Called Circulating Tumor Cells Research Articles

Abstract 3005: Low-density lipoprotein receptor-related protein 1B (LRP1B) depletion promotes tumorigenicity by modulating fibroblasts in the tumor microenvironment in esophageal squamous cell carcinoma

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is widespread worldwide with a poor survival rate. Tumor cells shedding from tumor tissues traveling to other organs through the blood circulatory system are called circulating tumor cells (CTCs). The CTCs in the blood of ESCC patients were enriched and then subjected to CTC mutational profiling by target sequencing. Our results suggested that CTC mutations in low-density lipoprotein receptor-related protein 1B (LRP1B) may have a potential prognostic role for ESCC. LRP1B belongs to the low-density receptor (LDLR) family with diverse roles in cellular functions and development, but the function of LRP1B in ESCC remains unclear. Therefore, this study aims to do a functional characterization of LRP1B. Methods: The CRISPR cas9 system was used to functionally knock down the candidate gene in ESCC cell lines with the highest endogenous level of LRP1B. The in vitro cell proliferation rate and viability were measured by MTT assay, colony formation assay, and wound healing assay. The in vivo tumorigenicity assay was performed to evaluate the tumor suppressive effect by subcutaneous injection. To examine the clinical relevance of LRP1B, we also performed immunohistochemistry (IHC) in tumor tissue microarray (TMA). Results: The reduction or loss of LRP1B mRNA was demonstrated in 60% of a panel of 15 ESCC cell lines. The depletion of LRP1B did not influence cell proliferation, cell clonogenic ability, or cell migration in ECSS cell lines. However, LRP1B depletion significantly promoted the growth of tumors in mouse xenograft models. Therefore, we hypothesize that LRP1B may be involved in modulating the tumor microenvironment (TME) by impacting various stromal components leading to an immuno-suppressive TME. We utilized RT-qPCR to investigate markers of various stromal components and observed that fibroblast markers (acta2, pdgfrb, and vimentin) had significantly higher expression levels after LRP1B downregulation in ESCC cell-derived mouse xenografts. The higher level of fibroblast cells was then verified by IHC staining. Further molecular mechanistic studies are underway and are expected to provide novel therapeutic insights by targeting the LRP1B genes. The LRP1B ESCC TMA data analysis is underway and statistical analysis will be presented. Conclusions: LRP1B plays a tumor-suppressive role in ESCC by modulating the tumor-associated fibroblasts in the TME. Acknowledgements: TBRS grant T12-701/17R and HMRF grant 05160926 to MLL Citation Format: KaiYan Xu, Josephine Mun-yee KO, Valen Zhuo you Yu, Maria Li LUNG. Low-density lipoprotein receptor-related protein 1B (LRP1B) depletion promotes tumorigenicity by modulating fibroblasts in the tumor microenvironment in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3005.

Epithelial-to-Mesenchymal Plasticity in Circulating Tumor Cell Lines Sequentially Derived from a Patient with Colorectal Cancer.

Simple SummaryMetastasis is a complex dynamic multistep process; however, our knowledge is still limited. Very few circulating tumor cells (CTCs) are metastatic precursor cells and represent the intermediate stage of metastasis. Epithelial–mesenchymal plasticity (EMP) has crucial roles in tissue development and homeostasis, and also in metastasis formation. In this study, we explored the EMP phenotype of a unique series of CTC lines, obtained from a patient with colon cancer during the disease course and treatment, by detecting markers involved in the epithelial–mesenchymal and mesenchymal–epithelial (MET) transitions. This study shows that these colon CTC lines have acquired only few mesenchymal features to migrate and intravasate, whereas an increase of MET-related markers was observed, suggesting that metastasis-competent CTCs need to revert quickly to the epithelial phenotype to reinitiate a tumor at a distant site.Metastasis is a complicated and only partially understood multi-step process of cancer progression. A subset of cancer cells that can leave the primary tumor, intravasate, and circulate to reach distant organs are called circulating tumor cells (CTCs). Multiple lines of evidence suggest that in metastatic cancer cells, epithelial and mesenchymal markers are co-expressed to facilitate the cells’ ability to go back and forth between cellular states. This feature is called epithelial-to-mesenchymal plasticity (EMP). CTCs represent a unique source to understand the EMP features in metastatic cascade biology. Our group previously established and characterized nine serial CTC lines from a patient with metastatic colon cancer. Here, we assessed the expression of markers involved in epithelial–mesenchymal (EMT) and mesenchymal–epithelial (MET) transition in these unique CTC lines, to define their EMP profile. We found that the oncogenes MYC and ezrin were expressed by all CTC lines, but not SIX1, one of their common regulators (also an EMT inducer). Moreover, the MET activator GRHL2 and its putative targets were strongly expressed in all CTC lines, revealing their plasticity in favor of an increased MET state that promotes metastasis formation.

CIRCULATING TUMOR CELLS: WHERE WE LEFT OFF?

Cancer metastasis and recurrence are the leading causes of cancer-related death. Tumor cells which leave the primary or secondary tumors and shed into the bloodstream are called circulating tumor cells (CTC). These cells are the key drivers of cancer dissemination to surrounding tissues and to distant organs. The use of CTC in clinical practice necessitates the deep insight into their biology, as well as into their role in cancer evasion of immune surveillance, tumor resistance to chemo- radio- and immunotherapies and metastatic dormancy. Aim. The purpose of the work was to review the current knowledge on the CTC biology, as well as the prospects for their use for the diagnosis and targeted treatment of metastatic disease. Methods. The work proposed the integrative literature review using MEDLINE, Biological Abstracts and EMBASE databases. Results. This review summarizes and discusses historical milestones and current data concerning СTС biology, the main stages of their life cycle, their role in metastatic cascade, clinical prospects for their use as markers for the diagnosis and prognostication of the disease course, as well as targets for cancer treatment. Conclusions. Significant progress in the area of CTC biology and their use in cancer theranostics convincingly proved the attractiveness of these cells as targets for cancer prognosis and therapy. The effective use of liquid biopsy with quantitative and phenotypic characteristics of CTCs is impeded by the imperfection of the methodology for taking biological material and by the lack of reliable markers for assessing the metastatic potential of CTCs of various origins. The variety of mechanisms of tumor cells migration and invasion requires the development of complex therapeutic approaches for anti-metastatic therapy targeting CTCs. Efforts to address these key issues could help developing new and effective cancer treatment strategies.

The combination of mammaglobin A and TWIST-1 increases detection of circulating tumor cells in breast cancer

ABSTRACT Objectives: Breast cancer cells that are released into the bloodstream are called circulating tumor cells (CTCs). CTCs can express different genes, like TWIST-1 and mammaglobin A (MGA). The aims of this study were to analyze the expression of TWIST-1 and MGA in the blood of breast cancer patients to detect CTCs and to assess the association between the presence of CTCs and prognostic parameters of breast cancer. Methods: Prospective study. Total ribonucleic acid (RNA) from blood mononucleated cells was obtained from breast cancer patients (n = 36; age: 51.5 ± 12.5 years) and healthy donors (n = 14; age: 49.4 ± 9.4 years). Real-time polymerase chain reaction (RT-PCR) was performed to analyze the expression of TWIST-1 and MGA. Results: Patient carcinomas - ductal (86.7%), other types (13.3%). MGA gene expression was not detected in the donors’ samples, while it was detected in 14% of the patient samples. Overexpression of TWIST-1 gene was observed in 17% of the patient samples. The combined analysis of both markers allowed the detection of CTCs in 27.8% of the samples, resulting in a significant (p 0.05) were found between expression of the analyzed genes and the breast cancer prognostic factors. Conclusion: Combined analysis of TWIST-1 and MGA increased the sensitivity of CTCs detection compared to the single analysis of each gene. The detection of CTCs was not associated with known prognostic factors, suggesting that it is able to provide clinical information in addition to routine breast cancer clinicopathological parameters.

Steps in metastasis: an updated review

Metastasis is the most complex and deadly event. Tumor-stromal interface is a place where invasion of tumor cells in the form of single-cell or collective migration occurs, with the latter being less common but more efficient. Initiation of metastasis relies on the tumor cell cross-talking with stromal cells and taking an epithelial-mesenchymal transition (EMT) in single cells, and a hybrid EMT in collective migratory cells. Stromal cross-talking along with an abnormal leaky vasculature facilitate intravasation of tumor cells, here the cells are called circulating tumor cells (CTCs). Tumor cells isolated from the primary tumor exploit several mechanisms to maintain their survival including rewiring metabolic demands to use sources available within the new environments, avoiding anoikis cell death when cells are detached from extracellular matrix (ECM), adopting flow mechanic by acquiring platelet shielding and immunosuppression by negating the activity of suppressor immune cells, such as natural killer (NK) cells. CTCs will adhere to the interstituim of the secondary organ/s, within which the newly arrived disseminative tumor cells (DTCs) undergo either dormancy or proliferation. Metastatic outgrowth is under the influence of several factors, such as the activity of macrophages, impaired autophagy and secondary site inflammatory events. Metastasis can be targeted by multiple ways, such as repressing the promoters of pre-metastatic niche (PMN) formation, suppressing environmental contributors, such as hypoxia, oxidative and metabolic stressors, and targeting signaling and cell types that take major contribution to the whole process. These strategies can be used in adjuvant with other therapeutics, such as immunotherapy.

Abstract 5383: Design, optimization, and validation of multiplex immunofluorescence assay for detecting biomarker expression on circulating tumor cells in breast cancer

Abstract Majority of breast cancer-related deaths occur due to metastasis. In the metastatic process, tumor cells travel from the primary site to distant organs. Tumor cells traveling in the blood stream are called circulating tumor cells (CTCs), which can serve as liquid biopsies as representative of the tumor bulk in patients. A major advantage of liquid biopsies for the analysis of tumor cells is its less-invasive collection, which allows more frequent and real-time monitoring of treatment response over conventional tissue biopsies. CTCs are being evaluated for early diagnosis and screening, treatment selection, and monitoring of treatment response. In our research, we are investigating CTCs as a tool to evaluate presence of prognostic and predictive markers in breast cancer. The purpose of this study was to design, optimize, and validate a multiplex immunofluorescence (IF) assay to identify a panel of unique biomarkers for CTCs. The panel of biomarkers to be tested on CTCs included estrogen receptor (ER), B-cell lymphoma (Bcl2), retinoblastoma protein (RB), and phosphorylated-RB (pRB). CTCs were defined as pan-cytokeratin-positive, CD45-negative, and nuclear stain-positive cells. First, antibodies for individual protein markers were tested on breast cancer cells spiked into control mouse blood. Positive and negative controls were used to test the selectivity and specificity of the antibodies against the key biomarkers. Multiple parameters including different fixative reagents, direct and indirect antibody conjugation, and varying antibody incubation time were examined to increase the sensitivity of the IF assay for individual markers as well as multiplexed assay. Our results indicated that various antigen staining was similar with both fixation reagents, 4% paraformaldehyde and 2% formaldehyde. In comparing the direct (single step) and indirect (two-step) antibody additions, a more intense signal for the CTCs and leukocytes were observed for the latter. Additionally, overnight incubation with primary antibodies allowed for better binding between the antibody and the antigen on cells as indicated by strong fluorescence signal with less exposure time. On the other hand, incubation for only one hour with primary antibodies had a weaker signal with high non-specific background from other blood components. The validated assay has been successfully applied to our ongoing CTC investigation using the blood from breast cancer patient-derived xenograft models. Citation Format: Tanya Kumar, Hariprasad Thangavel, Raksha Bhat, Noor M. Abdulkareem, Lacey E. Dobrolecki, Michael T. Lewis, Meghana V. Trivedi. Design, optimization, and validation of multiplex immunofluorescence assay for detecting biomarker expression on circulating tumor cells in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5383.

The biology and clinical potential of circulating tumor cells.

BackgroundTumor cells can shed from the tumor, enter the circulation and travel to distant organs, where they can seed metastases. These cells are called circulating tumor cells (CTCs). The ability of CTCs to populate distant tissues and organs has led us to believe they are the primary cause of cancer metastasis. The biological properties and interaction of CTCs with other cell types during intravasation, circulation in the bloodstream, extravasation and colonization are multifaceted and include changes of CTC phenotypes that are regulated by many signaling molecules, including cytokines and chemokines. Considering a sample is readily accessible by a simple blood draw, monitoring CTC levels in the blood has exceptional implications in oncology field. A method called the liquid biopsy allows the extraction of not only CTC, but also CTC products, such as cell free DNA (cfDNA), cell free RNA (cfRNA), microRNA (miRNA) and exosomes.ConclusionsThe clinical utility of CTCs and their products is increasing with advances in liquid biopsy technology. Clinical applications of liquid biopsy to detect CTCs and their products are numerous and could be used for screening of the presence of the cancer in the general population, as well as for prognostic and predictive biomarkers in cancer patients. With the development of better CTC isolation technologies and clinical testing in large prospective trials, increasing clinical utility of CTCs can be expected. The understanding of their biology and interactions with other cell types, particularly with those of the immune system and the rise of immunotherapy also hold great promise for novel therapeutic possibilities.

Detection of Circulating Tumor Cells Using Microfluidics.

Metastasis is the main cause of death in cancer patients worldwide. During metastasis, cancer cells detach from the primary tumor and invade distant tissue. The cells that undergo this process are called circulating tumor cells (CTCs). Studies show that the number of CTCs in the peripheral blood can predict progression-free survival and overall survival and can be informative concerning the efficacy of treatment. Research is now concentrated on developing devices that can detect CTCs in the blood of cancer patients with improved sensitivity and specificity that can lead to improved clinical evaluation. This review focuses on devices that detect and capture CTCs using different cell properties (surface markers, size, deformability, electrical properties, etc.). We also discuss the process of tumor cell dissemination, the biology of CTCs, epithelial-mesenchymal transition (EMT), and several challenges and clinical applications of CTC detection.

Fluid shear stress induces cancer stem cell-like phenotype in MCF7 breast cancer cell line without inducing epithelial to mesenchymal transition.

Metastasis is the leading cause of cancer deaths due to the spread of cancer cells through the blood vessels and the subsequent formation of secondary tumors. Metastasizing cancer cells in the human vasculature are called circulating tumor cells (CTCs) and are characterized to express the epithelial cell adhesion molecule (EpCAM). They are further known to survive physiological fluid shear stress (FSS) conditions. However, the effect of FSS on CTC molecular phenotype, such as the epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) expression, has not been extensively studied. Here, CTCs in FSS are evaluated in an invitro model system. MCF7 and MDA-MB-231 breast cancer cell lines were grown in adherent and suspension culture media. The cell lines were tested for EMT and CSC genetic and protein markers using qRT-PCR and flow cytometry, respectively. Suspension cells showed a significantly increased EMT signature compared to adherent cells (p<0.05), suggesting that they model cells detaching from primary tumors invivo. Upon application of FSS, MCF7 and MDA-MB-231 cells did not show a significant change in EMT expression (p>0.05), but there was a statistically significant increase of the CSC population in MCF7 suspension cultures (p<0.05). These results with MCF7 suggest that CTCs can be modeled invitro as non-adherent cancer cells in FSS and that they show an increased CSC-like signature during circulation, providing new insights to the importance of CSC-targeting strategies when treating metastatic patients.

Circulating and disseminated tumor cells: harbingers or initiators of metastasis?

Tumor cells leave the primary tumor and enter the circulation. Once there, they are called circulating tumor cells (CTCs). A fraction of CTCs are capable of entering distant sites and persisting as disseminated tumor cells (DTCs). An even smaller fraction of DTCs are capable of progressing toward metastases. It is known that the DTC microenvironment plays an important role in sustaining their survival, regulating their growth, and conferring resistance to therapy. But we still have much to learn about the nature of these rare cell populations to predict which will progress and what exactly should cause concern for future relapse. Although recent technological advances in our ability to detect and molecularly and functionally characterize CTCs and DTCs promise to unravel this ambiguity, the timing of dissemination and the precise source of CTCs and DTCs profiled will impact the conclusions that can be made from these endeavors. In this review, we discuss the biology of CTCs and DTCs; the technologies to detect, isolate, and profile these cells; and the exceptions we must apply to our understanding of what role these cells play in the metastatic process. We conclude that a greater effort to understand the unique biology of these cells in context will positively impact our ability to use these cells to predict outcome, monitor treatment efficacy, and reveal therapeutically relevant targets to deplete these populations and ultimately prevent metastasis.

Interaction between circulating cancer cells and platelets: clinical implication.

Metastasis is the main cause of cancer-associated mortality. During this complicated process, some cancer cells, also called circulating tumor cells (CTCs), detach from primary sites, enter bloodstream and extravasate at metastatic site. Thrombocytosis is frequently observed in patients with metastatic cancers suggesting the important role of platelets in metastasis. Therefore this review focuses on how platelets facilitate the generation of CTCs, protect them from various host attacks, such as immune assaults, apoptosis and shear stress, and regulate CTCs intravasation/extravasation. Platelet-derived cytokines and receptors are involved in this cascade. Identification the mechanisms underlie platelet-CTCs interactions could lead to the development of new platelet-targeted therapeutic strategy to reduce metastasis.

Sweeping lymph node micrometastases off their feet: an engineered model to evaluate natural killer cell mediated therapeutic intervention of circulating tumor cells that disseminate to the lymph nodes

Approximately 90% of cancer related deaths are due to metastasis. Cells from the primary tumor can metastasize through either the vascular or lymphatic circulation. Cancer cells in circulation are called circulating tumor cells (CTCs) and it has been shown that bone marrow is a niche for homing of blood borne CTCs from several epithelial tumors. Cancer cells found in bone marrow are termed disseminated tumor cells (DTCs). Likewise, CTCs in the lymphatic circulation are more often seeded in the sentinel lymph nodes (SLN) that drain the tumor. Micrometastases (<2 mm) occur after the arrest and implantation of DTCs in lymph nodes over time. This paper presents a cell culture platform termed microbubbles formed in polydimethylsiloxane (PDMS) from a microfabricated silicon wafer for mimicking lymph node micrometastases. We cultured lymph node seeking cancer cells in microbubbles to evaluate the efficacy of natural killer (NK) mediated therapy for targeting lymph node micrometastasis. The microbubble platform consists of an array of microcavities that provides a unique microenvironment for mimicking the deep cortical unit of the lymph nodes. We show that cancer cells cultured in microbubbles with therapeutic NK cells undergo apoptosis after 24 h in culture. Since lymph node metastases are prevalent across several types of cancer, this platform may be useful for developing improved cancer therapies for targeting lymph node micrometastases.

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.

Abstract 368: Detection of circulating tumor cells in peripheral blood of renal cell carcinoma patients is correlated with survival.

Abstract Purpose: The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood [so called circulating tumor cells (CTCs)] for renal cell carcinoma (RCC) patients after a median follow-up time of 81 months. Methods: 233 peripheral blood samples from 154 RCC patients were investigated for the presence of disseminated tumor cells (DTCs) by autoMACS technique and immunocytochemical (ICC) staining of cytokeratin. The frequency of CTCs was analyzed statistically for correlation with relevant clinical data. Results: Two kinds of tumor cells were detected: Those with expression of cytokeratin 8/18 (CK+), but also cells without a detectable CK+ expression, which we called large blue stained cells (Bl+) with a tumor-like morphology. We hypotesized, that the latter cells could be “sleeping” tumor cells. After following the CD45 autoMACS depletion protocol, we identified CTCs in 96 out of 233 peripheral blood samples (41%), which originated from 81 out of 154 (53%) RCC patients. An update to follow up the RCC patients was conducted in 2012. Besides a significant correlation between the detection of CTCs before and after primary tumor resection a significant association with prognosis was found (p &amp;lt; 0.002; Chi-squared test). RCC patients with CK+ CTCs in their peripheral blood had a 5 times risk of tumor-related death compared to patients without these cells. In a Kaplan-Meier-Analysis these results were confirmed (p &amp;lt; 0.001). Patients with large blue stained cells (Bl+) also had a shorter survival time. Conclusion: In summary, for RCC patients, the presence of CTCs was associated with a poor overall survival and significantly correlated with an increased risk of tumor-related death. It is important to evaluate CK+ tumor cells to determine the role of CTCs in tumor behavior and disease progression. Detection of CK+ CTCs in peripheral blood is a significant and independent prognostic factor for renal cell carcinoma patients. Citation Format: Steffen Goebel, Karen Bluemke, Udo Bilkenroth, Axel Meye, Susanne Fuessel, Christine Lautenschlaeger, Andres Melchior, Hans Heynemann, Paolo Fornara, Helge Taubert. Detection of circulating tumor cells in peripheral blood of renal cell carcinoma patients is correlated with survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 368. doi:10.1158/1538-7445.AM2013-368

Abstract 3196: In vivo drug sensitivity testing of CTCs isolated using the FMSA device

Abstract BACKGROUND: Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response; indeed, the enumeration of fixed CTCs with the Johnson and Johnson Veridex CellSearch System is FDA-approved for assessment of patient prognosis in metastatic breast, prostate, and colorectal cancer. The opportunity to isolate viable CTCs using the Flexible Micro Spring Array (FMSA) device – a novel microfluidic device that enriches CTCs by two physical parameters: size and flexibility – offers the possibility of testing cancer treatments in real time and rapidly providing information back to clinicians regarding the best course of treatment. OBJECTIVES: In this study, we evaluated the possibility of isolating viable CTCs from human blood using the FMSA device, growing these cells in a mouse xenograft model, and determining successful treatments for metastatic disease. METHODS: GFP-labeled HCT-116 cells were spiked into samples of blood from healthy donors to mimic the blood of patients with metastatic disease. This spiked blood was filtered using the FMSA device, and the recovered cells were implanted into the flanks of immunodeficient mice for study. A series of in vivo experiments were carried out to investigate the effect of chemotherapy on the resulting tumors. These tumors were molecularly characterized, compared to tumors derived from non-filtered cells, and analyzed for potential differences resulting from FMSA enrichment. Studies are ongoing to further investigate the use of this model and approach to culture CTCs from patients. RESULTS &amp; CONCLUSIONS: Cancer cells isolated from blood using the FMSA can proliferate when implanted subcutaneously into the flanks of immunodeficient mice – developing into tumors within two weeks. Although they grow at rates that depend on the number of cancer cells found in each mL of blood, these tumors provide a means for testing the effects of chemotherapy on CTCs in vivo. Using this model, treatments targeted towards CTCs could be assessed and provide real-time information to clinicians regarding treatment interventions for patients with metastatic cancer and detectable CTCs. It may also be possible to use this model as the primary mode for CTC culture as the in vivo environment may provide soluble factors, cell-to-cell interactions, and physical parameters that are not easily mimicked by in vitro culture. The approach has potential to help with efforts to develop personalized therapy options for patients with advanced cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3196. doi:1538-7445.AM2012-3196

Analysis and characterization of subpopulations of circulating tumor cells in patients with breast cancer.

e21090 Background: Distant metastasis is the main cause of death in patients with breast cancer. Disseminated tumor cells are found in peripheral blood, bone marrow, and lymph nodes. Tumor cells in peripheral blood, called circulating tumor cells (CTCs), are a strong negative prognostic factor for overall survival in breast cancer patients. The cancer stem cell (CSC) hypothesis proposes that a subpopulation of stem cell-like cells initiates and maintains tumor growth, metastasis and resistance to therapy. CSCs are characterized by their ability to self–renew, differentiate, and creat new tumors. Studies of tumor sections and bone marrow from breast cancer patients reveal that cells bearing the phenotype of EpCAM+CD44+CD24-/dim are putative tumor stem cells and may favor the development of distant metastasis. The objective of this study was to evaluate and characterize subpopulations of CTCs in breast cancer patients for CSC phenotypes by immunophenotyping and gene expression profiling. Methods: Breast cancer patients with more than 50 CTCs were selected for this study. Blood samples collected into CellSave tubes were enriched for CTCs using a Veridex Profile kit. CTCs were pulled down by magnetic beads conjugated with anti-EpCam antibody. Enriched samples were immunostained for various cell surface markers and then sorted by flow cytometric cell sorting. The immunophenotypes of CTCs were characterized by confocal microscopy. The gene expression profile of CTC subpopulations were analyzed by RT-PCR. Results: Different phenotypes of CTCs are found in breast cancer patients, including EpCAM+CD44-CD24-/dimCD45-, EpCAM+CD44+CD24-/dimCD45-, EpCAM+CD44+CD24+CD45-, and EpCAM+CD44+CD24+CD45dim. Of them, the putative cancer stem cell phenotype (EpCAM+CD44+CD24-/dimCD45-) accounts for 20-30 percent of the total CTCs. The expression of genes associated with stem cells, metastasis, and drug targets is assessed in different subsets of CTCs. Conclusions: These preliminary data support the existence of a CTC subset with a putative stem cell phenotype in breast cancer patients. The link between CTC subsets and clinical outcomes, metastasis, and response to therapy warrants further evaluation.

Abstract 5215: Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma

Abstract Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood, so called circulating tumor cells (CTCs), for renal cell carcinoma (RCC) patients. 233 peripheral blood samples from 154 RCC patients were investigated for the presence of disseminated tumor cells (DTCs) by autoMACS technique and immunocytochemical (ICC) staining of cytokeratin. The frequency of CTCs was analyzed statistically for correlation with relevant clinical data. Two kinds of tumor cells were detected: those with expression of cytokeratin 8/18 (CK+), but also cells without a detectable CK+ expression, which we called large blue stained (Bl+) cells with a tumor-like morphology. After following the CD45 autoMACS depletion protocol, we identified CTCs in 96 out of 233 peripheral blood samples (41%), which originated from 81 out of 154 (53%) RCC patients. A significant correlation between the detection of CTCs and positive lymph node status (p &amp;lt; 0.001; Chi-square test) and the presence of synchronous metastases at the time of primary tumor resection (p &amp;lt; 0.02; Chi-square test) was found. In a multivariate Cox's regression hazard model, presence of CK+ CTCs was significantly correlated with poor overall survival for RCC patients (RR=2.3; p &amp;lt; 0.05). The presence of CTCs correlated to lymph node status and presence of synchronous metastases in RCC. It is important to evaluate CK+ and Bl+ tumor cells together to determine the role of CTCs in tumor behavior and disease progression. Detection of CK+ CTCs in peripheral blood is a significant and independent prognostic factor for RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2011-5215

Abstract 2231: Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma correlates with prognosis

Abstract Purpose. The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood [so called circulating tumor cells (CTCs)] for renal cell carcinoma (RCC) patients. Methods. 233 peripheral blood samples from 154 RCC patients were investigated for the presence of disseminated tumor cells (DTCs) by autoMACS technique and immunocytochemical (ICC) staining of cytokeratin. The frequency of CTCs was analyzed statistically for correlation with relevant clinical data. Results. Two kinds of tumor cells were detected: those with expression of cytokeratin 8/18 (CK+), but also cells without a detectable CK+ expression, which we called large blue stained (Bl+) cells with a tumor-like morphology. After following the CD45 autoMACS depletion protocol, we identified CTCs in 96 out of 233 peripheral blood samples (41%), which originated from 81 out of 154 (53%) RCC patients. A significant correlation between the detection of CTCs and positive lymph node status (p&amp;lt;0.001; chi2-test) and the presence of synchronous metastases at the time of primary tumor resection (p=0.014; chi2-test) was found. In a multivariate Cox's regression hazard model, presence of CK+ CTCs was significantly correlated with poor overall survival for RCC patients (RR=2.3; P=0.048). Conclusions. The presence of CTCs correlated to lymph node status and presence of synchronous metastases in RCC. It is important to evaluate CK+ and Bl+ tumor cells together to determine the role of CTCs in tumor behavior and disease progression. Detection of CK+ CTCs in peripheral blood is a significant and independent prognostic factor for RCC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2231.

171 POSTER Integrin LFA-1 expression regulates angiogenesis-stimulating potential of colorectal carcinoma cells at premetastatic niches in the liver

During the process of metastasis, which is the leading cause of cancer-related death, cancer cells dissociate from primary tumors, migrate to distal sites, and finally colonize, eventually leading to the formation of metastatic tumors. The migrating tumor cells in circulation, e.g., those found in peripheral blood (PB) or bone marrow (BM), are called circulating tumor cells (CTCs). CTCs in the BM are generally called disseminated tumor cells (DTCs). Many studies have reported the detection and characterization of CTCs to facilitate early diagnosis of relapse or metastasis and improve early detection and appropriate treatment decisions. Initially, epithelial markers, such as EpCAM and cytokeratins (CKs), identified using immunocytochemistry or reverse transcription polymerase chain reaction (RT-PCR) were used to identify CTCs in PB or BM. Recently, however, other markers such as human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and immuno-checkpoint genes also have been examined to facilitate detection of CTCs with metastatic potential. Moreover, the epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSCs) have also received increasing attention as important CTC markers owing to their roles in the biological progression of metastasis. In addition to these markers, researchers have attempted to develop detection or capture techniques for CTCs. Notably, however, the establishment of metastasis requires cancer-host interactions. Markers from host cells, such as macrophages, mesenchymal stem cells, and bone marrow-derived cells, which constitute the premetastatic niche, may become novel biomarkers for predicting relapse or metastasis or monitoring the effects of treatment. Biological studies of CTCs are still emerging. However, recent technical innovations, such as next-generation sequencing, are being used more commonly and could help to clarify the mechanism of metastasis. Additionally, biological findings are gradually being accumulated, adding to our body of knowledge on CTCs. In this review, we will summarize recent approaches to detect or capture CTCs. Moreover, we will introduce recent studies of the clinical and biological importance of CTCs and host cells.