasts elongated, curved and formed circular patterns. Western blot and qRT-PCR analyses revealed that CPP effectively reduced the level of the dermal fibroblasts-marker Vimentin and increased levels of the vascular endothelial cells -markers CD31 and CD133. Detection of the percentage of CD31-positive cells from immunofluorescent staining confirmed that CPP efficiently induces dermal fibroblasts to differentiate into vascular endothelial cells. Matrigel assays showed that CPP-treated dermal fibroblasts have the functions of vascular endothelial cells. Western blot and qRT-PCR analyses of pro-angiogenic factors (VEGF, FGF-2 and PDGF-BB) showed that CPP induces dermal fibroblasts to vascular endothelial cells by promoting the expression of pro-angiogenic factors (VEGF, FGF-2 and PDGF-BB). Conclusions: Our results indicate that the small chemical molecule CPP efficiently induces the differentiation of dermal fibroblasts into vascular endothelial cells. Simultaneously, this new inducer provides a potential to develop new approaches to restore vascular function for the treatment of ischemic vascular diseases.

scholarly journals Classical swine fever virus and p7 protein induce secretion of IL-1β in macrophages

2014 ◽  
Vol 95 (12) ◽  
pp. 2693-2699 ◽  
Author(s):  
Zhi Lin ◽  
Wulong Liang ◽  
Kai Kang ◽  
Helin Li ◽  
Zhi Cao ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Channel Blocker ◽  
Inflammatory Responses ◽  
Vascular Endothelial Cells ◽  
Classical Swine Fever ◽  
Innate Immune ◽  
Fever Virus ◽  
Vascular Endothelial ◽  
Insight Into ◽  
Pro Inflammatory Cytokines

Classical swine fever virus (CSFV) has a tropism for vascular endothelial cells and immune system cells. The process and release of pro-inflammatory cytokines, including IL-1β and IL-18, is one of the fundamental reactions of the innate immune response to viral infection. In this study, we investigated the production of IL-1β from macrophages following CSFV infection. Our results showed that IL-1β was upregulated after CSFV infection through activating caspase-1. Subsequent studies demonstrated that reactive oxygen species may not be involved in CSFV-mediated IL-1β release. Recently, research has indicated a novel mechanism by which inflammasomes are triggered through detection of activity of viroporin. We further demonstrated that CSFV viroporin p7 protein induced IL-1β secretion which could be inhibited by the ion channel blocker amantadine and also discovered that p7 protein was a short-lived protein degraded by the proteasome. Together, our observations provided an insight into the mechanism of CSFV-induced inflammatory responses.

scholarly journals Circulating tumour cells at baseline and late phase of treatment provide prognostic value in breast cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuyun Pang ◽  
Hanjun Li ◽  
Shu Xu ◽  
Liying Feng ◽  
Xueping Ma ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Adjuvant Chemotherapy ◽  
Prognostic Value ◽  
Late Phase ◽  
Progression Free Survival ◽  
Breast Disease ◽  
Breast Tumour ◽  
Tumour Cells ◽  
Immunofluorescent Staining ◽  
Circulating Tumour Cells

AbstractTo determine the prognostic value of the timing of circulating breast tumour cell measurement during treatment, peripheral blood from 164 patients with breast disease was collected. Circulating tumour cells (CTCs) were enriched by using immunomagnetic nanospheres (IMNs) and were identified by using immunofluorescent staining. The CTC shows nuclear-positive, EpCAM-positive, CK19-positive, and CD45-negative. Patients with CTC positivity (> 19/7.5 mL blood) had shorter progression-free survival (PFS) and overall survival (OS) than those with negative results (≤ 19/7.5 mL blood) at baseline. Surgery caused an increase in the number and prevalence of CTCs, and the effect disappeared on day 14 after surgery. During adjuvant chemotherapy, CTCs detected before therapy was only correlated with PFS; however, CTCs at the end of adjuvant chemotherapy were correlated with both PFS and OS. The PFS and OS of the CTC-positive group were significantly shorter than those of the CTC-negative group at the end-point follow-up visit. The prognostic value of CTCs at different measurement time points was demonstrated during breast cancer treatment. Surgery and chemotherapy affected the prevalence of CTCs, leading to different prognostic relevance of CTCs at different treatment stages. CTCs detected at baseline or in the late phase of treatment are preferable for prognosis.

scholarly journals A Pilot Study of the Predictive Potential of Chemosensitivity and Gene Expression Assays Using Circulating Tumour Cells from Patients with Recurrent Ovarian Cancer

2020 ◽  
Vol 21 (13) ◽  
pp. 4813 ◽  
Author(s):  
Stefano Guadagni ◽  
Marco Clementi ◽  
Francesco Masedu ◽  
Giammaria Fiorentini ◽  
Donatella Sarti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovarian Cancer ◽  
Pilot Study ◽  
Stage Iv ◽  
Chemotherapeutic Agents ◽  
Tumour Cells ◽  
Response To Therapy ◽  
Circulating Tumour Cells ◽  
Rt Pcr ◽  
Liquid Biopsies

Circulating tumour cells (CTCs) from liquid biopsies are under current investigation in several cancers, including epithelial ovarian cancer (EOC) but face significant drawbacks in terms of non-standardised methodology, low viable cell numbers and accuracy of CTC identification. In this pilot study, we report that chemosensitivity assays using liquid biopsy-derived metastatic EOC CTCs, from 10 patients, nine with stage IIIC and one with stage IV disease, in progression after systemic chemotherapy, submitted for hypoxic isolated abdominal perfusion (HAP), are both feasible and useful in predicting response to therapy. Viable metastatic EOC CTCs (>5 cells/mL for all 10 blood samples), enriched by transient culture and identified by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence (IF), were subjected to flow cytometry-based Annexin V-PE assays for chemosensitivity to several chemotherapeutic agents and by RT-PCR for tumour gene expression profiling. Using a cut-off value of >80% cell death, CTC chemosensitivity tests were predictive of patient RECIST 1.1 responses to HAP therapy associated with 100% sensitivity, 50% specificity, 33% positive predictive, 100% negative predictive and 60% accuracy values. We propose that the methodology employed in this study is feasible and has the potential to predict response to therapy, setting the stage for a larger study.

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