Detection of circulating lymphendothelial cells in patients with solid tumors

20010 Background: Bone marrow derived endothelial progenitor cells (ECPs) contribute to neoangiogenesis in cancer patients. Elevated numbers of circulating endothelial cells and EPCs can be detected in the peripheral blood of tumor patients. Recently, proliferating lymphatic vessels have been described in human cancers such as melanoma and head and neck tumors. Therefore, we investigated whether circulating lymphatic endothelial cells can be detected in cancer patients. Methods: We developed a sensitive immunocytochemical approach using a monoclonal antibody against the lymphendothelial specific hyaluronic receptor LYVE. After enrichment by Ficoll density gradient centrifugation, 7 × 10(5) peripheral blood mononuclear cells (PBMNCs) from 23 patients with metastatic cancer (6 gastrointestinal, 4 lung, 3 thymus, 3 thyroid, 1 mamma, 1 pancreas, 2 renal, 1 adrenal, 2 urothelial cancer, 1 PNET, 1 NET) and healthy individuals (n = 7) were spun onto glass slides. Two million PBMNs from each patient were stained for LYVE using the APAP technique. Isotype antibodies were used as controls. Cytospins were analyzed with the automated cellular imaging system (ACIS; ChromaVision Medical Systems). The method was validated with spiked blood samples. Results: Circulating LYVE+ lymphatic endothelial cells could be detected in 4 of 7 healthy subjects (57%) and in 16 of 23 patients (69%) with metastatic cancer. The mean number of lymphendothelial cells was significantly higher in cancer patients (15 cells/1 × 10(6) PBMNCs [range 0–276] vs. 1.0 cells/1 × 10(6) PBMNCs [range 0–2]). As a control, circulating tumor cells were enumerated after staining with a cytokeratin antibody (A45-B/B3). Circulating tumor cells could not be detected in healthy controls but in 13 of 23 of cancer patients (mean 1.4 cells/1 × 10(6) PBMNCs range [0–9]). Conclusion: Circulating lymphendothelial cells can be detected in patients with metastatic cancer and healthy subjects. Tumor patients have higher levels of circulating lymphendothelial cells than normal controls. These cells may participate in the generation of lymphatic vessels within tumor manifestations. No significant financial relationships to disclose.

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Identification of Circulating Lymphendothelial Cells in the Blood of Patients with Solid Tumors.

Blood ◽

10.1182/blood.v108.11.3947.3947 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3947-3947

Author(s):

Sonja Loges ◽

Karin Suhrbier ◽

Jasmin Otten ◽

Sabine Riethdorf ◽

Gunter Schuch ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Progenitor Cells ◽

Cancer Patients ◽

Solid Tumors ◽

Peripheral Blood ◽

Healthy Subjects ◽

Metastatic Cancer ◽

Lymphatic Endothelial Cells ◽

Endothelial Progenitor

Abstract Angiogenesis and lymphangiogenesis promote tumor growth and metastasis. In addition to vessel sprouting circulating bone marrow derived endothelial progenitor (ECPs) and endothelial cells (CECs) contribute to neoangiogenesis in a process termed vasculogenesis. CECs can be detected in the blood of cancer patients and increased numbers are related to tumor progression. CECs and EPCs express endothelial cell surface markers as CD144 (VE-Cadherin) and VEGFR-2. Sprouting of new lymphatics from the preexisting lymphatic vessels has been described in solid tumors. Circulating lymphatic endothelial progenitor cells expressing CD133 and VEGFR3 were shown to differentiate into LYVE+ lymphatic endothelial cells (LECs) in vitro. Recently integration of circulating lymphatic endothelial progenitor cells in lymphatic neovessels was demonstrated in vivo. Therfore a distinct mechanism similar to vasculogenesis exists in lymphangiogenesis. Goal of our study was to identify and characterize circulating lymphatic endothelial cells in cancer patients. Presence of lymphatic endothelial cells in the peripheral blood was investigated in 26 patients with metastatic cancer and 10 healthy individuals. Additionally PBMNCs from G-CSF mobilised peripheral blood were analyzed (n=5). By developing a sensitive immunocytochemical approach we could identify a novel population of circulating lymphatic endothelial cells (CLECs) in patients with metastatic cancer. CLECs were LYVE+ and could be detected in 16 of 26 patients (62%) with metastatic cancer and in 4 of 10 healthy subjects (40%). The mean number of lymphendothelial cells was significantly higher in cancer patients (8.8 cells/1x106 PBMNCs [range 0–131] vs. 0.3 cells/1x106 PBMNCs [range 0–2]; p=0.03). We additionally investigated if CLECs were mobilised from bone marrow upon stimulation with G-CSF. 60% of analysed PBMNCs from leukapheresis products contained low numbers of circulating lymphatic endothelial cells comparable to normal peripheral blood (0.3 cells/1x106 PBMNCs [range 0–1]). Consequently CLECs unlike CEPs are not mobilised after cytokine stimulation from bone marrow. To better characterize CLECs, co-expression of VEGFR3 and LYVE was analyzed. We found that 90% of LYVE+ cells were positive for VEGFR3 which is consistent with a LEC phenotype previously described. Coexpression of vascular endothelial specific cell adhesion molecule CD144 (VE-Cadherin) was determined on LYVE+ cells by two-colour immunofluorescence. LYVE+ cells were negative for VE-Cadherin consistent with their lymphatic nature. We investigated whether LYVE+ cells represented a differentiated or a progenitor phenotype by double staining for LYVE and CD34 (n=3). All LYVE+ cells were negative for the progenitor marker CD34 indicating a differentiated lymphendothelial phenotype. Macrophages posess the capacity to transdifferentiate into lymphatic endothelial cells. We investigated whether circulating LYVE+ cells expressed CD11b and found a subfraction with a mean of 19% expressing LYVE and CD11b probably representing this macrophage subpopulation (range 13–25%, n=3). In summary our data show significantly higher levels of LYVE+ circulating lymphendothelial cells in patients with metastatic cancer compared to healthy subjects. Further studies are needed to clarify the origin of the circulating LECs and to analyse their potential as surrogate marker for lymphangiogenesis in cancer patients.

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Distribution and Clinical Analysis of EpCAM+/Vimentin+ Circulating Tumor Cells in High-Risk Population and Cancer Patients

Frontiers in Oncology ◽

10.3389/fonc.2021.642971 ◽

2021 ◽

Vol 11 ◽

Author(s):

Chunjin Huang ◽

Sheng Ding ◽

Chunyan Huang ◽

Feng Pan ◽

Xiaodong Liu ◽

...

Keyword(s):

High Risk ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Healthy Subjects ◽

Magnetic Bead ◽

High Risk Population ◽

Risk Population ◽

Blood Samples

Circulating Tumor Cells (CTCs) are already present in the peripheral blood of patients with early tumors and even precancerous lesions. The objective of this study was to determine the count of CTCs in peripheral blood from high-risk population(HRP), healthy subjects and patients with Pan-cancer. The CTCs in the peripheral blood from HRP and cancer patients were enriched and identified based on the positive sorting method by epithelial cell adhesion molecular (EpCAM) liposome magnetic bead (Ep-LMB) and Vimentin liposome magnetic bead (Vi-LMB). Simultaneously, further analysis was carried out focusing on the clinical characteristics of patients by collecting the peripheral blood samples from healthy subjects as the parallel control. According to the results, the prepared LMBs had high specificity and stability, resulting in an average (Av) proliferation rate of over 90% for each cell line, and the average capture rate of higher than 80%. In terms of CTCs count detection in clinical blood samples, the average count was 0.9 (Ep: Av=0.6, Vi: Av=0.3), 2.4 (Ep: Av=1.4, Vi: Av=0.8) and 7.3 (Ep: Av=4.0, Vi: Av=3.3) in healthy subjects, HRP and total cancer patients, respectively. Besides, there was no obvious difference in the average count of CTCs among patients with different cancer types. While count of CTCs in the aforementioned cancer patients was statistically different from that in healthy subjects and patients with HRP. The survival time of cancer patients whose number of CTCs is greater than the average is significantly increased. Collectively, the study confirmed that CTCs can achieve early tumor detection and auxiliary diagnosis, and its number is related to the occurrence and development of tumors, and CTCs can be detected in HRP and sub-health population.

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Circulating Tumor Cells in HER-2 Positive Metastatic Breast Cancer Patients Treated with Trastuzumab and Chemotherapy

The International Journal of Biological Markers ◽

10.1177/172460080902400101 ◽

2009 ◽

Vol 24 (1) ◽

pp. 1-10 ◽

Cited By ~ 4

Author(s):

Raquel A. Nunes ◽

Xiaochun Li ◽

Soonmo Peter Kang ◽

Harold Burstein ◽

Lisa Roberts ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Treatment Response ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Metastatic Breast ◽

Breast Cancer Patients ◽

Her 2

The detection of circulating tumor cells (CTCs) in peripheral blood may have important prognostic and predictive implications in breast cancer treatment. A limitation in this field has been the lack of a validated method of accurately measuring CTCs. While sensitivity has improved using RT-PCR, specificity remains a major challenge. The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2-positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Serial CTCs were measured at planned time points and clinical correlations were made. Immunomagnetic selection of circulating epithelial cells was used to address the specificity of tumor cell detection using cytokeratin 19 (CK19). In addition, the extracellular domain of the HER-2 protein (HER-2/ECD) was measured to determine if CTCs detected by CK19 accurately reflect tumor burden. The presence of CTCs at first restaging was associated with disease progression. We observed an association between CK19 and HER-2/ECD. The association of HER-2/ECD with clinical response followed a similar pattern to that seen with CK19. Finally, the absence of HER-2/ECD at best overall response and a change of HER-2/ECD from positive at baseline to negative at best overall response was associated with favorable treatment response. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients. The association between CK19 and markers of disease burden is in line with the concept that CTCs may be a reliable measure of tumor cells in the peripheral blood of patients with metastatic breast cancer. The association of CTCs at first restaging with treatment failure indicates that CTCs may have a role as surrogate markers to monitor treatment response.

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Comparison of cytokeratin 20 RT-PCR and immuncytochemistry for the detection of circulating tumor cells in the peripheral blood of colorectal cancer patients

Gastroenterology ◽

10.1016/s0016-5085(08)82983-6 ◽

2001 ◽

Vol 120 (5) ◽

pp. A600

Author(s):

Andras Ladanyi ◽

Bela Molnar ◽

Lajos Floro ◽

Lidia Sreter ◽

Zsolt Tulassay

Keyword(s):

Colorectal Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Cytokeratin 20 ◽

Rt Pcr ◽

Colorectal Cancer Patients

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Abstract 3604: Centrosome amplification in epcam-captured circulating tumor cells from metastatic cancer patients

10.1158/1538-7445.am2018-3604 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ashok Singh ◽

Ryan A. Denu ◽

Jamie M. Sperger ◽

Beth A. Weaver ◽

Mark E. Burkard ◽

...

Keyword(s):

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Metastatic Cancer ◽

Centrosome Amplification

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Efficient Propagation of Circulating Tumor Cells: A First Step for Probing Tumor Metastasis

Cancers ◽

10.3390/cancers12102784 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2784

Author(s):

Jerry Xiao ◽

Joseph R. McGill ◽

Kelly Stanton ◽

Joshua D. Kassner ◽

Sujata Choudhury ◽

...

Keyword(s):

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Metastatic Cancer ◽

Metastatic Breast ◽

Success Rates ◽

Breast Cancer Patients ◽

Metastatic Cells ◽

Healthy Donors

Circulating tumor cells (CTCs) represent a unique population of cells that can be used to investigate the mechanistic underpinnings of metastasis. Unfortunately, current technologies designed for the isolation and capture of CTCs are inefficient. Existing literature for in vitro CTC cultures report low (6−20%) success rates. Here, we describe a new method for the isolation and culture of CTCs. Once optimized, we employed the method on 12 individual metastatic breast cancer patients and successfully established CTC cultures from all 12 samples. We demonstrate that cells propagated were of breast and epithelial origin. RNA-sequencing and pathway analysis demonstrated that CTC cultures were distinct from cells obtained from healthy donors. Finally, we observed that CTC cultures that were associated with CD45+ leukocytes demonstrated higher viability. The presence of CD45+ leukocytes significantly enhanced culture survival and suggests a re-evaluation of the methods for CTC isolation and propagation. Routine access to CTCs is a valuable resource for identifying genetic and molecular markers of metastasis, personalizing the treatment of metastatic cancer patients and developing new therapeutics to selectively target metastatic cells.

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