Recovery Of Circulating Tumor Cells Research Articles

Novel organoid platform for drug screening of patients with breast cancer.

e13125 Background: Marketed methods are available for in vitro organoid growth for the purpose of translational science, evaluation of drug efficacy and patient treatment predictivity, but require needle biopsy of the tumor which is invasive to the patient. Non-invasive methods exist for isolating circulating tumor cells (CTC) from blood but require large volumes due to their low frequency (<1 in 105-106 PBMCs). Low CTC recovery limits the number of downstream analyses. To overcome these obstacles, Cellentia is introducing a novel in vitro system called R3CE (Rapid, Reproducible, Rare Cell Expansion), developed by AcroCyte Therapeutics Inc. (Taiwan), to the US market. The advantage of R3CE is that blood is used to generate viable representative cells of the patient’s tumor which can be expanded and banked, thus the number of characterization assays is flexible. The R3CE method allows for extensive drug screening within a rapid timeframe to inform patient treatment. This poster will show drug screening results on organoids derived from blood of breast cancer patients, and R3CE method transfer to Cellentia. Methods: For patient drug screening, 0.5 mL of peripheral blood was drawn from stage IV breast cancer patients. Cells were seeded onto proprietary plates, cultured for several days, passaged and screened for drug resistance or susceptibility to Cisplatin, Paclitaxel, 5-FU, Vinorelbine, and Gemcitabine by assessing the relative viability from luminescent measurements of drug treated organoids normalized to a mock control. For R3CE method transfer, frozen breast cancer organoids were thawed and seeded onto proprietary plates, grown for several days and used in the drug screen as described above. Results: Blood from five breast cancer patients (Patients A-E) were grown into organoids and used for screening drugs listed (Table). Relative viabilities were calculated. A threshold was set at 10% where relative viabilities above or below 10% showed the organoids were resistant (R) or susceptible (S) to killing by the drug candidate, respectively. The results of the R3CE drug test were compared to the clinical (Clin) outcome for each patient (clinical outcome is unknown (U) in some instances) and shown (Table). The drug test and the clinical outcome showed 100% concordance. Cellentia executed drug screening with the same drug panel utilizing frozen breast cancer organoids in the R3CE platform and demonstrated susceptibility in the drug screening. Conclusions: R3CE platform showed promising results as a personalized drug screening tool for breast cancer patients. Frozen breast cancer organoids for drug screening demonstrated successful method transfer to Cellentia. [Table: see text]

Single-Cell Enzymatic Screening for Epithelial Mesenchymal Transition with an Ultrasensitive Superwetting Droplet-Array Microchip.

The phenotypic changes of circulating tumor cells (CTCs) during the epithelial-mesenchymal transition (EMT) have been a hot topic in tumor biology and cancer therapeutic development. Here, an integrated platform of single-cell fluorescent enzymatic assays with superwetting droplet-array microchips (SDAM) for ultrasensitive functional screening of epithelial-mesenchymal sub-phenotypes of CTCs is reported. The SDAM can generate high-density, volume well-defined droplet (0.66 nL per droplet) arrays isolating single tumor cells via a discontinuous dewetting effect. It enables sensitive detection of MMP9 enzyme activities secreted by single tumor cells, correlating to their epithelial-mesenchymal sub-phenotypes. In the pilot clinical double-blind tests, the authors have demonstrated that SDAM assays allow for rapid identification and functional screening of CTCs with different epithelial-mesenchymal properties. The consistency with the clinical outcomes validates the usefulness of single-cell secreted MMP9 as a biomarker for selective CTC screening and tumor metastasis monitoring. Convenient addressing and recovery of individual CTCs from SDAM have been demonstrated for gene mutation sequencing, immunostaining, and transcriptome analysis, revealing new understandings of the signaling pathways between MMP9 secretion and the EMT regulation of CTCs. The SDAM approach combined with sequencing technologies promises to explore the dynamic EMT plasticity of tumors at the single-cell level.

Abstract 5589: The effect of morphine-based perioperative analgesia on circulating tumor cells dissemination in colorectal cancer patients

Abstract Purpose: Circulating tumor cells (CTCs) are primary or metastatic tumor cells shed into the bloodstream and are considered precursors of distant metastatic spread and can act as an independent prognostic and predictive biomarker. Colorectal cancer (CRC) is the leading cause of cancer-related deaths worldwide and metastasis is the major cause of death. Previous studies showed poorer survival in CRC patients treated perioperatively with morphine in comparison to piritramide. Patients and Methods: In total, 150 CRC stage I-III patients undergoing either radical or laparoscopic surgery were enrolled in this prospective multicentric randomized study (NCT03700411). Patients were randomized into three arms using different perioperative analgesia - morphine, piritramide, and epidural analgesia. Three peripheral blood samples were collected preoperatively, one day and one month after surgery respectively. To preserve the cellular content of specimens, Cell-Free DNA BCT® (Streck, Inc.) blood collection tubes were used. The CTCs were identified using CytoTrack CT11TM (2/C, Denmark), a semi-automated immunofluorescence microscopy detecting the pan-cytokeratin and EpCAM signals. The method recovery rate was analyzed using SW-480 cancer cell line by flow cytometry. Results: The CTCs recovery rate of the method was 72% and the method was certified by ISO 15189. The analyses of CTC presence revealed the highest positivity rate in the samples collected from patients treated with morphine on the first day after surgery (51.4 %). Also, a significant increase in positivity (from 23.5 % for the perioperatively collected samples) compared to samples from patients treated with epidural analgesia (31.1 % to 33.3 %) and piritramide (44.2% to 41.0%) was observed. The CTC presence was lower in control samples collected after one month irrespective of the analgesia type. The highest positivity among the control samples was in those associated with morphine analgesia (32.1 % versus 25.7% for epidural and 25.0% for piritramide analgesia). Conclusion: A significant increase in CTC levels was found in postoperative blood samples of CRC patients treated with morphine compared to piritramide and epidural analgesia. Enrollment in the study is ongoing. Acknowledgments: This study was supported by the Ministry of Health of the Czech Republic (NV18-03-00470), Palacky University Olomouc (LF 2022_012), European Union - Next Generation EU (LX22NPO5102) and European Regional Development Fund (ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868). Citation Format: Pavel Stejskal, Josef Srovnal, Emil Berta, Alona Rehulkova, Lubomír Večeřa, Tomas Gabrhelik, Filip Haiduk, Jan Maca, Jan Bruthans, Pavel Michalek, Pavla Kourilova, Marian Hajduch. The effect of morphine-based perioperative analgesia on circulating tumor cells dissemination in colorectal cancer patients. [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 5589.

Abstract 1040: Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry

Abstract The detection of liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have demonstrated promise as non-invasive assays for monitoring response to therapy for patients with cancer. While ctDNA cannot predict transcriptomic and proteomic changes in response to therapy, we can measure direct cell surface antigen and gene expression changes in CTCs. In pediatric settings, blood samples are often collected from multi-institutional trials. Therefore, to translate liquid biopsies into routine practice, it is important to explore variables most likely to impact the functionality of CTCs including time to process the sample, prospective cryopreservation, blood collection tube and fixative reagents. Cell stabilizer tubes such as Streck Cell-Free DNA, CellSave, CellRescue, or PAXgene ccfDNA have been designed to prevent cell lysis and release of genomic DNA into the plasma, often observed in standard EDTA tubes. In pediatrics, we are often limited by the volume of blood we can draw for research. To this end, we have established an innovative pipeline to collect ctDNA, CTCs, and matched germline DNA from a single blood sample. Blood samples were processed for plasma extraction and CTC isolation after 4h, 24h, and 48h. We isolated rare CTCs from whole blood by combining peripheral blood cell depletion with antigen-based CTC identification. We then evaluated CTC recovery rate and cell membrane integrity (cell-membrane permeabilization) across all blood collection tubes by flow or mass cytometry. Additionally, we integrated whole transcriptome analysis of RNA transcripts in individually sorted CTCs. In this study, we detected similar levels of cell-free DNA, germline DNA and ctDNA in healthy donors and patient samples collected in the DNA stabilizer tubes up to 48h after blood collection. Surprisingly, frequent clotting was observed in the cell layer of samples collected in PAXgene tubes only 24h after blood collection, preventing further analysis of CTC quality in these tubes. At 4h, EDTA tubes and stabilizer tubes seem to perform similarly in preserving CTC integrity. However, at later timepoints, CTC recovery rate, RNA quality and cell membrane integrity were significantly higher in CellRescue tubes. Finally, we showed that CTC count, and RNA quality were slightly lower in patient samples enumerated after cryopreservation compared to the paired-samples processed the same day in both EDTA and cell-stabilizing tubes. In conclusion, we validated the CellRescue tubes as the best candidate to reduce cell lysis and protect against cell membrane permeabilization and mRNA degradation for up to 48 hours, allowing sample collection from multi-institutional studies. To the best of our knowledge, this study is the first to compare the key steps in handling blood samples and how this affects transcriptomic-based assay performance. Citation Format: Anne-Florence Blandin, Sarah Finstuen-Magro, Samuel Abbou, Kelly Klega, Alejandra E. Aguilar, Mohammad Tanhaemami, Maria Ana Isabel C. De Los Santos, Joadly Duplan, Brian Crompton. Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry [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 1040.

Accurate isolation and detection of circulating tumor cells using enrichment-free multiparametric high resolution imaging.

The reliable and accurate detection of rare circulating tumor cells (CTCs) from cancer patient blood samples promises advantages in both research and clinical applications. Numerous CTC detection methods have been explored that rely on either the physical properties of CTCs such as density, size, charge, and/or their antigen expression profiles. Multiple factors can influence CTC recovery including blood processing method and time to processing. This study aimed to examine the accuracy and sensitivity of an enrichment-free method of isolating leukocytes (AccuCyte® system) followed by immunofluorescence staining and high-resolution imaging (CyteFinder® instrument) to detect CTCs. Healthy human blood samples, spiked with cancer cells from cancer cell lines, as well as blood samples obtained from 4 subjects diagnosed with cancer (2 pancreatic, 1 thyroid, and 1 small cell lung) were processed using the AccuCyte-CyteFinder system to assess recovery rate, accuracy, and reliability over a range of processing times. The AccuCyte-CyteFinder system was highly accurate (95.0%) at identifying cancer cells in spiked-in samples (in 7.5 mL of blood), even at low spiked-in numbers of 5 cells with high sensitivity (90%). The AccuCyte-CyteFinder recovery rate (90.9%) was significantly higher compared to recovery rates obtained by density gradient centrifugation (20.0%) and red blood cell lysis (52.0%). Reliable and comparable recovery was observed in spiked-in samples and in clinical blood samples processed up to 72 hours post-collection. Reviewer analysis of images from spiked-in and clinical samples resulted in high concordance (R-squared value of 0.998 and 0.984 respectively). The AccuCyte-CyteFinder system is as an accurate, sensitive, and clinically practical method to detect and enumerate cancer cells. This system addresses some of the practical logistical challenges in incorporating CTCs as part of routine clinical care. This could facilitate the clinical use of CTCs in guiding precision, personalized medicine.

Hybrid Microfluidic Device for High Throughput Isolation of Cells Using Aptamer Functionalized Diatom Frustules

Circulating tumor cells (CTCs), secreted from primary and metastatic malignancies, hold a wealth of essential diagnostic and prognostic data for multiple cancers. Significantly, the information contained within these cells may hold the key to understanding cancer metastasis, both individually and fundamentally. Accordingly, developing ways to identify, isolate and interrogate CTCs plays an essential role in modern cancer research. Unfortunately, CTCs are typically present in the blood in vanishingly low titers and mixed with other blood components, making their isolation and analysis extremely challenging. Herein, we report the design, fabrication and optimization of a microfluidic device capable of automatically isolating CTCs from whole blood. This is achieved in two steps, via the passive viscoelastic separation of CTCs and white blood cells (WBCs) from red blood cells (RBCs), and subsequent active magnetophoretic separation of CTCs from WBCs. We detail the specific geometries required to balance the elastic and inertial forces required for successful passive separation of RBCs, and the use of computational fluid dynamics (CFD) to optimize active magnetophoretic separation. We subsequently describe the use of magnetic biosilica frustules, extracted from Chaetoceros sp. diatoms, to fluorescently tag CTCs and facilitate magnetic isolation. Finally, we use our microfluidic platform to separate HepG2-derived CTCs from whole blood, demonstrating exceptional CTC recovery (94.6%) and purity (89.7%)

Abstract 1952: Validation of enhanced performance of the AccuCyte®-CyteFinder® platform for circulating tumor cell characterization

Abstract Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and stratification of cancer patients. AccuCyte® is a density-based unbiased isolation method that transfers nucleated cells from whole blood to slides for the characterization of CTCs and other rare cells. RarePlex® panel kits are IF staining reagents used on automated slide staining instruments to label cells to differentiate CTCs from white blood cells (WBC). CyteFinder® is a seven-channel automated fluorescent imaging system that rapidly scans microscope slides and applies machine learning algorithms to identify CTCs. Together, these technologies provide an end-to-end solution for CTC characterization. For analysis, blood is drawn into AccuCyte blood collection tubes (BCTs) containing a preservative which maintains cell properties prior to processing onto slides. Once slides are prepared, they can be stored at -20°C without significant biomarker degradation. This flexible workflow allows investigators to bank samples for batch analysis and to begin sample collection prior to validating the IF assay to be used. This study was designed to evaluate: (1) stability time between collection in the AccuCyte BCT and sample processing; (2) performance of an improved version of the AccuCyte kit with higher nucleated cell isolation capacity; and (3) storage time that AccuCyte prepared slides can be banked frozen prior to staining. The study was performed using model CTCs and cancer patient samples. Metrics to determine performance were CTC recovery and mean fluorescence intensity (MFI) of biomarker expression. Our results demonstrate that the AccuCyte BCT preserves blood components for at least 5 days after collection without significant effect on CTC recovery or biomarker expression. The latest version of the AccuCyte kit demonstrated a higher cell isolation capacity and could collect up to 60% more nucleated blood cells than the previous version, increasing CTC recovery. The increased capacity was demonstrated in patients treated with hematopoietic growth factors, whose WBC count was significantly higher than the normal range. Finally, accelerated-aging study results demonstrated that AccuCyte-prepared slides can be stored at -20°C for at least 4 years without significant effect on most biomarkers tested. In conclusion, enhancements to the AccuCyte-CyteFinder platform reported here increase flexibility and performance for analysis of CTCs in global clinical trials by allowing longer periods of time before collected blood samples need to be processed and by extending the length of time processed slides can be banked before they are stained. Citation Format: Arturo B. Ramirez, Lillian Costandy, Brady S. Gardner, Ryan H. Huston, A Anders Larson Tevis, Casey E. Helmicki, Alisa C. Clein, Daniel E. Sabath, Joshua J. Nordberg, Tad C. George. Validation of enhanced performance of the AccuCyte®-CyteFinder® platform for circulating tumor cell characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1952.

Abstract 5994: Separating cancer cells from neutrophils by gradient acoustic focusing

Abstract Introduction: A subset of aggressive prostate cancers (PCa) progress to invade surrounding tissue to form distant metastases and shed of circulating tumor cells (CTCs). However, due to a scarcity of CTC among the millions of nucleated blood cells, there is an unmet need to improve CTC assays. Acoustophoresis uses standing ultrasound waves in a flow-through microfluidic chamber to discriminate cells based on the size, density, and compressibility. The technique is label-free as opposed to assays that rely on the cell surface marker EpCAM to detect CTCs. We will use label-free two-step acoustophoresis method as the initial separation step followed by a density medium purging step, to obtain efficient recovery of CTC of very high purity. Experimental procedure: Acoustic cell separation of CTCs from blood is challenging in reference to overlap of acoustic properties between small-sized CTCs and densely granular WBC. To obtain high recovery of CTC, we must apply a sufficiently large acoustic focusing amplitude which leads to a contaminating cell population comprising 2% white blood cells, (WBCs) that mainly contain eosinophil cells. To obtain high purity cancer cell fractions there is a critical need for an additional cleaning step. Therefore, we used the density medium iodixanol in a purging step where the optimal iodixanol medium concentration is intended to provide one cell type with positive, and the other with negative acoustic contrast. Preliminary results: show that the granulocytes can be transferred by the ultrasound field into the high-density medium (iodixanol) and can thereby be removed through a first outlet. The cancer cells do not transfer into the iodixanol medium and will be collected in a pure PCa cell fraction in the low-density medium through a second outlet. The samples were processed at a flow rate of 75 μl/min. The limiting factor regarding flow rate when purging the sample from granulocytes is overheating caused by the ultrasound transducer. The separation efficiency for cancer cells was 99.5% with 2.2% contaminating granulocytes. Theoretically, by combining the two separation steps only 0,044% of the WBC would remain in the cancer cell fraction. Conclusion: It is unclear which are the functional properties that enable a primary tumor to shed CTCs, and comprehensive characterization is urgently needed to identify key features of these processes. CTC fractions of high purity will enable single cell analysis with validated Fluidigm-based RT-PCR panels, mass spectrometry, and RNA-sequencing. We propose to use a two-step acoustic separation method, with a primary acoustic separation step and a second high-density medium purging step. Citation Format: Cecilia Magnusson, Mahdi Rezayati Charan, Hans Lilja, Per Augustsson. Separating cancer cells from neutrophils by gradient acoustic focusing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5994.

Abstract LB117: Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry

Abstract Introduction: CTCs play a critical role in breast cancer (BCa) metastases. Rapid identification of live CTCs may help to predict BCa prognosis and identify which patients may derive treatment benefit, especially for those with metastatic disease. However, CTCs are present at very low concentrations in the peripheral blood, and CTC enumeration is costly and technically challenging. We previously reported that cell-size based device utilizing a microfluidic cassette could be used to isolate live blood cells (Zhang et al. 2019 AACR). Here we describe a pilot study using novel microfluidic Parsortix workflow to identify live CTCs for patients with Stage IV BCa. Methods: Four whole blood samples (7.5ml/each) were collected from each of 10 patients with stage IV BCa patients who had continued clinical progression of their disease on systemic therapy, under a Northwestern University IRB-approved study (NU20B03). All samples were stored at 15-30 °C in EDTA tubes. Live CTCs enrichment was performed using a cell size and deformability based Parsortix system (Angle) which utilizes a microfluidic based disposable cassette containing a step separation structure. The four samples from each patient were loaded onto the device and the separation procedure was started by using 4.5μm, 6.5μm 8μm and 10μm separation cassettes, respectively. After separation and cleaning, the CTCs captured by the Parsortix system were harvested by inverting the flow direction and then flushing from the cassette in 200μL PBS. The harvested CTCs were multi-stained directly by fluorescence labeled antibodies for Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes) (Menarini). Flow cytometry (FCM) was used to identify the CTCs which were classified as CK+, DAPI+ and CD45-. White blood cells from patient samples and the human breast cancer cell line MDA-MB-231 were used as negative and positive controls, respectively. Results: A total of 500 and 200 MDA-MB-231 cells were processed as controls, which were collected completely by Parsortix and identified by FCM confirming that Parsortix system could recover the CTCs effectively. CTCs were classified based on morphology and phenotype as CK+, DAPI+ and CD45-. Live CTCs (≥1) were detected in 8 out of 10 (80%), 9 out of 10 (90%), 7 out of 10 (70%) and 5 out of 10 (50%) samples by using 4.5μm (Group 1), 6.5μm (Group 2), 8μm (Group 3) and 10μm (Group 4) separation cassettes. Maximum CTCs isolated from each group were 18, 32, 12 and 8 from Group 1, 2, 3 and 4, respectively. The corresponding average CTCs isolated from each sample were 3.5 CTCs, 5.6 CTCs, 2.5 CTCs and 1.7 CTCs from Group 1, 2, 3, and 4, respectively. The 6.5μm cassettes (Group 2) using for CTCs collection had significantly higher efficacy on collection of CTCs compared to other three groups. Conclusions: In this pilot study, our findings demonstrated that the cell-sized dependent Parsortix system using multiple staining and FCM is an effective and specific approach for rapid recovery and identification of CTCs from Stage IV BCa patients. With further optimization, this strategy can help to accurately evaluate CTCs and offers a potential technique for diagnosis and treatmentmonitoring of patients with metastatic breast cancer. Citation Format: Qiang Zhang, Marwa Manai, Andrew Davis, Carolina Reduzzi, Paolo D’Amico, Saya Liz Jacob, Jianhua Jiao, Weijun Qin, Lisa Flaum, Amir Behdad, Massimo Cristofanilli, Ami Shad, Leonidas Platanias, William Gradishar. Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB117.

Abstract 1956: Perioperative opioid analgesia affects the circulating tumor cells levels in colorectal cancer patients

Abstract Purpose Colorectal cancer (CRC) is one of the most common cancers worldwide and metastases-related death is the main cause of its high mortality. Previous studies showed the poorer survival in colorectal cancer patients treated perioperatively with morphine in comparison to piritramide. Circulating tumor cells (CTCs) are considered as precursors of distant metastatic spread and can act as an independent prognostic and predictive biomarker. The hypothesis that different pain-killers used in perioperative period can affect the CTCs levels was tested. Patients and methods In total, 100 CRC stage I-III patients undergoing radical surgery were enrolled in this prospective multicentric randomized pilot study (NCT03700411). Three peripheral blood samples were collected into Cell-Free DNA BCT® (Streck, Inc.) preoperatively, one day and one month after surgery respectively. Patients were randomized into three arms using different perioperative analgesia - morphine, piritramide and epidural analgesia. The CTCs were identified using the CytoTrack CT11࣪ (2/C, Denmark), a semi-automated immunofluorescence microscopy detecting the pan-cytokeratin and EpCAM signals. The method recovery rate was analyzed using SW-480 cancer cell line by flow cytometry. Results The CTCs were found most frequent in the samples obtained at first day after surgery (44,9%) followed by the samples collected before surgery (36%). The lowest levels were observed in control samples one month after surgery (31%). The significant increase of CTCs levels was found in majority of patients treated with morphine (16,3%) compared to piritramide and epidural analgesia. However, the significant decrease of CTCs levels was observed in samples drawn one month after surgery in both, morphine and piritramide arm. There were no significant changes in CTCs levels of patients with epidural analgesia. The CTCs recovery rate of the method was 72%. Conclusion The significant increase of CTCs levels was found in postoperative blood samples of CRC patients treated with morphine compared to the piritamide and epidural analgesia. The enrollment into the study is ongoing. Acknowledgements This study was supported by Ministry of Health of the Czech Republic (NV18-03-00470), Ministry of Education, Youth and Sport of the Czech Republic (LM2018132), Palacky University Olomouc (LF 2021_019) and European Regional Development Fund (ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868, ACGT CZ.02.1.01/0.0/0.0/16_026/0008448). Citation Format: Pavel Stejskal, Josef Srovnal, Emil Berta, Alona Rehulkova, Lubomir Vecera, Filip Haiduk, Pavel Michalek, Marian Hajduch. Perioperative opioid analgesia affects the circulating tumor cells levels in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1956.

An ultra-thin silicon nitride membrane for label-free CTCs isolation from whole blood with low WBC residue

Microfiltration is the most straightforward and effective method to enrich circulating tumor cells (CTCs) from large numbers of background blood cells in the peripheral blood. It is still very challenging to achieve both high recovery of CTCs and low residue of white blood cells (WBCs) through one-time whole blood filtration. In this study, through a simulation, we revealed that reducing the thickness of the filter significantly reduces the WBC residue. A filter device was created using a 400-nm-thick Si3N4 membrane. The recovery rates of A549 and MCF-7 tumor cell lines were 82.04 ± 1.19% and 86.53 ± 3.27%, respectively, while the residual number of WBCs was largely reduced from 6.84 × 106 cells/mL cells to 604 ± 33.74 cells/mL. In addition, the assembled device could process whole blood without pretreatment at a high throughput of 1 mL/min. Utilizing as small volume of peripheral blood as 1 mL, CTCs were enriched by our device in four of six lung cancer patients and four of five breast cancer patients. The lower WBC residue is advantageous in CTC enumeration and subsequent proteomic or genomic characterization. Therefore, this technology enables label-free CTC isolation with a low WBC residue from whole blood, paving the way for a CTC analysis in clinical applications.

Single-Cell Phenotypic and Molecular Characterization of Circulating Tumor Cells Isolated from Cryopreserved Peripheral Blood Mononuclear Cells of Patients with Lung Cancer and Sarcoma.

The isolation of circulating tumor cells (CTCs) requires rapid processing of the collected blood due to their inherent fragility. The ability to recover CTCs from peripheral blood mononuclear cells (PBMCs) preserved from cancer patients could allow for retrospective analyses or multicenter CTC studies. We compared the efficacy of CTC recovery and characterization using cryopreserved PMBCs vs fresh whole blood from patients with non-small cell lung cancer (NSCLC; n = 8) and sarcoma (n = 6). Two epithelial cellular adhesion molecule (EpCAM)-independent strategies for CTC enrichment, based on Parsortix® technology or immunomagnetic depletion of blood cells (AutoMACS®) were tested, followed by DEPArray™ single-cell isolation. Phenotype and genotype, assessed by copy number alterations analysis, were evaluated at a single-cell level. Detection of target mutations in CTC-enriched samples from frozen NSCLC PBMCs was also evaluated by digital PCR (dPCR). The use of cryopreserved PBMCs from cancer patients allowed for the retrospective enumeration of CTCs and their molecular characterization, using both EpCAM-independent strategies that performed equally in capturing CTC. Cells isolated from frozen PBMCs were representative of whole blood-derived CTCs in terms of number, phenotype, and copy number aberration profile/target mutations. Long-term storage (≥3 years) did not affect the efficacy of CTC recovery. Detection of target mutations was also feasible by dPCR in CTC-enriched samples derived from stored PBMCs. Isolating CTCs from longitudinally collected PBMCs using an unbiased selection strategy can offer a wider range of retrospective genomic/phenotypic analyses to guide patients' personalized therapy, paving the way for sample sharing in multicenter studies.

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.

Evaluation of a marker independent isolation method for circulating tumor cells in esophageal adenocarcinoma.

The enrichment of circulating tumor cells (CTCs) from blood provides a minimally invasive method for biomarker discovery in cancer. Longitudinal interrogation allows monitoring or prediction of therapy response, detection of minimal residual disease or progression, and determination of prognosis. Despite inherent phenotypic heterogeneity and differences in cell surface marker expression, most CTC isolation technologies typically use positive selection. This necessitates the optimization of marker-independent CTC methods, enabling the capture of heterogenous CTCs. The aim of this report is to compare a size-dependent and a marker-dependent CTC-isolation method, using spiked esophageal cells in healthy donor blood and blood from patients diagnosed with esophageal adenocarcinoma. Using esophageal cancer cell lines (OE19 and OE33) spiked into blood of a healthy donor, we investigated tumor cell isolation by Parsortix post cell fixation, immunostaining and transfer to a glass slide, and benchmarked its performance against the CellSearch system. Additionally, we performed DEPArray cell sorting to infer the feasibility to select and isolate cells of interest, aiming towards downstream single-cell molecular characterization in future studies. Finally, we measured CTC prevalence by Parsortix in venous blood samples from patients with various esophageal adenocarcinoma tumor stages. OE19 and OE33 cells were spiked in healthy donor blood and subsequently processed using CellSearch (n = 16) or Parsortix (n = 16). Upon tumor cell enrichment and enumeration, the recovery rate ranged from 76.3 ± 23.2% to 21.3 ± 9.2% for CellSearch and Parsortix, respectively. Parsortix-enriched and stained cell fractions were successfully transferred to the DEPArray instrument with preservation of cell morphology, allowing isolation of cells of interest. Finally, despite low CTC prevalence and abundance, Parsortix detected traditional CTCs (i.e. cytokeratin+/CD45-) in 8/29 (27.6%) of patients with esophageal adenocarcinoma, of whom 50% had early stage (I-II) disease. We refined an epitope-independent isolation workflow to study CTCs in patients with esophageal adenocarcinoma. CTC recovery using Parsortix was substantially lower compared to CellSearch when focusing on the traditional CTC phenotype with CD45-negative and cytokeratin-positive staining characteristics. Future research could determine if this method allows downstream molecular interrogation of CTCs to infer new prognostic and predictive biomarkers on a single-cell level.

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.

EMT-independent detection of circulating tumor cells in human blood samples and pre-clinical mouse models of metastasis

Circulating tumor cells (CTCs) present an opportunity to detect/monitor metastasis throughout disease progression. The CellSearch® is currently the only FDA-approved technology for CTC detection in patients. The main limitation of this system is its reliance on epithelial markers for CTC isolation/enumeration, which reduces its ability to detect more aggressive mesenchymal CTCs that are generated during metastasis via epithelial-to-mesenchymal transition (EMT). This Technical Note describes and validates two EMT-independent CTC analysis protocols; one for human samples using Parsortix® and one for mouse samples using VyCap. Parsortix® identifies significantly more mesenchymal human CTCs compared to the clinical CellSearch® test, and VyCap identifies significantly more CTCs compared to our mouse CellSearch® protocol regardless of EMT status. Recovery and downstream molecular characterization of CTCs is highly feasible using both Parsortix® and VyCap. The described CTC protocols can be used by investigators to study CTC generation, EMT and metastasis in both pre-clinical models and clinical samples.

Multigene model for predicting metastatic prostate cancer using circulating tumor cells by microfluidic magnetophoresis

We aimed to isolate circulating tumor cells (CTCs) using a microfluidic technique with a novel lateral magnetophoretic microseparator. Prostate cancer–specific gene expressions were evaluated using mRNA from the isolated CTCs. A CTC‐based multigene model was then developed for identifying advanced prostate cancer. Peripheral blood samples were obtained from five healthy donors and patients with localized prostate cancer (26 cases), metastatic hormone‐sensitive prostate cancer (mHSPC, 10 cases), and metastatic castration‐resistant prostate cancer (mCRPC, 28 cases). CTC recovery rate and purity (enriched CTCs/total cells) were evaluated according to cancer stage. The areas under the curves of the six gene expressions were used to evaluate whether multigene models could identify mHSPC or mCRPC. The number of CTCs and their purity increased at more advanced cancer stages. In mHSPC/mCRPC cases, the specimens had an average of 27.5 CTCs/mL blood, which was 4.2 × higher than the isolation rate for localized disease. The CTC purity increased from 2.1% for localized disease to 3.8% for mHSPC and 6.7% for mCRPC, with increased CTC expression of the genes encoding prostate‐specific antigen (PSA), prostate‐specific membrane antigen (PSMA), and cytokeratin 19 (KRT19). All disease stages exhibited expression of the genes encoding androgen receptor (AR) and epithelial cell adhesion molecule (EpCAM), although expression of the AR‐V7 variant was relatively rare. Relative to each gene alone, the multigene model had better accuracy for predicting advanced prostate cancer. Our lateral magnetophoretic microseparator can be used for identifying prostate cancer biomarkers. In addition, CTC‐based genetic signatures may guide the early diagnosis of advanced prostate cancer.

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.

A Novel Microfluidic Method Utilizing a Hydrofoil Structure to Improve Circulating Tumor Cell Enrichment: Design and Analytical Validation.

Being one of the major pillars of liquid biopsy, isolation and characterization of circulating tumor cells (CTCs) during cancer management provides critical information on the evolution of cancer and has great potential to increase the success of therapies. In this article, we define a novel strategy to effectively enrich CTCs from whole blood based on size, utilizing a spiral microfluidic channel embedded with a hydrofoil structure at the downstream of the spiral channel. The hydrofoil increases the distance between the streams of CTCs and peripheral blood cells, which are already distributed about two focal axes by the spiral channel, thereby improving the resolution of the separation. Analytical validation of the system has been carried out using Michigan Cancer Foundation-7 (MCF7) breast cancer cell lines spiked into blood samples from healthy donors, and the performance of the system in terms of white blood cell (WBC) depletion, CTC recovery rate and cell viability has been shown in single or two-step process: by passing the sample once or twice through the microfluidic chip. Single step process yielded high recovery (77.1%), viable (84.7%) CTCs. When the collected cell suspension is re-processed by the same chip, recovery decreases to 65.5%, while the WBC depletion increases to 88.3%, improving the purity. Cell viability of >80% was preserved after two-step process. The novel microfluidic chip is a good candidate for CTC isolation applications requiring high recovery rate and viability, including functional downstream analyses for variety of cancer types.

Evaluation of Microfluidic Ceiling Designs for the Capture of Circulating Tumor Cells on a Microarray Platform

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.