Prostate Circulating Tumor Cells Research Articles

Unveiling the characteristics of D4 and R4 aptamers for their future use in prostate cancer clinical practice

The DNA and RNA aptamers D4 and R4, respectively, emerged from the modification of PC-3 cell-binding aptamer A4. Our objective was to characterize the aptamers in silico and in vitro and begin to identify their target molecules. We represented their structures using computational algorithms; evaluated their binding to several prostate cell lines and their effects on the viability and migration of these cells; and determined their dissociation constant by flow cytometry. We analyzed circulating prostate tumor cells from patients using D4, R4, anti-CD133 and anti-CD44. Finally, the target proteins of both aptamers were precipitated and identified by mass spectrometry to simulate their in silico docking. The aptamers presented similar structures and bound to prostate tumor cells without modifying the cellular parameters studied, but with different affinities. The ligand cells for both aptamers were CD44+, indicating that they could identify cells in the mesenchymal stage of the metastatic process. The possible target proteins NXPE1, ADAM30, and MUC6 need to be further studied to better understand their interaction with the aptamers. These results support the development of new assays to determine the clinical applications of D4 and R4 aptamers in prostate cancer.

Development of a multiplex assay to assess activated p300/CBP in circulating prostate tumor cells.

Reduced SIRT2 deacetylation and increased p300 acetylation activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in castration-resistant prostate cancer (CRPC). We examined whether circulating tumor cells (CTCs) identify patients with altered p300/CBP acetylation. CTCs were isolated from 13 advanced PC patients using Exclusion-based Sample Preparation (ESP) technology. Bound cells underwent immunofluorescent staining for histone modifying enzymes (HMEs) of interest and image capture with NIS-Elements software. Using the cBioPortal PCF/SU2C dataset, the response of CRPC to androgen receptor signaling inhibitors (ARSI) was analyzed in 50 subjects. Staining optimization and specificity revealed clear expression of acetyl-p300, acetyl-H3K18, and SIRT2 on CTCs (CK positive, CD45 negative cells). Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced p300 and H3K18 acetylation. In CRPC patients, a-p300 strongly correlated with its target acetylated H3k18 (Pearson's R = 0.61), and SIRT2 expression showed robust negative correlation with a-H3k18 (R = -0.60). A subgroup of CRPC patients (6/11; 55%) demonstrated consistent upregulation of acetylation based on these markers. To examine the clinical impact of upregulation of the CBP/p300 axis, CRPC patients with reduced deacetylase SIRT2 expression demonstrate shorter response times to ARSI therapy (5.9 vs. 12 mo; p = 0.03). A subset of CRPC patients demonstrate increased p300/CBP activity based on a novel CTC biomarker assay. With further development, this biomarker suite may be used to identify candidates for CBP/p300 acetylation inhibitors in clinical development.

Abstract B024: Nuclear size and nuclear shape instability in prostate cancer epithelial cells during metastatic progression

Abstract Very small nuclear (vsn) circulating tumor cells (CTCs) are detected in a subgroup of metastatic, castration resistant prostate cancer (mCRPC) patients who develop visceral metastasis following intensive androgen receptor suppression. In addition, we have previously observed that nuclear shape instability (characterized by the loss and/or mis-localization of the nuclear envelop protein emerin) is present in human prostate cancer tissues, and in CTCs from patients with metastatic disease. Herein, we investigated the biological and clinical implications of the vsn phenotype in prostate CTCs. Patients with vsnCTCs had poor overall survival and low PSA response to androgen receptor suppression compared with non-vsnCTC patients. The vsnCTCs exhibited a loss/downregulation of emerin (EMD) and we found a direct correlation between emerin loss and nuclear size reduction in CTC from mCRPC patients. Further, in vitro studies revealed that the vsn phenotype was characterized by high invasive capacity, enhanced cellular plasticity, and resistance to AR target therapy. Finally, depletion of EMD in prostate tumor cells resulted in the vsn phenotype. Together, these findings demonstrate that the vsnCTC phenotype is characterized by deregulation of EMD expression, and the presence of these cells result in poor clinical outcomes in prostate cancer. Citation Format: Karen Cavassani, Edwin Posadas, Pai-Chi Teng, Sadaf Pustchi Pustchi, Minhyung Kim, Neil Bhowmick, Sungyong You, Michael Freeman, Leigh Ellis, Dolores Di Vizio, Hsian-Rong Tseng, Leland W. Chung, Susan Yao, Catherine Grasso. Nuclear size and nuclear shape instability in prostate cancer epithelial cells during metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B024.

SFRP2 Overexpression Induces an Osteoblast-like Phenotype in Prostate Cancer Cells

Prostate cancer bone metastasis is still one of the most fatal cancer diagnoses for men. Survival of the circulating prostate tumor cells and their adaptation strategy to survive in the bone niche is the key point to determining metastasis in early cancer stages. The promoter of SFRP2 gene, encoding a WNT signaling modulator, is hypermethylated in many cancer types including prostate cancer. Moreover, SFRP2 can positively regulate osteogenic differentiation in vitro and in vivo. Here, we showed SFRP2 overexpression in the prostate cancer cell line PC3 induces an epithelial mesenchymal transition (EMT), increases the attachment, and modifies the transcriptome towards an osteoblast-like phenotype (osteomimicry) in a collagen 1-dependent manner. Our data reflect a novel molecular mechanism concerning how metastasizing prostate cancer cells might increase their chance to survive within bone tissue.

Telomerase positive CTCs with PSMA high expression associated with prostate cancer metastasis.

BackgroundWhether circulating tumor cells (CTCs) with prostate-specific membrane antigen (PSMA) high expression was related to the metastatic progress in prostate cancer (PCa) remains explored. This study aimed to provide evidence to elucidate this relationship via the telomerase reverse transcriptase (TERT)-based CTC detection method.MethodsA total of 71 patients were enrolled and divided into the local PCa group (n=44) and metastatic PCa group (n=27). TERT-based CTC detection (TBCD) was used to detect CTCs. CTCs single-cell sequencing data were analyzed using gene ontology (GO) functional classification and enrichment.ResultsThe mean ‘TERT+ CTCs’ number was 6.11±9.63 in the metastatic group and 4.09±3.41 in the local group. GO enrichment analysis for 77 prostate CTCs single-cell sequencing confirmed that proliferation-related terms were enriched in the PSMA-high expression group, and 27 metastasis-related gene panels also had high expression in this group. Then, PSMA antibody was applied to mark the ‘TERT+ CTCs’. The proportion of patients with ‘TERT+ PSMA+ CTCs’ was positively associated with the Gleason score. Furthermore, the proportion of ‘TERT+ PSMA+ CTCs’ patients was 48.15% in the metastatic group, significantly higher than 22.72% in the local group.ConclusionsThis study suggested that TERT positive CTCs with high PSMA expression were associated with the PCa metastatic progress.

Can we infer tumor presence of single cell transcriptomes and their tumor of origin from bulk transcriptomes by machine learning?

There is a growing need to build a model that uses single cell RNA-seq (scRNA-seq) to separate malignant cells from nonmalignant cells and to identify tumor of origin of single cells and/or circulating tumor cells (CTCs). Currently, it is infeasible to build a tumor of origin model learnt from scRNA-seq by machine learning (ML). We then wondered if an ML model learnt from bulk transcriptomes is applicable to scRNA-seq to infer single cells’ tumor presence and further indicate their tumor of origin. We used k-nearest neighbors, one-versus-all support vector machine, one-versus-one support vector machine, random forest and introduced scTumorTrace to conduct a pioneering experiment containing leukocytes and seven major cancer types where bulk RNA-seq and scRNA-seq data were available. 13 ML models learnt from bulk RNA-seq were all reliable to use (F-score > 96%) shown by a validation set of bulk transcriptomes, but none of them was applicable to scRNA-seq except scTumorTrace. Making inferences from bulk RNA-seq to scRNA-seq was impaired by feature selection and improved by log2-transformed TPM units. scTumorTrace with transcriptome-wide 2-tuples showed F-score beyond 98.74 and 94.29% in inferring tumor presence and tumor of origin at single-cell resolution and correctly identified 45 single candidate prostate CTCs but lineage-confirmed non-CTCs as leukocytes. We concluded that modern ML techniques are quantitative and could hardly address the raised questions. scTumorTrace with transcriptome-wide 2-tuples is qualitative, standardization-free and not subject to log2-transformed quantities, enabling us to infer tumor presence of single cell transcriptomes and their tumor of origin from bulk transcriptomes.

TRAIL-coated leukocytes to kill circulating tumor cells in the flowing blood from prostate cancer patients

BackgroundRadical surgery is the first line treatment for localized prostate cancer (PC), however, several studies have demonstrated that surgical procedures induce tumor cell mobilization from the primary tumor into the bloodstream.MethodsThe number and temporal fluctuations of circulating tumor cells (CTC), cancer associated fibroblasts (CAF) and CTC cluster present in each blood sample was determined.ResultsThe results show that both CTC and CTC cluster levels significantly increased immediately following primary tumor resection, but returned to baseline within 2 weeks post-surgery. In contrast, the CAF level decreased over time. In patients who experienced PC recurrence within months after resection, CTC, CAF, and cluster levels all increased over time. Based on this observation, we tested the efficacy of an experimental TNF-related apoptosis-inducing ligand (TRAIL)-based liposomal therapy ex-vivo to induce apoptosis in CTC in blood. The TRAIL-based therapy killed approximately 75% of single CTCs and CTC in cluster form.ConclusionCollectively, these data indicate that CTC cluster and CAF levels can be used as a predictive biomarker for cancer recurrence. Moreover, for the first time, we demonstrate the efficacy of our TRAIL-based liposomal therapy to target and kill prostate CTC in primary patient blood samples, suggesting a potential new adjuvant therapy to use in combination with surgery.

A Direct Comparison between the Lateral Magnetophoretic Microseparator and AdnaTest for Isolating Prostate Circulating Tumor Cells.

Circulating tumor cells (CTCs) are important biomarkers for the diagnosis, prognosis, and treatment of cancer. However, because of their extreme rarity, a more precise technique for isolating CTCs is required to gain deeper insight into the characteristics of cancer. This study compares the performance of a lateral magnetophoretic microseparator (“CTC-μChip”), as a representative microfluidic device, and AdnaTest ProstateCancer (Qiagen), as a commercially available specialized method, for isolating CTCs from the blood of patients with prostate cancer. The enumeration and genetic analysis results of CTCs isolated via the two methods were compared under identical conditions. In the CTC enumeration experiment, the number of CTCs isolated by the CTC-μChip averaged 17.67 CTCs/mL, compared to 1.56 CTCs/mL by the AdnaTest. The number of contaminating white blood cells (WBCs) and the CTC purity with the CTC-μChip averaged 772.22 WBCs/mL and 3.91%, respectively, whereas those with the AdnaTest averaged 67.34 WBCs/mL and 1.98%, respectively. Through genetic analysis, using a cancer-specific gene panel (AR (androgen receptor), AR-V7 (A\\androgen receptor variant-7), PSMA (prostate specific membrane antigen), KRT19 (cytokeratin-19), CD45 (PTPRC, Protein tyrosine phosphatase, receptor type, C)) with reverse transcription droplet digital PCR, three genes (AR, AR-V7, and PSMA) were more highly expressed in cells isolated by the CTC-μChip, while KRT19 and CD45 were similarly detected using both methods. Consequently, this study showed that the CTC-μChip can be used to isolate CTCs more reliably than AdnaTest ProstateCancer, as a specialized method for gene analysis of prostate CTCs, as well as more sensitively obtain cancer-associated gene expressions.

Abstract 5362: Analysis of circulating tumor cells from prostate cancer patients

Abstract Background: Intermediate and high-risk prostate cancer patients are candidates for radiotherapy (RT). Evidence has shown that the presence of circulating tumor cells (CTCs) correlates with poor overall survival for patients with metastatic prostate cancer. CTCs have been reported as a better earlier predictor of treatment response than evaluation of prostate-specific antigen (PAS). The aim of our study was to identify and quantify circulating tumor cells from the peripheral blood of patients enrolled in two randomized Phase II radiotherapy clinical trials (BLaStM, NCT02307058 and CoMBINe, NCT02997709). Moreover, we investigated the genomic profiling of single CTCs using 10X Genomics to explore prognostic factors. Design/Methods: CTCs were successfully harvested and enumerated from more than 215 patients and 500 blood samples using a microfilter system, the Circulogix FaCTChecker, and the cells of interest enriched onto CyteCatch slides. Circulating tumor cells were visualized by staining, identified and quantified for epithelial and prostate specific markers. The number of single CTCs, CTC clusters and their sum were recorded. Unfixed CTCs were enriched from whole blood from 13 prostate cancer patients at high risk of metastasis (median age of 63.5 years and median CTC value of 12 (range 0-42)) using The Parsortix™ Cell Separation System (ANGLE Inc.), a microfluidic-based platform able to capture and harvest CTCs, and single-cell RNA sequencing was then performed with Chromium (10X Genomics) and Illumina NGS sequencing technologies. Initial bioinformatic and data analysis was performed by the Biostatistics and Bioinformatics Shared Resource (BBSR) at SCCC. Subsequent principal component analysis (PCA) and gene set enrichment analysis (GSEA) were performed. Meanwhile, patient demographic and disease characteristic variables (PSA, Gleason Score (GS), T stage, % tumor in biopsies, age, race/ethnicity) were also collected and compared. Results: Single CTCs or CTC clusters were evident in over 70% of patients analyzed using Immunofluorescence staining, with a median number of 30 (range 0-346). CTC transcriptomes at the single cell level was performed using the 10X platform, and principal component analysis discovered 16 distinct cell clusters of differential gene signatures within these cells in a t-SNE plot. Gene ontology analysis found some of these cell clusters contain differential expression pathways such as mTOR signaling (p=5.82E-05), EIF2 signaling (p=7.90E-05) and regulation of eIF4 and p70S6K signaling (p= 1.04E-04) which have been associated with prostate cancer. Gene Set Enrichment Analysis also identified distinct differential expression pathways in prostate CTCs comparing with non-CTCs. Conclusion: We have developed a novel pipeline to harvest CTCs and analyze transcriptomes at the single-cell level. Our results suggest that gene expression profiling of CTCs can be a promising way to explore prognostic markers for predicting radiation sensitivity and metastatic risk. Citation Format: Teresa Giret, Wensi Tao, Robert Suter, Saba Ansari, Sion Williams, Nagi Ayad, Radka Stoyanova, Brian Marples, Alan Pollack. Analysis of circulating tumor cells from prostate cancer patients [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 5362.

Initial detection of circulating tumor cells from metastatic prostate cancer patients with a novel small device.

BackgroundVarious devices for isolating and detecting circulating tumor cells (CTCs) have been developed, whereas the CellSearch® system has been clinically used in numerous prostate CTC studies. CTCs might become more useful surrogate markers of prostate cancer, and they should be measured in all settings, but a smaller, low-cost CTC capture system is required.MethodsAn inexpensive and highly sensitive microfluidic CTC-capture polymeric chip, developed by the Toyama Industrial Technology Center, as described in the following text, was used to assess the number of CTCs from patients with metastatic prostate cancer. After verifying that cultured human prostate cancer cells (PC3 and LNCaP) could be captured with the chip coated with anti–epithelial cell adhesion molecule (CD326) antibody, whole blood samples of 14 patients with prostate cancer were screened.ResultsThe average capture efficacy of PC3 cells was 94.60% in phosphate-buffered saline (PBS) and 83.82% in whole blood. The average capture efficacy of LNCaP cells was 82.73% in PBS and 75.78% in whole blood. CTCs were detected by the chip device in all 14 patients with metastatic prostate cancer using 2-mL blood samples. Although fewer CTCs were detected in patients with oligometastases, all patients with multiple distant metastases had CTCs. The average CTC count was 48 cells/mL (range 1–81 cells/mL).ConclusionThis CTC-chip will be able to capture CTCs and be useful to check CTCs as a surrogate marker in prostate cancer with smaller samples and lower cost in any small institution.

Abstract B070: Epithelial-marker absent and disease-marker specific circulating and disseminated tumor cells are rarely detected in men with localized prostate cancer

Abstract Introduction and Objectives: Epithelial-marker negative circulating tumor cells (CTCs) have recently been discovered in metastatic prostate cancer (PCa). Many popular CTC detection methods, including the AdnaTest, utilize a selection-based first step of bead pull down against an epithelial marker such as EpCAM. It has previously been shown that disseminated tumor cells (DTCs) are rare at the time of localized PCa using the AdnaTest. Here we queried the matched epithelial-marker depleted buffy coat (DBC) fraction of the AdnaTest for the presence of DTCs and CTCs in a cohort of localized patients undergoing radical prostatectomy (RP). Methods: 8 mL of bone marrow (BM) were collected from 28 patients (5 controls, 2 metastatic, and 21 localized) along with 8 mL of matched peripheral blood (PB) from 11 patients (3 controls, 2 metastatic, 6 localized). 5 mL of BM and PB were evaluated with the AdnaTest ProstateCancer Select kit (Qiagen) to enrich for EpCAM positive cells using immunomagnetic beads. The DBCs having the remaining cells after AdnaTest enrichment were isolated and queried for the presence of DTCs and CTCs. Cells were lysed and reverse transcribed into cDNA followed by real-time qPCR for the expression of the prostate-specific markers, NKX3.1, androgen receptor (AR), prostate specific antigen (PSA), and HOXB13, as well as the epithelial marker EpCAM. The buffy coats from the remaining 3 mL of nonenriched BM and PB were isolated and stored in -80°C for future evaluation. Results: EpCAM, NKX3.1, and AR were expressed in localized, control, and metastatic BM and PB and thus were nonspecific to identifying prostate CTCs and DTCs. PSA and HOXB13 positive cells were only identified in the metastatic setting. With the AdnaTest, PSA and HOXB13 positive DTCs were detected in 0/21 (0%) localized patients, but were identified in 2/2 (100%) metastatic samples. Positive PSA and HOXB13 matched in all cases. No controls expressed PSA or HOXB13. The DBC fraction was contaminated with EpCAM positive cells in 27/28 (96%) BM samples in addition to 8/11 (73%) PB specimens. PSA or HOXB13 expression were not detected in any DBC, demonstrating the absence of epithelial-marker negative DTCs. In two instances, EpCAM was detected in the PB depleted fraction but not with the AdnaTest. Conclusion: EpCAM, NKX3.1, and AR are nonspecific, while PSA and HOXB13 are prostate-specific markers in the PB and BM. PSA and HOXB13 positive prostate CTCs and DTCs are rarely identified in localized men undergoing RP. Epithelial-marker negative DTCs and CTCs were not detected in localized men. The AdnaTest bead selection step did not capture all epithelial cells, and ongoing investigation into a selection-free CTC/DTC detection platform is necessary. Citation Format: Stephanie Glavaris, Heather Chalfin, Changxue Lu, Yan Chen, Emily Caruso, Kenneth Valkenburg, Jun Luo, Kenneth Pienta. Epithelial-marker absent and disease-marker specific circulating and disseminated tumor cells are rarely detected in men with localized prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B070.

Abstract 5230: Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells

Abstract Background. Androgen receptor (AR) signaling is the primary driver of prostate cancer. Expression of the AR splice variant AR-V7, is predictive of resistance to anti-androgen therapies enzalutimide and abiratirone. There is great interest to non-invasively identify circulating tumor cells (CTCs) and investigate their expression of AR and AR-V7 and monitor them over time. RareCyte has developed a platform for automated visual identification of CTCs by immunofluorescence (IF) that allows up to 6-parameter assessment. We have developed a set of prototype assays using tyramide amplification to detect AR and AR-V7 individually and in combination with prostate membrane-specific antigen (PSMA). Using a novel method, we also developed an assay to identify AR and AR-V7 simultaneously. Methods. Normal human whole blood samples were spiked with prostate cancer lines serving as model CTCs (mCTCs). Prostate cancer blood from a patient with advanced and known high CTC count were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer with CTC detection assays incorporating 4 core parameters (nuclear dye, CD45, pan-cytokeratin, EpCAM) plus the following markers: (1) AR; (2) AR-V7; (3) AR and PSMA; (4) AR-V7 and PSMA; (5) AR and AR-V7. Each assay was applied to the cancer cell lines PC3, LNCaP, and 22RV1 and to the patient sample. An antibody denaturing process was used to sequentially amplify two rabbit monoclonals for the assay containing both AR and AR-V7. No-primary (diluent only) staining controls were run to confirm success of the denaturing step. Results. Staining of spike-in CTC models confirmed the reported AR and AR-V7 phenotype of the cell lines, supporting the specificity of the assays. When applied to an equivalent volume of the patient sample, the assays identified a mean (SD) of 36 (11) CTCs. In the assays staining AR, the mean percent of AR+ CTCs was 70% (range 61 - 86) and for the assays staining AR-V7, the percent of AR-V7+ CTCs was 30% (range 26 - 39). In the assay staining both AR and AR-V7, 10 of 38 CTCs were AR-V7+ (26%); all of these were AR+; 15 CTCs were only AR+ (39%). Conclusions. We have developed multi-parameter IF assays for detection of AR and AR-V7 in prostate CTCs, including the first assay we are aware of to simultaneously visualize AR and AR-V7. It has been validated in prostate cancer CTC models. The percentages of AR and AR-V7-positive CTCs identified in a patient sample with the assay are consistent with the percentages observed when AR and AR-V7 are detected in independent assays, supporting the accuracy of the simultaneous detection assay. All AR-V7-positive CTCs were also AR-positive, supporting specificity of AR-V7 staining. Citation Format: Yao Sun, Daniel Campton, Arturo Ramirez, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel Sabath, Stephen Plymate, Eric Kaldjian. Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells [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 5230.

Novel method for rapid enrichment of high purity circulating tumor cells (CTCs) for prostate cancer (PCa) gene expression profiling.

271 Background: CTC RNA analysis currently involves single cell recovery that is laborious and expensive, or alternatively lysis of preserved whole blood which yields RNA predominantly from leukocytes which vastly outnumber CTCs. To effectively characterize gene expression in large patient cohorts, new enrichment methodologies are needed that yield high purity CTC populations while preserving RNA integrity. Here we describe a simple yet robust method for enrichment of prostate CTCs for gene expression analysis. Methods: Blood was drawn with informed consent under an IRB-approved protocol. For initial optimization, CFSE-stained PCa cells were spiked into healthy blood and recovered using various combinations of 2 methods: microfluidic enrichment (Parsortix™ system) and CD45 depletion. For assay qualification, a prostate-specific multiplexed qRT-PCR gene expression panel was developed. Enrichment and gene expression were tested initially using PCa cell lines spiked into healthy blood, then metastatic castrate resistant prostate cancer (mCRPC) blood samples in parallel with CellSearch enumeration. Results: Optimal enrichment of live cells was achieved with CD45 depletion followed by microfluidic enrichment, resulting in an average spiked cell recovery of 30% and approximately 100 contaminating background leukocytes. Using this enrichment method, prostate specific genes were detectable by multiplexed qRT-PCR down to 25 cells spiked into 7.5 ml whole blood, and transcripts were not measurable in matched healthy blood controls. When applied to mCRPC patient blood containing CTCs by CellSearch, multiplexed qRT-PCR successfully detected prostate specific genes in all samples. Conclusions: We developed a novel enrichment method capable of rapidly and efficiently recovering live CTCs with high purity, free of magnetic beads and with very few background leukocytes. Captured cells yielded high-quality RNA with high sensitivity and specificity for prostate-specific transcripts. This approach is applicable to high throughput gene expression profiling assays and offers an alternative to laborious single cell recovery or non-cancer-specific whole blood fixation.

Prostate Cancer Disseminated Tumor Cells are Rarely Detected in the Bone Marrow of Patients with Localized Disease Undergoing Radical Prostatectomy across Multiple Rare Cell Detection Platforms

Prostate circulating tumor cells escape into peripheral blood and enter bone marrow as disseminated tumor cells, representing an early step before conventionally detectable metastasis. It is unclear how frequently this occurs in localized disease and existing detection methods rely on epithelial markers with low specificity and sensitivity. We used multiple methodologies of disseminated tumor cell detection in bone marrow harvested at radical prostatectomy. Bone marrow was harvested from 208 clinically localized cases, 16 controls and 5 metastatic cases with peripheral blood obtained from 37 metastatic cases. Samples were evaluated at 4 centers with 4 distinct platforms using antibody enrichment with the AdnaTest (Qiagen®) or VERSA (versatile exclusion based rare sample analysis), or whole sample interrogation with the RareCyte platform (Seattle, Washington) or HD-SCA (high definition single cell assay) using traditional epithelial markers and prostate specific markers. We investigated the sensitivity and specificity of these markers by evaluating expression levels in control and metastatic cases. EpCAM, NKX3.1 and AR were nonspecifically expressed in controls and in most samples using AdnaTest with no relation to perioperative variables. Only 1 patient with localized disease showed positive results for the prostate specific marker PSA. With the VERSA platform no localized case demonstrated disseminated tumor cells. With the RareCyte and HD-SCA platforms only a single patient had 1 disseminated tumor cell. Evaluation across multiple platforms revealed that epithelial markers are nonspecific in bone marrow and, thus, not suitable for disseminated tumor cell detection. Using prostate specific markers disseminated tumor cells were typically not detected in patients with localized prostate cancer.

Label-free isolation of prostate circulating tumor cells using Vortex microfluidic technology

There has been increased interest in utilizing non-invasive “liquid biopsies” to identify biomarkers for cancer prognosis and monitoring, and to isolate genetic material that can predict response to targeted therapies. Circulating tumor cells (CTCs) have emerged as such a biomarker providing both genetic and phenotypic information about tumor evolution, potentially from both primary and metastatic sites. Currently, available CTC isolation approaches, including immunoaffinity and size-based filtration, have focused on high capture efficiency but with lower purity and often long and manual sample preparation, which limits the use of captured CTCs for downstream analyses. Here, we describe the use of the microfluidic Vortex Chip for size-based isolation of CTCs from 22 patients with advanced prostate cancer and, from an enumeration study on 18 of these patients, find that we can capture CTCs with high purity (from 1.74 to 37.59%) and efficiency (from 1.88 to 93.75 CTCs/7.5 mL) in less than 1 h. Interestingly, more atypical large circulating cells were identified in five age-matched healthy donors (46–77 years old; 1.25–2.50 CTCs/7.5 mL) than in five healthy donors <30 years old (21–27 years old; 0.00 CTC/7.5 mL). Using a threshold calculated from the five age-matched healthy donors (3.37 CTCs/mL), we identified CTCs in 80% of the prostate cancer patients. We also found that a fraction of the cells collected (11.5%) did not express epithelial prostate markers (cytokeratin and/or prostate-specific antigen) and that some instead expressed markers of epithelial–mesenchymal transition, i.e., vimentin and N-cadherin. We also show that the purity and DNA yield of isolated cells is amenable to targeted amplification and next-generation sequencing, without whole genome amplification, identifying unique mutations in 10 of 15 samples and 0 of 4 healthy samples.

Ninjurin1 Is Up-regulated in Circulating Prostate Tumor Cells and Plays a Critical Role in Prostate Cancer Cell Motility.

Ninjurin1 is a 17-kDa membrane protein that is highly expressed in circulating tumor cells (CTCs) obtained from locally-advanced prostate cancer patients. As CTCs are implicated in the initiation of distant metastasis, we examined the potential contribution of Ninjurin1 to the motility of prostate cancer cells. Ninjurin1 expression was evaluated in CTCs harvested from seven locally advanced patients with no metastatic hallmarks using real-time polymerase chain reaction (PCR). The role of Ninjurin1 in cell motility was investigated using small interfering RNA (siRNA), neutralizing antibodies against Ninjurin1 and Ninjurin1-overexpressing adenoviruses. Ninjurin1 was ranked as the most significantly up-regulated adhesion protein identified by RNA-Seq analysis. Both Ninjurin1 down-regulation by siRNA and neutralizing antibodies blocking Ninjurin1 homophilic interactions effectively inhibited cell motility. In contrast, cell motility was enhanced in prostate cancer cells infected with adenovirus enabling Ninjurin1 overexpression. Ninjurin1-neutralizing antibodies or Ninjurin1-targeting siRNA merit further development for patients with metastatic potential.

Analysis of DNA methylation in single circulating tumor cells.

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAMpos/CKpos/CD45neg/DAPIpos) and subsequent FACS sorting. In contrast to CD45pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

Accession of Tumor Heterogeneity by Multiplex Transcriptome Profiling of Single Circulating Tumor Cells.

Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. CTCs were enriched based on their EpCAM expression (CellSearch®) or by size and deformability (ParsortixTM), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

Abstract 4967: Label-free collection of prostate circulating tumor cells using microfluidic Vortex technology

Abstract BACKGROUND Prostate cancer is among the most common cancers in men worldwide. Better markers than Prostate Specific Antigen (PSA) are still needed for the detection and monitoring of disease progression. Circulating Tumor Cells (CTCs) are shed into the blood stream from primary tumor(s) and may play key roles in the metastatic process. Liquid biopsies have emerged as a promising approach, with a correlation between the CTC numbers and patient prognosis for prostate cancer. CTCs have also been shown to enable early detection of recurrence, and could be potential candidates for guiding cancer therapy in real-time [1]. Current CTC enrichment technologies, including immuno-affinity and size-based filtration methods, have focused on high capture efficiency with sometimes tedious sample preparation and overall low purity. METHOD Here, we describe the use of the microfluidic Vortex Chip [2] for rapid and size-based isolation of CTCs from the blood of 23 patients with advanced prostate cancer, and 10 healthy donors; 5 being &amp;lt;30 years old, 5 age-matched with the patient cohort. Requiring no upstream sample preparation, blood was diluted 10-fold and processed through the highly parallelized Vortex Chip at 8 mL/min (800 μL/min whole blood). Larger cells (predominantly CTCs) were captured in microscale vortices produced on the Chip, released into a small volume and collected off-chip for CK, PSA, CD45 and DAPI immunostaining and enumeration. RESULTS Preliminary work with LNCaP prostate cancer cells spiked in blood showed a 29% capture efficiency and 50% purity. In vitro cell assays confirmed that cells enriched with Vortex chip were alive and proliferating for up to 7 days. For 23 patient samples, CTCs were captured (0.5 - 20 CTCs/mL) with high purity (3.6 - 72.3%), in less than 1H, without prior sample preparation. 11.5% of the cells collected were CK and PSA-negative, but some were identified as undergoing epithelial-mesenchymal transition (EMT) following staining for vimentin and N-cadherin. Few atypical cells were also isolated from age-matched healthy donors (0.7 - 2.8 CTCs/mL), while none was detected in younger healthy donors. Using a threshold calculated from the age-matched healthy donors (3.31 CTCs/mL = mean + 2CV), 70% of the patients were characterized as “positive for CTCs”. No correlation was found between CTC counts and elevated PSA level. CONCLUSION These results demonstrate the ability to rapidly collect pure populations of CTCs in metastatic prostate cancer, independent of surface marker expression, without prior sample preparation. Future studies will use chips with optimized capture performance, sample recycling, and will include CTC molecular analysis by targeted panel sequencing. A larger cohort of healthy donors is also being examined to determine a statistically-robust CTC baseline for this size-based capture approach. [1] Scher Hi, et al., J. Clin. Oncol. 2015 [2] Sollier E, et al., Lab Chip 2014 Citation Format: Edward Pao, Corinne Renier, Clementine Lemaire, James Che, Melissa Matsumoto Di Carlo, Melanie Triboulet, Sandy Srivinas, Stefanie S. Jeffrey, Rajan P. Kulkarni, Matthew Rettig, Elodie Sollier, Dino Di Carlo. Label-free collection of prostate circulating tumor cells using microfluidic Vortex technology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4967.

Studying the gene expression profile among circulating tumor cells.

e23099 Background: Circulating tumor cells (CTCs) are cells that have detached from the primary tumor and flow into the blood or lymphatic circulation creating a metastasis. Despite their rare kind of cancer cells in the blood, CTCs are widely used as a predictive marker for cancer progression. The last years there have been made great efforts in isolating and characterizing the above cell population. However, it is not clear understood the mechanism by which CTCs contribute to metastasis and response to treatment. The present study aimed to identify the gene expression profile of CTCs in different cancer types. Methods: Blood samples were collected from 20 patients suffered from breast, colorectal, lung and prostate cancer, in different stages of the disease. CTCs were isolated from the above samples using enrichment isolation protocol. RNA was extracted and used as template for DNA microarray experiments, using the the Human MI ReadyArray platform. A commercial RNA, representing different human tissues, was used as reference sample. The microarray data were normalized using background subscription and analysis of variance was used. Genes with log2ratios of means > 2 were selected for further analysis. Results: In breast cancer more than 160 genes were over-expressed in all samples. The number was equal in prostate CTCs and lower on the other two types. The clustering analysis revealed that there was no gene which was over-expressed in all cases. The clustering analysis in pairs revealed that in all groups there were observed genes correlated with signal transduction pathways (NDRG2, ATF7, TAOK2, RNF24) and cytoskeleton (ACE, KLHL33). When the clustered group contained three types of cancer, the number of genes was quite lower; however, genes correlated with signaling were over-expressed. In clustering analysis of all samples, no gene was observed with log2ratio > 2. Conclusions: The present study demonstrated that CTCs from different cancer types have a different gene expression pattern. Furthermore, the identification of specific genes expressed in all samples, might contribute to new drug-able targets discovery.Further studies, in more samples, are needed to confirm all the above, so that it can be validate them in order to be used at clinical level.