Model Of Circulating Tumor Cells Research Articles

Simulating the Effect of Removing Circulating Tumor Cells (CTCs) from Blood Reveals That Only Implantable Devices Can Significantly Reduce Metastatic Burden of Patients.

Circulating tumor cells (CTCs) are cells that have separated from a solid cancerous lesion and entered the bloodstream. They play a crucial role in driving the metastatic spread to distant organs, which is the leading cause of cancer-related deaths. Various concepts for blood purification devices aiming to remove CTCs from the blood and prevent metastases have been developed. Until now, it is not clear if such devices can indeed reduce new metastasis formation in a significant way. Here, we present a simple theoretical model of CTCs in the bloodstream that can be used to predict a reduction in metastatic burden using an extracorporeal or intracorporeal blood purification device. The model consists of a system of ordinary differential equations that was numerically solved and simulated. Various simulations with different parameter settings of extracorporeal and intracorporeal devices revealed that only devices implanted directly in tumor-draining vessels can reduce the metastatic burden significantly. Even if an extracorporeal device is used permanently, the reduction in metastases is only 82%, while a permanently operating implanted device in the tumor-draining vessel would achieve a reduction of 99.8%. These results are mainly due to the fact that only a small fraction of CTCs reaches peripheral circulation, resulting in a proportionally small amount of purified blood in extracorporeal devices.

Engineered Leukocyte Biomimetic Colorimetric Sensor Enables High-Efficient Detection of Tumor Cells Based on Bioorthogonal Chemistry.

Accurate detection of heterogeneous circulating tumor cells (CTCs) is critical as they can make tumor cells more aggressive, drug-resistant, and metastasizing. Although the leukocyte membrane coating strategy is promising in meeting the challenge of detecting heterogeneous CTCs due to its inherent antiadhesive properties, it is still limited by the reduction or loss of expression of known markers. Bioorthogonal glycol-metabolic engineering is expected to break down this barrier by feeding the cells with sugar derivatives with a unique functional group to establish artificial targets on the surface of tumor cells. Herein, an engineered leukocyte biomimetic colorimetric sensor was accordingly fabricated for high-efficient detection of heterogeneous CTCs. Compared with conventional leukocyte membrane coating, the sensor could covalently bound to the heterogeneous CTCs models fed with Ac4ManNAz in vitro through the synergy of bioorthogonal chemistry and metabolic glycoengineering, ignoring the phenotypic changes of heterogeneous CTCs. Meanwhile, a sandwich structure composed of leukocyte biomimetic layer/CTCs/MoS2 nanosheet was formed for visual detection of HeLa cells as low as 10 cells mL-1. Overall, this approach can overcome the dependence of conventional cell membrane biomimetic technology on specific cell phenotypes and provide a new viewpoint to highly efficiently detect heterogeneous CTCs.

Unique Cohorts of Salivary Gland Cancer Cells as an in-vitro Model of Circulating Tumor Cells.

The characterization of circulating tumor cells (CTC) and circulating tumor microemboli (CTM) has emerged as both a challenge to the standard view of metastasis, and as a valuable means for understanding genotypic and phenotypic variability shown even within the same cancer type. However, in the case of salivary gland neoplasms, limited data are available for the role that CTCs and CTMs play in metastasis and secondary tumor formation.ru.AQ1 In response to this, we propose that similarities between in vitro clusters of cultured salivary gland cancer cells may act as a surrogate model for in vivo CTCs and CTMs isolated from patients. Using techniques in immunofluorescence, immunoblotting, and 2-dimensional migration, we isolated and characterized a group of cohort cells from a commercially available cell line (HTB-41). Results: Here, cells exhibited a hybrid phenotype with simultaneous expression of both epithelial and mesenchymal markers (E-cadherin, vimentin, and α-SMA). Cohort cells also exhibited increased migration in comparison to parental cells. Data suggest that these isolated cell clusters may fucntion as a potential in vitro model of CTCs and CTMs.

All-in-One Microfluidic Chip for Online Labeling, Separating, and Focusing Rare Circulating Tumor Cells from Blood Samples Followed by Inductively Coupled Plasma Mass Spectrometry Detection.

Circulating tumor cell (CTC) detection is essential for early cancer diagnosis and evaluating treatment efficacy. Despite the growing interest in isolating CTCs and further quantifying surface biomarkers at the single-cell level, highly efficient separation of rare CTCs from massive blood cells is still a big challenge. Here, we developed an all-in-one microfluidic chip system for the immunolabeling, magnetic separation, and focusing of HepG2 cells (as a CTC model) and online combined it with single cell-inductively coupled plasma mass spectrometry (SC-ICP-MS) for quantitative analysis of the asialoglycoprotein receptor (ASGPR) on single HepG2 cells. Lanthanide-labeled anti-ASGPR monoclonal antibody and antiepithelial cell adhesion molecule-modified magnetic beads were prepared as signal and magnetic probes, respectively. Target cells were highly efficiently labeled with signal and magnetic probes in the mixing zone of the microfluidic chip and then focused and sorted in the separation zone by specific magnetic separation techniques to avoid matrix contamination. The average cell recovery of HepG2 cells was derived to be 94.1 ± 5.7% with high separation efficiency and purity. The sorted cells with signal probes were detected for enumeration and quantification of ASGPR on their surface by SC-ICP-MS. The developed method showed good specificity and high sensitivity, detecting an average of (1.0 ± 0.2) × 105 ASGPR molecules per cell surface. This method can be used for absolute quantitative analysis of ASGPR on the surface of single hepatocellular carcinoma cells in real-world samples, providing a highly efficient analytical platform for studying targeted drug delivery in cancer therapy.

Propagated Circulating Tumor Cells Uncover the Potential Role of NFκB, EMT, and TGFβ Signaling Pathways and COP1 in Metastasis.

Circulating tumor cells (CTCs), a population of cancer cells that represent the seeds of metastatic nodules, are a promising model system for studying metastasis. However, the expansion of patient-derived CTCs ex vivo is challenging and dependent on the collection of high numbers of CTCs, which are ultra-rare. Here we report the development of a combined CTC and cultured CTC-derived xenograft (CDX) platform for expanding and studying patient-derived CTCs from metastatic colon, lung, and pancreatic cancers. The propagated CTCs yielded a highly aggressive population of cells that could be used to routinely and robustly establish primary tumors and metastatic lesions in CDXs. Differential gene analysis of the resultant CTC models emphasized a role for NF-κB, EMT, and TGFβ signaling as pan-cancer signaling pathways involved in metastasis. Furthermore, metastatic CTCs were identified through a prospective five-gene signature (BCAR1, COL1A1, IGSF3, RRAD, and TFPI2). Whole-exome sequencing of CDX models and metastases further identified mutations in constitutive photomorphogenesis protein 1 (COP1) as a potential driver of metastasis. These findings illustrate the utility of the combined patient-derived CTC model and provide a glimpse of the promise of CTCs in identifying drivers of cancer metastasis.

Exploring the Role of Hypoxia-Inducible Carbonic Anhydrase IX (CAIX) in Circulating Tumor Cells (CTCs) of Breast Cancer

Circulating tumor cells (CTCs) in the peripheral blood are believed to be the source of metastasis and can be used as a liquid biopsy to monitor cancer progression and therapeutic response. However, it has been challenging to accurately detect CTCs because of their low frequency and the heterogeneity of the population. In this study, we have developed an in vitro model of CTCs by using non-adherent suspension culture. We used this model to study a group of breast cancer cell lines with distinct molecular subtypes (TNBC, HER2+, and ER+/PR+). We found that, when these breast cancer cell lines lost their attachment to the extracellular matrix, they accumulated a subtype of cancer stem cells (CSC) that expressed the surface markers of stem cells (e.g., CD44+CD24-). These stem-like CTCs also showed high expressions of hypoxia-inducible gene products, particularly the hypoxia-inducible carbonic anhydrase IX (CAIX). Inhibition of CAIX activity was found to reduce CAIX expression and stem cell phenotypes in the targeted CTCs. Further studies are needed, using CTC samples from breast cancer patients, to determine the role of CAIX in CTC survival, CSC transition, and metastasis. CAIX may be a useful surface marker for the detection of CSCs in the blood, and a potential target for treating metastatic breast cancers.

Liquid biopsy approaches and immunotherapy in colorectal cancer for precision medicine: Are we there yet?

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths globally, with nearly half of patients detected in the advanced stages. This is due to the fact that symptoms associated with CRC often do not appear until the cancer has reached an advanced stage. This suggests that CRC is a cancer with a slow progression, making it curable and preventive if detected in its early stage. Therefore, there is an urgent clinical need to improve CRC early detection and personalize therapy for patients with this cancer. Recently, liquid biopsy as a non-invasive or nominally invasive approach has attracted considerable interest for its real-time disease monitoring capability through repeated sample analysis. Several studies in CRC have revealed the potential for liquid biopsy application in a real clinical setting using circulating RNA/miRNA, circulating tumor cells (CTCs), exosomes, etc. However, Liquid biopsy still remains a challenge since there are currently no promising results with high specificity and specificity that might be employed as optimal circulatory biomarkers. Therefore, in this review, we conferred the plausible role of less explored liquid biopsy components like mitochondrial DNA (mtDNA), organoid model of CTCs, and circulating cancer-associated fibroblasts (cCAFs); which may allow researchers to develop improved strategies to unravel unfulfilled clinical requirements in CRC patients. Moreover, we have also discussed immunotherapy approaches to improve the prognosis of MSI (Microsatellite Instability) CRC patients using neoantigens and immune cells in the tumor microenvironment (TME) as a liquid biopsy approach in detail.

Designing a Peptide‐modified Screen‐printed Gold Electrode as a Sensor for the Human Monocytic Leukemia Cell Line

AbstractWe designed a screen‐printed gold electrode (SPGE) with a peptide to detect cancer cells. The ability to sense circulating tumor cells (CTCs) and monitor their movement in blood vessels is important for cancer diagnosis. Human monocytic leukemia cells were selected as a model of CTCs because of their origin. The cell‐recognition and cell‐sensing peptide moieties were EICADP and Y4C. The SPGE was modified with the cell‐recognition moieties of the CY4EICADPY4C in a bridge‐type construction due to the cysteine residue at both terminals. The calibration curve was linear and ranged from 25 to 2,000 cells/mL with a LOD of 8 cells/mL.

An in vivo zebrafish model reveals circulating tumor cell targeting capacity of serum albumin nanoparticles

Nanoparticles are promising tools of drug delivery in modern medicine. There is a need for fast and reliable models for in vivo validation of newly developed nanocarriers. Here, we report a fast and easy zebrafish larval model to study the biodistribution and cancer cell targeting capacity of serum albumin nanoparticles in vivo. Fluorescently tagged Bovine Serum Albumin Nanoparticles (BSA-NPs) delivered intravenously to the zebrafish larvae, can be used to study the biodistribution via live imaging. We showed that the BSA-NPs were instantly distributed to the larval vasculature including the brain, without causing any toxicity. The clearance of nanoparticles from the body occurred within few days, which gives sufficient time to study anti-cancer efficiency of the BSA-NPs. Next, we asked whether the BSA-NPs can target the cancer cells in circulation. We established a circulating tumor cell (CTC) xenograft model and described a quantitative method for colocalization and cancer cell death analysis in the intact live organism. We showed that BSA-NPs effectively found and localized to MCF7 cells in vasculature which were killed upon doxorubicin delivery. Interestingly, folic acid coating of BSA-NPs caused faster colocalization but did not increase the overall cell death. This is the first report of the biodistribution, toxicity and anti-cancer effectiveness of serum albumin-based nanoparticles in the zebrafish model. Moreover, here we report for the first time that BSA-NPs are able to target the CTCs in an in vivo model. The zebrafish CTC model and the analysis protocol reported here can be used to assess CTC targeting capacity of nanoparticles and devise patient specific CTC targeting tests.

Abstract 5120: Deciphering melanoma CTC signatures leading to immune escape and brain metastasis: The first MRI CTC xenograft model

Abstract Melanoma brain metastasis (MBM) is a devastating disease which is diagnosed in up to 60% of melanoma patients and 80% of patients at autopsy. While microarray studies identified many genes differentially expressed between MBM and extracranial metastasis, magnetic resonance imaging (MRI) is the established technology to evaluate clinical MBM. Circulating Tumor Cells (CTCs) are “seeds” of fatal metastasis and smallest functional units of cancer. Defining mechanisms which regulate the spatial and temporal competence of patient-isolated CTCs driving MBM thus presents the opportunity to suppress its occurrence. Hypothesis is that MRI has fundamental translational relevance to validate preclinical CTC models and that MBM onset depends upon the absence or presence of human immune system cells (Hu-mice), and mice gender and age. We isolated CTCs from blood of primary/metastatic melanoma patients, and confirmed CTC detection by CellSearch࣪, Parsortix࣪, high-definition microscopy and RNA-Seq profiling. Second, we injected melanoma CTCs in immunodeficient but also immunocompetent xenografts (NBSGW female/male mice generated by multiple donors CD34+ cells), and assessed tumor progression by IVIS and MRI with parallel monitoring of CTC gene-specific transcriptional activation. Third, we discovered specific differences in number of CTCs/CTC clustering patterns and CTC signatures which were dependent upon MBM status. Quantitation of melanoma CTC-derived signatures enabled serial noninvasive monitoring of tumor burden. Lastly, we found that these differences and the occurrence, severity and spatial distribution of MBM highly depended from gender, age, and immunocompetency status of injected animals. Thus, by reporting the first MRI CTC xenograft model, its use for melanoma CTC interrogation can provide a platform for early monitoring of response to immunotherapeutic and/or targeted interventions in melanoma, and contribute to rational therapy applications, notably for MBM prediction or prevention. Citation Format: Tetiana Bowley, Dario Marchetti. Deciphering melanoma CTC signatures leading to immune escape and brain metastasis: The first MRI CTC xenograft model [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 5120.

Label-free enrichment of MCF7 breast cancer cells from leukocytes using continuous flow dielectrophoresis.

Circulating tumor cells (CTCs) present in the bloodstream are strongly linked to the invasive behavior of cancer; therefore, their detection holds great significance for monitoring disease progression. Currently available CTC isolation tools are often based on tumor-specific antigen or cell size approaches. However, these techniques are limited due to the lack of a unique and universal marker for CTCs, and the overlapping size between CTCs and regular blood cells. Dielectrophoresis (DEP), governed by the intrinsic dielectric properties of the particles, is a promising marker-free, accurate, fast, and low-cost technique that enables the isolation of CTCs from blood cells. This study presents a continuous flow, antibody-free DEP-based microfluidic device to concentrate MCF7 breast cancer cells, a well-established CTC model, in the presence of leukocytes extracted from human blood samples. The enrichment strategy was determined according to the DEP responses of the corresponding cells, obtained in our previously reported DEP spectrum study. It was based on the positive-DEP integrated with hydrodynamic focusing under continuous flow. In the proposed device, the parylene microchannel with two inlets and outlets was built on top of rectangular and equally spaced isolated planar electrodes rotated certain degree relative to the main flow (13°). The recovery of MCF7 cells mixed with leukocytes was 74%-98% at a frequency of 1MHz and a magnitude of 10-12Vpp . Overall, the results revealed that the presented system successfully concentrates MCF7 cancer cells from leukocytes, ultimately verifying our DEP spectrum study, in which the enrichment frequency and separation strategy of the microfluidic system were determined.

Dual-Aptamer-Targeted Immunomagnetic Nanoparticles to Accurately Explore the Correlations between Circulating Tumor Cells and Gastric Cancer.

It has been acknowledged that circulating tumor cells (CTCs) are promising biomarkers in liquid biopsy for cancer diagnosis and prognosis. However, the relationship between the CTC number and gastric cancer has scarcely been quantitatively investigated. Moreover, the single criterion of epithelial cell adhesion molecule (EpCAM) antibody/aptamer to specifically recognize epithelial CTCs cannot be universally applied for clinical applications, as it fails to recognize EpCAM-negative CTCs. Herein, we propose simple, low-cost, dual-aptamer (EpCAM and PTK7)-modified immunomagnetic Fe3O4 particles (IMNs) for efficient capture of heterogeneous CTCs and downstream analysis in gastric cancer patients. High PTK7 expression and a significant negative correlation between PTK7 and EpCAM expression were observed in primary gastric cancer tissues. Taking MGC-803 and BGC-823 cells as CTC models, the obtained dual-targeting IMNs could distinguishably recognize these cells with both high or low EpCAM and PTK7 expressions, which enhanced the accuracy of CTC recognition in gastric cancer. More than 95% of these two kinds of cells could be captured within 20 min of incubation, which was significantly more efficient than that of single EpCAM- or PTK7-modified IMNs. With this strategy, as low as five CTCs could be captured from phosphate-buffered saline (PBS), a cell mixture containing THP-1 cells, and lysed blood mediums. Moreover, the obtained CTCs can be used for subsequent gene analysis. Finally, the fabricated IMNs were successfully applied for CTC capture in 1.0 mL of peripheral blood samples from patients with gastric cancer. The detected CTC numbers in 72 participants were found to have close relationships with chemotherapy sensitivity, diagnosis, stage, and distant metastasis of patients. This work provides important references for further investigations on CTC-related diagnosis and individualized treatment.

Process simplification and structure design of parallelized microslit isolator for physical property-based capture of tumor cells.

Numerous attempts have been made to develop efficient systems to purify trace amounts of circulating tumor cells (CTCs) from blood samples. However, current technologies are limited by complexities in device fabrication, system design, and process operability. Here we describe a facile, scalable, and highly efficient approach to physically capturing CTCs using a rationally designed microfluidic isolator with an array of microslit channels. The wide but thin microslit channels with a depth of several micrometers selectively capture CTCs, which are larger and less deformable than other blood cells, while allowing other blood cells to just flow through. We investigated in detail the effects of the microchannel geometry and operating parameters on the capture efficiency and selectivity of several types of cultured tumor cells spiked in blood samples as the CTC model. Additionally, in situ post-capture staining of the captured cells was demonstrated to investigate the system's applicability to clinical cancer diagnosis. The presented approach is simple in operation but significantly effective in capturing specific cells and hence it may have great potential in implementating cell physics-based CTC isolation techniques for cancer liquid biopsy.

PH-Sensitive Dye-Based Nanobioplatform for Colorimetric Detection of Heterogeneous Circulating Tumor Cells.

The efficient capture and sensitive detection of circulating tumor cells (CTCs) play a vital role in cancer diagnosis and prognosis. However, CTCs in the peripheral blood are very rare and heterogeneous, which make them difficult to isolate and detect. Herein, a novel colorimetric nanobioplatform was successfully developed for the highly efficient capture and highly sensitive detection of heterogeneous CTCs, which consisted of two parts: the multivalent aptamer-modified gold nanoparticles as the capture unit and two kinds of aptamer-functionalized pH-sensitive allochroic dyes (thymolphthalein and curcumin) @ molybdenum disulfide nanoflakes (MoS2 NFs) acting as the visual simultaneous detection of heterogeneous CTCs. Using MCF-7 and HeLa cells as the CTC models, the capture unit can effectively isolate the CTCs due to the multivalent probe with improved affinity. The two allochroic dyes can display obvious color changes under alkaline conditions (pH 12.5) in the presence of MCF-7 and HeLa cells, which provided a rapid and sensitive strategy for visualizing simultaneous detection of heterogeneous CTCs as low as 5 cells mL-1. This nanoplatform possessed a high sensitivity toward CTC detection owing to high dye loading capacity of MoS2 NFs and allochroic dyes with excellent pH sensitivity. It can successfully distinguish and quantitatively detect the targeted heterogeneous CTCs from numerous interfering cells in diluted whole blood. It can also be used to detect CTCs from lysed blood samples from cancer patients, indicating promising application for cancer diagnosis.

The Mechanical Fingerprint of Circulating Tumor Cells (CTCs) in Breast Cancer Patients.

Simple SummaryDetection of circulating tumor cells (CTCs) in the blood of cancer patients is a challenging issue, since they adapt to the biochemical and physical landscape of the bloodstream. We approached the issue of CTC identification on a biophysical level. For the first time, we recorded the mechanical deformation profiles of potential CTCs, which were isolated from the blood of breast cancer patients, at the force regime of the deforming blood flow. Mechanical fingerprints of CTCs were significantly different from healthy white blood cells. We used machine learning to further evaluate the differences and identify discrimination criteria. Our results suggest that mechanical characterization of CTCs at low forces is a promising path towards CTC detection.Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

Biostable Double-Strand Circular Aptamers Conjugated Onto Dendrimers for Specific Capture and Inhibition of Circulating Leukemia Cells.

Background/ObjectiveCirculating tumor cells (CTCs) are known as the root of cancer metastasis. Capture and inhibition of CTCs may prevent metastasis. Due to the rarity of CTCs in vivo, the current technology about CTCs capture is still challenging. The aim of our study was to conjugate the enhanced biostable double-strand (ds) circular aptamer (dApR) with dendrimers for capturing and restraining CTCs in vitro and in vivo.MethodsCEM-targeting aptamer (Ap) was looped by ligation after phosphorylation to form circular ds aptamer dApR, which was then conjugated to dendrimers by biotin-streptavidin affinity reaction and named as G-dApR. The physicochemical properties of G-dApR were characterized by using PAGE gel electrophoresis, UV, DLS, AFM, fluorophotometer and laser confocal microscope. Biostability of G-dApR was also analyzed by gel electrophoresis. Confocal microscopy and flow cytometry were then performed to determine the binding specificity of G-dApR to CEM cells and the captured CTCs in mice and in human blood. Apoptosis of the captured cells was finally evaluated by using MTT assay, DAPI staining, AO/EB staining, cell cycle analysis and Annexin V-FITC/PI staining.ResultsPhysicochemical characterization demonstrated the entity of dApR and G-dApR, and the nano-size of G-dApR (about 180 nm in aqueous phase). G-dApR exhibited the excellent biostability that confers their resistance to nuclease-mediated biodegradation in serum for at least 6 days. In our established CTCs model, we found that G-dApR could specifically and sensitively capture CTCs not non-target cells even in the presence of millions of interfering cells (108), in mice and in human blood. Finally, the activity of captured CTCs was significantly down-regulated by G-dApR, resulting in apoptosis.ConclusionWe created the enhanced biostable dApR-coated dendrimers (G-dApR) that could specifically capture and restrain CTCs in vitro and in vivo for preventing CTC-mediated cancer metastasis.

Abstract 4604: Can breast cancer cells be distinguished from blood cells by mechanical parameters? A label-free CTC detection approach

Abstract Background: Even years after successful treatment of the primary tumor about one third of breast cancer patients are suffering from metastatic relapse. A possible reason might be hematogenous spread. Therefore circulating tumor cells (CTCs) in the blood might be interesting and easy accessible surrogate markers for disease monitoring. Usually, detection methods are based on cellular parameters like cell surface markers, size, density or flexibility. Due to phenotypical, mechanical and functional changes during cancer progression and treatment response, isolation of CTC subpopulations remains very challenging. Here we focused on the characterization of CTC mechanics to evaluate the utility of mechanical parameters for CTC separation from blood. Methods: For the first time, we investigated the active and passive mechanical CTC properties such as elasticity, viscosity and contractile force exertion. In an optical rheometer, cells are deformed non-invasively by a dual beam trap. Thus, the relative deformation of a single cell can be measured while phase contrast and fluorescent images are taken. For proof of premise we used peripheral blood mononuclear cells (PBMC) isolated from a healthy donor and human GFP-expressing MDA-MB 231 breast cancer cells to mimic a CTC model system and to measure cell type specific mechanical deformation profiles. For translational experiments blood samples were collected from breast cancer patients. Hematopoietic cells were depleted using CD45. The cell suspensions were applied to the optical stretcher and rheological parameters were measured in the same manner. Phase contrast images of potentially interesting cells were analyzed manually to eliminate dead cells and to identify possible CTC candidates. Results: We were able to reveal distinct mechanical profiles from hematopoietic cells and breast cancer cells, respectively. The optical deformability at the end of the step stress creep experiment (EOS) was significantly different in both, the model and the translational, systems comparing healthy PPMC to MDA-MB 231 cells respective possible CTC from breast cancer patients. Evaluation of secondary parameters like viscosity and activity using a machine learning algorithm allowed for a clear distinction between hematopoietic and cancer cells in the model system. Preliminary results of the translational system suggest that identification of CTC candidates exclusively based on mechanical parameters might be possible. Conclusion: Together with morphology and cell size, the cellular deformation pattern might be a proper tool for marker-free CTC detection in the peripheral blood of breast cancer patients. The optical stretcher device is not suitable for high throughput CTC measurement. However, it allows recommendations for pore size or flow rates which play an important role in the development of label-free isolation approaches including filtration by size or separation by cellular compressibility. Citation Format: Ivonne Nel, Erik W. Morawetz, Josef A. Kaes, Bahriye Aktas. Can breast cancer cells be distinguished from blood cells by mechanical parameters? A label-free CTC detection approach [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 4604.

Abstract PR11: Multimodal analysis of circulating tumor cell RNA, circulating cell-free DNA, and genomic DNA from a single blood sample collected into a PAXgene Blood ccfDNA Tube*

Abstract Background: High demand for liquid biopsy tests in cancer research increases the need for combined approaches that allow multimodal testing from a limited blood volume. Although crosslinking-based fixatives allow for efficient preservation of the circulating cell-free DNA (ccfDNA) profile in whole blood, analysis of mRNA from circulating tumor cells (CTCs) is hampered. Currently, there are no blood collection tubes available that allow RNA analysis from CTCs after prolonged (>48 hours) storage. In this research study, we present a newly developed combined workflow for blood stabilization and subsequent analysis of CTC RNA, ccfDNA, and gDNA after prolonged (≥72 hours) storage. Methods: To establish the multimodal workflow, blood was collected from healthy donors into PAXgene Blood ccfDNA Tubes*, aliquoted and manually spiked with 20 individual LNCaP95 cells/5 mL blood as a CTC model or 20 µL PBS as a control. Blood samples were spiked and stored at 2–8°C for 3, 24, 48, and 72 hours before processing. CTCs were enriched and detected using the AdnaTest ProstateCancerPanel AR-V7† (QIAGEN). The plasma and cellular fraction of the CTC-depleted blood was then used to extract ccfDNA and gDNA, respectively. As a control for ccfDNA and gDNA yield measurements, plasma and the cellular fraction were also generated from whole samples (not CTC-depleted) spiked with 20 LNCaP95 cells/5 mL. The resulting multimodal workflow was tested: 1) in comparison to Streck Cell-Free DNA BCT; 2) to assess the impact of room temperature. Results: Tumor cells spiked into PAXgene Blood ccfDNA Tubes could be enriched and detected at experimental time points 3, 24, 48, and 72 hours after spiking with 100% test sensitivity within 24 hours storage and 91% test sensitivity after 72 hours storage at 2–8°C. In unspiked samples, the PAXgene stabilization solution did not cause false positive signals at any experimental time point. Yields of ccfDNA and gDNA were not statistically significantly affected by CTC capture (p>0.05). RNA from spiked tumor cells was detected in blood samples collected and stored in Streck Cell-Free DNA BCTs for 3 hours, but not at later time points. Storage of PAXgene stabilization solution-treated samples at room temperature is feasible, with 100% test sensitivity for CTC detection within 24 hours storage (similar to storage at 2–8°C) and 80% after 72 hours storage. Conclusions: Blood collected into PAXgene Blood ccfDNA Tubes and stored for up to 72 hours at 2–8°C or at room temperature can be used for multimodal analysis of CTC RNA, ccfDNA, and gDNA from a single blood sample. *For research use only. Not for use in diagnostic procedures. †For molecular biology applications. Not intended for the diagnosis, prevention, or treatment of a disease. This abstract is also being presented as Poster B18. Citation Format: Eric Provencher, Maike Remmel, Siegfried Hauch, Michael Otte, Andrea Ullius, Daniel Groelz, Anna Babayan. Multimodal analysis of circulating tumor cell RNA, circulating cell-free DNA, and genomic DNA from a single blood sample collected into a PAXgene Blood ccfDNA Tube* [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR11.

Can tumor cells be distinguished from blood cells by mechanical parameters? A label-free CTC detection approach.

e15526 Background: Even years after successful treatment of the primary tumor about 30% of breast cancer patients are suffering from metastatic relapse. One reason might be hematogenous spread. Therefore circulating tumor cells (CTC) in the blood might be interesting and easy accessible surrogate markers for disease monitoring. Due to phenotypical, mechanical and functional changes during cancer progression and treatment response, isolation of CTC subpopulations remains very challenging. Here we focused on the characterization of CTC mechanics to evaluate the utility of mechanical parameters for CTC separation from blood. Methods: For the first time, we investigated the active and passive mechanical CTC properties such as elasticity, viscosity and contractile force exertion using a dual beam trap. For proof of premise we used peripheral blood mononuclear cells (PBMC) from a healthy donor and human GFP-expressing MDA-MB 231 breast cancer cells to mimic a CTC model system and to measure cell type specific mechanical deformation profiles using an optical rheometer. For translational experiments blood samples were collected from patients with mamma or vulvar carcinoma. Hematopoietic cells were depleted using CD45. The remaining cell suspensions were applied to the optical stretcher and rheological parameters were measured in the same manner. Results: We were able to reveal distinct mechanical profiles from hematopoietic cells and breast cancer cells, respectively. The optical deformability was significantly different in both the model and the translational, systems comparing healthy PPMC to MDA-MB 231 cells, respective possible CTC candidates from breast and vulvar carcinoma patients. Evaluation of secondary parameters like viscosity and activity using a machine learning algorithm allowed for a clear distinction between hematopoietic and cancer cells in the model system. CD45-positive and -negative cells from patients with mamma or vulvar carcinoma (n = 7) showed distinct differences in the rheological behavior of both cell populations. Surprisingly, the mechanical profiles of CD45-negative cells from mamma carcinoma samples were severely different from vulvar carcinoma. Conclusions: Together with morphology and cell size, the cellular deformation pattern might be a proper tool for marker-free CTC detection in the peripheral blood of breast cancer patients.

Nanoemulsions to support ex vivo cell culture of breast cancer circulating tumor cells

The culture and expansion of circulating tumor cells (CTCs) for ex vivo assays plays an important role in precision medicine. However, it still represents a big challenge in translational research. Generating knowledge about the characteristics of CTCs can help to shed light about the metastasis process. Furthermore, ex vivo culture of CTCs might allow performing functional analyses and testing different drugs, to guide clinical therapies. In this work, we present a new methodology based on the use of nanosystems to support ex vivo culture of CTCs. We have formulated oil-in-water (O/W) nanoemulsions (NEs) composed by lipids and fatty acids, and have demonstrated that they can help increasing cell viability on different breast cancer cell lines. Moreover, we have generated a CTC model from breast cancer mice xenografts, to prove the ability of the NEs to facilitate their culture and expansion. Additionally, we have postulated a mechanism of action based on the cell consumption of the NEs, which are acting as energy suppliers, driving proliferation. This work corroborates the potential of nanotechnology to provide valuable tools for precision oncology, and the ability of our NEs to improve proliferation of breast cancer CTCs for the establishment of CTCs culture protocols.