Detection and Quantitation of Circulating Tumor Cells, Cancer Stem Cells and Endothelial Progenitor Cells Using Multiparameter Flow Cytometry

Abstract Abstract 2984 Background: Multiple myeloma (MM), a neoplasm of committed B-lymphocytes within the bone marrow (BM), is the second most common hematologic malignancy in the U.S. Despite prolonged median survival with anti-myeloma strategies aimed at the tumor and its BM microenvironment, MM remains invariably fatal. Endothelial progenitor cells (EPCs) are CD133+/KDR+ cells that originate in the BM and play a key role in supporting tumor growth and MM progression. Using X-chromosome inactivation and RT-PCR analyses, we previously found EPCs from MM patients to be clonally restricted and to display clonotypic IG heavy-chain gene rearrangements identical to the same patients' tumor cells (Braunstein et al., 2006). Based on the shared genetic identity that we and others have demonstrated between tumor cells and EPCs in MM patients, the present study explored the hypothesis that, similar to hemangioblasts, which are CD133-expressing precursors to adult hematopoietic and endothelial cells, EPCs may be a source of vascular and MM progenitor cells. Since hemangioblasts are known to exist predominately in the quiescent phases of the cell cycle, in this study we also examined the cell cycle status of CD133-expressing BM cells from MM patients in order to gain insight into their hemangioblastic traits. Methods: BM aspirates were acquired from MM patients under IRB approval. EPCs (>98% vWF/CD133/KDR+ and CD38-) from BM aspirates of MM patients were outgrown on laminin-coated flasks as previously described. The spleen colony assay was used to determine the stem cell capacity within BM-derived EPCs by i.v. injection into NOD/SCID mice. The spleens and BM of mice were harvested 2–4 weeks later. Cells were analyzed by immunofluorescence (IF) and flow cytometry. Hoechst 33342 (Hst) and Pyronin Y (PY) were used to measure the cell cycle status of CD133+ cells using FACS analysis. Results: Two to four weeks following i.v. injection of MM EPCs, human cell surface marker expression detected by flow cytometry within mouse BM and spleen cells shifted from CD133+/CD45-lo, a progenitor cell phenotype, to CD133−/CD45-hi, a more differentiated phenotype, suggesting the ability of MM EPCs to differentiate in vivo. Cell cycle analysis of the CD133+ population in BM cells of MM patients showed that these cells were predominantly non-cycling. On average, 60.5% of CD133+ cells were found to be in the G0/G1 phase of the cell cycle, as indicated by low levels of IF staining with Hst and PY. Conclusions: CD133+ cells are strong candidates as precursors to MM tumor and vascular progenitor cells. As quiescent cells are non-dividing, they often escape cytotoxic effects of chemotherapy, resulting in relapse, and therefore, identification of these cells is critical. Ongoing studies include the engraftment of CD133+ cells in the subcutaneous NOD/SCID gamma xenotransplant model and their growth in response to anti-myeloma strategies; results of these studies will be discussed. Disclosures: No relevant conflicts of interest to declare.

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Generation of Endothelial Progenitor Cells from Amniotic Fluid Stem Cells

Blood ◽

10.1182/blood.v112.11.5403.5403 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5403-5403

Author(s):

Jose E. Cardier ◽

Olga Wittig ◽

Jose Alonso ◽

Egidio Romano

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Amniotic Fluid ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Adult Stem Cell ◽

Stem Cell Markers ◽

Adherent Cells ◽

Endothelial Progenitor

Abstract Recently it has been shown that human amniotic fluid contains stem cells of fetal origin (AFS) that express embryonic and adult stem cell markers. AFS can be expanded extensively in culture and give rise to multiple differentiated cells such those of myogenic, osteogenic, neurogenic and endothelial lineages, all of them with potential therapeutic value. In this study, we investigated the generation of endothelial progenitor cells (EPC) from AFS. For this purpose, remnant amniotic fluid was obtained from mothers undergoing amniocentesis for medical reasons at 18 weeks of pregnancy. Cells were collected by centrifugation and cultured in DMEM, supplemented with Chang medium and fetal bovine serum. Adherent cells were expanded and characterized by flow cytometry at various times using antibodies for CD34, CD45, CD133, CD117, KDR (VEGFR-2) and CD31. Cultures were also carried out in endothelial growth medium and analyzed for EPC markers (CD133, KDR, CD31). Adherent cells showed, at 8 and 17 days of culture, fibroblastoid features. Flow cytometry analysis, at 2 weeks of culture, showed cells expressing CD 133 (37%), CD 34 (1%), CD 117 (3%) and KDR (12%). Cultures carried out in endothelial differentiation medium (EDM), at 33 days of culture, showed a significant increase of cells expressing EPC markers: CD133 (40%), CD34 (3%) KDR (24%), KDR/CD31 (9%). These cells formed blood vessel-like structures on collagen-coated plates. The in vivo angiogenic capacity of these cells was demonstrated by inoculating them into collagen microspheres and implanting them subcutaneously into immunocompromised mice. Histologic examination revealed that these cells formed microvessels on implantation. Our data indicates that EPC can be generated and expanded from AFS, and they might be useful for therapeutic angiogenesis in ischemic tissues.

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The Inhibitory Effect of Lysophosphatidylcholine on Proangiogenesis of Human CD34+ Cells Derived Endothelial Progenitor Cells

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.682367 ◽

2021 ◽

Vol 8 ◽

Author(s):

Haijun Zhao ◽

Yanhui He

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Umbilical Vein ◽

Vascular Inflammation ◽

Flow Cytometry Analysis ◽

Endothelial Progenitor ◽

Hematopoietic Stem

Increasing evidence reveals that lysophosphatidylcholine (LPC) is closely related to endothelial dysfunction. The present study aimed to investigate the mechanism of LPC in inhibiting the proangiogenesis and vascular inflammation of human endothelial progenitor cells (EPCs) derived from CD34+ cells. The early EPCs were derived from CD34+ hematopoietic stem cells whose purity was identified using flow cytometry analysis. The surface markers (CD34, KDR, CD31; VE-cadherin, vWF, eNOS) of EPCs were examined by flow cytometry analysis and immunofluorescence. RT-qPCR was used to detect the mRNA expression of inflammatory cytokines (CCL2, IL-8, CCL4) and genes associated with angiogenesis (VEGF, ANG-1, ANG-2) in early EPCs after treatment of LPC (10 μg/ml) or phosphatidylcholine (PC, 10 μg/ml, control). The angiogenesis of human umbilical vein endothelial cells (HUVECs) incubated with the supernatants of early EPCs was detected by a tube formation assay. The mRNA and protein levels of key factors on the PKC pathway (phosphorylated PKC, TGF-β1) were measured by RT-qPCR and western blot. The localization of PKC-β1 in EPCs was determined by immunofluorescence staining. We found that LPC suppressed the expression of CCL2, CCL4, ANG-1, ANG-2, promoted IL-8 expression and had no significant effects on VEGF expression in EPCs. EPCs promoted the angiogenesis of HUVECs, which was significantly inhibited by LPC treatment. Moreover, LPC was demonstrated to promote the activation of the PKC signaling pathway in EPCs. In conclusion, LPC inhibits proangiogenesis of human endothelial progenitor cells derived from CD34+ hematopoietic stem cells.

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Circulating tumor cells and circulating cancer stem cells and their detection by the method of flow cytometry

Proceedings of the National Academy of Sciences of Belarus Biological Series ◽

10.29235/1029-8940-2021-66-3-370-384 ◽

2021 ◽

Vol 66 (3) ◽

pp. 370-384

Author(s):

T. A. Pozniak ◽

A. Y. Hancharou ◽

V. M. Abashkin ◽

A. I. Stanovaya ◽

A. V. Prokhorov ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Cancer Stem Cells ◽

Early Diagnosis ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Specific Cell ◽

Early Stage Disease ◽

Recurrent Cancer ◽

Mesenchymal Transition

This review describes the circulating cancer stem cells (CCSCs) and circulating tumor cells (CTCs). CCSCs are one of the main initiators of recurrent cancer and thus make them an important target for the development of new treatment methods. CTCs are relatively new biomarkers for the early diagnosis of metastasis. CTCs provide doctors with valuable information about each stages of cancer treatments: diagnostic of early-stage disease, early detection of recurrent cancer, the efficiency of chemotherapy, and makes it possible to select an individual sensitive drug.The most informative and frequently used markers for the detection of CSCs and CSCs were described. The mechanism of two models of tumor formation is considered: clonal and hierarchical. The known mechanisms of epithelial-mesenchymal transition of tumor cells are described. The most widely used specific cell surface markers for the detection and isolation of CTCs and CCSCs are described. The efficiency of a sensitive high-precision method of multicolor flow cytometry using specific fluorescent dye-labeled monoclonal antibodies for the detection of CCSCs and CTCs in the blood of cancer patients is analyzed. Detection of CTCs and CCSCs provides important information for the early diagnosis of metastasis and open a possibility to personalized treatment, and to monitoring of all stages cancers.

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Endothelial Progenitor Cell Marker CD133 Identifies Tumor Endothelium

Blood ◽

10.1182/blood.v112.11.5449.5449 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5449-5449

Author(s):

Muhamed Baljevic ◽

Sergey V Shmelkov ◽

Daniel J Nolan ◽

Andrea T Hooper ◽

Adilia Hormigo ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Cancer Stem Cells ◽

Brain Tumors ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Tumor Vasculature ◽

Significant Heterogeneity ◽

Endothelial Progenitor ◽

Hematopoietic Stem

Abstract CD133, a pentaspan surface antigen, is considered a marker of tissue-specific stem cells, including hematopoietic stem cells and endothelial progenitors. We and others have previously shown that CD133 expression is high on endothelial progenitor cells; that it is downregulated during their differentiation and that it is not found on mature endothelium. In addition, CD133+ cells in brain tumors were shown to exhibit features of cancer stem cells. Recent report showed that CD133+ tumor cells are located in perivascular niches in astrocytomas and glioblastomas. It was also demonstrated that in glioblastomas, CD133+ cancer stem cells are tightly associated with tumor vasculature and that signals from endothelial cells are the key factors in self-renewal and proliferation of cancer stem cells. However, the contribution of CD133+ endothelial progenitor cells to tumor angiogenesis remains unknown. We sought to investigate the role of CD133+ cells in human brain tumors and their relation to endothelial progenitor cells and to the mature endothelium. To this end we examined tumor samples resected from patients with glioblastomas. Histological and immunohistochemical analyses revealed that CD133+ cells in these tumors are predominantly endothelial cells as demonstrated by co-staining with CD31 and CD133. We further analyzed a population of endothelial cells using multicolor flow cytometry analysis and we demonstrated that on average 60% of endothelial cells, as defined by the expression of CD31 and VE-cadherin, also express CD133. However, we also found significant heterogeneity between glioblastomas obtained from different patients. Additionally, CD133 was not expressed on CD45−CD31+ subpopulation in meningiomas. Taken together, we demonstrated that high percentage of endothelial cells in glioblastomas expresses CD133, while CD133 could also be found on other cell types within the tumor. Furthermore, our data suggest that tumor vasculature might be enriched with bone marrow derived hemangiogenic progenitors.

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Improved Bone Formation by Differentiated Mesenchymal Stem Cells and Endothelial Progenitor Cells Seeded on High Concentrated Bioglass-Polylactic Acid Composite in Calvarial Rat Bone Defect

Stem Cells Research Development & Therapy ◽

10.24966/srdt-2060/100004 ◽

2015 ◽

Vol 2 (1) ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Caroline Seebach ◽

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Polylactic Acid ◽

Bone Defect ◽

Endothelial Progenitor ◽

Rat Bone

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Elevated Inflammatory Parameter Levels Negatively Impact Populations of Circulating Stem Cells (CD133+), Early Endothelial Progenitor Cells (CD133+/VEGFR2+), and Fibroblast Growth Factor in Stroke Patients

Current Neurovascular Research ◽

10.2174/1567202616666190129164906 ◽

2019 ◽

Vol 16 (1) ◽

pp. 19-26 ◽

Cited By ~ 7

Author(s):

Monika Golab-Janowska ◽

Edyta Paczkowska ◽

Boguslaw Machalinski ◽

Dariusz Kotlega ◽

Agnieszka Meller ◽

...

Keyword(s):

Risk Factors ◽

Stem Cells ◽

Growth Factor ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Plasma Levels ◽

Fibroblast Growth ◽

Vascular Risk ◽

Endothelial Progenitor ◽

Inflammatory Parameters

Background: Endothelial Progenitor Cells (EPCs) are important players in neovascularization, mobilized through signalling by Angiogenic Growth Factors (AGFs) such as Vascular Endothelial Growth Factor (VEGF) and fibroblast growth factor (FGF). In vitro, inflammatory parameters impair the function and influence of EPCs on AGFs. However, this connection is not clear in vivo. To understand the mechanisms of augmented arteriogenesis and angiogenesis in acute ischemic stroke (AIS) patients, we investigated whether circulating stem cells (CD133+), early endothelial progenitor cells (CD133+/VEGFR2+), and endothelial cells (ECs; CD34¯/CD133¯/VEGFR2+) were increasingly mobilized during AIS, and whether there were correlations between EPC levels, growth factor levels and inflammatory parameters. Methods: Data on demographics, classical vascular risk factors, neurological deficit information (assessed using the National Institutes of Health Stroke Scale), and treatment were collected from 43 consecutive AIS patients (group I). Risk factor control patients (group II) included 22 nonstroke subjects matched by age, gender, and traditional vascular risk factors. EPCs were measured by flow cytometry and the populations of circulating stem cells (CD133+), early EPCs (CD133+/VEGFR2+), and ECs (CD34¯/CD133¯/VEGFR2+) were analysed. Correlations between EPC levels and VEGF and FGF vascular growth factor levels as well as the influence of inflammatory parameters on EPCs and AGFs were assessed. Results: Patient ages ranged from 54 to 92 years (mean age 75.2 ± 11.3 years). The number of circulating CD34¯/CD133¯/VEGF-R2+ cells was significantly higher in AIS patients than in control patients (p < 0.05). VEGF plasma levels were also significantly higher in AIS patients compared to control patients on day 7 (p < 0.05). FGF plasma levels in patients with AIS were significantly higher than those in the control group on day 3 (p < 0.05). There were no correlations between increased VEGF and FGF levels and the number of CD133+, CD133+/VEGFR2+, or CD34¯/CD133¯/VEGFR2+ cells. Leukocyte levels, FGF plasma levels, and the number of early EPCs were negatively correlated on day 3. High sensitivity C-reactive protein levels and the number of CD133+ and CD133+/VEGFR2+ cells were negatively correlated on day 7. In addition, there was a negative correlation between fibrinogen levels and FGF plasma levels as well as the number of early EPCs (CD133+/VEGFR2+). Conclusion: AIS patients exhibited increased numbers of early EPCs (CD133+/VEGFR2+) and AGF (VEGF and FGF) levels. A negative correlation between inflammatory parameters and AGFs and EPCs indicated the unfavourable influence of inflammatory factors on EPC differentiation and survival. Moreover, these correlations represented an important mechanism linking inflammation to vascular disease.

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