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Yadav A, Kumar A, Siddiqui MH. Detection of circulating tumour cells in colorectal cancer: Emerging techniques and clinical implications. World J Clin Oncol 2021; 12:1169-1181. [PMID: 35070736 PMCID: PMC8716996 DOI: 10.5306/wjco.v12.i12.1169] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Revised: 07/15/2021] [Accepted: 11/15/2021] [Indexed: 02/06/2023] Open
Abstract
Despite several advances in oncological management of colorectal cancer, morbidity and mortality are still high and devastating. The diagnostic evaluation by endoscopy is cumbersome, which is uncomfortable to many. Because of the intra- and inter-tumour heterogeneity and changing tumour dynamics, which is continuous in nature, the diagnostic biopsy and assessment of the pathological sample are difficult and also not adequate. Late manifestation of the disease and delayed diagnosis may lead to relapse or metastases. One of the keys to improving the outcome is early detection of cancer, ease of technology to detect with uniformity, and its therapeutic implications, which are yet to come. "Liquid biopsy" is currently the most recent area of interest in oncology, which may provide important tools regarding the characterization of the primary tumour and its metastasis as cancer cells shed into the bloodstream even at the early stages of the disease. By using this approach, clinicians may be able to find out information about the tumour at a given time. Any of the following three types of sampling of biological material can be used in the "liquid biopsy". These are circulating tumour cells (CTCs), circulating tumour DNA, and exosomes. The most commonly studied amongst the three is CTCs. CTCs with their different applications and prognostic value has been found useful in colorectal cancer detection and therapeutics. In this review, we will discuss various markers for CTCs, the core tools/techniques for detection, and also important findings of clinical studies in colorectal cancer and its clinical implications.
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Affiliation(s)
- Alka Yadav
- Department of Surgical Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow 226014, Uttar Pradesh, India
| | - Ashok Kumar
- Department of Surgical Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow 226014, Uttar Pradesh, India
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Kitz J, Goodale D, Postenka C, Lowes LE, Allan AL. EMT-independent detection of circulating tumor cells in human blood samples and pre-clinical mouse models of metastasis. Clin Exp Metastasis 2021; 38:97-108. [PMID: 33415568 PMCID: PMC7882592 DOI: 10.1007/s10585-020-10070-y] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2020] [Accepted: 12/25/2020] [Indexed: 01/31/2023]
Abstract
Circulating tumor cells (CTCs) present an opportunity to detect/monitor metastasis throughout disease progression. The CellSearch® is currently the only FDA-approved technology for CTC detection in patients. The main limitation of this system is its reliance on epithelial markers for CTC isolation/enumeration, which reduces its ability to detect more aggressive mesenchymal CTCs that are generated during metastasis via epithelial-to-mesenchymal transition (EMT). This Technical Note describes and validates two EMT-independent CTC analysis protocols; one for human samples using Parsortix® and one for mouse samples using VyCap. Parsortix® identifies significantly more mesenchymal human CTCs compared to the clinical CellSearch® test, and VyCap identifies significantly more CTCs compared to our mouse CellSearch® protocol regardless of EMT status. Recovery and downstream molecular characterization of CTCs is highly feasible using both Parsortix® and VyCap. The described CTC protocols can be used by investigators to study CTC generation, EMT and metastasis in both pre-clinical models and clinical samples.
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Affiliation(s)
- Jenna Kitz
- London Regional Cancer Program, London Health Sciences Centre, London, Canada
- Department of Anatomy & Cell Biology, Western University, London, Canada
| | - David Goodale
- London Regional Cancer Program, London Health Sciences Centre, London, Canada
| | - Carl Postenka
- London Regional Cancer Program, London Health Sciences Centre, London, Canada
| | - Lori E Lowes
- Flow Cytometry, London Health Sciences Centre, London, Canada
| | - Alison L Allan
- London Regional Cancer Program, London Health Sciences Centre, London, Canada.
- Department of Anatomy & Cell Biology, Western University, London, Canada.
- Department of Oncology, Western University, London, Canada.
- Lawson Health Research Institute, London, ON, Canada.
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Karimi N, Oloomi M, Orafa Z. Circulating Tumor Cells Detection in Patients with Early Breast Cancer Using MACS Immunomagnetic Flow Cytometry. Avicenna J Med Biotechnol 2020; 12:148-156. [PMID: 32695277 PMCID: PMC7368115] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
BACKGROUND Circulating Tumor Cells (CTCs) detection in peripheral blood of epithelial cancer patients is an indicator of the presence of primary tumors and metastasis. The CTC phenotype detection uses epithelial markers in defining, detecting, and isolating CTCs. Circulating cell-separation technologies, with the epithelial origin, can be identified by epithelial biomarkers, with different techniques such as flow cytometry. The purpose of this study was to evaluate the expression of molecular Cytokeratins (CKs), CK7, CK8, CK18, CK19 (Pan-CK) and Epithelial Cell Adhesion Molecule (EpCAM) markers for CTC detection. METHODS The Magnetic Activated Cell Sorting (MACS) was used to identify CTCs in the blood of patients. Specific antibodies to EpCAM and Pan-CK were used and analyzed by flow cytometry. In this study, 35 blood samples of patients with breast cancer were assessed before any treatment and 35 healthy blood samples as the control were evaluated. RESULTS Expression of CK markers in the peripheral blood of breast cancer patients was statistically significant with p≤0.05, specifically at stages II-IV, but it was not significant in patients at stage I and healthy controls. Biomarkers expression in the blood of patients and healthy controls was assessed along with the pathologic characteristics of patients. CONCLUSION CTC assessment by flow cytometry in patients with breast cancer could not only be used for detection but also can be considered as a source of specific and subjective evaluation for monitoring the therapy. Besides, the sensitivity and specificity of CTC detection were shown that could be enhanced by specific CK markers.
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Affiliation(s)
| | - Mana Oloomi
- Corresponding author: Mana Oloomi, Ph.D., Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran, Tel: +98 21 66953311, Fax: +98 21 66465132, E-mail:
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Ebeed SA, Abd El-Moneim NA, Saad A, Zaher ERE, Yassin OG, Khamis SA. Diagnostic and prognostic value of circulating tumor cells in female breast cancer patients. ALEXANDRIA JOURNAL OF MEDICINE 2019. [DOI: 10.1016/j.ajme.2012.02.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Affiliation(s)
- Samia A. Ebeed
- Medical Research Institute, Radiation Sciences , 165 Elhorreya Avenue, Alexandria, Egypt
| | - Nadia A. Abd El-Moneim
- Medical Research Institute, Radiation Sciences , 165 Elhorreya Avenue, Alexandria, Egypt
| | - Ahmed Saad
- Medical Research Institute, Radiation Sciences , 165 Elhorreya Avenue, Alexandria, Egypt
| | - Ebtsam RE. Zaher
- Medical Research Institute, Radiation Sciences , 165 Elhorreya Avenue, Alexandria, Egypt
| | - Omayma G. Yassin
- Medical Research Institute, Radiation Sciences , 165 Elhorreya Avenue, Alexandria, Egypt
| | - Shadwa A. Khamis
- Medical Research Institute, Radiation Sciences , 165 Elhorreya Avenue, Alexandria, Egypt
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The increased adhesion of tumor cells to endothelial cells after irradiation can be reduced by FAK-inhibition. Radiat Oncol 2019; 14:25. [PMID: 30717801 PMCID: PMC6360706 DOI: 10.1186/s13014-019-1230-3] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2018] [Accepted: 01/23/2019] [Indexed: 12/30/2022] Open
Abstract
Background Radiotherapy is administered in more than 60% of all solid tumors. Most patients are cured but a significant number develops local recurrences or distant metastases. The question arises if irradiation might influence the metastatic process. In the present study we examined whether the adhesion of glioblastoma or breast cancer cells to endothelial cells, an important step in metastasis, is affected by photon irradiation. Methods U-87 MG, U-373 MG and MDA-MB-231 cancer cells as well as primary human endothelial cells were irradiated with 0, 2, 4, or 8 Gy photons at a dose rate of 5 Gy/min. The adhesion of cancer cells to endothelial cells was tested either with the Vybrant based assay via fluorescent labelling or with an ibidi pump system able to mimic the physiological blood flow in vitro. In addition, the impact of FAK (focal adhesion kinase) inhibitor PF-573, 228 on the adhesion of non-irradiated and irradiated tumor cells was analyzed. Adhesion related and regulated proteins were analyzed by Western blotting. Results The cellular adhesion was increased after irradiation regardless of which cell type was irradiated. The FAK-inhibitor was able to reduce the adhesion of non-irradiated cells but also the irradiation-induced increase in adhesion of tumor cells to endothelium. Adhesion related proteins were enhanced after irradiation with 4 Gy or 8 Gy in both cells types. The increased adhesion after irradiation is accompanied by the phosphorylation of src (Y416), FAK (Y397) and increased expression of paxillin. Conclusion Irradiation with photons in therapeutic doses is able to enhance the interaction between tumor cells and endothelial cells and by that might influence important steps of the metastatic process.
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Kölbl AC, Jeschke U, Andergassen U. The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells. Int J Mol Sci 2016; 17:E1308. [PMID: 27529216 PMCID: PMC5000705 DOI: 10.3390/ijms17081308] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Revised: 08/02/2016] [Accepted: 08/04/2016] [Indexed: 12/12/2022] Open
Abstract
Epithelial to mesenchymal transition (EMT) is a process involved in embryonic development, but it also plays a role in remote metastasis formation in tumor diseases. During this process cells lose their epithelial features and adopt characteristics of mesenchymal cells. Thereby single tumor cells, which dissolve from the primary tumor, are enabled to invade the blood vessels and travel throughout the body as so called "circulating tumor cells" (CTCs). After leaving the blood stream the reverse process of EMT, the mesenchymal to epithelial transition (MET) helps the cells to seed in different tissues, thereby generating the bud of metastasis formation. As metastasis is the main reason for tumor-associated death, CTCs and the EMT process are in the focus of research in recent years. This review summarizes what was already found out about the molecular mechanisms driving EMT, the consequences of EMT for tumor cell detection, and suitable markers for the detection of CTCs which underwent EMT. The research work done in this field could open new roads towards combating cancer.
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Affiliation(s)
- Alexandra C Kölbl
- Department of Gynecology and Obstetrics, LMU Munich, Maistrasse 11, 80337 Munich, Germany.
| | - Udo Jeschke
- Department of Gynecology and Obstetrics, LMU Munich, Maistrasse 11, 80337 Munich, Germany.
| | - Ulrich Andergassen
- Department of Gynecology and Obstetrics, LMU Munich, Maistrasse 11, 80337 Munich, Germany.
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Keyvani S, Karimi N, Orafa Z, Bouzari S, Oloomi M. Assessment of Cytokeratin-19 Gene Expression in Peripheral Blood of Breast Cancer Patients and Breast Cancer Cell Lines. BIOMARKERS IN CANCER 2016; 8:57-63. [PMID: 27147896 PMCID: PMC4852760 DOI: 10.4137/bic.s38229] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/15/2015] [Revised: 02/21/2016] [Accepted: 02/26/2016] [Indexed: 11/24/2022]
Abstract
Detection of cytokeratin-19 (CK19) expression as an epithelial-specific marker in circulating tumor cells (CTCs) of breast cancer patients can be important for diagnostic purposes. Comparison of CK19 expression in breast cancer cell lines can indicate that expression of this marker is different in various breast cancer cell lines based on their category. Thirty-five breast cancer patients were evaluated for detection of CK19 mRNA in their peripheral blood using CK19-specific primers and a nested reverse transcriptase polymerase chain reaction (RT-PCR) technique. CK19 expression levels were detected in MCF7, T47D, SK-BR-3, and MDA-MB-231 cell lines by semiquantitative RT-PCR and Western blot analyses. Statistical analysis of our data indicates that there is no significant difference between CK19 expression and histopathological parameters and some molecular markers, including Ki-67, HER-2, and P53, but there are statistically significant correlations between estrogen receptor (P = 0.040) and progesterone receptor (P = 0.046) with CK19 expression. CK19 expression was detected in MCF7, T47D, and SK-BR-3 cell lines but not in MDA-MB-231 cell line. More studies are needed to determine the relationship between this marker and other markers in the diagnosis and treatment of breast cancer. On the other hand, the study of different markers using breast cancer cell lines as experimental models of breast cancer could have an impact on improving the health outcomes of patients with breast cancer.
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Affiliation(s)
- Saeideh Keyvani
- Molecular Biology Department, Pasteur Institute of Iran, Tehran, Iran
| | - Nasrin Karimi
- Molecular Biology Department, Pasteur Institute of Iran, Tehran, Iran
| | - Zahra Orafa
- Molecular Biology Department, Pasteur Institute of Iran, Tehran, Iran
| | - Saeid Bouzari
- Molecular Biology Department, Pasteur Institute of Iran, Tehran, Iran
| | - Mana Oloomi
- Molecular Biology Department, Pasteur Institute of Iran, Tehran, Iran
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Vilalta M, Rafat M, Giaccia AJ, Graves EE. Recruitment of circulating breast cancer cells is stimulated by radiotherapy. Cell Rep 2014; 8:402-9. [PMID: 25017065 DOI: 10.1016/j.celrep.2014.06.011] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2013] [Revised: 05/07/2014] [Accepted: 06/10/2014] [Indexed: 12/31/2022] Open
Abstract
Radiotherapy (RT) is a localized therapy that is highly effective in killing primary tumor cells located within the field of the radiation beam. We present evidence that irradiation of breast tumors can attract migrating breast cancer cells. Granulocyte-macrophage colony stimulating factor (GM-CSF) produced by tumor cells in response to radiation stimulates the recruitment of migrating tumor cells to irradiated tumors, suggesting a mechanism of tumor recurrence after radiation facilitated by transit of unirradiated, viable circulating tumor cells to irradiated tumors. Data supporting this hypothesis are presented through in vitro invasion assays and in vivo orthotopic models of breast cancer. Our work provides a mechanism for tumor recurrence in which RT attracts cells outside the radiation field to migrate to the site of treatment.
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Affiliation(s)
- Marta Vilalta
- Division of Radiation and Cancer Biology, Department of Radiation Oncology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA 94305, USA
| | - Marjan Rafat
- Division of Radiation and Cancer Biology, Department of Radiation Oncology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA 94305, USA
| | - Amato J Giaccia
- Division of Radiation and Cancer Biology, Department of Radiation Oncology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA 94305, USA
| | - Edward E Graves
- Division of Radiation and Cancer Biology, Department of Radiation Oncology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA 94305, USA; Division of Radiation Physics, Department of Radiation Oncology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA 94305, USA.
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Riahi R, Yang YL, Kim H, Jiang L, Wong PK, Zohar Y. A microfluidic model for organ-specific extravasation of circulating tumor cells. BIOMICROFLUIDICS 2014; 8:024103. [PMID: 24803959 PMCID: PMC3987064 DOI: 10.1063/1.4868301] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/23/2013] [Accepted: 02/28/2014] [Indexed: 05/08/2023]
Abstract
Circulating tumor cells (CTCs) are the principal vehicle for the spread of non-hematologic cancer disease from a primary tumor, involving extravasation of CTCs across blood vessel walls, to form secondary tumors in remote organs. Herein, a polydimethylsiloxane-based microfluidic system is developed and characterized for in vitro systematic studies of organ-specific extravasation of CTCs. The system recapitulates the two major aspects of the in vivo extravasation microenvironment: local signaling chemokine gradients in a vessel with an endothelial monolayer. The parameters controlling the locally stable chemokine gradients, flow rate, and initial chemokine concentration are investigated experimentally and numerically. The microchannel surface treatment effect on the confluency and adhesion of the endothelial monolayer under applied shear flow has also been characterized experimentally. Further, the conditions for driving a suspension of CTCs through the microfluidic system are discussed while simultaneously maintaining both the local chemokine gradients and the confluent endothelial monolayer. Finally, the microfluidic system is utilized to demonstrate extravasation of MDA-MB-231 cancer cells in the presence of CXCL12 chemokine gradients. Consistent with the hypothesis of organ-specific extravasation, control experiments are presented to substantiate the observation that the MDA-MB-231 cell migration is attributed to chemotaxis rather than a random process.
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Affiliation(s)
- R Riahi
- Department of Aerospace and Mechanical Engineering, The University of Arizona, Tucson, Arizona 85719, USA
| | - Y L Yang
- Department of Aerospace and Mechanical Engineering, The University of Arizona, Tucson, Arizona 85719, USA
| | - H Kim
- Department of Molecular and Cellular Biology, The University of Arizona, Tucson, Arizona 85719, USA
| | - L Jiang
- Department of Aerospace and Mechanical Engineering, The University of Arizona, Tucson, Arizona 85719, USA ; College of Optical Science, The University of Arizona, Tucson, Arizona 85719, USA
| | - P K Wong
- Department of Aerospace and Mechanical Engineering, The University of Arizona, Tucson, Arizona 85719, USA ; Department of Biomedical Engineering, The University of Arizona, Tucson, Arizona 85719, USA ; BIO5 Institute, The University of Arizona, Tucson, Arizona 85719, USA
| | - Y Zohar
- Department of Aerospace and Mechanical Engineering, The University of Arizona, Tucson, Arizona 85719, USA ; Department of Biomedical Engineering, The University of Arizona, Tucson, Arizona 85719, USA ; BIO5 Institute, The University of Arizona, Tucson, Arizona 85719, USA ; Arizona Cancer Center, The University of Arizona, Tucson, Arizona 85719, USA
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Perez EA, Cortés J, Gonzalez-Angulo AM, Bartlett JM. HER2 testing: Current status and future directions. Cancer Treat Rev 2014; 40:276-84. [DOI: 10.1016/j.ctrv.2013.09.001] [Citation(s) in RCA: 102] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2013] [Revised: 08/19/2013] [Accepted: 09/02/2013] [Indexed: 11/30/2022]
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Andergassen U, Kölbl AC, Hutter S, Friese K, Jeschke U. Detection of Circulating Tumour Cells from Blood of Breast Cancer Patients via RT-qPCR. Cancers (Basel) 2013; 5:1212-20. [PMID: 24202442 PMCID: PMC3875936 DOI: 10.3390/cancers5041212] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2013] [Revised: 08/20/2013] [Accepted: 09/11/2013] [Indexed: 12/25/2022] Open
Abstract
Breast cancer is still the most frequent cause of cancer-related death in women worldwide. Often death is not caused only by the primary tumour itself, but also by metastatic lesions. Today it is largely accepted, that these remote metastases arise out of cells, which detach from the primary tumour, enter circulation, settle down at secondary sites in the body and are called Circulating Tumour Cells (CTCs). The occurrence of such minimal residual diseases in the blood of breast cancer patients is mostly linked to a worse prognosis for therapy outcome and overall survival. Due to their very low frequency, the detection of CTCs is, still a technical challenge. RT-qPCR as a highly sensitive method could be an approach for CTC-detection from peripheral blood of breast cancer patients. This assumption is based on the fact that CTCs are of epithelial origin and therefore express a different gene panel than surrounding blood cells. For the technical approach it is necessary to identify appropriate marker genes and to correlate their gene expression levels to the number of tumour cells within a sample in an in vitro approach. After that, samples from adjuvant and metastatic patients can be analysed. This approach may lead to new concepts in diagnosis and treatment.
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Affiliation(s)
- Ulrich Andergassen
- Department of Obstetrics and Gynaecology, Ludwig Maximilians University of Munich, Munich, Maistrasse 11, D-80337 Munich, Germany.
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Osisami M, Keller ET. Mechanisms of Metastatic Tumor Dormancy. J Clin Med 2013; 2:136-50. [PMID: 26237067 PMCID: PMC4470233 DOI: 10.3390/jcm2030136] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2013] [Revised: 08/20/2013] [Accepted: 09/10/2013] [Indexed: 12/29/2022] Open
Abstract
Tumor metastasis can occur years after an apparent cure due to a phenomenon known as metastatic tumor dormancy; in which tumor masses or individual tumor cells are growth restricted for extended periods of time. This period of dormancy is induced and maintained by several mechanisms, including: (1) Tumor microenvironment factors such as cytokine expression, immunosurveillance and angiogenesis; (2) Metastasis suppressor gene activity; and (3) Cancer therapeutics. Disseminated tumor cells (DTC) are the key cells that result in dormant tumors. However, many challenges exist towards isolating DTCs for mechanistic studies. The main DTC that may represent the dormant cell is the cancer stem cells (CSC) as they have a slow proliferation rate. In addition to limited knowledge regarding induction of tumor dormancy, there are large gaps in knowledge regarding how tumors escape from dormancy. Emerging research into cancer stem cells, immunotherapy, and metastasis suppressor genes, may lead to new approaches for targeted anti-metastatic therapy to prevent dormancy escape. Overall, an enhanced understanding of tumor dormancy is critical for better targeting and treatment of patients to prevent cancer recurrence.
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Affiliation(s)
- Mary Osisami
- Department of Urology, University of Michigan Medical School, 5111 CCGC1500 E. Medical Center, Ann Arbor, MI 48109-0940, USA.
| | - Evan T Keller
- Department of Urology, University of Michigan Medical School, 5111 CCGC1500 E. Medical Center, Ann Arbor, MI 48109-0940, USA.
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Dannull J, Haley NR, Archer G, Nair S, Boczkowski D, Harper M, De Rosa N, Pickett N, Mosca PJ, Burchette J, Selim MA, Mitchell DA, Sampson J, Tyler DS, Pruitt SK. Melanoma immunotherapy using mature DCs expressing the constitutive proteasome. J Clin Invest 2013; 123:3135-45. [PMID: 23934126 DOI: 10.1172/jci67544] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2012] [Accepted: 05/01/2013] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Many cancers, including melanoma, exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast, mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules, we hypothesized that mature melanoma antigen-loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo. METHODS Subjects with metastatic melanoma were vaccinated with mature DCs transfected with RNAs encoding melanoma antigens MART1, MAGE-3, gp100, and tyrosinase. These DCs were derived from monocytes that were untransfected (Arm A; n = 4), transfected with control siRNA (Arm B; n = 3), or transfected with siRNAs targeting the 3 inducible iP subunits (Arm C; n = 5). RESULTS Vaccination stimulated antigen-specific T cell responses in all subjects, which peaked after 3-4 vaccinations, but remained elevated in Arm C subjects. Also in Arm C, circulating melanoma cell levels (as detected by quantitative PCR) fell, and T cell lytic activity against autologous melanoma was induced. In HLA-A2⁺ subjects, CD8⁺ T cells that bound tetramers loaded with cP-derived melanoma antigenic peptides were found in the peripheral blood only in Arm C subjects. Of 2 subjects with active disease (both in Arm C), one had a partial clinical response, while the other, who exhibited diffuse dermal and soft tissue metastases, had a complete response. CONCLUSION These results suggest that the efficacy of melanoma DC-based immunotherapy is enhanced when tumor antigen-loaded DCs used for vaccination express cPs. TRIAL REGISTRATION Clinicaltrials.gov NCT00672542. FUNDING Duke Clinical Research Institute/Duke Translational Medicine Institute, Duke Melanoma Consortium, and Duke University Department of Surgery.
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Affiliation(s)
- Jens Dannull
- Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
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Saucedo-Zeni N, Mewes S, Niestroj R, Gasiorowski L, Murawa D, Nowaczyk P, Tomasi T, Weber E, Dworacki G, Morgenthaler NG, Jansen H, Propping C, Sterzynska K, Dyszkiewicz W, Zabel M, Kiechle M, Reuning U, Schmitt M, Lücke K. A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire. Int J Oncol 2012; 41:1241-50. [PMID: 22825490 PMCID: PMC3583719 DOI: 10.3892/ijo.2012.1557] [Citation(s) in RCA: 143] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2012] [Accepted: 04/03/2012] [Indexed: 12/25/2022] Open
Abstract
The isolation of circulating tumor cells (CTCs) from the blood of patients afflicted with solid malignant tumors becomes increasingly important as it may serve as a 'liquid biopsy' with the potential of monitoring the course of the cancer disease and its response to cancer therapy, with subsequent molecular characterization. For this purpose, we functionalized a structured medical Seldinger guidewire (FSMW), normally used to obtain safe access to blood vessels and other organ cavities, with a chimeric monoclonal antibody directed to the cell surface expressed epithelial cell surface adhesion molecule (EpCAM). This medical device was optimized in vitro and its biocompatibility was tested according to the regulations for medical devices and found to be safe with no noteworthy side effects. Suitability, specificity and sensitivity of the FSMW to catch and enrich CTCs in vivo from circulating peripheral blood were tested in 24 breast cancer or non-small cell lung cancer (NSCLC) patients and in 29 healthy volunteers. For this, the FSMW was inserted through a standard venous cannula into the cubital veins of healthy volunteers or cancer patients for the duration of 30 min. After removal, CTCs were identified by immuno-cytochemical staining of EpCAM and/or cytokeratins and staining of their nuclei and counted. The FSMW successfully enriched EpCAM-positive CTCs from 22 of the 24 patients, with a median of 5.5 (0-50) CTCs in breast cancer (n=12) and 16 (2-515) CTCs in NSCLC (n=12). CTCs could be isolated across all tumor stages, including early stage cancer, in which distant metastases were not yet diagnosed, while no CTCs could be detected in healthy volunteers. In this observatory study, no adverse effects were noted. Evidently, the FSMW has the potential to become an important device to enrich CTCs in vivo for monitoring the course of the cancer disease and the efficacy of anticancer treatment.
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SERRANO MARÍAJOSÉ, ROVIRA PEDROSÁNCHEZ, MARTÍNEZ-ZUBIAURRE I, RODRIGUEZ MIGUELDELGADO, FERNÁNDEZ MÓNICA, LORENTE JOSEA. Dynamics of circulating tumor cells in early breast cancer under neoadjuvant therapy. Exp Ther Med 2012; 4:43-48. [PMID: 23060920 PMCID: PMC3460281 DOI: 10.3892/etm.2012.540] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2011] [Accepted: 01/20/2012] [Indexed: 12/25/2022] Open
Abstract
At present, the majority of patients with breast cancer are diagnosed at early stages of disease development. However, a considerable number of such cases develop secondary malignancies after a relatively short period of time. The presence of circulating tumor cells (CTCs) has been proposed as a strong biomarker to predict disease recurrence in metastatic breast cancer. However, the prognostic significance is not clear in early breast cancer. We present results on CTC determination in peripheral blood in non-metastatic breast cancer patients in the context of neoadjuvant treatment. Twenty-six breast cancer patients, scheduled for neoadjuvant therapy, were enrolled in a prospective study, of which 24 were able to complete therapy. CTC assessment was performed by sorting out cytokeratin-positive cells from 10 ml of peripheral blood using immunomagnetic separation, followed by immunocytochemical characterization of cells. Seventeen blood samples out of 24 patients were CTC-positive when collected prior to neoadjuvant chemotherapy. No significant correlations were found between the presence of CTCs and lymph node status (p=0.1), histological type (p=0.802), stage (p=0.43) or overall survival (OS) (p=0.599). Thirteen CTC-positive samples were observed in blood samples collected after treatment. Univariate analyses revealed that the presence of CTCs was related to OS when the detection was positive both before and after treatment (p=0.023). CTCs can be a strong prognostic marker in early breast cancer. The persistence of CTCs before and after treatment can identify a subpopulation of patients with an increased risk of recurrence.
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Affiliation(s)
- MARÍA JOSÉ SERRANO
- Centro Pfizer, Universidad de Granada, Junta de Andalucía de Genómica e Investigación Oncológica (GENYO), Granada
| | | | | | | | | | - JOSE A. LORENTE
- Centro Pfizer, Universidad de Granada, Junta de Andalucía de Genómica e Investigación Oncológica (GENYO), Granada
- Laboratory of Genetic Identification – UGR, Department of Legal Medicine, University of Granada, Granada,
Spain
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Hwang SB, Bae JW, Lee HY, Kim HY. Circulating Tumor Cells Detected by RT-PCR for CK-20 before Surgery Indicate Worse Prognostic Impact in Triple-Negative and HER2 Subtype Breast Cancer. J Breast Cancer 2012; 15:34-42. [PMID: 22493626 PMCID: PMC3318172 DOI: 10.4048/jbc.2012.15.1.34] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2011] [Accepted: 01/15/2012] [Indexed: 01/07/2023] Open
Abstract
Purpose Circulating tumor cells (CTC) clearly correlate with unfavorable outcomes for patients with metastatic breast cancer, but the long-term prognostic implications of CTC for molecular subtypes of operable breast cancer are not yet known. We explored the relationships between previously established prognostic factors and CTC in operable breast cancer, and the significance of CTC by breast cancer molecular subtype. Methods We retrospectively evaluated 166 patients with operable breast cancer (stage I-IIIA) diagnosed from April 1997 to May 2003. CTC were detected using cytokeratin-20 (CK-20) mRNA expression in peripheral blood samples that were collected just prior to surgery under general anesthesia. Clinicopathological characteristics of the cancer were analyzed according to CTC status. Metastasis-free survival (MFS) and overall survival (OS) were analyzed according to CTC status and breast cancer molecular subtype. Results CK-20 mRNA-positive CTC was detected in 37 of 166 patients (22.3%) and was not correlated with any previous clinical factors in univariate analysis (p>0.05). After a median follow-up of 100 months, the patients with CK-20 mRNA-positive CTC had less favorable outcomes in terms of MFS and OS than those without detectable CTC (log-rank p<0.05). Among molecular subtypes of operable breast cancer, the patients with CK-20 mRNA-positive CTC had shorter MFS and OS in triple negative and human epidermal growth factor 2 (HER2) breast cancer subtype (log-rank, p<0.05). Conclusion CK-20 mRNA-positive CTC may lend insight into tumor progression as a prognostic indicator especially in the triple negative and HER2 subtypes of operable breast cancer.
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Affiliation(s)
- Seong Bae Hwang
- Division of Breast-Endocrine Surgery, Department of Surgery, Korea University Anam Hospital, Seoul, Korea
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