Purification Of Circulating Tumor Cells Research Articles

Purification of Circulating Tumor Cells Based on Multiantibody-Modified Magnetic Nanoparticles and Molecular Analysis toward Epithelial Ovarian Cancer Detection.

Circulating tumor cells (CTCs) are valuable circulating biomarkers of cancer, which carry primary tumor information and may provide real-time assessment of tumor status as well as treatment response in cancer patients. Herein, we developed a novel assay for accurate diagnosis and dynamic monitoring of epithelial ovarian cancer (EOC) using CTC RNA analysis. Multiantibody-modified magnetic nanoparticles were prepared for purification of EOC CTCs from whole blood samples of clinical patients. Subsequently, nine EOC-specific mRNAs of purified CTCs were quantified using droplet digital PCR. The EOC CTC Score was generated using a multivariate logistic regression model for each sample based on the transcripts of the nine genes. This assay exhibited a distinguishing diagnostic performance for the detection of EOC (n = 17) from benign ovarian tumors (n = 30), with an area under the receiver operating characteristic curve (AUC) of 0.96 (95% CI = 0.91-1.00). Moreover, dynamic changes of the EOC CTC Score were observed in patients undergoing treatment, demonstrating the potential of the assay for monitoring EOC. In conclusion, we present an accurate assay for the diagnosis and monitoring of EOC via CTC RNA analysis, and the results suggest that it may provide a promising solution for the detection and treatment response assessment of EOC.

Abstract 2311: Extensive molecular heterogeneity in circulating tumor cells: A key player in oral squamous cell carcinoma prognosis

Abstract Background: Oral Squamous cell carcinoma (OSCC) often results in unfavorable outcomes due to locoregional relapse. While circulating tumor cells (CTCs) may be attributed to these locoregional metastases, detecting CTCs and their molecular profiling remains challenging. Here we report a novel strategy for isolating CTCs in OSCC. Secondly, we characterized the CTCs to prognosticate the disease outcome using their transcriptomic signature. Methods: Keeping in view the inherent disadvantages of currently available CTC-isolation techniques, like the use of single marker or size-based isolation, we developed a novel FACS–based strategy, combining pre-enrichment by immunomagnetic depletion of CD45+ cells, followed by positive selection of CTCs using multiple FITC-conjugated epithelial markers (EpCAM, EGFR, CK) and EMT marker (Vimentin), with a novel gating strategy for isolating pure CTCs. Isolated CTCs were reconfirmed using various methods. CTCs and Primary tumors were isolated in 63 OSCC patients, and their transcriptomic signature was correlated with disease outcome. Results: Seventy-one percent (45/63) of patients were positive for CTCs, and associated with poor OS and DFS, while there is no difference between the proportion of CTC-positivity between early (23/33;69%) and late (22/30;73%) TNM stage. CTC-specific markers (EpCAM, EGFR, CK, and Vim) are expressed in various combinations, indicating extensive phenotypic heterogeneity. Importantly, EpCAM-positive CTCs were detected only in 46% (29/63) patients, and patients with EpCAM-negative CTCs had worse OS (p=0.044*), indicating the importance of a multi-marker approach. On unsupervised hierarchical clustering of RNAseq data of isolated CTCs and primary tumors, we found that CTCs and primary tumors clustered separately, indicating that the CTCs are transcriptionally different from the primary tumors. Moreover, CTCs were clustered into two distinct sub-clusters with significantly different OS (p=0.0004***) and DFS (p=0.0097***), suggesting that the inter-patient transcriptomic heterogeneity of CTCs is an important prognostic factor. We found coagulation, IL-6 JAK STAT3 signaling, allograft rejection, complement, EMT, inflammatory response, heme metabolism, UV response, and ECM receptor interaction pathways significantly enriched in CTCs compared to the primary tumor. TNF-alpha signaling via NF-kB and KRAS signaling pathways were also positively enriched in CTCs with poor prognostic traits. Interestingly, for the first time, we found various Cancer/Testis (CT) antigens upregulated in CTCs, while not detected in primary tumors. Conclusion: Here we report a novel, technologically less difficult, cost-effective CTC isolation technique suitable for developing countries. We explored the prognostic implications of the extensive heterogeneity in CTCs isolated from OSCC patients for the first time. Citation Format: Anshika Chauhan, Geeta S. Boora, Sahana Ghosh, Monil Parsana, Subrata K. Patra, Roshan K. Verma, Jaimanti Bakshi, Debajyoti Chatterjee, Radhika Srinivasan, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Extensive molecular heterogeneity in circulating tumor cells: A key player in oral squamous cell carcinoma prognosis [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 2311.

An ultra-compact acoustofluidic device based on the narrow-path travelling surface acoustic wave (np-TSAW) for label-free isolation of living circulating tumor cells

Obtaining highly purified intact living cells from complex environments has been a challenge, such as the isolation of circulating tumor cells (CTCs) from blood. In this work, we demonstrated an acoustic-based ultra-compact device for cell sorting, with a chip size of less than 2 × 1.5 cm2. This single actuator device allows non-invasive and label-free isolation of living cells, offering greater flexibility and applicability. The device performance was optimized with different-sized polystyrene (PS) particles and blood cells spiked with cancer cells. Using the narrow-path travelling surface acoustic wave (np-TSAW), precise isolation of 10 μm particles from a complex mixture of particles (5, 10, 20 μm) and separation of 8 μm and 10 μm particles was achieved. The purified collection of 10 μm particles with high separation efficiency (98.75%) and high purity (98.1%) was achieved by optimizing the input voltage. Further, we investigated the isolation and purification of CTCs (MCF-7, human breast cancer cells) from blood cells with isolation efficiency exceeding 98% and purity reaching 93%. Viabilities of the CTCs harvested from target-outlet were all higher than 97% after culturing for 24, 48, and 72 h, showing good proliferation ability. This novel ultra-miniaturized microfluidic chip demonstrates the ability to sorting cells with high-purity and label-free, providing an attractive miniaturized system alternative to traditional sorting methods.

Reversible capture and release of circulating tumor cells on a three‐dimensional conductive interface to improve cell purity for gene mutation analysis

AbstractRare circulating tumor cells (CTCs) are always harvested with large numbers of white blood cells (WBCs) by current CTC isolation techniques, which influences the accuracy of CTC‐related gene and protein analysis. Therefore, it is very urgent to develop a method for efficient isolation of CTCs with high purity. In this work, we fabricated a reversibly assembled interface by layer‐by‐layer assembly of biotinylated poly‐(L‐lysine‐graft‐ethylene glycol) (PLL‐g‐PEG‐biotin), streptavidin, and CTC‐specific antibody on a three‐dimensional conductive scaffold, and further embedded it into a customized microchip for CTC capture. The assembled multilayers increased the roughness of the scaffold, which improved the capture efficiency. Importantly, the PLL‐g‐PEG‐biotin covalently coupled with CTCs could be detached from the scaffold by electrostatic repulsion, and the released CTCs could be recaptured on the conductive scaffold via electrostatic attraction while the WBCs were continuously drifted away. This reversible capture and release strategy significantly improved the purity of CTCs, and pure CTCs were obtained from cancer patients’ blood samples for gene mutation assay, which will be of great significance for assisting cancer diagnosis and treatment in a noninvasive way.

MinimuMM-seq: Genome Sequencing of Circulating Tumor Cells for Minimally Invasive Molecular Characterization of Multiple Myeloma Pathology.

In this study, we established an approach enabling the enumeration and sequencing of CTCs to replace standard molecular cytogenetics. CTCs harbored the same pathognomonic MM abnormalities as BM plasma cells. Longitudinal sampling of serial CTCs was able to track clonal dynamics over time and detect the emergence of high-risk genetic subclones. This article is highlighted in the In This Issue feature, p. 247.

Circulating tumour cell gene expression and chemosensitivity analyses: predictive accuracy for response to multidisciplinary treatment of patients with unresectable refractory recurrent rectal cancer or unresectable refractory colorectal cancer liver metastases

BackgroundPatients with unresectable recurrent rectal cancer (RRC) or colorectal cancer (CRC) with liver metastases, refractory to at least two lines of traditional systemic therapy, may receive third line intraarterial chemotherapy (IC) and targeted therapy (TT) using drugs selected by chemosensitivity and tumor gene expression analyses of liquid biopsy-derived circulating tumor cells (CTCs).MethodsIn this retrospective study, 36 patients with refractory unresectable RRC or refractory unresectable CRC liver metastases were submitted for IC and TT with agents selected by precision oncotherapy chemosensitivity assays performed on liquid biopsy-derived CTCs, transiently cultured in vitro, and by tumor gene expression in the same CTC population, as a ratio to tumor gene expression in peripheral mononuclear blood cells (PMBCs) from the same individual. The endpoint was to evaluate the predictive accuracy of a specific liquid biopsy precision oncotherapy CTC purification and in vitro culture methodology for a positive RECIST 1.1 response to the therapy selected.ResultsOur analyses resulted in evaluations of 94.12% (95% CI 0.71–0.99) for sensitivity, 5.26% (95% CI 0.01–0.26) for specificity, a predictive value of 47.06% (95% CI 0.29–0.65) for a positive response, a predictive value of 50% (95% CI 0.01–0.98) for a negative response, with an overall calculated predictive accuracy of 47.22% (95% CI 0.30–0.64).ConclusionsThis is the first reported estimation of predictive accuracy derived from combining chemosensitivity and tumor gene expression analyses on liquid biopsy-derived CTCs, transiently cultured in vitro which, despite limitations, represents a baseline and benchmark which we envisage will be improve upon by methodological and technological advances and future clinical trials.

MagPure chip: an immunomagnetic-based microfluidic device for high purification of circulating tumor cells from liquid biopsies.

The isolation of circulating tumor cells (CTCs) directly from blood, as a liquid biopsy, could lead to a paradigm shift in cancer clinical care by providing an earlier diagnosis, a more accurate prognosis, and personalized treatment. Nevertheless, CTC-specific challenges, including their rarity and heterogeneity, have hampered the wider use of CTCs in clinical studies. Microfluidic-based isolation technologies have emerged as promising tools to circumvent these limitations but still fail to meet the constraints of high purity and short processing time required to ensure compatibility with clinical follow-up. In this study, we developed an immunomagnetic-based microfluidic device, the MagPure chip, to achieve the negative selection of CTCs through the depletion of white blood cells (WBCs) and provide highly purified samples for subsequent analysis. We demonstrate that the MagPure chip depletes all magnetically labeled WBCs (85% of WBCs were successfully labeled) and ensures a CTC recovery rate of 81%. In addition, we show its compatibility with conventional biological studies, including 2D and 3D cell culture, as well as phenotypic and genotypic analyses. Finally, we successfully implemented a two-step separation workflow for whole blood processing by combining a size-based pre-enrichment system (ClearCell FX1®) with the MagPure chip as a subsequent purification step. The total workflow led to high throughput (7.5 mL blood in less than 4 h) and high purity (947 WBCs per mL remaining, 99.99% depletion rate), thus enabling us to quantify CTC heterogeneity in size and tumor marker expression level. This tumor-marker-free liquid biopsy workflow could be used in a clinical context to assess phenotype aggressiveness and the prognosis rate.

Circulating tumor cell proportion scoring (CTPS) based PD-L1 assessment and clinical application of circulating tumor cells (CTCs) on stage IV non-small cell lung cancer (NSCLC) through liquid biopsy.

e15031 Background: Stage IV lung cancer is the most advanced lung cancer state accompanied by metastasized to the area around the lungs or distant major organs. The most common type of lung cancer is non-small cell lung cancer, which is more aggressive and may spread quickly due to organ-specific complex networks such as lymph and major blood vessels. Thus, only precise diagnostic strategy approaches will determine the effectiveness of the actual and successful clinical treatment. Until a recent date, Immunohistochemistry (IHC) for programmed death-ligand 1(PD-L1) test is the only available biomarker test that purpose diagnostics (CDx) and guide the treatment with immune checkpoint inhibitors in NSCLC. Methods: Given that CDx strategy, tissue biopsy has inevitable limitations, including patient risk, repetitive examination, sample preparation, sensitivity, and accuracy. For this reason, our research team contrived the best strategy for biomarker, PD-L1-specific CTCs in stage IV NSCLC group (N = 30) compared to pulmonary inflammatory patient groups (N = 30) CytoGen Smart biopsy platform. Herein, we removed false-positive cells for the first strategy of distinguishing between lymphoid/myeloid cells and the enriched-CTCs. And the second strategic approach is to calculate the pure CTCs (without false-positive cells) and then CTPS) as measured by the PD-L1 expression among pure-CTCs. That application is the percentage of viable CTCs showing partial or complete stained cells at the deducted cut-off value in each fluorescence, respectively. Results: Consequently, we demonstrated over 80% of the concordance rate between VENTANA PD-L1(SP263) and DAKO PD-L1(SP263) assay tested by the PD-L1 expression on stage IV NSCLC in tissue and pure-CTCs based CTPS from the blood. In contrast, pure-CTCs based CTPS in the pulmonary inflammatory group were all negative (recorded as zero). Conclusions: Conclusively, this study implicates that pure-CTCs based CTPS could be deployed for innovative diagnosis strategies as alternatives for tissue biopsy. Our clinical study's data suggested that the possibility for prompt decision for diagnosis and gain powerful insights to guide the personalized treatment in NSCLCs. Clinical trial information: 2020-0553.

A Three-Dimensional Conductive Scaffold Microchip for Effective Capture and Recovery of Circulating Tumor Cells with High Purity.

Effective acquirement of highly pure circulating tumor cells (CTCs) is very important for CTC-related research. However, it is a great challenge since abundant white blood cells (WBCs) are always co-collected with CTCs because of nonspecific bonding or low depletion rate of WBCs in various CTC isolation platforms. Herein, we designed a three-dimensional (3D) conductive scaffold microchip for highly effective capture and electrochemical release of CTCs with high purity. The conductive 3D scaffold was prepared by dense immobilization of gold nanotubes (Au NTs) on porous polydimethylsiloxane and was functionalized with a CTC-specific biomolecule facilitated by a Au-S bond before embedding into a microfluidic device. The spatially distributed 3D macroporous structure compelled cells to change migration from linear to chaotic and the densely covered Au NTs enhanced the topographic interaction between cells and the substrate, thus synergistically improving the CTC capture efficiency. The Au NT-coated 3D scaffold had good electrical conductivity and the Au-S bond was breakable by voltage exposure so that captured CTCs could be specifically released by electrochemical stimulation while nonspecifically bonded WBCs were not responsive to this process, facilitating recovery of CTCs with high purity. The 3D conductive scaffold microchip was successfully applied to obtain highly pure CTCs from cancer patients' blood, benefiting the downstream analysis of CTCs.

Abstract 6130: Zebrafish: A prominent tool for cancer drug screening and discovery

Abstract Zebrafish xenotransplants have been exploited to study a myriad of cancers. Due to high genetic and physiological similarities with humans, zebrafish represents a powerful and cost-effective animal model. The main advantages of using this model are transparency, easy manipulation, larger sample size, and short testing period. We have utilized this model system to study lung cancer by performing high throughput drug screening, genetic knockout, xenotransplant of human cancer cells, and circulating tumor cells (CTCs). Among the various applications mentioned, we discuss here,1) Cisplatin resistance in NSCLC (Non-small cell lung cancer). 2) CTC growth with single cell sequencing to identify gene signature of CTCs. 1) NSCLC is the most commonly diagnosed form of lung cancer (>85% of cases). Cisplatin resistance remains a major hurdle in treating advanced NSCLC. To understand the mechanistic basis of how drug resistance evolves we studied cancer progression and group behavior of cisplatin sensitive vs cisplatin resistant cancer cells in zebrafish. Cells were stained with DiI Red (H23-sensitive) and DiI Green dyes (H2009-resistant) respectively. Single culture and co-culture microinjections were done with 50-100 cells and the growth of tumor cells was monitored using live imaging for 72 hours at 30°C. In single culture injections we identified that H2009 cells tend to grow in larger groups where 90% of the cells grow in perivitelline space whereas H23 grow as single cells and are able travel farther in the animal faster. For H23 cells some grow in punctate groups in perivitelline space and about 30% of the cells travel to tail region. Next steps are to establish co-culture phenotypes by injecting different ratios (1:1; 1:2; 1:4; 1:8) of two cell types and assay drugs. 2) CTCs are major players of metastasis which leads to progression of cancer at secondary sites. With disease progression and treatment, it is known that the CTC repertoire changes with enrichment of certain populations of CTCs termed sub-clonal evolution. Therefore, monitoring CTC populations to study changes in gene expression profiles, especially in mutation status -before and after treatment, is a great tool to study development of resistance mechanisms. However, isolating a pure CTC population is not trivial due to their extreme rarity in peripheral blood. We utilize the ANGEL Parsotix technology which works on the differences in physical properties of CTCs and blood cells, like size and deformability. But still we retrieve merely 5-10 CTCs from 8ml of blood. Therefore, we allow these CTCs to grow by xenotransplanting them in zebrafish to get enough population of cells for single cell sequencing. Based on this information we plan to study CTC population genotype evolution -before and after treatment and comparison of gene expression profiles/mutation status of CTCs in primary tumor and metastatic lesions. Citation Format: Saumya Srivastava, Linlin Guo, Atish Mohanty, Michael Nelson, Brian Armstrong, Prakash Kulkarni, Ravi Salgia. Zebrafish: A prominent tool for cancer drug screening and discovery [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 6130.

Abstract 774: Highly effective isolation of circulating tumor cells using lateral magnetophoretic technology for precise genetic analysis of prostate cancer

Abstract Introduction Prostate cancer is the second leading cancer among men in the world and known as the most frequently diagnosed cancer in more than half of countries with estimated 1.3 million new emerging cases. Because of low specificity of serum PSA screening for prostate cancer, it raised concerns in keep management of clinical prognosis and therapeutic decision and thereby, there are still needed precise real-time biomarkers. Many recent studies have reported that circulating tumor cells (CTCs) can be used as important biomarkers for prostate cancer at cellular and molecular levels. However, due to their extreme rarity in the circulatory system and the lack of technology to isolate them precisely, it is still difficult to isolate them with high purity and efficiency that can be applied for clinical purposes. In this study, we introduce a microfluidic technology using lateral magnetophoresis for isolating CTCs from blood of prostate cancer patients (n=62) with high purity and efficiency and demonstrate clinical usefulness of isolated CTCs at cellular and molecular levels. Materials and methods By the lateral magnetophoretic technology using immunomagnetic nanobeads, highly pure CTCs were isolated from 5 ml blood, drawn from patients with localized (n=25) and metastatic (n=37) prostate cancer. Then, prostate cancer related genes (AR, AR-V7, EpCAM, KRT-19, PSA and PSMA) were quantified by droplet digital PCR (ddPCR) method using mRNA in CTCs. Results and discussion CTCs were detected from all blood samples (n=62). In localized cases, the average number of isolated CTCs and purity were 6.58 per ml and 2.09%. In metastatic cases, the average number of isolated CTCs and purity were 27.55 per ml and 6.00%, higher than those of localized cases. Then, mRNA was extracted from isolated CTCs and used for detecting prostate cancer specific genes. Their detection rate and expression level were increased from localized to metastatic stages. AR was detected at a similar rate of 76.00% in both localized and mHSPC stages and increased to 100% in mCRPC stage. AR-V7 was detected only in metastatic stages, mainly in mCRPC stages. EpCAM showed the highest detection rate in all stages, such as 96.00% in localized, 88.89% in mHSPC and 100% in mCRPC. This result indicates that CTCs are isolated from all blood of patients. KRT-19 showed slightly lower detection rate than EpCAM, but tend to be similar to EpCAM expression for cancer stages. PSA and PSMA were expressed very high in metastatic stage. PSA expression level in metastatic stage was 198.28 times higher than that in localized stage. Interestingly, the expression of PSA mRNA measured from CTCs was linearly proportional to serum PSA protein level, which is a commonly used diagnostic biomarker for prostate cancer. Conclusions In all stages of prostate cancer patients, the proposed microfluidic technology using the lateral magnetophoresis is a high-performance method for isolating CTCs with high detection rate and purity that can be used for precision genetic analysis. Citation Format: Hyungseok Cho, Ki-Ho Han, Jae-Seung Chung. Highly effective isolation of circulating tumor cells using lateral magnetophoretic technology for precise genetic analysis of prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 774.

Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells.

Well-preserved mRNA in circulating tumor cells (CTCs) offers an ideal material for conducting molecular profiling of tumors, thereby providing a noninvasive diagnostic solution for guiding treatment intervention and monitoring disease progression. However, it is technically challenging to purify CTCs while retaining high-quality mRNA.Here, we demonstrate a covalent chemistry-based nanostructured silicon substrate ("Click Chip") for CTC purification that leverages bioorthogonal ligation-mediated CTC capture and disulfide cleavage-driven CTC release. This platform is ideal for CTC mRNA assays because of its efficient, specific, and rapid purification of pooled CTCs, enabling downstream molecular quantification using reverse transcription Droplet Digital polymerase chain reaction. Rearrangements of ALK/ROS1 were quantified using CTC mRNA and matched with those identified in biopsy specimens from 12 patients with late-stage non-small cell lung cancer. Moreover, CTC counts and copy numbers of ALK/ROS1 rearrangements could be used together for evaluating treatment responses and disease progression.

Abstract 453: A circulating tumor cell assay for dynamic assessment of drug sensitivity in metastatic castration-resistant prostate cancer

Abstract Background: Tissue-based gene signatures can predict clinical behavior in prostate cancer (PC). Our objective was to extend their application to circulating tumor cells (CTCs) and to show that changes in the signature were associated with changes in clinical behavior. Methods: Our approach combined the Thermoresponsive(TR)-NanoVelcro CTC purification system with the Nanostring nCounter system for cellular purification and transcriptomic analysis. The Prostate Cancer Classification System (PCS) panel was modified for use in CTCs. We selected 31 blood samples from 23 PC patients receiving androgen receptor signaling inhibitors (ARSI) and measured the PCS1 Z score (probability). These findings were compared with clinical outcome data (responsiveness/resistance). Results: A modified, 16-gene PCS1 signature was established and validated through a rigorous bioinformatics process. We performed analytical validation of our combined CTC-RNA system to ensure reproducibility and specificity. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). The analyzed bloods contained samples from 8 patients who developed resistance to an ARSI allowing for dynamic measurement of gene expression. Our analysis found that the PCS1 Z score increased at the time that ARSI-resistance emerged (Pairwise T-test, P=0.016). Conclusions:Using this new methodology, contemporary, clinically-relevant gene signatures such as PCS could be measured non-invasively in CTCs. These findings can be used to relate gene expression to clinical drug response. This approach also allowed for measurement of dynamic variations of gene expression in individual patients over time that correlated to ARSI sensitivity. Citation Format: Pai-Chi Teng, Yu Jen Jan, Junhee Yoon, Jie-Fu Chen, Pin-Jung Chen, Nu Yao, Shirley Cheng, Amber Lozano, Michael R. Freeman, Sungyong You, Hsian-Rong Tseng, Edwin M. Posadas. A circulating tumor cell assay for dynamic assessment of drug sensitivity in metastatic castration-resistant prostate cancer [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 453.

A circulating tumor cell RNA assay for dynamic assessment of androgen receptor signaling inhibitors sensitivity in metastatic castration-resistant prostate cancer.

157 Background: Tissue-based gene signatures can predict clinical behavior in prostate cancer (PC). Our objective was to extend their application to circulating tumor cells (CTCs) and to show that changes in the signature were associated with changes in clinical behavior. Methods: Our approach combined the Thermoresponsive(TR)-NanoVelcro CTC purification system with the Nanostring nCounter system for cellular purification and transcriptomic analysis. The Prostate Cancer Classification System (PCS) panel was modified for use in CTCs. We selected 31 blood samples from 23 PC patients receiving androgen receptor signaling inhibitors (ARSI) and measured the PCS1 Z score (probability). These findings were compared with clinical outcome data (responsiveness/resistance). Results: A modified, 16-gene PCS1 signature was established and validated through a rigorous bioinformatics process. We performed analytical validation of our combined CTC-RNA system to ensure reproducibility and specificity. In patient bloods, ARSI-resistant samples (ARSI-R, n = 14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n = 17) (Rank-sum test, P = 0.003). The analyzed bloods contained samples from 8 patients who developed resistance to an ARSI allowing for dynamic measurement of gene expression. Our analysis found that the PCS1 Z score increased at the time that ARSI-resistance emerged (Pairwise T-test, P = 0.016). Conclusions: Using this new methodology, contemporary, clinically-relevant gene signatures such as PCS could be measured non-invasively in CTCs. These findings can be used to relate gene expression to clinical drug response. This approach also allowed for measurement of dynamic variations of gene expression in individual patients over time that correlated to ARSI sensitivity.

Isolation of circulating tumor cells and detection of EGFR mutations in patients with non-small-cell lung cancer.

The aim of the present study was to develop a procedure for the isolation of circulating tumor cells (CTCs), and to evaluate its application in the detection of epidermal growth factor receptor (EGFR) mutations, and potential heterogeneity in patients with non-small-cell lung cancer (NSCLC). Peripheral blood samples were collected from 91 patients with lung cancer, 10 patients with benign disease and 10 healthy volunteers. CTCs were enriched by positive immunomagnetic separation, detected by immunocytochemistry, and processed for single-cell capture. Pure CTC DNA was amplified, and the EGFR gene was analyzed using the amplification refractory mutation system (ARMS) and digital polymerase chain reaction (dPCR). The CTC capture rate in patients with lung cancer was 61.5% (56/91), whereas no CTCs were detected in patients with benign lung disease or in healthy volunteers. The CTC-positive detection rates were 69.3% (52/75) and 25.0% (4/16) in patients with TNM stage III and IV disease, respectively. Markedly more CTCs were captured from patients with small-cell lung cancer compared with patients with other types of cancer. In patients who were positive for EGFR mutations, the detection rate of these mutations was low (16.67%, 2/12), at the single CTC level. The sensitivity increased as the number of CTCs per sample increased. A total of four patients displayed consistent detection of EGFR mutations at the 10-cell level, and one patient exhibited a clear, inconsistent and rare mutation (G719×) between CTCs. A simplified technique for isolating CTCs from blood was established, though multiple CTCs were required to sensitively detect mutations in these cells. The detection of EGFR mutations in CTCs and tissue specimens was generally homogeneous, and therefore, the CTC-level mutation analysis may potentially contribute to the discovery of heterogeneous mutations.

Blood-Based Cancer Biomarkers in Liquid Biopsy: A Promising Non-Invasive Alternative to Tissue Biopsy.

Cancer is one of the greatest threats facing our society, being the second leading cause of death globally. Currents strategies for cancer diagnosis consist of the extraction of a solid tissue from the affected area. This sample enables the study of specific biomarkers and the genetic nature of the tumor. However, the tissue extraction is risky and painful for the patient and in some cases is unavailable in inaccessible tumors. Moreover, a solid biopsy is expensive and time consuming and cannot be applied repeatedly. New alternatives that overcome these drawbacks are rising up nowadays, such as liquid biopsy. A liquid biopsy is the analysis of biomarkers in a non-solid biological tissue, mainly blood, which has remarkable advantages over the traditional method; it has no risk, it is non-invasive and painless, it does not require surgery and reduces cost and diagnosis time. The most studied cancer non-invasive biomarkers are circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes. These circulating biomarkers play a key role in the understanding of metastasis and tumorigenesis, which could provide a better insight into the evolution of the tumor dynamics during treatment and disease progression. Improvements in isolation technologies, based on a higher grade of purification of CTCs, exosomes, and ctDNA, will provide a better characterization of biomarkers and give rise to a wide range of clinical applications, such as early detection of diseases, and the prediction of treatment responses due to the discovery of personalized tumor-related biomarkers.

Platelet–Leukocyte Hybrid Membrane‐Coated Immunomagnetic Beads for Highly Efficient and Highly Specific Isolation of Circulating Tumor Cells

AbstractBiomimetic cell‐membrane‐camouflaged nanoparticles with desirable features have been widely used for various biomedical applications. However, the current research focuses on single cell membrane coating and using multiple cell membranes for nanoparticle functionalization is still challenging. In this work, platelet (PLT) and leukocyte (WBC) membranes are fused, PLT–WBC hybrid membranes are coated onto magnetic beads, and then their surface is modified with specific antibodies. The resulting PLT–WBC hybrid membrane‐coated immunomagnetic beads (HM‐IMBs) inherit enhanced cancer cell binding ability from PLTs and reduce homologous WBC interaction from WBCs, and are further used for highly efficient and highly specific isolation of circulating tumor cells (CTCs). By using spiked blood samples, it is found that, compared with commercial IMBs, the cell separation efficiency of HM‐IMBs is improved to 91.77% from 66.68% and the cell purity is improved to 96.98% from 66.53%. Furthermore, by using the HM‐IMBs, highly pure CTCs are successfully identified in 19 out of 20 clinical blood samples collected from breast cancer patients. Finally, the robustness of HM‐IMBs is verified in downstream CTC analysis such as the detection of PIK3CA gene mutations. It is anticipated that this novel hybrid membrane coating strategy will open new possibilities for overcoming the limitations of current theranostic platforms.

Abstract 1578: NanoVelcro CTC Purification Systems for expressional analysis of circulating tumor cells from prostate cancer patients

Abstract Background: Circulating tumor cells (CTCs) are blood borne tumor cells shed from all present disease sites, including primary and metastatic tumors. Researchers have been exploring the use of CTCs to identify important transcriptomic features such as androgen receptor splicing variants in prostate cancer (PCa). Newer transcriptomic profiles such as the prostate cancer classification system (PCS) have been developed that may be useful clinically. Over the past decade, we have been developing the NanoVelcro CTC purification system which includes 3 different stimuli-responsive strategies (temperature-dependent, glycan-stimulated, click chemistry-mediated) to capture and release viable CTCs with intact RNA. The purified CTCs can then be subjected to transcriptomic analysis such as quantitative PCR (qPCR), Droplet Digital PCR (ddPCR) and NanoString's nCounter system. In this study, we benchmarked the efficiency of these platforms for purification of CTCs from blood specimens and the feasibility for detection of PCa-related RNA signatures from purified CTCs. Methods: NanoVelcro CTC purification system is combined with qPCR and ddPCR platform to detect PCa-specific RNA targets such as full length AR (AR-FL), AR splicing variant 7 (AR-V7), KLK3 (prostate-specific antigen, PSA), FOLH1 (prostate-specific membrane antigen, PSMA), and long-noncoding RNA SChLAP1 (second chromosome locus associated with prostate-1) in purified CTCs from patients' blood samples. We also developed a modified PCS panel of 30 genes adapted to the nCounter platform and tested purified CTCs from PCa patient blood samples and performed differential expression analysis for PCa disease profiling. Results: PCa-related RNA signals were detected in 16/17 CTC(+) PCa patients, including 3 of 4 non-metastatic patients. We also tested the gene expressions of the modified PCS panel using 32 patient blood samples. Upon performing clustering of samples based on the RNA signatures, we found the differential expression of CTC-target genes grouped patients in a manner which strongly related to drug response and clinical status. Conclusions: With the evolution of our NanoVelcro CTC purification system, we have shown the capability of purifying CTCs with high efficiency, while retaining cell viability and molecular integrity, allowing for detection of PCa-specific RNA signatures from CTCs. Our non-invasive, blood-based assay will be useful for detecting and continuous monitoring molecular alterations related to disease evolvement, addressing an unmet need for prostate cancer clinical care. Citation Format: Yu Jen Jan, Jie-Fu Chen, Sungyong You, Junhee Yoon, Nu Yao, Shirley Cheng, Michael R. Freeman, Hsian-Rong Tseng, Edwin M. Posadas. NanoVelcro CTC Purification Systems for expressional analysis of circulating tumor cells from prostate cancer patients [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 1578.

NanoVelcro CTC purification systems for expressional analysis of circulating tumor cells from prostate cancer patients.

295 Background: Circulating tumor cells (CTCs) are blood borne tumor cells shed from all present disease sites, including primary and metastatic tumors. Researchers have been exploring the use of CTCs to identify important transcriptomic features such as androgen receptor splicing variants in prostate cancer (PCa). Newer transcriptomic profiles such as the prostate cancer classification system (PCS) have been developed that may be useful clinically. Over the past decade, we have been developing the NanoVelcro CTC purification system which includes different stimuli-responsive strategies to capture and release viable CTCs with high efficiency and preserve intact RNA. The purified CTCs can then be subjected to transcriptomic analysis such as qPCR, ddPCR and NanoString’s nCounter system. In this study, we benchmarked the efficiency of these platforms for purification of CTCs from blood specimens and the feasibility for detection of PCa-related RNA signatures from purified CTCs. Methods: NanoVelcro CTC purification system is combined with qPCR and ddPCR platform to detect PCa-specific RNA targets in purified CTCs from patients' blood samples. We also developed a modified PCS panel of 30 genes adapted to the nCounter platform and tested purified CTCs from PCa patient blood samples and performed differential expression analysis for PCa disease profiling. Results: PCa-related RNA signals were detected in 16/17 CTC(+) PCa patients, including 3 of 4 non-metastatic patients. We also tested the gene expressions of the modified PCS panel using 48 patient blood samples. Upon performing clustering of samples based on the RNA signatures, we found the differential expression of CTC-target genes grouped patients in a manner which strongly related to drug response and clinical status. Conclusions: We have shown the capability of purifying CTCs with high efficiency using our system, while retaining cell viability and molecular integrity, allowing for detection of PCa-specific RNA signatures from CTCs. Our non-invasive, blood-based assay will be useful for detecting and continuous monitoring molecular alterations related to disease evolvement, addressing an unmet need for PCa clinical care.

NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells.

Circulating tumor cells (CTCs) are cancer cells shredded from either a primary tumor or a metastatic site and circulate in the blood as the potential cellular origin of metastasis. By detecting and analyzing CTCs, we will be able to noninvasively monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status, thus realizing the concept of "tumor liquid biopsy". However, it is technically challenging to identify CTCs in patient blood samples because of the extremely low abundance of CTCs among a large number of hematologic cells. In order to address this challenge, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with remarkable efficiency. Four generations of NanoVelcro CTC assays have been developed over the past decade for a variety of clinical utilities. The 1st-gen NanoVelcro Chips, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, were created for CTC enumeration. The 2nd-gen NanoVelcro Chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation in conjunction with the use of the laser microdissection (LMD) technique. By grafting thermoresponsive polymer brushes onto SiNS, the 3rd-gen Thermoresponsive NanoVelcro Chips have demonstrated the capture and release of CTCs at 37 and 4 °C respectively, thereby allowing for rapid CTC purification while maintaining cell viability and molecular integrity. Fabricated with boronic acid-grafted conducting polymer-based nanomaterial on chip surface, the 4th-gen NanoVelcro Chips (Sweet chip) were able to purify CTCs with well-preserved RNA transcripts, which could be used for downstream analysis of several cancer specific RNA biomarkers. In this review article, we will summarize the development of the four generations of NanoVelcro CTC assays, and the clinical applications of each generation of devices.