Circulating Tumor Cells Isolation Methods Research Articles

Abstract 5166: A multiplexed immunofluorescence assay to assess Rb and phospho-Rb as predictive biomarkers for CDK4/6 inhibitors in HR+/HER2- breast cancer

Abstract Introduction: Cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) are used as the first-line treatment of advanced/metastatic or high-risk HR+/HER2- breast cancer (BC) that make up about 60-70% of all cases. However, nearly 50% of patients exhibit drug resistance. Therefore, biomarkers that predict CDK4/6i response are needed to spare patients from toxicities and high costs. Resistance to CDK4/6i [palbociclib, ribociclib, and abemaciclib] is associated with the aberrant or loss of Retinoblastoma (Rb). A decline in phospho-Rb (pRb) levels on therapy is also associated with CDK4/6i response. We aimed to develop a multiplex immunofluorescence (IF) assay to assess Rb and pRb levels that can be used on circulating tumor cells (CTCs) to predict tumor response to CDK4/6i. Here, we report the selection of internal controls for Rb and pRb, assay repeatability and reproducibility, as well as compatibility of the CTC isolation protocol with the assay. Methods: Rb and pRb expression was evaluated by western blotting (WB) and IF in parental (P), estrogen deprivation-resistant (EDR), palbociclib-resistant (PalboR), and EDR/PalboR derivatives of two HR+/HER2- cell lines (MCF7 and T47D), Rb1 KO cells of MCF7 and T47D, as well as triple-negative cell lines, BT549 and MDA-MB-468. IF-stained cells were imaged using Leica STED SP8 confocal microscope and Zeiss Axioscan 7. The concordance between IF and WB results was assessed using a Bland-Altman plot. Selectivity and specificity of the assay as well as repeatability and reproducibility between investigators were also assessed. Blood from healthy subjects was used to test the compatibility of the ScreenCell® filtration system to isolate CTC with the IF assay. Results: Our initial approach utilized indirect IF staining for Rb and pRb using the same primary antibodies as WB. On the Bland-Altman plot, most data points were relatively close to the bias line, suggesting that the difference between the two methods was acceptable. The analytical range for mean Rb and pRb staining intensity was 0.744 - 46.779 and 1.001 - 52.919, respectively. Based on WB and IF data, we nominated T47D PalboR and MDA-MB-468 cells as negative controls for Rb and pRb expression. The selectivity and specificity were 100% and 70%, respectively, for the Rb expression and 99% and 61% for pRb, respectively. The IF assay was repeatable and reproducible between two investigators. The ScreenCell® CTC isolation method was not compatible with the assay, as Rb and pRb expression was either not detected or limited when processed using FC2 or PBTFC buffer, irrespective of the antigen retrieval. Conclusion: We have selected internal cell line controls for Rb and pRb and are in the process of optimizing signal-to-noise ratio and specificity using conjugated antibodies. We are also testing the compatibility of other CTC isolation methods with the IF assay. Citation Format: Mantasha Tabassum, Shivaani Suresh Kanna, Wangjia Cao, Mothaffar Rimawi, George E Miles, Meghana V Trivedi. A multiplexed immunofluorescence assay to assess Rb and phospho-Rb as predictive biomarkers for CDK4/6 inhibitors in HR+/HER2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5166.

Size effect of dynamic bioactive magnetic particles on regulated isolation of tumor cells

Circulating tumor cells (CTCs) are critical biomarkers in cancer metastasis, yet their isolation from the bloodstream remains a challenging task due to their low abundance. This study introduces a method for the efficient isolation of CTCs, utilizing dynamic biointerface-based magnetic particles (DBMPs), with a particular emphasis on the role of particle size. We have developed three distinct sizes DBMPs specifically tailored for effective CTCs interaction, leveraging the unique properties of polydopamine (PDA) coatings and phenylboronic acid (PBA)-modified capture peptides through reversible catechol-boronate interactions. Comparative analysis of DBMPs of varying sizes was assessed under uniform experimental conditions for their capture and release efficiencies. The findings reveal a clear distinction: smaller particles (DBMP1 and DBMP2) achieved greater efficiency in capturing CTCs, whereas the larger particles (DBMP3) were more effective in releasing them. This difference is ascribed to the varied interaction dynamics between the particles and CTCs. Furthermore, we have fine-tuned DBMP3 to optimize CTC capture while still maintaining a high efficiency in cell release. The integration of these DBMPs into a magnetic isolation framework is poised to significantly enhance CTCs isolation methods, marking a pivotal advancement in cancer diagnostics and management.

Abstract 3689: Optimizing CTC isolation techniques for molecular characterization of circulating tumor cells in clear cell renal cell carcinoma: A comparative study of EpCAM-based and density-based methods

Abstract Introduction: Renal cell carcinoma (RCC) belongs to the most invasive cancers with the high molecular intrapatient heterogeneity. Circulating tumor cells (CTCs) are cells that detach from the tumor, reflecting the activity of the metastatic procedure. The most popular CTCs isolation methods are based on EpCAM signal detection on epithelial tumor cells. The clear cell RCC (ccRCC) originate from epithelial entity, since that is the EpCAM-based method suitable to isolate CTCs in this cancer. However, the prone of CTCs to EMT is known, which may in consequence downregulate the EpCAM expression and change the cell profile (hybrid phenotype). This is one of the points challenging the CTCs isolation by RCC disease. In our study, we compare the effectivity of antibody-based and density-based isolations technics in ccRCC patients to detect potential markers in CTCs. Purpose: Our aim was to find a precise and costs effective method to isolate and characterize CTCs from ccRCC patients. Materials and Methods: Prospective blood samples (3mL) from pulmonary and/or osseous metastatic ccRCC patients were collected during the immunotherapy and from the healthy donors. CTCs were isolated using EpCAM beads (RCC n=13; healthy n=15) or density-based (RCC n=13; healthy n=15) method. Expression of potential RCC biomarkers (mucin-1 (Muc1), androgen receptor (AR)) was identified using qPCR (Real-Time PCR) followed by dPCR (digital PCR), the results status received from both methods was evaluated. Summary: Based on our prior results, we first isolated CTCs based on EpCAM beads. Using qPCR we analyzed expression of genes, which we previously associated to the RCC (Muc1, PD-L1, AR). The results revealed differences in Muc1 (40% of tested patients positive but none of the healthy donors), when PD-L1 did not differed and AR was not detected. Applying more precise dPCR did neither show any expression. We continued our investigations with isolation of CTCs using density-based method. We confirmed more frequent expression of Muc1 by RCC Patients than by donors, as in our EpCAM cohort. PD-L1 was similar in both groups. Results of AR obtained using qPCR were confusing, showing deviations within same patients. The use of dPCR displayed significant differences in expression between RCC patients (77%; median [range] 0.28 [0.00 - 0.62] and donors (60%; 0.04 [0.00 - 0.51] confirming dPCR as optimal tool for molecular CTC characterization. Conclusions: Based on our results we demonstrate higher expression of Muc1 and AR in CTCs of metastatic ccRCC patients. The isolation technic should be adapted to the disease status and treatment choice. Further investigations confirming Muc1 and AR as ccRCC biomarkers are required. Citation Format: Joanna Bialek, Anne Muthe, Stefan Yankulov, Felix Kawan, Georgios Gakis, Gerit Theil. Optimizing CTC isolation techniques for molecular characterization of circulating tumor cells in clear cell renal cell carcinoma: A comparative study of EpCAM-based and density-based methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3689.

Microfluidic System Consisting of a Magnetic 3D-Printed Microchannel Filter for Isolation and Enrichment of Circulating Tumor Cells Targeted by Anti-HER2/MOF@Ferrite Core-Shell Nanostructures: A Theranostic CTC Dialysis System.

Low number of circulating tumor cells (CTCs) in the blood samples and time-consuming properties of the current CTC isolation methods for processing a small volume of blood are the biggest obstacles to CTC usage in practice. Therefore, we aimed to design a CTC dialysis system with the ability to process cancer patients' whole blood within a reasonable time. Two strategies were employed for developing this dialysis setup, including (i) synthesizing novel in situ core-shell Cu ferrites consisting of the Cu-CuFe2O4 core and the MIL-88A shell, which are targeted by the anti-HER2 antibody for the efficient targeting and trapping of CTCs; and (ii) fabricating a microfluidic system containing a three-dimensional (3D)-printed microchannel filter composed of a polycaprolactone/Fe3O4 nanoparticle composite with pore diameter less than 200 μm on which a high-voltage magnetic field is focused to enrich and isolate the magnetic nanoparticle-targeted CTCs from a large volume of blood. The system was assessed in different aspects including capturing the efficacy of the magnetic nanoparticles, CTC enrichment and isolation from large volumes of human blood, side effects on blood cells, and the viability of CTCs after isolation for further analysis. Under the optimized conditions, the CTC dialysis system exhibited more than 80% efficacy in the isolation of CTCs from blood samples. The isolated CTCs were viable and were able to proliferate. Moreover, the CTC dialysis system was safe and did not cause side effects on normal blood cells. Taken together, the designed CTC dialysis system can process a high volume of blood for efficient dual diagnostic and therapeutic purposes.

Isolation of Viable Epithelial and Mesenchymal Circulating Tumor Cells from Breast Cancer Patients.

Circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) may exhibit more aggressive features than epithelial CTCs and are more frequently observed during disease progression. Therefore, detection and characterization of both epithelial and mesenchymal CTCs in cancer patients are urgently needed to allow for a better understanding of the metastatic process and more effective treatment. Here we describe a method for detection and isolation of viable epithelial and mesenchymal CTCs from peripheral blood of breast cancer patients. The method is based on density gradient centrifugation, multiplex immunofluorescent staining, and negative anti-CD45 selection. Cells obtained after the procedure are suitable for genomic or transcriptomic profiling, and they can also be isolated by micromanipulation for single-cell analysis.

352. GENERATION OF PATIENT-DERIVED ESOPHAGEAL ADENOCARCINOMA ORGANOIDS FROM CIRCULATING TUMOR CELLS

Abstract Background Esophageal adenocarcinoma (EAC) is diagnosed in nearly 40% of patients when their cancer has already metastasized. Circulating tumor cells (CTCs) play a critical role in the metastatic cascade and need to be investigated in the context of EAC, but these must be expanded due to their scarcity in blood. Organoid models have been shown to recapitulate tumor heterogeneity and in vivo drug sensitivity. Thus, we aim to generate and characterize CTC-derived organoids from EAC patients. Methods CTCs were isolated from 32 blood samples obtained from 13 EAC patients using two methods: (1) Ficoll-based density gradient centrifugation followed by CD45+ cell depletion using magnetic-activated cell sorting (MACS) and/or (2) immunodensity separation using the RosetteSep CTC Enrichment Cocktail (Stemcell Technologies). Isolated CTCs were embedded and grown in Matrigel domes. Cultures were dissociated into single cells which were magnetically labelled and captured on a microfluidic chip. The captured cells were stained with DAPI, anti-CD45, anti-CK11, anti-CK13, and anti-CK18 antibodies. These cells were then visualized and quantified using fluorescence microscopy. Results Organoids were generated from twenty-one out of thirty-two samples, after two to six weeks in culture, with an average of 20.76 ± 6.435 organoids per sample. These EAC CTC derived organoids underwent a growth arrest once they reached an arbitrary size but were able to regenerate upon dissociation and re-seeding. However, no increase in the size or number of organoids was observed post-passaging. Of the nine CTC-derived organoid samples characterized using microfluidic capture and immunostaining, all nine had the presence of CD45 negative, CK positive, and DAPI positive cells. Conclusion In this study, we validated commonly used CTC isolation methods and were able to demonstrate that CTCs isolated can be cultured to generate organoids. Additional investigation is required to overcome the growth arrest which occurs during culture.

Effective Circulating Tumor Cell Isolation Using Epithelial and Mesenchymal Markers in Prostate and Pancreatic Cancer Patients

Circulating tumor cells (CTCs) display antigenic heterogeneity between epithelial and mesenchymal phenotypes. However, most current CTC isolation methods rely on EpCAM (epithelial cell adhesion molecule) antibodies. This study introduces a more efficient CTC isolation technique utilizing both EpCAM and vimentin (mesenchymal cell marker) antibodies, alongside a lateral magnetophoretic microseparator. The effectiveness of this approach was assessed by isolating CTCs from prostate (n = 17) and pancreatic (n = 5) cancer patients using EpCAM alone, vimentin alone, and both antibodies together. Prostate cancer patients showed an average of 13.29, 11.13, and 27.95 CTCs/mL isolated using EpCAM alone, vimentin alone, and both antibodies, respectively. For pancreatic cancer patients, the averages were 1.50, 3.44, and 10.82 CTCs/mL with EpCAM alone, vimentin alone, and both antibodies, respectively. Combining antibodies more than doubled CTC isolation compared to single antibodies. Interestingly, EpCAM antibodies were more effective for localized prostate cancer, while vimentin antibodies excelled in metastatic prostate cancer isolation. Moreover, vimentin antibodies outperformed EpCAM antibodies for all pancreatic cancer patients. These results highlight that using both epithelial and mesenchymal antibodies with the lateral magnetophoretic microseparator significantly enhances CTC isolation efficiency, and that antibody choice may vary depending on cancer type and stage.

Baits-trap chip for accurate and ultrasensitive capture of living circulating tumor cells.

Accurate analysis of living circulating tumor cells (CTCs) plays a crucial role in cancer diagnosis and prognosis evaluation. However, it is still challenging to develop a facile method for accurate, sensitive, and broad-spectrum isolation of living CTCs. Herein, inspired by the filopodia-extending behavior and clustered surface-biomarker of living CTCs, we present a unique baits-trap chip to achieve accurate and ultrasensitive capture of living CTCs from peripheral blood. The baits-trap chip is designed with the integration of nanocage (NCage) structure and branched aptamers. The NCage structure could "trap" the extended filopodia of living CTCs and resist the adhesion of filopodia-inhibited apoptotic cells, thus realizing the accurate capture (∼95% accuracy) of living CTCs independent of complex instruments. Using an in-situ rolling circle amplification (RCA) method, branched aptamers were easily modified onto the NCage structure, and served as "baits" to enhance the multi-interactions between CTC biomarker and chips, leading to ultrasensitive (99%) and reversible cell capture performance. The baits-trap chip successfully detects living CTCs in broad-spectrum cancer patients and achieves high diagnostic sensitivity (100%) and specificity (86%) of early prostate cancer. Therefore, our baits-trap chip provides a facile, accurate, and ultrasensitive strategy for living CTC isolation in clinical. STATEMENT OF SIGNIFICANCE: A unique baits-trap chip integrated with precise nanocage structure and branched aptamers was developed for the accurate and ultrasensitive capture of living CTCs. Compared with the current CTC isolation methods that are unable to distinguish CTC viability, the nanocage structure could not only "trap" the extended-filopodia of living CTCs, but also resist the adhesion of filopodia-inhibited apoptotic cells, thus realizing the accurate capture of living CTCs. Additionally, benefiting from the "baits-trap" synergistic effects generated by aptamer modification and nanocage structure, our chip achieved ultrasensitive, reversible capture of living CTCs. Moreover, this work provided a facile strategy for living CTC isolation from the blood of patients with early-stage and advanced cancer, exhibiting high consistency with the pathological diagnosis.

Genomic characteristics and clinical significance of CD56+ circulating tumor cells in small cell lung cancer

Circulating tumor cells (CTC) have been studied in various solid tumors but clinical utility of CTC in small cell lung cancer (SCLC) remains unclear. The aim of the CTC-CPC study was to develop an EpCAM-independent CTC isolation method allowing isolation of a broader range of living CTC from SCLC and decipher their genomic and biological characteristics. CTC-CPC is a monocentric prospective non-interventional study including treatment-naïve newly diagnosed SCLC. CD56+ CTC were isolated from whole blood samples, at diagnosis and relapse after first-line treatment and submitted to whole-exome-sequencing (WES). Phenotypic study confirms tumor lineage and tumorigenic properties of isolated cells for the 4 patients analyzed with WES. WES of CD56+ CTC and matched tumor biopsy reveal genomic alteration frequently impaired in SCLC. At diagnosis CD56+ CTC were characterized by a high mutation load, a distinct mutational profile and a unique genomic signature, compared to match tumors biopsies. In addition to classical pathways altered in SCLC, we found new biological processes specifically affected in CD56+ CTC at diagnosis. High numeration of CD56+ CTC (> 7/ml) at diagnosis was associated with ES-SCLC. Comparing CD56+ CTC isolated at diagnosis and relapse, we identify differentially altered oncogenic pathways (e.g. DLL3 or MAPK pathway). We report a versatile method of CD56+ CTC detection in SCLC. Numeration of CD56+ CTC at diagnosis is correlated with disease extension. Isolated CD56+ CTC are tumorigenic and show a distinct mutational profile. We report a minimal gene set as a unique signature of CD56+ CTC and identify new affected biological pathways enriched in EpCAM-independent isolated CTC in SCLC.

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.

EP16.02-004 Clinical Significance and Genomic Characteristics of CD56+ Circulating Tumor Cells in Small Cell Lung Carcinoma

Circulating Tumor Cells (CTC) have been studied in various solid tumors. However, clinical utility of CTC in Small Cell Lung Cancer (SCLC) remains poorly understood. Most published studies are based on Cellsearch® isolation methods that did not capture CTC undergoing epithelial-mesenchymal transition. The aim of the CTC-CPC study was to develop an EpCAM-independent CTC isolation method allowing isolation of living CTC from SCLC and decipher their genomic characteristics.

Verification of A New Filter for Isolation of Circulating Tumor Cells by Only Blood Filtration.

Since circulating tumor cells (CTCs) are precursors of metastatic lesions, extracting CTCs from whole blood is useful in obtaining information for cancer treatment. One of the CTC isolation methods is the size selection method; however, since the conventional methods are expensive and cumbersome, we developed an affordable and simple filter, whose usefulness is verified in this study. The new filter [hereafter, soft micropore filter (S-MPF)] is made up of a polyethylene film with a thickness of 15 μm and conical pores having a diameter of 8-10 μm, which are opened uniformly (opening rate, 20%). This filter can filter whole blood by free-falling under gravity. The possibilities of the filter's usage for model CTC isolation, immunostaining, short-term cell culture, and gene mutation detection in extracted model CTCs were verified. S-MPF was able to extract model CTCs with an isolation rate of up to 15%. These model CTCs were detected by cytology, immunostaining, and culture by short-term incubation of filtered cells. Furthermore, genetic mutations were identified in the cultured cells. In addition, CTC isolation from the peripheral blood of patients with lung cancer was demonstrated by setting the volume of collected blood to 15 ml to prevent a low recovery rate. The S-MPF can be used to extract model CTCs quickly and easily. Moreover, cytological diagnosis, immunostaining, short-term culture, and gene mutation search are possible with this filter. Given its proven applicability in clinical samples, this filter can be used in clinical settings.

A Laboratory-Friendly CTC Identification: Comparable Double-Immunocytochemistry with Triple-Immunofluorescence.

Simple SummaryTumor cells that circulate in the peripheral blood of patients with solid tumors are called circulating tumor cells. Since the source of circulating tumor cells are from primary cancer sites, metastatic sites, and/or a disseminated tumor cell pool, these cells have clinical significance. The circulating tumor cells offer a rare glimpse of the evolution of the tumor and its response/resistance to treatment in a real-time non-invasive manner. Although the clinical relevance of circulating tumor cells is undeniable, the routine use of these cells remains limited due to the elusive nature of the cells, which demands highly sophisticated and costly instrumentation. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of circulating tumor cells from peripheral blood of 91 consented and enrolled patients with tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Our user-friendly cost-effective circulating tumor cells detection technique has the potency to facilitate the routine use of circulating tumor cells detection even in community-based cancer centers for prognosis, before and after surgery, which will provide a unique opportunity to move cancer diagnostics forward.The source of circulating tumor cells (CTC) in the peripheral blood of patients with solid tumors are from primary cancer, metastatic sites, and a disseminated tumor cell pool. As 90% of cancer-related deaths are caused by metastatic progression and/or resistance-associated treatment failure, the above fact justifies the undeniable predictive and prognostic value of identifying CTC in the bloodstream at stages of the disease progression and resistance to treatment. Yet enumeration of CTC remains far from a standard routine procedure either for post-surgery follow-ups or ongoing adjuvant therapy. The most compelling explanation for this paradox is the absence of a convenient, laboratory-friendly, and cost-effective method to determine CTC. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of CTC from peripheral blood of 91 consented and enrolled patients with various malignant solid tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Using a pressure-guided method, we used the size-based isolation to capture CTC on a commercially available microfilter. CTC identification was carried out by two expression marker-based independent staining methods, double-immunocytochemistry parallel to standard triple-immunofluorescence. The choice of markers included specific markers for epithelial cells, EpCAM and CK8,18,19, and exclusion markers for WBC, CD45. We tested the method’s specificity based on the validation of the staining method, which included positive and negative spiked samples, blood from the healthy age-matched donor, healthy age-matched leucopaks, and blood from metastatic patients. Our user-friendly cost-effective CTC detection technique may facilitate the regular use of CTC detection even in community-based cancer centers for prognosis, before and after surgery.

Abstract B24: Integration of filtration with immunoaffinity for isolating circulating tumor cells from pancreatic and colorectal cancer patients

Abstract We report two microfluidic devices that combine filtration and immunoaffinity for the detection of circulating tumor cells (CTCs) from peripheral blood samples of pancreatic and colorectal cancer patients. Our approach contrasts with most CTC isolation methods that are based either on cells’ physical property (e.g., size-based filtration) or on biologic property (e.g., immunoaffinity). The combined approach addresses the challenges related to CTCs’ heterogeneity in their properties. For instance, immunoaffinity-based methods such as CellSearch® that employ antibodies against epithelial cell adhesion molecule (EpCAM) cannot isolate those CTCs expressing little or no EpCAM. Filtration-based methods cannot detect CTCs of a size smaller than the predefined filter pore diameter while many normal blood cells of a size larger than the pore are retained by the filter. Our microfluidic devices contain a serpentine main channel and an array of lateral microfilters. The key difference of our devices from the conventional filtration platforms is that our filters are not in the direction of the main flow. The unique design of the device layout leads to a two-dimensional flow that allows the majority of a sample to pass by while all cells have opportunities to interact with filters, resulting in a larger throughput, reduced cell clogging, and increased purity of cells isolated. Another advantage of our devices is that a series of filter sizes can be created in one device to accommodate different CTC diameters, which is difficult to achieve in traditional filtration platforms. Our devices are further functionalized by immobilizing antibodies on the surfaces of microfilters to combine filtration with immunoaffinity for CTC isolation. We have designed one device with filter sizes of 6-10 μm and another device with filter sizes of 12-24 μm, and we tested them for the isolation of L3.6pl cells (pancreatic cancer cells) spiked in a buffer or blood. We compared an antibody-immobilized device with the same device containing filters only. Without antibody, the device has a capture efficiency from 69.8% to 83.9%, depending on the flow rate used. With antibody, the device with combined CTC isolation mechanisms can accomplish a capture efficiency of (98.7 ± 1.2)% at a flow rate of 1.8 mL/h. Other tumor cells with different sizes have also been evaluated in the devices. Further, we employed the devices for enumerating CTCs from blood samples of pancreatic and colorectal cancer patients. We compared our integration devices with a previously reported device containing herringbone-based micromixers. We found that the integration devices generally detected a higher number of CTCs. In summary, microfluidic devices have been developed to integrate filtration with immunoaffinity for CTC isolation. The devices offer better performance than those based on one isolation mechanism only, with a potential to address CTC heterogeneity and detect CTCs with different sizes and various expression levels. Citation Format: Hugh Fan, Kangfu Chen, Pablo Dopico, Jacob Amontree, Thomas George. Integration of filtration with immunoaffinity for isolating circulating tumor cells from pancreatic and colorectal cancer patients [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 B24.

A Review on Microdevices for Isolating Circulating Tumor Cells.

Cancer metastasis is the primary cause of high mortality of cancer patients. Enumeration of circulating tumor cells (CTCs) in the bloodstream is a very important indicator to estimate the therapeutic outcome in various metastatic cancers. The aim of this article is to review recent developments on the CTC isolation technologies in microdevices. Based on the categories of biochemical and biophysical isolation approaches, a literature review and in-depth discussion will be included to provide an overview of this challenging topic. The current excellent developments suggest promising CTC isolation methods in order to establish a precise indicator of the therapeutic outcome of cancer patients.

Microfluidic applications on circulating tumor cell isolation and biomimicking of cancer metastasis

The prognosis of malignant tumors is challenged by insufficient means to effectively detect tumors at early stage. Liquid biopsy using circulating tumor cells (CTCs) as biomarkers demonstrates a promising solution to tackle the challenge, because CTCs play a critical role in cancer metastatic process via intravasation, circulation, extravasation, and formation of secondary tumor. However, the effectiveness of the solution is compromised by rarity, heterogeneity, and vulnerability associated with CTCs. Among a plethora of novel approaches for CTC isolation and enrichment, microfluidics leads to isolation and detection of CTCs in a cost-effective and operation-friendly way. Development of microfluidics also makes it feasible to model the cancer metastasis in vitro using a microfluidic system to mimick the in vivo microenvironment, thereby enabling analysis and monitor of tumor metastasis. This paper aims to review the latest advances for exploring the dual-roles microfluidics has played in early cancer diagnosis via CTC isolation and investigating the role of CTCs in cancer metastasis; the merits and drawbacks for dominating microfluidics-based CTC isolation methods are discussed; biomimicking cancer metastasis using microfluidics are presented with example applications on modelling of tumor microenvironment, tumor cell dissemination, tumor migration, and tumor angiogenesis. The future perspectives and challenges are discussed.

Nano Meets Micro-Translational Nanotechnology in Medicine: Nano-Based Applications for Early Tumor Detection and Therapy.

Nanomaterials have great potential for the prevention and treatment of cancer. Circulating tumor cells (CTCs) are cancer cells of solid tumor origin entering the peripheral blood after detachment from a primary tumor. The occurrence and circulation of CTCs are accepted as a prerequisite for the formation of metastases, which is the major cause of cancer-associated deaths. Due to their clinical significance CTCs are intensively discussed to be used as liquid biopsy for early diagnosis and prognosis of cancer. However, there are substantial challenges for the clinical use of CTCs based on their extreme rarity and heterogeneous biology. Therefore, methods for effective isolation and detection of CTCs are urgently needed. With the rapid development of nanotechnology and its wide applications in the biomedical field, researchers have designed various nano-sized systems with the capability of CTCs detection, isolation, and CTCs-targeted cancer therapy. In the present review, we summarize the underlying mechanisms of CTC-associated tumor metastasis, and give detailed information about the unique properties of CTCs that can be harnessed for their effective analytical detection and enrichment. Furthermore, we want to give an overview of representative nano-systems for CTC isolation, and highlight recent achievements in microfluidics and lab-on-a-chip technologies. We also emphasize the recent advances in nano-based CTCs-targeted cancer therapy. We conclude by critically discussing recent CTC-based nano-systems with high therapeutic and diagnostic potential as well as their biocompatibility as a practical example of applied nanotechnology.

Electrostatic reaction for the detection of circulating tumor cells as a potential diagnostic biomarker for metastasis in solid tumor.

The detection of circulating tumor cells (CTCs) from blood samples is important to predict metastatic spread of cancer cells. However, effective quantification and identification of CTCs in solid tumors remain a challenge. Aerobic glycolysis is a hallmark of cancer cells, which makes cancer cells have more negative membrane potentials than that of normal cells. Herein, we reported a CTC isolation method with 80.7% capture efficiency based on electrostatic reaction, which was accomplished within 30 min in mimic clinical samples. Following in vitro verification using Lewis lung carcinoma (LLC1) (EpCAM-positive) and B16F10 (EpCAM-negative) melanoma cells, syngeneic tumor models were used to evaluate specificity and sensitivity of the surface charged nanoparticles. After subcutaneous implantation, blood was drawn from mice every three days, and CTCs were successfully detected in all implanted subjects. From 100 µl blood samples, the minimum amount found in blood was 9-34 CTCs on 3 day and the maximum was 94-107 CTCs on 15 day. Besides, the isolated CTCs cells remained viable and verified by re-implantation. This study confirms that our multifunctional nanoparticles are highly efficient in detecting CTCs in tumor metastasis and has huge potential in translational medicine.

Abstract 1383: Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis

Abstract Purpose: The presence of circulating tumor cells (CTC) in blood is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. For non-metastatic PCa patients and patients with biochemical recurrence (BCR), it is extremely difficult to find CTCs in 5-10 ml of peripheral blood (PB). The purpose of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by other enrichment technologies to increase the CTC yield by using a large screening blood volume. Methods: Blood samples were collected from one healthy donor and one mPCa patient. During LP procedure, different blood elements were separated to different layers based on their densities. The mononuclear cell layer (that also contains CTCs) was collected. To assess recovery rate of different CTC isolation and detection methods, the healthy donor LP was spiked with a known number of PCa cell line LNCaP cells. Three CTC isolation and detection methods were used, including FAST (Clinomics, Ulsan, South Korea), a size-selection platform followed by immunofluorescence (IF); the AccuCyte-CyteFinder system (RareCyte, Inc., Seattle, WA), a selection-free method that utilizes IF; the AlereTM CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method followed by RT-qPCR. The criteria for defining a CTC are DAPI+, CK/EpCAM+, and CD45-. The Ct value thresholds for positive CTC signal by RT-qPCR were EpCAM 36.4, PSA 36.3, HOXB13 40, AR 35.4. Results: For the healthy donor LP with spiked LNCaP cells, the CTC recovery rate from FAST was 53.7%~67.2%. For Adna test, 0, 44, 95 and 1000 LNCaP cells were spiked in 5 ml of LP respectively. The Ct values of EpCAM were 36.28, 29.32, 28.22 and 24.76. For the mPCa patient, two tubes of 7.5 ml of PB were collected for FAST and RareCyte. The FAST found 3 CTCs while the RareCyte found 1 CTC. An LP sample was also collected from the same mPCa patient. The LP sample volume was 162 ml (WBC concentration was 83×109/L). Triplicates of 5 ml of samples were sent through AdnaTest and CTCs were identified in all samples (Ct: EpCAM 34.21±0.77, PSA 33.86±1.81, HOXB13 36.25±0.34, AR 35.29±0.22). Then the remainder of the mPCa LP was subjected to Ficoll-Paque density centrifugation to further concentrate mononuclear cells to a 90 ml volume. 1 ml samples were then applied to FAST in triplicate. The CTC numbers were 22 and 15. The density of cells on one of the filters was too high to be successfully scanned. Conclusion: Finding CTCs in LP samples by size-based technology or AdnaTest is feasible and offers a promising method to do liquid biopsy for mPCa patients. Screening a larger volume of PB by LP has significantly increased the CTC yield compared to using the same capture technology on 7.5 ml of PB. Citation Format: Liang Dong, Zhongyuan Zhang, Changxue Lu, Diane Reyes, Amber de Groot, Sarah R. Amend, Jun Luo, Kenneth J. Pienta. Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis [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 1383.

Abstract 2216: Circulating tumor cell isolation based on both physical & biological properties

Abstract Isolation and detection of circulating tumor cells (CTCs) in peripheral blood have been used for cancer prognosis and treatment monitoring. Most CTC isolation methods are based on either physical property (e.g. size) or biological property (e.g. immunoaffinity) of CTCs. However, it is well known that CTCs are very heterogeneous and their properties are not uniform. For immunoaffinity-based approaches such as the FDA-approved CellSearch® that employs antibodies against epithelial cell adhesion molecule (EpCAM), they cannot detect those CTCs expressing little or no EpCAM (due partially to epithelial-to-mesenchymal transition). For size-based methods such as those using microfilters, CTCs with a smaller size are not detected while a large number of normal blood cells are retained by the filters. To address the challenge, isolation methods based on both physical and biological properties of CTCs have been explored. We report here a microfluidic device that contains a serpentine main channel and an array of lateral microfilters. The key difference of the device from the conventional microfilter devices is that our filters are not in the direction of the main flow. The unique design of the device layout produces a two-dimensional flow that allows the majority of a sample to pass by while all cells have opportunities to interact with filters, resulting in a larger throughput, reduced cell clogging, and increased purity of cells isolated. The device is further functionalized by immobilizing antibodies on the surfaces of microfilters to achieve a platform that combines filtration-based CTC isolation with immunoaffinity-based isolation. We first tested the device for the isolation of L3.6pl cells (pancreatic cancer cells) spiked in a buffer or blood, and compared the antibody-immobilized device with the same device containing filters only. Without antibody, the device has a capture efficiency from 69.8% to 83.9%, depending on the flow rate we used. With antibody, the device with combined CTC isolation mechanisms can accomplish a capture efficiency of (98.7 ± 1.2)% at a flow rate of 1.8 mL/h. Other tumor cells with different sizes have also been evaluated in the device. Further we employed the device for enumerating CTCs from blood samples of pancreatic and colorectal cancer patients. We compared this integrated device with a previously reported device containing herringbone-based micromixers. We found that the new device generally detected a higher number of CTCs. In summary, a microfluidic device has been developed to integrate filtration-based CTC isolation with immunoaffinity-based isolation. The device offers better performance than those based on one isolation mechanism only, with a potential to address CTC heterogeneity and detect CTCs with different size (single versus cluster) and/or various expression level (epithelial versus mesenchymal). Citation Format: Hugh Fan, Kangfu Chen, Pablo Dopico, Thomas George. Circulating tumor cell isolation based on both physical & biological properties [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 2216.