Cancer Circulating Tumor Cells Research Articles

Abstract A003: Characterization of circulating tumor cells from patients with metastatic bladder cancer

Abstract Assessing the molecular profile of bladder tumors from patients with metastatic disease is crucial for implementing personalized treatment. Current standards are based on highly invasive biopsies. Alternatively, liquid biopsies of tumor cells, proteins, and nucleic acids allows longitudinal profiling in a less invasive manner. Circulating tumor cells (CTCs) isolated from blood are a key analyte of liquid biopsy. In this study, we developed a protein and transcriptomic co-analysis workflow for bladder cancer CTCs utilizing the graphene oxide (GO) microfluidic chip conjugated with antibodies against both EpCAM and EGFR to isolate CTCs from blood of patients with metastatic bladder cancer. Following CTC capture, protein and mRNA were analyzed using immunofluorescent staining and ion-torrent whole transcriptome sequencing. This is the first time that whole transcriptome sequencing was used in a downstream application for the GO microfluidic platform. We found that elevated CTC counts were significantly associated with patient disease status at the time of blood draw and that greater than 2 CTCs per mL were associated with shorter overall survival. Invasive markers, including EGFR, HER-2, CD31, and ADAM15, were detected in subpopulations of CTCs. Targeted whole transcriptome sequencing showed distinct expression profiles from patients with metastatic burden at the time of blood draw. In these patients, we found significant upregulation of several metastasis-related and chemotherapy-resistant genes demonstrating the capability of identifying tumor-related RNA signatures from circulating CTCs using immune-affinity GO chip-based assays. In addition, this workflow highlights the prognostic potential of CTCs in metastatic bladder cancer patients. Citation Format: Zeqi Niu, Molly Kozminski, Kathleen C. Day, Luke Broses, Marian L. Henderson, Sophia Merajver, Aaron Udager, Phillip L. Palmbos, Sunitha Nagrath, Mark L. Day. Characterization of circulating tumor cells from patients with metastatic bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A003.

Rapid Isolation and Detection of Breast Cancer Circulating Tumor Cells Using Microfluidic Sequential Trapping Array

AbstractCirculating tumor cells (CTCs) have garnered special attention as promising cancer biomarkers. Phenotypic changes of CTCs reveal invaluable information for oncologists in disease prognosis and adjusting their treatment options. Microfluidic technology has emerged as a promising tool for CTC isolation; however, two major hurdles remain to be solved in employing them in CTC analysis. First, a rapid CTC isolation scheme is needed to allow immediate use of patient samples for point‐of‐care treatment monitoring. Second, multiplexed and streamlined CTC imaging is needed to facilitate CTC detection. Here, a microfluidic CTC sequential trapping array (STA) is proposed which addresses these hurdles enabling pipette‐based CTC isolation and simultaneous profiling of multiple CTC protein expressions. The STA device isolates CTCs based on their size difference from blood cells and increases sample processing throughput through its parallel design configuration. It successfully isolates CTC from a depleted peripheral blood mononuclear cells sample of breast cancer patients with a high recovery rate of 80% and discriminates the number and types of CTCs in breast cancer based on their disease stage. These findings will open a new avenue in clinical translation of CTC profiling technologies. It will be an example for future translational developments in CTC‐based cancer management.

Colorimetric detection of circulating tumor cells in breast cancer based on ladder-branch hybridization chain reaction and DFs/AuNCs nanozyme

Breast cancer is the most common malignant tumor in women, which accounts for 6.9% of all cancer-related deaths. Early diagnosis is crucial for making the best clinical decision and improving the prognosis of patients. Circulating tumor cells (CTCs) have been regarded as significant tumor biomarkers. Herein, we designed a colorimetric biosensor for breast cancer CTCs quantification based on ladder-branch hybridization chain reaction (HCR) and DNA flowers/gold nanoclusters (DFs/AuNCs) nanozyme. With the assistance of complementary DNA labeled on magnetic beads (MBs), the cleavage products of RNA-cleaving DNAzymes (RCDs) could be rapidly captured, subsequently triggering ladder-branch HCR. In addition, the DFs/AuNCs nanozyme was applied for colorimetric analysis, which further improved the sensitivity for the detection of target CTCs. Benefiting from specific RCDs, ladder-branch HCR and DFs/AuNCs, we achieved a superior detection limit of 3 cells/mL as well as a broad linear range of 10 cells/mL to 104 cells/mL. Conclusively, this colorimetric biosensor achieved sensitively and selectively detection of breast cancer CTCs without the participation of enzymes at room temperature, which might provide new insight into the early detection of breast cancer.

Abstract 2757: Investigating regulatory drivers of prostate cancer metastasis in circulating tumor cells

Abstract Prostate cancer is the most common cancer in men and the second-leading cause of death. Most prostate cancer deaths are due to metastasis, particularly to the bone. Metastasis occurs when cells from the primary tumor travel through the blood as circulating tumor cells (CTCs) and settle in metastatic sites. CTCs are difficult to collect from patients, and understanding the relationships between CTCs and the tumor phenotypes of origin could reveal mechanisms of cancer metastasis. Hence, we utilized several genetically engineered mouse models (GEMMs) of metastatic prostate cancer to evaluate tumor phenotypes and CTCs. We compared the tumor phenotypes of several GEMMs of metastatic prostate cancer and identified a level of heterogeneity among tumors, particularly focusing on a GEMM that demonstrated severe metastasis, especially to the bone. Next, we analyzed CTCs from this GEMM and focused our analysis on transcriptional states that are regulated by proteins called master regulators (MRs). MRs control the expression of groups of genes called regulons, and pharmacologically inhibiting these MRs may lead to reversal of cancer phenotypes. We hypothesized that these MRs may be mechanistic drivers of CTC cell state in prostate cancer metastasis. We investigated MRs enriched in CTCs from mice and screened and validated Lap2, the lamina-associated polypeptide 2 protein, as a regulatory driver in mice with metastatic prostate cancer. Analysis of bulk RNA-seq profiles from primary and metastatic prostate samples in The Cancer Genome Atlas and Stand Up to Cancer cohorts, respectively, revealed that the regulon-based activity of Lap2 was significantly higher in the latter group. Moreover, analysis of single-cell RNA-seq profiles of CTCs isolated from prostate cancer patients confirmed aberrant Lap2 activity. Finally, the role of Lap2 was assessed in human tissue from an engineered multi-organ-on-a-chip model of metastasis. Silencing of Lap2 production in cells dissociated from mouse CTC-derived organoids inhibited extravasation to the bone in the model, suggesting a critical role for Lap2 in metastasis. Our findings illustrate the unique transcriptional profile of prostate cancer CTCs associated with their metastatic potential. We identified Lap2 as a novel driver of CTC dissemination and metastasis in mice and validated its activity in human prostate cancer, which can aid future therapies to prevent metastatic progression in patients. Citation Format: Aunika Zheng, Aleksandar Obradovic, Kacey Ronaldson-Bouchard, Gordana Vunjak-Novakovic, Andrea Califano, Peter A. Sims, Cory Abate-Shen, Arianna Giacobbe. Investigating regulatory drivers of prostate cancer metastasis in circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2757.

Functional requirement of alternative splicing in epithelial-mesenchymal transition of pancreatic circulating tumor

Circulating tumor cells (CTCs) that undergo epithelial-to-mesenchymal transition (EMT) can provide valuable information regarding metastasis and potential therapies. However, the current studies on the EMT overlook the alternative splicing. Here, we used the single-cell full-length transcriptome data and mRNA-sequencing of CTCs to identify stage-specific alternative splicing of partial EMT and mesenchymal states during pancreatic cancer metastasis. We classified definitive tumor and normal epithelial cells via genetic aberrations and demonstrated dynamic changes in the epithelial-mesenchymal continuum in both epithelial cancer cells and CTCs. We provided the landscape of alternative splicing in CTCs at different stages of EMT, uncovering cell-type-specific splicing patterns and splicing events in cell-surface proteins suitable for therapies. We showed that MBNL1 governs cell fate through alternative splicing independently of changes in gene expression and affects the splicing pattern during EMT. We found a high frequency of events that contained multiple premature termination codons and were enriched with C and G nucleotides in close proximity, which influence the likelihood of stop codon readthrough and expand the range of potential therapeutic targets. Our study provides insights into the EMT transcriptome's dynamic changes and identifies potential diagnostic and therapeutic targets in pancreatic cancer.

Intracellular MicroRNA Imaging and Specific Discrimination of Prostate Cancer Circulating Tumor Cells Using Multifunctional Gold Nanoprobe-Based Thermophoretic Assay.

Circulating tumor cells (CTCs) have emerged as powerful biomarkers for diagnosis of prostate cancer. However, the effective identification and concurrently accurate imaging of CTCs for early screening of prostate cancer have been rarely explored. Herein, we reported a multifunctional gold nanoprobe-based thermophoretic assay for simultaneous specific distinguishing of prostate cancer CTCs and sensitive imaging of intracellular microRNA (miR-21), achieving the rapid and precise detection of prostate cancer. The multifunctional gold nanoprobe (GNP-DNA/Ab) was modified by two types of prostate-specific antibodies, anti-PSMA and anti-EpCAM, which could effectively recognize the targeting CTCs, and meanwhile linked double-stranded DNA for further visually imaging intracellular miR-21. Upon the specific internalization of GNP-DNA/Ab by PC-3 cells, target aberrant miR-21 could displace the signal strand to recover the fluorescence signal for sensitive detection at the single-cell level, achieving single PC-3 cell imaging benefiting from the thermophoresis-mediated signal amplification procedure. Taking advantage of the sensitive miR-21 imaging performance, GNP-DNA/Ab could be employed to discriminate the PC-3 and Jurkat cells because of the different expression levels of miR-21. Notably, PC-3 cells were efficiently recognized from white blood cells, exhibiting promising potential for the early diagnosis of prostate cancer. Furthermore, GNP-DNA/Ab possessed good biocompatibility and stability. Therefore, this work provides a great tool for aberrant miRNA-related detection and specific discrimination of CTCs, achieving the early and accurate diagnosis of prostate cancer.

Enumeration and Molecular Characterization of Circulating Tumor Cell Using an Epithelial Cell Adhesion Molecule/Vimentin/Epidermal Growth Factor Receptor Joint Capture System in Lung Cancer.

Detection rate and isolation yield of circulating tumor cells (CTCs) are low in lung cancer with approaches due to CTC invasiveness and heterogeneity. In this study, on the basis of the epithelial cell adhesion molecule (EpCAM) phenotype, markers of vimentin and epidermal growth factor receptor (EGFR) phenotype were added to jointly construct a precise and efficient CTC capture system for capture of lung cancer CTCs. A CTC capture system combined with EpCAM lipid magnetic bead (Ep-LMB)/vimentin lipid magnetic bead (Vi-LMB)/EGFR lipid magnetic bead (EG-LMB) was constructed, and its performance was tested. The amount of CTC captured in the blood of patients with lung cancer was detected by immunofluorescence identification and analyzed for clinical relevance. The constructed CTC capture system has low cytotoxicity. The capture efficiency of lung cancer cells in phosphate belanced solution (PBS) system was 95.48%. The capture efficiency in the blood simulation system is 94.55%. The average number of CTCs in the blood of patients with lung cancer was 9.73/2 mL. The quantity distribution of CTCs is significantly correlated with tumor staging and metastasis. The area under the curve (AUC) of CTCs for the diagnosis of lung cancer was 0.9994 (95% CI = 0.9981-1.000, P < .0001). The cutoff value was 4.5/2 mL. The sensitivity was 99.39%, and the specificity was 96.88%. The EpCAM/vimentin/EGFR combined capture system has feasibility and high sensitivity in the detection of lung cancer CTC typing, which can be used as an auxiliary diagnostic indicator for lung cancer and is expected to promote the clinical application of CTCs.

Controllable self-assembled DNA nanomachine enable homogeneous rapid electrochemical one-pot assay of lung cancer circulating tumor cells

A homogeneous rapid (45 min) one-pot electrochemical (EC) aptasensor was established to quantitatively detect circulating tumor cells (CTCs) in lung cancer patients using mucin 1 as a marker. The core of this study is that the three single-stranded DNA (Y1, Y2, and Y3) could be hybridized to form Y-shaped DNA (Y-DNA) and further self-assemble to form DNA nanosphere. The aptamer of mucin 1 could be complementary and paired with Y1, thus disrupting the conformation of the DNA nanosphere. When mucin 1 was present, the aptamer combined specifically with mucin 1, thus preserving the DNA nanosphere structure. Methylene blue (MB) acted as a signal reporter, which could be embedded between two base pairs in the DNA nanosphere to form a DNA nanosphere-MB complex, reducing free MB and resulting in a lower electrochemical signal. The results demonstrated that the linear ranges for mucin 1 and A549 cells were 1 ag/mL-1 fg/mL and 1–100 cells/mL, respectively, with minimum detectable concentrations were 1 ag/mL and 1 cell/mL, respectively. The quantitative analysis of CTCs in 44 clinical blood samples was performed, and the results were consistent with the computerized tomography (CT) images, pathological findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve exhibited an area under the curve (AUC) value of 0.970. The assay revealed 100% specificity and 94.1% sensitivity. It is believed that this electrochemical aptasensor could provide a new approach to detect CTCs.

CTC, ctDNA, and Exosome in Thyroid Cancers: A Review.

Thyroid cancer has become more common in recent years all around the world. Many issues still need to be urgently addressed in the diagnosis, treatment, and prognosis of thyroid cancer. Liquid biopsy (mainly circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and circulating exosomes) may provide a novel and ideal approach to solve these issues, allows us to assess the features of diseases more comprehensively, and has a function in a variety of malignancies. Recently, liquid biopsy has been shown to be critical in thyroid cancer diagnosis, treatment, and prognosis in numerous previous studies. In this review, by testing CTCs, ctDNA, and exosomes, we focus on the possible clinical role of liquid biopsy in thyroid cancer, including diagnostic and prognostic biomarkers and response to therapy. We briefly review how liquid biopsy components have progressed in thyroid cancer by consulting the existing public information. We also discuss the clinical potential of liquid biopsy in thyroid cancer and provide a reference for liquid biopsy research. Liquid biopsy has the potential to be a useful tool in the early detection, monitoring, or prediction of response to therapies and prognosis in thyroid cancer, with promising clinical applications.

Identification and gene expression analysis of circulating tumor cells with high expression of KRAS in pancreatic cancer

This study aims to establish a highly sensitive and specific method for single cell screening and identification of KRAS+/epithelial cell adhesion molecules (EpCAMs)/vimentin+/pancreatic cancer circulating tumor cells (CTCs) and to explore the clinical implications in the diagnosis of pancreatic cancer. A single cell separation and identification system for pancreatic cancer CTCs was constructed based on the preparation of KRAS immunolipid magnetic spheres. The function of the system was evaluated, the physical parameters of the immunoliposomes (IMLs) were characterized, and the isolation effect of the IMLs on CTCs was studied and compared with EpCAM and vimentin phenotypic CTCs. CTCs in the peripheral blood of 50 patients with pancreatic cancer confirmed by preoperative CT diagnosis and postoperative pathology were isolated and identified. The isolated CTCs were stained with CK immune antibody, observed, and identified, the number of CTCs was counted, and the relationship between the CTC count and the clinical malignant grade markers and grade indicators was examined. The consistency of the KRAS mutation rate between CTCs and tissues was analyzed and compared. It was found that the circulating tumor cell sorting system with a high expression of KRAS-ML could effectively enrich the circulating tumor cells with a positive expression of KRAS in pancreatic cancer. The pancreatic cancer CTC sorting and single-cell gene detection system can be used as a new detection technology of peripheral blood CTCs in patients with pancreatic cancer. As a minimally invasive liquid biopsy method, this technology has significant clinical practice implications for pancreatic cancer detection and effectiveness assessment.

Improved Characterization of Circulating Tumor Cells and Cancer-Associated Fibroblasts in One-Tube Assay in Breast Cancer Patients Using Imaging Flow Cytometry.

Circulating tumor cells (CTCs) and circulating cancer-associated fibroblasts (cCAFs) have been individually considered strong indicators of cancer progression. However, technical limitations have prevented their simultaneous analysis in the context of CTC phenotypes different from epithelial. This study aimed to analyze CTCs and cCAFs simultaneously in the peripheral blood of 210 breast cancer patients using DAPI/pan-keratin (K)/vimentin (V)/alpha-SMA/CD29/CD45/CD31 immunofluorescent staining and novel technology-imaging flow cytometry (imFC). Single and clustered CTCs of different sizes and phenotypes (i.e., epithelial phenotype K+/V- and epithelial-mesenchymal transition (EMT)-related CTCs, such as K+/V+, K-/V+, and K-/V-) were detected in 27.6% of the samples and correlated with metastases. EMT-related CTCs interacted more frequently with normal cells and tended to occur in patients with tumors progressing during therapy, while cCAFs coincided with CTCs (mainly K+/V- and K-/V-) in seven (3.3%) patients and seemed to correlate with the presence of metastases, particularly visceral ones. This study emphasizes the advantages of imFC in the field of liquid biopsy and highlights the importance of multimarker-based analysis of different subpopulations and phenotypes of cancer progression-related cells, i.e., CTCs and cCAFs. The co-detection of CTCs and cCAFs might improve the identification of patients at higher risk of progression and their monitoring during therapy.

Microfluidic isolation of breast cancer circulating tumor cells from microvolumes of mouse blood.

Liquid biopsy has shown significant research and clinical implications in cancer. Particularly, the isolation of circulating tumor cells (CTCs) in preclinical studies can provide crucial information about disease progression and therefore may guide treatment decisions. Microfluidic isolation systems have played a considerable role in CTC isolation for cancer studies, disease diagnosis, and prognosis. CTCs are often studied using preclinical animal models such as xenografts or syngeneic models. However, most isolation systems are tested on human cell lines and human blood, whereas less validation studies are done on preclinical samples such as CTCs from mouse models. Here, we demonstrate and evaluate a complete workflow of a sized-based inertial microfluidic device to isolate CTCs from blood using exclusively mouse blood and mouse cancer cell lines. We then incorporate the cytospin, a commonly used method for enumeration of small number of cells in a glass slide to quantify the total cell yield of our workflow.

Analysis of Diagnostic Value of CTC and CTDNA in Early Lung Cancer.

This paper is the exploration and analysis of the diagnostic value of circulating tumor cells (CTC) and circulating tumor deoxyribonucleic acid (ctDNA) in early non-small cell lung cancer (NSCLC).A total of 125 NSCLC patients that hospitalized from March 2019 to December 2020 were selected, of which 64 subjects with stage I-II were listed as the early-stage group, and 61 patients with stage III-IV were classified as the intermediate-advanced group; In addition, 47 patients with benign pulmonary nodules hospitalized within same period were selected as a group of benign pulmonary nodules, and 50 healthy subjects were enrolled as a control group. The levels of CTC and ctDNA, serum tumor markers carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cytokeratin 21-1 (CYFRA21-1) and gastrin-releasing peptide precursor (pro-GRP) were detected. The diagnostic value of CTC and ctDNA in early NSCLC patients was analyzed by ROC curve. The area under the curve of CTC count for early-stage lung cancer was 0. 949 (P=0. 000, 95%CI: 0. 918-0. 980), and ctDNA was 0. 914 (P=0. 000, 95%CI: 0. 866~0. 963). There were significant differences in CTC count and ctDNA among subjects in each group (P<0. 05). CTC count and ctDNA of the early and intermediate-advanced group were remarkably higher than those of the control group and group of benign pulmonary nodules (P<0. 05), and CTC count and ctDNA of patients in the middle-late group were critically higher than those in the early-stage group (P<0. 05). There were significant differences in serum CEA, NSE, CYFRA21-1 and pro-GRP levels among all groups (P<0. 05). The results showed that CTC and ctDNA in NSCLC patients were significantly positively correlated with CEA, NSE, CYFRA21-1 and pro-GRP (P<0. 05). Peripheral blood CTC and ctDNA levels are significantly increased in NSCLC patients. They are positively correlated with serum tumor markers CEA, NSE, CYFRA21-1 and pro-GRP levels, and are related to tumor progression. CTC and ctDNA have high value in the clinical diagnosis of early NSCLC.

Abstract PR013: Unlocking the proteome of metastatic prostate cancer circulating tumor cells

Abstract In recent years, liquid biopsies have emerged to provide improved insights on the current disease state of a patient, but they generally rely on a small number of protein markers from immunofluorescent staining or cell-free DNA, which provides limited information especially around drug targets. As more targeted therapies and immunotherapies enter the market, selecting the optimal drug target could improve patient outcomes drastically. One way to achieve this goal is to generate multi-omic datasets from the cancer cells in circulation, known as circulating tumor cells (CTCs). In this study, we collaborated with Astrin Biosciences and used their patented isolation technology to enrich CTCs with high purity from the blood of 15 metastatic castration resistant prostate cancer (mCRPC) patients. The enriched CTCs and a patient matched control sample consisting primarily of white blood cells were divided such that RNA and protein could be interrogated from the same patient. Label free unbiased mass spectrometry-based proteomics was used to identify the CTC-enriched proteins and marks the first time a global proteomic assessment has been performed on CTCs from cancer patients. Initial analysis identified over 1,500 peptides corresponding to greater than 500 proteins from each patients’ CTCs; including increased epithelial cell markers in the CTC-enriched sample and increased WBC markers in the control sample. Hallmarks of androgen response and detection of the androgen receptor were also identified in the CTC-enriched proteome. Overall, this study was designed to show the feasibility of obtaining large scale proteomic data from a liquid biopsy and future studies will provide more insight into the clinical utility of such data with a focus on cancer signaling mechanisms and biomarkers. Importantly, as we analyze more patients, we plan to compare the proteomic data with future RNA signatures across mCRPC patients to eventually be able to correlate them with drug response with the overall goal to improve treatment selection and patient outcomes. Citation Format: Justin M. Drake, Zoi Sychev, Alec Horrmann, Kaylee Judith Smith, Catalina Galeano-Garces, Ali Arafa, Nicholas Heller, Mahdi Ahmadi, Jiarong Hong, Megan Ludwig, Justin Hwang, Emmanuel S. Antonarakis, Jayant Parthasarathy. Unlocking the proteome of metastatic prostate cancer circulating tumor cells [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 PR013.

Development and initial clinical testing of a multiplex assay evaluating histone modifier p300 acetylation in prostate cancer circulating tumor cells.

e17020 Background: Histone modifying enzymes (HMEs) hold promise as targets for drug discovery in cancer. Histone acetyltransferases and histone deacetylases dictate the presence of acetyl marks, which regulate chromatin and transcriptional activation. We previously developed a novel ‘histone array on a chip’ to measure HME dysregulation associated with the development of castration resistant prostate cancer (CRPC) (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC xenografts. These alterations were subsequently validated in castration-resistant tissues (BMC Cancer 2017). The goal of the current study was to determine if circulating tumor cells (CTCs) could be used as a therapeutic biomarker to identify patients with altered p300 acetylation activity. Methods: Blood was collected from 11 CRPC and 3 hormone-sensitive patients with advanced PC under an IRB-approved protocol. PBMCs were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled coated magnetic beads. Bound cells were isolated with Exclusion-based Sample Preparation (ESP) technology. Bound cells were moved through staining and washing wells prior to imaging at 20x on a fluorescent microscope. Images were analyzed with NIS Elements software. Results: We initially optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in single cell prostate cancer cell cultures. Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced levels of p300 and H3K18 acetylation confirming assay specificity. CTCs from patient samples were then captured by EpCAM antibody binding and target staining revealed clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Acetyl-p300 displayed a strong positive correlation with its target acetylated H3k18 (Pearson’s r = 0.61), and SIRT2 expression showed robust negative correlation with H3k18 (r = -0.60). A distinct group of 6/11 (55%) of CRPC patients demonstrated altered expression. In one subject with an untreated CSPC sample and subsequent CRPC sample ( &gt; 12mo) a loss of SIRT2 expression, increased p300 activity, and histone H3 hyperacetylation are associated with the transition from hormone-sensitive to CRPC. Conclusions: Acetyl-p300, H3K18ac, and SIRT2 increase in a subset of CTC from patients with CRPC. These biomarkers are associated with increased CBP/p300 activity. This biomarker suite could potentially be used for noninvasive monitoring of outcomes and predicting tumor responses to CBP/p300 acetylation inhibitors in clinical development.

An Image-Based Identification of Aggressive Breast Cancer Circulating Tumor Cell Subtypes.

Using previously established CTC lines from breast cancer patients, we identified different morphometric subgroups of CTCs with one of them having the highest tumorigenic potential in vivo despite the slowest cell proliferation in vitro. This subgroup represents 32% of all cells and contains cells with small cell volume, large nucleus to cell, dense nuclear areas to the nucleus, mitochondria to cell volume ratios and rough texture of cell membrane and termed "Small cell, Large mitochondria, Rough membrane" (SLR). RNA-seq analyses showed that the SLR group is enriched in pathways and cellular processes related to DNA replication, DNA repair and metabolism. SLR upregulated genes are associated with poor survival in patients with ER+ breast cancer based on the KM Plotter database. The high tumorigenic potential, slow proliferation, and enriched DNA replication/repair pathways suggest that the SLR subtype is associated with stemness properties. Our new findings provide a simple image-based identification of CTC subpopulations with elevated aggressiveness, which is expected to provide a more accurate prediction of patient survival and therapy response than total CTC numbers. The detection of morphometric and transcriptomic profiles related to the SLR subgroup of CTCs also opens opportunities for potential targeted cancer treatment.

Feasibility study of expressing epcam + /vimentin + CTC in prostate cancer diagnosis.

Prostate cancer (PCa) is one of the most common malignancies in men and one of the leading causes of cancer-related deaths; circulating tumor cells (CTC) are malignant cells that have broken off from original tumor or metastatic sites and extravasated into the blood vessels either naturally or maybe as a consequence of surgical procedures. This study aims to explore the feasibility of liquid biopsy technique to diagnose prostate cancer. We constructed an assay platform integrating magnetic separation and fluorescence in situ hybridization (FISH) to effectively capture prostate cancer CTCs and evaluate the distribution between healthy volunteers and prostate cancer patients, respectively. There was a significant difference in the number of CTCs between the healthy population and prostate cancer patients (P < 0.001). The results of the study showed that the CTCs capture identification system has good sensitivity and specificity in identifying prostate cancer patients. The CTCs test allows us to accurately identify patients who are at high risk for prostate cancer, allowing for early intervention and treating patients effectively.

Abstract LB163: A microfluidic platform for developing circulating tumor cells (CTCs) organoids for precision medicine in pancreatic cancer

Abstract Introduction Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second leading cause of cancer death in the US by 2030. Besides the late detection and lack of effective treatments, the greatest treatment hurdle to PDAC is the ineffectiveness of patient-derived tumor models. Precision medicine-based treatment decisions require the generation of consistent and trackable complex organoid models that is not achievable by tissue biopsies. Liquid biopsies such as blood, instead, contain circulating biomarkers released by the tumor, among which especially interested in for this study are circulating tumor cells (CTCs). This work develops microfluidic 3D organoids from CTCs to be used as a precision medicine tool to predict and guide the drug response of early stage I-IIB (PDAC) patients. Experimental Methods CTCs are isolated from PDAC patient blood samples using microfluidic Labyrinth following removal of red blood cells with 6% dextran. Collected cells post-isolation are immunofluorescence (IF) stained with DAPI (nuclei), CD45 (leukocyte), pancytokeratin (type I/II cytokeratin 1-8, 10, 14- 16 and 19), Vimentin, and Epithelial cell adhesion molecule (EpCAM) to identify CTCs and characterize their phenotypes. The mold of the microfluidic platform for CTC organoids is fabricated with the standard SU-8 photolithography. The PDMS-based device is tested with a low seeding number of a pancreatic cancer CTC cell line labeled fluorescently. Decellularization of cancer associated fibroblasts (CAFs) generates the in vitro matrix that supplies amino-acid precursors for PDAC metabolism, promoting CTC organoid formation. Serial images of cell growth are obtained throughout the course using a Ti-20 microscope. After each 7-day culture, organoids are dissociated from the device and IF stained using a similar panel as above (with Ki67 marker instead of CD45 for cell proliferation). Preliminary gemcitabine dosage-performance analysis is performed on-chip. Results CTC count from the tested patient panel (n=16) averages 17.0±8.4/mL of blood. CAFs are successfully decellularized on-chip and the embedded pancreatic CTC cell line growth curve is generated. IF staining of the dissociated cells from the organoids confirms the proliferation of the loaded pseudo-CTCs. Conclusion The work above demonstrates the successful isolation of CTCs from PDAC patient blood samples and an engineered microfluidic platform to expand low abundance of cells into organoids. It has a potential to be tested and employed to expand CTCs from PDAC into organoids for drug screening. Citation Format: Yu Zhang, Scott Smith, Minal Nenwani, Valerie Gunchick, Vaibhav Sahai, Sunitha Nagrath, Deepak Nagrath. A microfluidic platform for developing circulating tumor cells (CTCs) organoids for precision medicine in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB163.

Abstract 2309: Unlocking the proteome of metastatic prostate cancer circulating tumor cells

Abstract In recent years, liquid biopsies have emerged to provide improved insights on the current disease state of a patient, but they generally rely on a small number of protein markers from immunofluorescent staining or cell-free DNA, which provides limited information especially around drug targets. As more targeted and immune therapies enter the market, selecting the optimal drug target could improve patient outcomes drastically. One way to achieve this goal is to generate multi-omic datasets from the cancer cells in circulation, known as circulating tumor cells (CTCs). In this study, Astrin Biosciences’ patented isolation technology was used to enrich CTCs with high purity from the blood of metastatic castration resistant prostate cancer (mCRPC) patients. The enriched CTCs and a patient matched control sample consisting primarily of white blood cells were divided such that RNA and protein could be interrogated from the same patient. Label free unbiased mass spectrometry was used to identify proteins and marks the first time a global proteomic assessment has been performed in CTCs from cancer patients. Initial analysis identified over 1,500 peptides corresponding to greater than 500 proteins with increased epithelial cell markers in the CTC-enriched sample and increased WBC markers in the control sample. Hallmarks of androgen response and detection of the androgen receptor were also identified in the CTC proteome. In addition to the proteome, RNA sequencing will also be performed to generate a multi-omic platform to detect cancer signaling mechanisms and biomarkers. Overall, this study was designed to show the feasibility of obtaining multi-omic data from a liquid biopsy and future studies will provide more insight into the clinical utility of such data. As we analyze more patients, we plan to compare the protein and RNA signatures across mCRPC patients and eventually be able to map patient outcomes and correlate them with drug response with the overall goal to improve treatment selection and patient outcomes. Citation Format: Justin M. Drake, Zoi Sychev, Alec Horrmann, Kaylee J. Smith, Catalina Galeano-Garces, Ali Arafa, Nicholas Heller, Mahdi Ahmadi, Jiarong Hong, Megan Ludwig, Justin Hwang, Emmanuel S. Antonarakis, Jayant Parthasarathy. Unlocking the proteome of metastatic prostate cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2309.

In vivo detection of circulating tumor cells predicts high-risk features in patients with bladder cancer.

Previous studies have suggested the potential diagnostic value of circulating tumor cells (CTCs). This study aims to validate the efficacy of in vivo detection of CTCs in bladder cancer (BC) patients. A total of 216 BC patients were enrolled in this study. All patients had one in vivo detection of CTCs before initial treatment as a baseline parameter. The results of CTCs were associated with different clinicopathological features including molecular subtypes. PD-L1 expression on CTCs was also assessed and compared with its expression on tumors. CTC positive was defined as > 2 CTCs detected. Among all 216 patients, 49 (23%) were detected as CTC positive (> 2 CTCs) at baseline. Positive detection of CTCs was associated with multiple high-risk clinicopathological features including the multiplicity of the tumor (P = 0.02), tumor size (P < 0.01), tumor stage (P < 0.01), tumor grade (P < 0.01) and tumor PD-L1 expression (P = 0.01). The expression of PD-L1 on tumor and CTCs were not coordinated. Only 55% (74/134) matched the same status of PD-L1 expression on tumor and CTCs, along with 56 CTC (+) Tissue (-) and 4 CTC (-) Tissue (+) (P < 0.01). Our study has demonstrated the efficacy of in vivo detection of CTCs. The positive detection of CTCs is associated with multiple clinicopathological features. PD-L1 expression on CTCs has the potential to be a supplementary biomarker for immunotherapy.