Stem-like Circulating Tumor Cells Research Articles

Exploring the Role of Hypoxia-Inducible Carbonic Anhydrase IX (CAIX) in Circulating Tumor Cells (CTCs) of Breast Cancer

Circulating tumor cells (CTCs) in the peripheral blood are believed to be the source of metastasis and can be used as a liquid biopsy to monitor cancer progression and therapeutic response. However, it has been challenging to accurately detect CTCs because of their low frequency and the heterogeneity of the population. In this study, we have developed an in vitro model of CTCs by using non-adherent suspension culture. We used this model to study a group of breast cancer cell lines with distinct molecular subtypes (TNBC, HER2+, and ER+/PR+). We found that, when these breast cancer cell lines lost their attachment to the extracellular matrix, they accumulated a subtype of cancer stem cells (CSC) that expressed the surface markers of stem cells (e.g., CD44+CD24-). These stem-like CTCs also showed high expressions of hypoxia-inducible gene products, particularly the hypoxia-inducible carbonic anhydrase IX (CAIX). Inhibition of CAIX activity was found to reduce CAIX expression and stem cell phenotypes in the targeted CTCs. Further studies are needed, using CTC samples from breast cancer patients, to determine the role of CAIX in CTC survival, CSC transition, and metastasis. CAIX may be a useful surface marker for the detection of CSCs in the blood, and a potential target for treating metastatic breast cancers.

Stem-like markers in the circulating tumor cells assessment in ovarian cancer.

e17542 Background: Circulating tumor cells (CTCs) are a potential source of dissemination and relapse in ovarian cancer (OC). Stem cell properties can provide a survival advantage for CTCs. The clinical significance of stem-like CTCs in OC remains to be studied. We aimed to assess the quantities of the stem, epithelial, mesenchymal CTCs and their relationships with the clinical parameters in the OC. Methods: Peripheral blood samples (7.5 ml) were obtained from patients with primary epithelial OC before treatment. CTCs were isolated by flow cytometry (Cytoflex S (Beckman Coulter, USA)) using antibodies to CD45 (BioLegend, USA); CD44 (BioLegend, USA), CD133 (Miltenyi biotec, Germany), ALDH (Stemcell, Canada) to detect the stem markers; EpCAM (BioLegend, USA), cytokeratins 8, 18 (Abcam plc., UK), vimentin (BioLegend, USA) for epithelial and mesenchymal markers. Blood samples from patients with benign ovarian tumors served as a control. Informed voluntary consent was obtained from all the women. Statistical processing included Mann-Whitney U-test, linear regression, Cox proportional hazards model for progression-free survival (PFS) (Statistica 13.0 (TIBCO, USA)). Results: The study included 30 patients, median age 64 (34-76) years. 15 patients had a FIGO stage IV, 12 - stage III, 1 – stage II and 1 – stage I. The content of CTCs populations is presented in the table. The CTCs counts did not differ depending on age, platelet count, and stage 3 or 4. The amount of CD45-CK+Vim- was higher in the presence of ascites (p = 0.035). We found a regression relationship between the serum CA-125 and the number of CD45-CD44+CD133+ (R2= 0.220, p = 0.016); the leukocyte count in blood and CD45-CD44+ALDHhigh (R2= 0.234, p = 0.017); the number of CD45-Vim+ and CD45-CD44+CD133+ALDH+ (R2= 0.305, p = 0.014); CD45-CK-Vim+ and CD45-EpCAM+CK+ (R2= 0.717, p < 0.001). The Cox regression model for PFS included the number of CD45-CD44+CD133+ALDH+ (HR 1.51 95% CI 1.01-2.24 p = 0.043) and the cytoreductive surgery performance (HR 0.09 95% CI 0.01-0.89 p = 0.039) during the first line of treatment. Conclusions: Various populations of circulating tumor cells coexist in ovarian cancer patients. The use of a combination of stem markers in the CTCs detection can increase their prognostic value in OC. This work was supported by the RFBR grant No. 19-315-90011.[Table: see text]

2002P - CXCR4, CCR2 and CCR5 expression in subsets of tumor cells with stem and/or EMT features

BackgroundNumerous investigations have been focused on the role of CXCR4, CCR2 and CCR5 in solid tumors, including breast cancer. These chemokine receptors make cancer cells move out of the circulation and traffic into organs with high amounts of chemokines, and thus forming metastases. In this connection, the objective of our stady was to investigate CXCR4, CCR2 and CCR5 expression in subsets of tumor cells with stem and/or EMT features in primary tumor and peripheral blood in breast cancer patients. MethodsTwenty-three patients with breast cancer, between 29 and 69 years of age, were included in this study. Five-color confocal microscopy was used to investigate tumor cells with stem and/or EMT features, CXCR4, CCR2 and CCR5 expression in primary tumor. Flow cytometry was used to analyze the similar phenotypes of circulating tumor cells (CTC). All patients signed an informed consent for voluntary participation. ResultsIn the primary tumor, CXCR4 was expressed on subsets of non-stem and stem-like tumor cells with EMT features. CCR5 was more often expressed by tumor cells with stem and EMT features. The count of such cells in the tumor decreased after neoadjuvant chemotherapy (NACT). CK7+CD45-CD44-CCR2+ and CK7+CD45-CD44-CXCR4+ CTC (without stem features) were detected in breast cancer patients with and without NACT. At the same time, the count of non-stem CXCR4+ CTC was significantly higher in the blood of patients after NACT. CK7+CD45-CD44-N-cadh+CCR5+ CTC (with EMT features) were more frequent in the blood of breast cancer patients after NACT, and its count was significantly higher. It is interesting to note that stem-like CTC didn’t express CCR2 and CCR5. Stem-like CXCR4+ CTC were rarely detected and had no EMT features in patients, regardless of the NACT performance. The count of CCR5+ tumor cells without stem and EMT features correlates with the count of non-stem CCR5+ CTC. The count of CXCR4+ tumor cells with stem and EMT features correlates with the count of non-stem CXCR4+ CTC with EMT features. ConclusionsThus, the potency for recruitment into the circulation is found in tumor cells with EMT features, regardless of the stemness status. The study was supported by the Russian Science Foundation (grant 19-75-30016). Legal entity responsible for the studyThe authors. FundingRussian Science Foundation. DisclosureAll authors have declared no conflicts of interest.

Abstract PD9-10: Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis

Abstract Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Cancer associated fibroblasts (CAFs) are the majority of stroma in BC and critical players in BC malignancy. For example, CAFs are the main source of SDF-1, a prominent chemokine in the tumor microenvironment (TME) that also imparts stem cell-like characteristics to BC cells. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. Stem-like CTCs and clusters have a greater propensity to establish metastasis. We recently identified circulating CAFs (cCAFs) in blood from patients with BC and in spontaneous, syngeneic, and xenograft mouse models of BC. cCAFs not only circulate individually, but are also found in clusters with CTCs. In this study, we examine the role of CAFs in promoting egress of stem-like CTCs (cCSCs), determine the capacity of stem-like CTCs to cluster with CAFs, and evaluate the involvement of CTC/cCAF clustering in augmenting BC metastasis. Methods: Our model employs NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines: the non-metastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis, and we used BALB/c mice injected with syngeneic 4T1 or 67nR cells to evaluate cCAFs, CTCs, and cluster egress in preclinical models. Mice were sacrificed at specific time points, and cardiac blood was collected. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were stained for IF and cCAFs, CTCs, cCSCs, and clusters were enumerated. Tumors from CAF co-injected mice were evaluated for their stem cell-like phenotype and re-implanted in mice to evaluate tumorigenicity and metastasis. Results: In spontaneous, syngeneic, and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs co-clusters appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. Co-inoculation of CAFs with BC cells resulted in a significant increase in tumor progression, metastasis, and in a substantially higher number of both individual cells and clusters in circulation. Dissociated tumor cells from CAF co-injected tumors had a higher proportion of CD44+stem cell-like cells (CSCs), enhanced ALDH-1 expression, and enhanced mammosphere formation. CD44+ CSCs, individually and in clusters, are found early on in the circulation of mice injected with dissociated tumor cells from CAF co-injected tumors. Upon re-implantation of CAF co-injected dissociated tumor cells without CAFs, dissociated tumor cells showed enhanced tumorigenicity and malignancy. Conclusion: CAFs are highly motile and cCAFs precede CTCs into circulation and can do so independently of tumor cells. CAFs sustain egress of tumor cells by augmenting malignancy and stemness of BC cells. cCAF clusters with the highly metastatic stem cell-like subset of CTCs bolster metastatic colonization. Targeting primary CAF function and/or cCAF/cCSC co-clusters may provide novel avenues to abrogate BC metastasis. Citation Format: Sharma U, Miller P, Medina Saenz K, Picon-Ruiz M, Morata-Tarifa C, Spartz A, Troness B, Park DN, Seagroves TN, Slingerland JM, Lippman ME, El-Ashry D. Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-10.

Comprehensive characterization of single circulating rare cells through automated picking and high-throughput mRNA profiling in patients with pancreatic cancer.

271 Background: Circulating rare cells (CRCs) such as circulating tumor cells (CTCs), tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression. However, there are limited reports characterizing single CRCs comprehensively because of technical limitations including isolation and analysis of rare cells. We aimed to test the feasibility of the automated picking and mRNA profiling platforms of CRCs for evaluating the phenotypic heterogeneity of CRCs and the relationship between clinical outcomes and CRCs in patients with pancreatic cancer. Methods: CRCs were collected from 3ml of whole blood in 4 patients with pancreatic cancer using filter-based lab-on-a-disc platform. The filter membrane was transferred to ALS CellCelector (ALS, Germany) which is picking single nucleated CD45 negative cells to 96-well plate. The single cells were analyzed with the BioMark HD (Fluidigm, USA) which is digital PCR-based mRNA profiling for 48 markers such as epithelial, mesenchymal, stem-like, macrophage, fibroblast, oncogenic and immunogenic markers. Results: CRCs were detected in 3 of 4 patients (75%). Sixty-six-year-old female who had large pancreatic cancer with liver and lung metastasis presented 13 single rare cells including 7 epithelial CTCs with hematopoietic features (CD45 and CD14 positive), 3 mesenchymal CTCs, 2 stem-like CTCs, and 1 CAF. One epithelial CTC with hematopoietic features and one stem-like CTC were detected in 51-year-old male and 61-year-old female with metastatic pancreatic cancer, respectively. There was no CTC in 80-year-old male with localized pancreatic cancer. Conclusions: The automated picking and mRNA profiling platforms showed the feasibility of showing tumor heterogeneity and predicting clinical outcomes in patients with pancreatic cancer.

Abstract 3402: Castration resistance transcriptome in prostate cancer revealed by single-cell RNA-seq

Abstract Fatal metastatic castration-resistant prostate cancer (mCRPC) remains without sensitive early detection biomarkers and effective therapeutic targets. Among 2.5 millions of prostate cancer patients, the majority will face a dilemma, to treat or not to treat, at a point as cancer progresses. Biomarkers for mRCPC at an early stage represent an unmet need. With early identification, clinicians could design new treatment strategies to reduce metastasis-related morbidity and to extend survival of patients. In this study, we deployed single-cell RNA-seq on prostate cancer cells (LNCaP, ABL and PC3) to determine the transcriptome in androgen independency and castration resistance of prostate cancer. We identified potential 336 androgen-independence specific genes and 2396 castration resistance specific genes in ABL and PC3 cells respectively, while only 136 genes were shared in both cells. These genes, mostly upregulated were enriched in 43 and 166 signaling pathways that implicated the complexity of the castration resistance transcriptomic systems and networks. The signaling pathways are involved in advanced and metastatic malignancies including WNT, TGFB, ITGA/B, STAT, EPH, focal adhesion, adherens junction, regulation of actin cytoskeleton, gap junction, tight junction and EMT. Malignant potencies of ~ 40 pathways were validated by in silico analysis of the RNA-seq data from the prostate cancer cohort of The Cancer Genomic Atlas (TCGA) using Kaplan-Meier disease free and survival curve analyses. The transcriptomic regulation of these genes was further validated and correlated with ATAC-seq data. In order to further verify the functions of those signaling pathways in castration resistance, 9 major signaling pathways were evaluated using small molecule inhibitors. Castration resistant prostate cancer cells showed significant defective cell proliferation, migration, invasion and sphere formation in the presence of inhibitors, whereas LNCaP and ABL cells displayed limited or non-significant changes. Interestingly, 4 small molecule inhibitors showed significant suppression on the growth of stem-like circulating tumor cells that were derived from clinical blood samples of prostate cancer patients. Our data suggest that those castration resistance specific genes and signaling pathways revealed by single-cell RNA-seq may serve as potential markers and therapeutic targets. Citation Format: Aaron M. Horning, Che-Kuang Lin, Yao Wang, Brandon Lieberman, Devalingam Mahalingam, Ming Gao, Pei Wang, Chiou-Miin Wang, Zhijie Liu, Jianhua Ruan, Michael A. Liss, Victor X. Jin, Tim H-M Huang, Chun-Liang Chen. Castration resistance transcriptome in prostate cancer revealed by single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3402.

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OBJECTIVES/SPECIFIC AIMS: The goal of this research is to use circulating tumor cells (CTC) enumeration and characterization to monitor anticancer treatment response. Emerging evidence strongly suggests the implications that epithelial-to-mesenchymal transition may have in cancer metastasis. Consequently, we hope to elucidate the significance of mesenchymal and stem-like CTCs in the peripheral blood of metastatic pancreatic cancer patients by analyzing the prevalence and frequency trends of CD133+ CTCs, as they relate to clinical events. We also hope to determine if there is a correlation between EpCAM+ CTCs and CD133+ CTCs numbers with tumor size, disease stage, and patient clinical outcome. METHODS/STUDY POPULATION: Blood samples of patients with metastatic pancreatic cancer (stage IV) were obtained from the University of Florida Health Cancer Center after informed consent through an IRB-approved protocol. CTC capture, characterization, and enumeration was performed on the blood of these cancer patients during their anticancer treatment. Patients had blood drawn for this purpose at time points aligned with clinical phlebotomy (every 2 weeks). CTC capture was performed by introducing treated patient blood samples into antibody-functionalized microdevices. The PDMS devices were functionalized by immobilizing either anti-EpCAM or anti-CD133, through an avidin-biotin complex. After capture, cells were fixated and permeabilized with 4% paraformaldehyde and 0.2% Triton X-100, respectively. Three-color immunocytochemistry (anti-cytokeratin-FITC, anti-CD45-PE, and DAPI) was performed to identify CTCs from nonspecifically captured blood cells. To be counted as a CTC, based on the FDA-approved technical definition, a cell with the appropriate cell size and morphology must be nucleated (DAPI+), express cytokeratin (CK+), and lack the leukocytic CD45 marker (CD45−). RESULTS/ANTICIPATED RESULTS: We tested the clinical utility of the device for monitoring the response of patients with advanced pancreatic cancer to a chemotherapy treatment consisting of anticancer drugs including 5-fluorouracil, leucovorin, oxaliplatin, and dasatinib. We have detected EpCAM+ CTCs in 47/47 (100%) and CD133+ CTCs in 41/47 (87.2%) of blood samples, coming from a cohort of 16 patients. We studied the correlation between the CTC numbers and the clinical result of patients in the study. We found that the size and changes in the size of the primary tumor (confirmed by CT scans) correlated with the frequency and increase/decrease trends in the number of CTCs detected. We expect to find some relationship between the number of detected CD133+ CTCs, or rather stem-like CTCs, and the clinical outcome of patients (eg, disease progression leading to withdrawal from study). DISCUSSION/SIGNIFICANCE OF IMPACT: Enumeration of patient CTCs and stem-like CTCs at different stages of a patient’s treatment may correlate with disease stage and prognosis, and prove useful in monitoring early recurrence, patient-specific treatment response, and newly acquired resistances; all of which would aid in providing guidance for the next step in clinical intervention. This type of liquid biopsy technology has great potential to make an impact in the future of personalized medicine and point-of-care diagnostics, as well as become a sturdy tool for translational research.

Abstract P1-01-18: A multiplexed immunofluorescence identifies phenotypic heterogeneity of circulating tumor cells in breast cancer

Abstract Introduction: Circulating Tumor Cells (CTCs) have attracted significant attention as a new class of "liquid biopsy", enabling longitudinal and non-invasive disease monitoring to capture an overall snapshot of individual disease. Primary breast tumors are highly heterogeneous due to their genetic instability. Thus, the presence of heterogeneous populations of CTCs is expected. A number of attempts have been made to address CTC's phenotypic heterogeneity using different methods and approaches. For comprehensive understanding of distinct CTC phenotypes, transcriptomic and cell surface marker studies using single cell analysis separated CTCs into different subgroups with epithelial-, mesenchymal-, and stem-like signatures. These studies concluded that the prevalence of stem-like CTCs is associated with poor prognosis. Immunofluorescence is an ideal approach to understand the heterogeneity of 3 - 4 markers in a large number of distinct CTCs; however, such approach does not address the presence of overlapping phenotypic signatures. The collection method of CTCs adds another layer of complexity since the most commonly adopted CTC enumeration strategy relies on affinity-based selection using antibody against EpCAM, which, although abundantly expressed on epithelial cancer cells, automatically eliminates a large fraction of non-epithelial CTCs. To better understand the whole landscape of this heterogeneous disease, the use of marker-independent unbiased methods to collect CTCs is critical to obtain the overall landscape of phenotypic heterogeneity. Aim: We aimed to investigate the heterogeneity of breast CTCs on a single cell level using size exclusion- based enrichment principal. Methods: Whole blood was collected from metastatic breast cancer patients under IRB approved protocol. CTCs were isolated on porous membrane (8 µm) under negative pressure (ISET®) from 10 mL of blood. CTCs were fixed on the porous membrane and used for multiplexed immunofluorescence to investigate the expression of epithelial (CK and EPCAM), mesenchymal (Vimentin, Snail and TWIST), and stem-like markers (CD44, ALDH1, and CD133) on CTCs. Results: CTCs were isolated on ISET filter from all subtypes, Stage IV patients. CTCs were identified based on their morphological and phenotypical characteristics with our CTC criteria of size of nucleus ≥16 µm, triple negative for α-smooth muscle actin, fibroblast associated protein, and CD45. The CTCs were further characterized based on their mesenchymal and stem-like signatures to understand their heterogeneity. Conclusions: Multiplex immunofluorescence staining of CTCs collected using the size-based enrichment approach enabled us to identify the heterogeneity of CTCs. The phenotypic heterogeneity represents a corner stone for future studies to identify a subset of CTCs that may be associated with breast cancer patient prognosis. Citation Format: Kamal M, Zhang R, Walker M, Squires R, Talbert B, Dooley W, Razaq W, Tanaka T. A multiplexed immunofluorescence identifies phenotypic heterogeneity of circulating tumor cells in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-18.

Abstract IA20: Molecular and genomic characterization of invasive circulating tumorcells (iCTCs) from men with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Molecular characterization and genomic analysis of circulating tumor cells (CTCs) may allow for a better understanding of the mechanisms of resistance to therapies in metastatic castration resistant prostate cancer (mCRPC). The Vitatex VitaAssay platform captures invasive CTCs (iCTCs) in a cell surface marker-independent fashion based on their ability to invade a fluorescently-labeled cell adhesion matrix (CAM), allowing for the analysis of multiple CTC subpopulations. We recently demonstrated our ability to detect EMT and stem-like CTC subpopulations with the CAM platform (Friedlander et al., Int J Cancer 2014). We hypothesize that CTC subpopulation diversity is associated with a shorter period of time to resistance for men with mCRPC. Here we sought to estimate epithelial, mesenchymal, stem-like and neuroendocrine iCTC subpopulation diversity in men with CRPC starting abiraterone acetate or enzalutamide therapy and to report the genomic profiles of iCTCs and matched metastatic biopsies. Methods: Blood is being obtained with informed consent from men with mCRPC partaking in either a dose escalation abiraterone trial or the West Coast Dream Team (WCDT) trial. iCTCs are being isolated from the blood samples using the CAM platform coupled with FACS. Paired metastatic biopsies are being obtained as part of the WCDT, when available. For CTC enrichment, iCTCs are defined as CAM+/CD45-/CD14-. For subpopulation characterizations, mesenchymal iCTCs are defined as vimentin+, stem-like iCTCs as CD44+ and neuroendocrine iCTCs as chomogranin (CgA)+. Agilent array comparative genomic hybridization (aCGH) of iCTCs and paired biopsies is being performed. Results: iCTCs were isolated using the CAM platform from 96 men, to date. The average baseline iCTC count per 7.5ml was 160 (range 0-1255). In a subset of 69 subjects, the average baseline CD44+ iCTC count per 7.5ml was 153 (range 0 -1423), and in a subset of 31 patients the median vimentin+ iCTC count per 7.5ml was 116 (range 5-538) and in a subset of 5 patients the median CgA+ iCTC counts per 7.5ml was 79 (range 46 - 185). Using aCGH, copy number aberration is detectable in iCTCs. Some iCTC aCGH profiles resemble paired metastatic biopsy aCGH profiles. Conclusions: Multiple CRPC iCTC subpopulations are identifiable from men with mCRPC, reflecting the heterogeneity of the disease. Data will continue to be collected as part of these two ongoing clinical trials to identify biomarkers of resistance and shed light on resistance pathways. Citation Format: Terence W. Friedlander, Gayatri Premasekharan, Archana Dilip, Jaime Tawney, Vy Ngo, Elizabeth Gilbert, Charles J. Ryan, Eric Small, Pamela L. Paris. Molecular and genomic characterization of invasive circulating tumorcells (iCTCs) from men with metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA20.

Abstract A153: Isolation and characterization of high-purity invasive CTCs from men with metastatic castration-resistant prostate cancer (mCRPC).

Abstract Introduction: As metastatic biopsies are often difficult and impractical to obtain; isolation, characterization, and genomic profiling of circulating tumor cells (CTCs) from men with metastatic castration resistant prostate cancer (mCRPC) is critical for understanding tumor progression and mechanisms of resistance to treatment. The Vitatex cell-adhesion matrix (CAM) platform captures invasive CTCs in both a cell surface marker- and size-independent manner based on the ability of CTCs to invade a fluorescently-labeled CAM. This platform allows for isolation and characterization of viable CTC sub-populations from individual patients with mCRPC. In this study, we sought to optimize CTC isolation and purification for downstream genomics and to explore epithelial-mesenchymal and stem-like CTC subtypes, all in the context of treatment resistance to Abiraterone, a novel inhibitor of androgen synthesis. Methods: Forty ml of blood was collected from 23 patients with mCRPC. The CTCs were isolated using the VitaAssay platform and paired metastatic biopsies were obtained for a subset of men. Fluorescence-activated cell sorting (FACS) was coupled with the VitaAssay platform to increase cell purity. Invasive CTCs having ingested the fluorescent CAM were sorted based on CAM+/CD45-/CD14-/DAPI- staining. To complement the FACS data and for quality control, a subset of cases (N=11) were also enumerated using Immunocytochemistry (ICC), where CTCs were identified as CAM+/CD45-/CD14-/DAPI+. Healthy donor blood was used as a control. FACS-sorted stem-like CTCs were defined as CD44+/CAM+/CD45-/CD14-/DAPI- and mesenchymal CTCs were identified with ICC as vimentin+/CAM+/CD45-/DAPI+. Array comparative genomic hybridization (aCGH) of FACS-sorted CAM-avid amplified CTCs using Agilent microarrays was performed to obtain the CTC and matched biopsy genomic profiles. Results: Enumeration of CTCs by VitaAssay coupled with FACS yielded an average of 72 CTCs per 7.5 ml blood (range 1 to 338) for the 23 patients. Only 4 of the 23 patients had ≤ 2 CTCs per 7.5 ml blood. No CTCs were detected in the healthy control subject after cell sorting. The CTC purity increased post-FACS to ∼90% from ∼1% pre-FACS. Enumeration along with genomic analysis (copy number aberrations) of CTCs and their subpopulations, comparison with matched biopsies and clinical data are currently being investigated. Conclusions: Combining the VitaAssay platform with FACS allows for recovery of high purity mCRPC CTC subpopulations suitable for downstream molecular and genomic analysis. Ultimately, studies incorporating these optimized CTC methods will allow for better understanding of mechanisms of treatment resistance, disease progression and potentially identify new drug targets for CRPC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A153. Citation Format: Gayatri Premasekharan, Terence W. Friedlander, Vy T. Ngo, Elizabeth Gilbert, Priya Sathaye, Anna Harris, Charles J. Ryan, Pamela L. Paris. Isolation and characterization of high-purity invasive CTCs from men with metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A153.