Analysis Of Single Circulating Tumor Cells Research Articles

Deciphering the Biology of Circulating Tumor Cells through Single-Cell RNA Sequencing: Implications for Precision Medicine in Cancer.

Circulating tumor cells (CTCs) hold unique biological characteristics that directly involve them in hematogenous dissemination. Studying CTCs systematically is technically challenging due to their extreme rarity and heterogeneity and the lack of specific markers to specify metastasis-initiating CTCs. With cutting-edge technology, single-cell RNA sequencing (scRNA-seq) provides insights into the biology of metastatic processes driven by CTCs. Transcriptomics analysis of single CTCs can decipher tumor heterogeneity and phenotypic plasticity for exploring promising novel therapeutic targets. The integrated approach provides a perspective on the mechanisms underlying tumor development and interrogates CTCs interactions with other blood cell types, particularly those of the immune system. This review aims to comprehensively describe the current study on CTC transcriptomic analysis through scRNA-seq technology. We emphasize the workflow for scRNA-seq analysis of CTCs, including enrichment, single cell isolation, and bioinformatic tools applied for this purpose. Furthermore, we elucidated the translational knowledge from the transcriptomic profile of individual CTCs and the biology of cancer metastasis for developing effective therapeutics through targeting key pathways in CTCs.

Abstract B045: Single cell proteomics as a method to analyze circulating tumor cells in prostate cancer patients

Abstract The purpose of this study was to develop a single-cell proteomic protocol capable of analyzing single circulating tumor cells (CTCs). Despite the numerous benefits possible from the analysis of CTCs they have not migrated into clinical use due to difficulty of purification and their minute numbers. Combined, this results in only miniscule amounts of sample, if any, for analysis. Most clinical assays primarily focus on genomic and/or transcriptomic assays, while only a select number of proteomic assays are used and these analyze a handful of targets at most. Recent developments in single-cell proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS) promise to solve this latter issue and increase the depth of proteome coverage. To analyze CTCs we adapted previously developed single-cell methodologies to be amenable for the analysis of CTCs. Our novel method uses a separately prepared carrier channel as opposed to traditional methods which rely on a large pool of the single-cells of interest. This is necessary since collected CTC’s will not have a large pool of similar cells to draw upon to form a carrier. As the carrier is prepared in bulk it allows for improved quality control measurements to be implemented. This is critical as for clinical use these metrics will be vital for ensuring repeatability and reliability of results. Using our developed protocol, we are able to analyze hundreds of proteins from each simulated CTC. Additionally, we demonstrate that a separately prepared carrier composed of cells from a different lineage still permits the identification of a wide array of proteins within single-cells as well as allows for better quality control over the digestion and TMT labeling efficiencies. In conclusion, our protocol demonstrates its capability to detect and quantify hundreds of proteins from individual cells. If this method was applied towards the analysis of CTCs it would represent by far the most comprehensive method for analysis of the proteome of single CTCs. Together, this provides a foundation for translating proteomic analysis of single CTCs towards guiding individualized cancer treatment in the clinic. Citation Format: Alec Horrmann, Zoi Sychev, Justin M. Drake. Single cell proteomics as a method to analyze circulating tumor cells in prostate cancer patients [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 B045.

Single-Circulating Tumor Cell Whole Genome Amplification to Unravel Cancer Heterogeneity and Actionable Biomarkers.

The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. In this regard, analyzing single circulating tumor cells (CTCs) is becoming particularly attractive due to the easy access to CTCs from simple blood samples called “liquid biopsies”. Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response. However, single-CTC analysis is restricted by the low amounts of DNA in a single cell genome. Whole genome amplification (WGA) techniques have emerged as a key step, enabling single-cell downstream molecular analysis. Here, we provide an overview of recent advances in WGA and their applications in the genetic analysis of single CTCs, along with prospective views towards clinical applications. First, we focus on the technical challenges of isolating and recovering single CTCs and then explore different WGA methodologies and recent developments which have been utilized to amplify single cell genomes for further downstream analysis. Lastly, we list a portfolio of CTC studies which employ WGA and single-cell analysis for genetic heterogeneity and biomarker detection.

Integrated Workflow for the Label-Free Isolation and Genomic Analysis of Single Circulating Tumor Cells in Pancreatic Cancer.

As pancreatic cancer is the third deadliest cancer in the U.S., the ability to study genetic alterations is necessary to provide further insight into potentially targetable regions for cancer treatment. Circulating tumor cells (CTCs) represent an especially aggressive subset of cancer cells, capable of causing metastasis and progressing the disease. Here, we present the Labyrinth–DEPArray pipeline for the isolation and analysis of single CTCs. Established cell lines, patient-derived CTC cell lines and freshly isolated CTCs were recovered and sequenced to reveal single-cell copy number variations (CNVs). The resulting CNV profiles of established cell lines showed concordance with previously reported data and highlight several gains and losses of cancer-related genes such as FGFR3 and GNAS. The novel sequencing of patient-derived CTC cell lines showed gains in chromosome 8q, 10q and 17q across both CTC cell lines. The pipeline was used to process and isolate single cells from a metastatic pancreatic cancer patient revealing a gain of chromosome 1q and a loss of chromosome 5q. Overall, the Labyrinth-DEPArray pipeline offers a validated workflow combining the benefits of antigen-free CTC isolation with single cell genomic analysis.

Transcriptomic profiling of single circulating tumor cells provides insight into human metastatic gastric cancer

Transcriptome analysis of circulating tumor cells (CTCs), which migrate into blood vessels from primary tumor tissues, at the single-cell level offers critical insights into the biology of metastasis and contributes to drug discovery. However, transcriptome analysis of single CTCs has only been reported for a limited number of cancer types, such as multiple myeloma, breast, hepatocellular, and prostate cancer. Herein, we report the transcriptome analysis of gastric cancer single-CTCs. We utilized an antigen-independent strategy for CTC isolation from metastatic gastric cancer patients involving a size-dependent recovery of CTCs and a single cell isolation technique. The transcriptomic profile of single-CTCs revealed that a majority of gastric CTCs had undergone epithelial-mesenchymal transition (EMT), and indicated the contribution of platelet adhesion toward EMT progression and acquisition of chemoresistance. Taken together, this study serves to employ CTC characterization to elucidate the mechanisms of chemoresistance and metastasis in gastric cancer.

Circulating Tumor Cell Clusters Are Cloaked with Platelets and Correlate with Poor Prognosis in Unresectable Pancreatic Cancer

Simple SummaryDespite recent advances, some patients with pancreatic cancer are refractory to treatment and the disease rapidly progresses, resulting in early death. The potential prognostic value of circulating tumor cells (CTCs) has been demonstrated in other cancer types, but the clinical validity in pancreatic cancer remains elusive. Here, we show that CTC clusters, which show mesenchymal characteristics and platelet marker expression, are highly correlated with poor prognosis in patients with unresectable pancreatic cancer.Circulating tumor cells (CTCs) are known to be heterogeneous and clustered with tumor-associated cells, such as macrophages, neutrophils, fibroblasts, and platelets. However, their molecular profile and clinical significance remain largely unknown. Thus, we aimed to perform a comprehensive gene expression analysis of single CTCs and CTC clusters in patients with pancreatic cancer and to identify their potential clinical relevance to provide personalized medicine. Epitope-independent, rapid (>3 mL of whole blood/min) isolation of single CTCs and CTC clusters was achieved from a prospective cohort of 16 patients with unresectable pancreatic cancer using a centrifugal microfluidic device. Forty-eight mRNA expressions of individual CTCs and CTC clusters were analyzed to identify pancreatic CTC phenotype. CTC clusters had a larger proportion of mesenchymal expression than single CTCs (p = 0.0004). The presence of CTC clusters positively correlated with poor prognosis (progression-free survival, p = 0.0159; overall survival, p = 0.0186). Furthermore, we found that most CTCs in these patients (90.7%) were cloaked with platelets and found the presence of a positive correlation between the increase in CTC clusters and rapid disease progression during follow-ups. Efficient CTC cluster isolation and analysis techniques will enhance the understanding of complex tumor metastasis processes and can facilitate personalized disease management.

Application of microfluidics-based technologies in the analysis of circulating tumor cells heterogeneity

<p indent=0mm>Circulating tumor cells (CTCs) play a critical role in the fluid biopsy, which has great potential for the low-invasive or even non-invasive analysis of tumor cells in body fluids. During the process of proliferation and metastasis, variation among tumor cells frequently occurs, including gene mutations, proteomic and epigenetic changes, bringing in great challenges to clinical diagnosis and treatment. Single CTCs analysis can reveal the intrinsic heterogeneity among the tumor cells, which is significant for early cancer diagnosis, therapeutic response and prognosis prediction. Microfluidic-based technologies for CTCs analysis have been considered as a skillful way because of its small size, easy manipulation, low cost and so on. In this review, we mainly discuss the application of microfluidics involved single-cell technology for providing novel insights into cellular heterogeneity and cancer evolution.

In Situ Single Cell Proteomics Reveals Circulating Tumor Cell Heterogeneity during Treatment.

Cancer is a dynamic disease with heterogenic molecular signatures and constantly evolves during the course of the disease. Single cell proteomic analysis could offer a suitable pathway to monitor cancer cell heterogeneity and deliver critical information for the diagnosis, recurrence, and drug-resistant mechanisms in cancer. Current standard techniques for proteomic analysis such as ELISA, mass spectrometry, and Western blots are time-consuming, expensive, and often require fluorescence labeling that fails to provide accurate information about the multiple protein expression changes at the single cell level. Herein, we report a surface-enhanced Raman spectroscopy-based simple microfluidic device that enables the screening of single circulating tumor cells (CTC) in a dynamic state to precisely understand the heterogeneous expression of multiple protein biomarkers in response to therapy. It further enables identifying intercellular heterogeneous expression of CTC surface proteins which would be highly informative to identify the cancer cells surviving treatment and potentially responsible for drug resistance. Using a bead and cell line-based model system, we successfully detect single bead and single cell spectra when flowed through the device. Using SK-MEL-28 melanoma cells, we demonstrate that our system is capable of monitoring heterogeneous expressions of multiple surface protein markers (MCSP, MCAM, and LNGFR) before and during drug treatment. Integrating a label-free electrochemical system with the device, we also monitor the expression of an intracellular protein (here, BRAFV600E) under drug treatment. Finally, we perform a longitudinal study with 15 samples from five different melanoma patients who underwent therapy. We find that the average expression of receptor proteins in a patient fails to determine the therapy response particularly when the disease progresses. However, single CTC analysis with our device shows a high level of intercellular heterogeneity in the receptor expression profiles of patient-derived CTCs and identifies heterogeneity within CTCs. More importantly, we find that a fraction of CTCs still shows a high expression of these receptor proteins during and after therapy, indicating the presence of resistant CTCs which may evolve after a certain time and progress the disease. We believe this automated assay will have high clinical importance in disease diagnosis and monitoring treatment and will significantly advance the understanding of cancer heterogeneity on the single cell level.

Androgen receptor variant shows heterogeneous expression in prostate cancer according to differentiation stage

Quantitation of androgen receptor variant (AR-V) expression in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) has great potential for treatment customization. However, the absence of a uniform CTC isolation platform and consensus on an analytical assay has prevented the incorporation of these measurements in routine clinical practice. Here, we present a single-CTC sensitive digital droplet PCR (ddPCR) assay for the quantitation of the two most common AR-Vs, AR-V7, and AR-v567es, using antigen agnostic CTC enrichment. In a cohort of 29 mCRPC patients, we identify AR-V7 in 66% and AR-v567es in 52% of patients. These results are corroborated using another gene expression platform (NanoStringTM) and by analysis of RNA-Seq data from patients with mCRPC (SU2C- PCF Dream Team). We next quantify AR-V expression in matching EpCAM-positive vs EpCAM-negative CTCs, as EpCAM-based CTC enrichment is commonly used. We identify lower AR-V prevalence in the EpCAM-positive fraction, suggesting that EpCAM-based CTC enrichment likely underestimates AR-V prevalence. Lastly, using single CTC analysis we identify enrichment for AR-v567es in patients with neuroendocrine prostate cancer (NEPC) indicating that AR-v567es may be involved in lineage plasticity, which warrants further mechanistic interrogation.

Tumor Evolution and Therapeutic Choice Seen through a Prism of Circulating Tumor Cell Genomic Instability.

Circulating tumor cells (CTCs) provide an accessible tool for investigating tumor heterogeneity and cell populations with metastatic potential. Although an in-depth molecular investigation is limited by the extremely low CTC count in circulation, significant progress has been made recently in single-cell analytical processes. Indeed, CTC monitoring through molecular and functional characterization may provide an understanding of genomic instability (GI) molecular mechanisms, which contribute to tumor evolution and emergence of resistant clones. In this review, we discuss the sources and consequences of GI seen through single-cell analysis of CTCs in different types of tumors. We present a detailed overview of chromosomal instability (CIN) in CTCs assessed by fluorescence in situ hybridization (FISH), and we reveal utility of CTC single-cell sequencing in identifying copy number alterations (CNA) oncogenic drivers. We highlight the role of CIN in CTC-driven metastatic progression and acquired resistance, and we comment on the technical obstacles and challenges encountered during single CTC analysis. We focus on the DNA damage response and depict DNA-repair-related dynamic biomarkers reported to date in CTCs and their role in predicting response to genotoxic treatment. In summary, the suggested relationship between genomic aberrations in CTCs and prognosis strongly supports the potential utility of GI monitoring in CTCs in clinical risk assessment and therapeutic choice.

Development of High Throughput Single-Cell Analysis System for Circulating Tumor Cells Based on Digital Micromirror Device

Circulating tumor cells (CTCs) are widely known as useful biomarkers in the liquid biopsies of cancer patients. Recently, in addition to counting the number of CTCs, genetic analysis of CTCs provides critical insights into cancer metastases and add detailed clinical information that enhances patient care. It is widely known that CTCs are genetically heterogeneous, the molecular characterization of CTCs should be performed at the single-cell level. In fact, several studies have identified inter- and intra-patient heterogeneity in the mutational status of CTCs in metastatic breast and lung cancers. These studies also reported that CTCs related to drug resistance are novel therapeutic targets. Thus, genetic analysis of single CTCs has the potential to be widely applicable in personalized medicine and drug discovery. However, CTCs are extremely rare cells that only 1-100 cells are contained in 1 ml of blood, so the recovery of such extremely rare CTCs from the peripheral blood is technically challenging. So far, we have developed a CTC recovery system using a microcavity array (MCA), which demonstrates highly efficient recovery of cancer cells based on differences in cell size and deformability. Furthermore, we have proposed a novel cell manipulation method through the visualization of single cells through hydrogel encapsulation for subsequent genetic analysis of single CTCs. Subsequently, recovered CTCs can be subjected to a hydrogel-encapsulation for the following genetic analysis of single cells. Clinical studies indicated that the genetic analysis of CTCs from lung, pancreatic and gastric cancer patients was successfully achieved. However, this hydrogel encapsulation has limitations in throughput. In this study, we demonstrate a high-throughput single-cell analysis for CTCs using a multiple single-cell encapsulation system with a digital micromirror device (DMD). The novel multiple single-cell encapsulation systemwas composed of two optical systems: the wide-field fluorescence imaging system and the photopolymerization system equipped with DMD. The wide-field fluorescence imaging system using a CMOS sensor was designed to visualize 2D fluorescence imaging of the entire MCA (6.0 × 6.0 mm2). We evaluated the sensitivity of fluorescence detection of the wide-field fluorescence imaging system using the calibration slide. The limit of detection of the wide-field fluorescence imaging system was 2.1 × 102 molecules-Cy3/μm2, which is comparable to conventional fluorescence microscopy (1.0 × 102 molecules-Cy3/μm2). Generally, the expression of cellular marker protein, such as cytokeratin, was estimated to be 1.3 × 103 molecules/μm2. Therefore, our proposed system had enough sensitivity for the detection of antibody-stained CTCs. Furthermore, this wide-field fluorescence imaging system for CTC detection enables the rapid visualization of all stained cells within the field of the MCA via one-shot imaging. This rapid detection method also allows for the rapid completion of single-CTC manipulation. The photopolymerization system for single-cell encapsulation was controlled by the DMD. The curing light (λmax = 365 nm) modulated by the DMD was projected onto an MCA for hydrogel generation, and single-cell isolation by hydrogel-photopolymerization was examined at the optimized conditions. After recovery of cancer cells from whole blood onto MCA and staining by the fluorescent-labeled antibodies, cancer cells were identified by the wide-field imaging system. Then light irradiation (3 sec) was performed by the photopolymerization system. To minimize the area of the hydrogel reserved for a single cell, we newly designed the convex-type hydrogel. In the spike-in experiment, single-cell isolation rates were 97.6% (41/42) for NCI-H1975 cells (non-small cell lung cancer cell) and 94.4% (51/54) for NCI-N87 (gastric cancer cell) that were comparable with our previous study using fluorescence microscopy. With this proposed system, more than 50 single cells were encapsulated simultaneously within 1 min and isolated with an efficiency. Furthermore, single cells were successfully isolated from adjacent cells on the same microcavity without any contamination. Single cancer cell was partially encapsulated on the hydrogel, and the partial encapsulation allows us to subject them to whole genome amplification (WGA). Light irradiation and photopolymerizaion did not affect the quality of the amplified WGA products for single-cell genomic analyses. We are currently investigating single cell genetic analysis of metastatic cancer patients, and this developed multiple single-cell encapsulation system will improve the high throughput single-cell genetic analysis of CTCs in clinical samples. Our system provides an attractive application for other targets such as adherent cells, tissue samples, and microorganisms, in addition to widespread use in the isolation of CTCs for liquid biopsy.

Self-Assembled GO Honeycomb Microarray for Selective Cancer Cell Capture and Single Cell Analysis of Proteolytic Expression.

Proteolytic enzymes expressed by circulating tumor cells are proved to facilitate their invasion into multiple organs via cleaving natural ECM networks, leading to consequent metastasis colonization and malignant lethality. Recent evidence suggests the rare metastasis initiating cells with higher proteolytic levels among circulating tumor cells (CTCs) may strongly increase the risk of metastasis. Beyond selective CTC capture, the heterogeneity in proteases expression provides a promising indicator for metastasis happening. To this end, the graphene oxide (GO) honeycomb microarray with single CTC matched sizes is fabricated via the self-assembly breath figure approach, which serves as an integrated protocol for selective CTC capture and single-cell analysis of protease activity. Contributing to synergistic effects of structure and chemistry, CTCs can be efficiently isolated and individually trapped in each honeycomb hole. Meanwhile, the crosstalk among CTCs can be erased by blocking direct cell-to-cell contact, which offers promising potentials in the single-cell analysis of protease expression. Integrating specific capture and in situ analysis of single CTCs on GO micropatterned surface is of significant importance in various biological and clinical applications such as cancer diagnostics and cancer therapeutic evaluation.

Genomic Analysis of Circulating Tumor Cells at the Single-Cell Level

Circulating tumor cells (CTCs) have a great potential for noninvasive diagnosis and real-time monitoring of cancer. A comprehensive evaluation of four whole genome amplification (WGA)/next-generation sequencing workflows for genomic analysis of single CTCs, including PCR-based (GenomePlex and Ampli1), multiple displacement amplification (Repli-g), and hybrid PCR- and multiple displacement amplification–based [multiple annealing and loop-based amplification cycling (MALBAC)] is reported herein. To demonstrate clinical utilities, copy number variations (CNVs) in single CTCs isolated from four patients with squamous non–small-cell lung cancer were profiled. Results indicate that MALBAC and Repli-g WGA have significantly broader genomic coverage compared with GenomePlex and Ampli1. Furthermore, MALBAC coupled with low-pass whole genome sequencing has better coverage breadth, uniformity, and reproducibility and is superior to Repli-g for genome-wide CNV profiling and detecting focal oncogenic amplifications. For mutation analysis, none of the WGA methods were found to achieve sufficient sensitivity and specificity by whole exome sequencing. Finally, profiling of single CTCs from patients with non–small-cell lung cancer revealed potentially clinically relevant CNVs. In conclusion, MALBAC WGA coupled with low-pass whole genome sequencing is a robust workflow for genome-wide CNV profiling at single-cell level and has great potential to be applied in clinical investigations. Nevertheless, data suggest that none of the evaluated single-cell sequencing workflows can reach sufficient sensitivity or specificity for mutation detection required for clinical applications.

An Integrated Microfluidic Chip and Its Clinical Application for Circulating Tumor Cell Isolation and Single-Cell Analysis.

Circulating tumor cells (CTCs) represent invasive tumor cell populations and provide a noninvasive solution to the clinical management and research of tumors. Characterization of CTCs at single-cell resolution enables the comprehensive understanding of tumor heterogeneity and may benefit the diagnosis and treatment of cancer patients. However, most efforts have been made on enumeration and detection of CTCs, while little focus has been directed to single-cell study. Herein, an integrated microfluidic platform for single-cell isolation and analysis was established. After validating this platform on lung cancer cell lines, we detected and isolated single CTCs from the peripheral blood samples of 20 cancer patients before and after one treatment cycle. Furthermore, we performed single-cell whole-exome DNA sequencing on a single CTC from the peripheral blood sample of a representative early stage lung cancer patient. Among the blood samples of 20 patients, 15 of them were positive for CTC detection (75.0% detectable rate). Single-cell analysis revealed detailed genetic variations of the CTC, while six new gene mutations were detected in both single CTC and surgical specimen. This study provides a useful tool for the isolation and analysis of single CTCs from peripheral blood samples, which not only facilitates the early diagnosis of cancers but also helps to unravel the genetic information of tumor at a single-cell level. © 2019 International Society for Advancement of Cytometry.

The application of nano-enrichment in CTC detection and the clinical significance of CTCs in non-small cell lung cancer (NSCLC) treatment.

Circulating tumor cells (CTCs) are an independent prognostic marker in non-small cell lung cancer (NSCLC). CTC numbers are closely related to early diagnosis, clinical stage, therapy surveillance, and prognosis of NSCLC. We used a more efficient nano-enrichment method to detect CTCs in NSCLC patients and explored the clinical value of CTCs. The results showed that CTC numbers in stage IV cases were significantly higher than those in stage I, II or III cases. The number of CTCs in poorly-differentiated cases was significantly higher than that in well-differentiated cases. During six chemotherapy cycles, the average CTC number decreased from 5.8/7.5 ml in cycle #1 to 2.4/7.5 ml in cycle #4 and remained at almost the same level from 4 to 6 cycles. CTC numbers in patients with EGFR mutations was significantly higher than those in patients with no mutations. The average progression free survival (PFS) in the favorable group (CTC ≤ 5/7.5 ml) was 11.3 months, which was longer than that in the unfavorable group (CTC > 5/7.5 ml, 7.2 months). In conclusion, the assessment of NSCLC cannot be performed using a single CTC analysis. The clinical value is more significant in the continuous analysis of CTC data, as well as the cross-validation of other indexes and imaging results.

Abstract 451: Transferrin Receptor identifies a distinct pool of circulating tumor cells from metastatic prostate cancer patients with unique molecular profiles enriched in AR variants

Abstract Androgen receptor (AR) drives prostate cancer (PC) progression, even in castrate-conditions due to expression of AR variants (AR-V), which are constitutively active in the nucleus. Castration-resistant prostate cancer (CRPC) is currently lethal, due to the development of drug resistance. The molecular basis of clinical drug resistance remains poorly elucidated, due to tumor heterogeneity and limited availability of serial tumor sampling as disease progresses. Circulating tumor cells (CTCs), isolated from patient peripheral blood, have emerged as a reliable, easy to obtain source of tumor cells which can be and used to interrogate the molecular disease evolution. To date, EpCAM-based CTC enrichment is commonly used but has important shortcomings as EpCAM is often lost during EMT, thus, missing the more aggressive mesenchymal CTCs. On the other hand, Transferrin Receptor (TfR) which is overexpressed in cancer cells, is a cell surface protein required for iron uptake and is critical for several biological processes such as cell proliferation and metabolism. Mining large gene expression databases in PC we observed that TfR expression increased with disease stage, from adenocarcinoma (TCGA) to CRPC (SU2C) to the more aggressive neuroendocrine PC (NEPC), while EpCAM expression remained unchanged. We also found that TfR expression was increased in EMT cell models, while EpCAM expression was lost, suggesting that TfR may perform well in CTC isolation across the EMT spectrum. We then tested TfR- and EpCAM-based CTC enrichment in a cohort of 31 CRPC patients, where we observed a significantly higher number of TfR+ CTCs (median, 304) than EpCAM+-CTCs (median, 21) (p&amp;lt;0.01). RNA-sequencing of TfR+ CTCs in comparison to EpCAM+ CTCs, followed by gene-set enrichment analyses revealed that the TfR+ subpopulation was significantly enriched in prostate specific oncogenic pathways including EMT, suggesting distinct molecular profiles between the two subsets. To investigate the clinical relevance of the two CTC subsets, we quantitated AR-FL and splice variants AR-V7 and AR-v567es expression by digital droplet PCR (ddPCR) in pools of TfR+-CTCs vs EpCAM+-CTCs. Our results from 16 CRPC patients showed that while expression of AR-FL was similar in the two CTC subsets, both AR variants were enriched in TfR+-CTCs. To better map the tumor heterogeneity of AR-V expression we isolated 102 single TfR+- and 63 single EpCAM+-CTCs from 3 CRPC patients and quantified AR-V expression by ddPCR. The single CTC analysis revealed that AR-V7 and AR-v567es were expressed in 21% and 18% of TfR+-CTCs, respectively, vs 0% in EpCAM+-CTCs. These data identify TfR as a promising marker for CTC identification across the EMT gradient, and provide a unique insight into tumor cell heterogeneity, which was not appreciated before, and which may have significant clinical impact. [A. Gjyrezi and G. Galletti contributed equally to this work.] Citation Format: Ada Gjyrezi, Giuseppe Galletti, Jiaren Zhang, Rohan Bareja, Ahmed Halima, Daniel Worroll, David Nanus, Scott Tagawa, Himisha Beltran, Paraskevi Giannakakou. Transferrin Receptor identifies a distinct pool of circulating tumor cells from metastatic prostate cancer patients with unique molecular profiles enriched in AR variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 451.

Highly multiplexed profiling of cell surface proteins on single circulating tumor cells based on antibody and cellular barcoding

Circulating tumor cells (CTCs) are extraordinarily rare in blood samples and represent a real-time "liquid biopsy" of tumors. Although genetic and transcriptional sequencing of single CTCs has been reported, these methods fail to provide phenotypic and functional information of CTCs such as protein levels of surface proteins. Studies ofsingle-cell proteomic assays of CTCs have been rare because of a lack of single-cell proteomic methods to handle and analyze rare cells in a high background of non-target cells with high sensitivity, throughput, and multiplexing capacity. Here, we develop a microchip-assisted single-cell proteomic method for profiling surface proteins of CTCs based on antibody and cellular DNA barcoding strategy. We combine DNA-encoded antibody tags and cell indexes to profile 15 proteins in ~ 100 single rare cells simultaneously, and use high-throughput sequencing as the readout to generate surface protein profiles of CTCs according to their cell indexes and antibody-derived protein barcodes. A 6400-well microchip and the automated puncher are used to rapidly retrieve single CTCs from enriched CTC population with minimal cell loss (~ 10%). This technological platform integrates reliable isolation and proteomic analysis of single CTCs and can be extendable to ~ 100 proteins in hundreds of rare cells with single-cell precision.

Abstract P3-01-09: Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient

Abstract Background: Copy number variation analysis has been shown as a valuable tool to infer clonal evolution of tumor cells. While bulk genomic analysis of FFPE tissue can provide information about the characteristics of a primary tumor or a metastatic nodule, these biopsies are rarely repeated due to their invasive nature. Liquid biopsies on the other hand, provide minimally invasive access to tumor derived cells and DNA and can therefore be used to monitor genomic changes of the tumor longitudinally and in real time. Here we evaluate the kinetics of liquid biopsy biomarkers and monitor genomic changes in single circulating tumor cells (CTCs) and cell-free DNA (cfDNA) from 17 longitudinal blood draws collected from a metastatic breast cancer patient over seven treatment regimes. FFPE tissue from the primary tumor and two metastatic sites in liver and bone are available for comparative analysis. Methods: We used the high definition – single cell assay (HD-SCA) platform to detect and enumerate CTCs of the blood of one metastatic breast cancer patient followed for 4 years. CTCs were characterized morphometrically and single CTCs of each draw were sequenced for copy number variation (CNV). cfDNA was extracted from plasma and tissue samples of the primary breast, bone and liver metastasis were micro-dissected. Extracted DNA from tissue samples and plasma underwent CNV analysis and were compared with CNV data of CTCs. Results: CTCs were identified in 14/17 draws and varied from 0 to &amp;gt;3000 cells/ml, spiking at two different times concordant with clinical progression. Changes in cell free tumor DNA fraction (ctDNA) correlated strongly with CTC count. Comparative CNV analysis of CTCs, cfDNA and FFPE tissue confirmed the tumorous origin of both cells and cfDNA. Single cell CNV analysis found four genomically related sub-clones in the patient's blood, three of which were present at time of enrollment, while the fourth clone emerged 3.5 years later concordant with the decline in the patient's health. Genomic copy number losses included tumor suppressor genes such as ARID1A, FOXP1, ATM, BRCA2, RB1, CDH11 and CDH1. Genomic gains were limited but included the proto-oncogenes BCL9, ABL2 and MDM4. It is striking that the sub-clonal structure persisted nearly unchanged for 3.5 years throughout various hormone and cytotoxic treatments, indicating high genomic stability. Despite their genomic differences, cells from the different clones are indistinguishable by immunofluorescence or morphometric analysis. CNV profiles of cfDNA reflect the most abundant CTC clone at each time-point. Conclusion: Using the HD-SCA assay cellular (CTC), and soluble (cfDNA) fractions of liquid biopsies can be compared at the molecular level over a long time course, and verified by comparison with tissue biopsy. This work confirms that liquid biopsies can be used to access the molecular state of a patient's cancer in near real-time and provide insight into the response to treatment years after the original characterization of the primary tissue. Here we show in a single patient example the power of single CTC analysis and highlight how CNV analysis of liquid biopsies could serve as minimally invasive tool to monitor tumor evolution longitudinally. Citation Format: Welter L, Xu L, McKinley D, Dago AE, Restrepo-Vassalli S, Rodriguez Lee M, Kolatkar A, Nieva J, Hicks J, Kuhn P. Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient [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 P3-01-09.

Beyond Capture: Circulating Tumor Cell Release and Single‐Cell Analysis

AbstractAs the most promising “liquid biopsy”, analysis of circulating tumor cells (CTCs) provides noninvasive access to obtain biological information of solid tumors. It not only advances the understanding of the mechanisms underlying tumor metastasis, relapse, and chemoresistance, but also promotes the development of precision medicine. Over the past decade, the rapid advances in micro/nanomaterials and microfluidics have overcome the technical challenges of capturing CTCs, allowing efficient enrichment and sensitive detection. Beyond the capture of CTCs, major challenges of in‐depth CTC analysis are the release of CTCs from capture platforms and the inherent heterogeneity in CTCs. A variety of technologies have now emerged for release and single‐cell analysis of CTCs. This review focuses on recent progress along this direction. First, different release strategies are outlined and discussed, including their design principles and characteristics (merits and limitations). Then, existing methods for single CTC analysis at the genomic, transcriptomic, proteomic, and functional level are summarized. Finally, some perspectives are provided on current development trends, future research directions, and challenges of CTC studies.

Rare Event Phenotyping and Molecular Characterization: Circulating Tumor Cells.

Noninvasive isolation of circulating tumor cells (CTCs) from patient blood samples allows for interrogation of valuable molecular and phenotypic information useful for disease diagnosis and monitoring response to therapy. However, CTCs are extremely rare relative to red and white blood cells (R/WBC), thus making CTC isolation from unmanipulated whole blood very time-consuming. Moreover, single CTC analysis often requires hand-picking, a step that can result in more CTC loss and compromised cell integrity. Here we describe an automated flow cytometry-based approach for isolation and analysis of single, viable CTCs that combines gentle RBC lysis and magnetic, no-wash negative-depletion of WBCs, followed by a highly adaptable sorting protocol for rare cells of interest. Multiparametric flow-cytometric panels allow probing of numerous extracellular markers for immunophenotyping, while whole transcriptome analysis contributes to molecular characterization of individual CTCs. Index sorting links single CTC proteogenomics information.