Higher Number Of Circulating Tumor Cells Research Articles

Platelets and circulating (and tumor) cells: partners in promoting metastatic cancer.

Despite being discovered decades ago, metastasis remains a formidable challenge in cancer treatment. During the intermediate phase of metastasis, tumor cells detach from primary tumor or metastatic sites and travel through the bloodstream and lymphatic system to distant tissues. These tumor cells in the circulation are known as circulating tumor cells (CTCs), and a higher number of CTCs has been linked to poor prognoses in various cancers. The blood is an inhospitable environment for any foreign cells, including CTCs, as they face numerous challenges, such as the shear stress within blood vessels and their interactions with blood and immune cells. However, the exact mechanisms by which CTCs survive the hostile conditions of the bloodstream remain enigmatic. Platelets have been studied for their interactions with tumor cells, promoting their survival, growth, and metastasis. This review explores the latest clinical methods for enumerating CTCs, recent findings on platelet-CTC crosstalk, and current research on antiplatelet therapy as a potential strategy to inhibit metastasis, offering new therapeutic insights. Laboratory and clinical data have provided insights into the role of platelets in promoting CTC survival, while clinical advancements in CTC enumeration offer improved prognostic tools. CTCs play a critical role in metastasis, and their interactions with platelets aid their survival in the hostile environment of the bloodstream. Understanding this crosstalk offers insights into potential therapeutic strategies, including antiplatelet therapy, to inhibit metastasis and improve cancer treatment outcomes.

A comparative study on ctDNA and tumor DNA mutations in lung cancer and benign cases with a high number of CTCs and CTECs

BackgroundLiquid biopsy provides a non-invasive approach that enables detecting circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) using blood specimens and theoretically benefits early finding primary tumor or monitoring treatment response as well as tumor recurrence. Despite many studies on these novel biomarkers, their clinical relevance remains controversial. This study aims to investigate the correlation between ctDNA, CTCs, and circulating tumor-derived endothelial cells (CTECs) while also evaluating whether mutation profiling in ctDNA is consistent with that in tumor tissue from lung cancer patients. These findings will help the evaluation and utilization of these approaches in clinical practice.Methods104 participants (49 with lung cancer and 31 with benign lesions) underwent CTCs and CTECs detection using integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy. The circulating cell-free DNA (cfDNA) concentration was measured and the mutational profiles of ctDNA were examined by Roche AVENIO ctDNA Expanded Kit (targeted total of 77 genes) by next generation sequencing (NGS) in 28 patients (20 with lung cancer and 8 with benign lesions) with highest numbers of CTCs and CTECs. Mutation validation in matched tumor tissue DNA was then performed in 9 patients with ctDNA mutations using a customized xGen pan-solid tumor kit (targeted total of 474 genes) by NGS.ResultsThe sensitivity and specificity of total number of CTCs and CTECs for the diagnosis of NSCLC were 67.3% and 77.6% [AUC (95%CI): 0.815 (0.722–0.907)], 83.9% and 77.4% [AUC (95%CI): 0.739 (0.618–0.860)]. The concentration of cfDNA in plasma was statistically correlated with the size of the primary tumor (r = 0.430, P = 0.022) and CYFRA 21–1 (r = 0.411, P = 0.041), but not with the numbers of CTCs and CTECs. In this study, mutations were found to be poorly consistent between ctDNA and tumor DNA (tDNA) in patients, even when numerous CTCs and CTECs were present.ConclusionDetection of CTCs and CTECs could be the potential adjunct tool for the early finding of lung cancer. The cfDNA levels are associated with the tumor burden, rather than the CTCs or CTECs counts. Moreover, the poorly consistent mutations between ctDNA and tDNA require further exploration.

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.

P-136 Prognostic implication of portal venous circulating tumor cells in resectable pancreatic cancer

Circulating tumor cells (CTCs) may be promising prognostic biomarkers in patients with advanced cancer. However, the paucity of CTCs in peripheral blood is the major challenge of clinical implication especially in early-stage cancer. Our study aimed to investigate whether the portal venous CTC can be a biomarker for early recurrence and poor prognosis in pancreatic cancer. Patients who were scheduled to undergo upfront curative resection for pancreatic cancer from September, 2017 to June, 2019 at a single tertiary hospital were consecutively enrolled in this prospective study. Intraoperatively, 7.5 mL blood was collected from the portal vein by direct puncture with a syringe and 7.5 mL peripheral venous blood was also collected. The detection and identification of circulating tumor cell (CTC) were performed using immunofloresence staining and examined under fluorescene microscope. We used 4-color staining protocol for CTC identification; DAPI (blue) for nucleated cells, CD45 (red) as a leukocyte marker, EpCAM or CK (yellow) as an epithelial marker, and vimentin (green) as a mesenchymal cell marker. During study period, peripheral blood CTC sampling was performed in 33 patients, of which portal vein CTC sampling was performed in 28. The median portal venous CTCs (2.5, interquartile ranges [IQR] 1-7.75) were significantly higher than the median peripheral venous CTCs (1, IQR 0-2, p < 0.001). Higher stage and regional lymph node metastasis were related with higher number of CTCs (≥3) in portal venous blood. Patient with low portal venous CTCs (≤ 2) showed better overall (not reached vs. 16.5 months, p=0.002) and recurrence free (13.4 vs. 7.0 months, p=0.007) survival than those with high portal venous CTCs (≥3). The number of peripheral venous CTCs were not related to overall and recurrence free survival after resection of pancreatic cancer. The phenotype (epithelial vs. mesenchymal) of CTC was not associated with cancer characteristics or prognosis. Portal venous CTCs can be used as a biomarker to determine prognosis after resection of pancreas cancer. In addition, the Portal CTC acquisition prior to surgery may be useful in determining neoadjuvant chemotherapy in patients with radiologically resectable pancreatic cancer.

Rapid and efficient capturing of circulating tumor cells from breast cancer Patient's whole blood via the antibody functionalized microfluidic (AFM) chip

Accurate enumeration of circulating tumor cells (CTCs) in cancer patient's blood functions as a form of “liquid biopsy”, which is pivotal for cancer screening, prognosis, and diagnosis. Herein, we demonstrate a novel antibody functionalized microfluidic (AFM) chip that rapidly and accurately qualifies CTCs from breast cancer patient's whole blood. The AFM chip consists of three buffering zones, and four main capturing zones filled with equilateral triangular pillars and periodically distributed obstacles. We validate the AFM chip with three Epithelial cell adhesion molecule (EpCAM) positive cancer cell lines, including breast (MCF-7), prostate (PC3), and lung cancer cell lines (A549), achieving capture efficiencies of 99.5%, 98.5%, and 96.72%, respectively, at a flow rate of 0.6 mL/hour. We further confirm the efficacy of the AFM chip with five advanced breast cancer patients' whole blood to capture EpCAM+/CK19+/CD45-/DAPI + CTCs. Interestingly, high number of CTCs were identified from each patient's 1 mL whole blood (595–2270), The AFM chip is highly efficient at rapidly capturing CTCs from cancer patients' whole blood without requiring extra equipment, which is critically beneficial for clinical application.

Positive Correlation Between the Number of Circulating Tumor Cells in the Pulmonary Vein and Tumor Spread Through Air Spaces in Resected Non-small Cell Lung Cancer.

Circulating tumor cells (CTCs) is one of the promising markers that predict dissemination and metastases. This study aimed to identify the relationship between CTCs in pulmonary vein (PuV) and spread through air space (STAS) in non-small cell lung cancers. We applied a cytology-based microfluidic platform for rare cell isolation. Twenty-four patients were enrolled. The rate of CTC detection in PuV was 79.2%, and STAS was observed in 54.2% of the samples. When the definitive cut-off value was 1 CTC/1 ml, of the 14 CTC-PuV-high cases, 11 (78.6%) were STAS-positive, whereas 2 of the 10 (20.0%) CTC-PuV-low cases were STAS-positive, and the difference between the two groups was statistically significant (p=0.02). CTC-PuV-high exhibited a significantly poorer survival (p<0.01). The higher frequency of STAS is significantly associated with a higher number of CTCs in PuV, and the combination of STAS and CTC was significantly associated with poor prognosis.

Abstract 585: A robust method for expanding patient-derived circulating tumor cells ex vivo

Abstract In the United States, an estimated 280,000 people will be diagnosed with invasive breast cancer, which has a 5-year survival rate of 27% and 22% in females and males, respectively. Despite the clinical burden of metastasis, the underlying machinery of metastasis remains unclear. This is largely due to a lack of available technologies that can robustly expand patient-derived metastatic cell populations for study ex vivo. One such population is circulating tumor cells (CTCs), which are cells that escape from the primary tumor, intravasate through blood vessel endothelium, and circulate throughout the body. Eventually, CTCs will extravasate at the metastatic site and become the seeds for metastatic colonization. Based on their role in metastasis, CTCs yield great promise for identifying key mechanistic drivers of metastasis. Unfortunately, CTCs exist in low levels within the blood (typically &amp;lt; 100 cells/10mL of blood). Previous methods for culturing CTCs ex vivo have reported low success rates (~ 20%), and often depend on the presence of a high number of CTCs for success. To better interrogate the mechanisms of metastasis, robust methods to propagate CTCs for study are needed. Recently, our lab developed a method using an unbiased selection criterion for isolating and propagating CTCs derived from metastatic breast cancer, yielding 100% success in twelve distinct individuals. This method, based on a density gradient centrifugation, extracts not only CTCs from blood plasma, but also includes potential cancer-associated neutrophils and macrophages. All cultures under our conditions demonstrated growth for &amp;gt; 30 days and were confirmed to have an epithelial and breast phenotype. Notably, 6/12 samples grew longer and were associated with increased CD45 expression using qRT-PCR. We have also recently demonstrated further success in culturing CTCs derived from a variety of other cancers, including colorectal, lung, and pancreatic cancers. This study therefore not only defines a novel technology for expanding CTCs, but also suggests a deficiency in modern isolation methods, which rely on CD45 exclusion, that is limiting culture success in previous methods. Citation Format: Jerry Xiao, Joshua D. Kassner, Kelly Stanton, Richard Schlegel, Paula R. Pohlmann, Seema Agarwal. A robust method for expanding patient-derived circulating tumor cells ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 585.

Abstract 596: Single circulating tumor cell enumeration and targeted sequencing in non-small cell lung cancer

Abstract Purpose: Circulating tumor cells (CTCs) may be novel biomarkers to predict cancer prognosis and monitor therapeutic efficacy. In the current study, we investigated the use of the time-dependent enumeration and targeted DNA sequensing of single CTCs as liquid biopsy based biomarkers of non-small cell lung cancer (NSCLC) patients for diagnosis, prognosis, therapeutic choice and outcome monitoring. Experimental Design: In a prospective study, we tested the AccuCyte® system (RareCyte®, Inc. Seattle, WA) for CTC enumeration in NSCLC using FDA criteria (CK/EpCAM+/CD45- with a nucleus). Whole blood (7.5 ml) from healthy donors was spiked with human lung adenocarcinoma (A549 cell line) cells at specific numbers (N=0, 100, 200, 1000). CTC detection and single-CTC picking was performed. NSCLC subjects' (N=20) and chronic smokers without cancer on screening low-dose CT subjects' (N=10) blood were collected (amount, method, biotechnology). For all blood samples, CTC detection and single CTC picking was performed. DNA was isolated from CTCs (N=3) and white blood cells (N=2) per sample. DNA libraries were prepared for targeted oncogene characterization using the CleanPlex® OncoZoom® panel. Somatic variant analysis and comparisons were performed. Results: AccuCyte enumeration of A549 cells spiked at four levels in healthy subjects' whole blood showed (Spiked #, Detected #): (0, 0); (100, 76); (200, 208); (1000, 1223). NSCLC subjects' (N=20; age: 64.85±6.64; BMI: 28.00±8.15sex ratio: 0.42) blood had a mean CTC count of 13.4 (SD=52, median=0, range [0, 237]). Ten screening controls without cancer (64.2±5.81, 1, 28.50±6.13, 0.66) had a mean CTC count of 0.20 (0.42, 0, [0, 1]). Significant association was noted regarding a higher number of CTCs found in NSCLC compared to screening subjects without cancer according to the (p= 0.020, Wilcoxon signed rank). CTC numbers increased with NSCLC AJCC stage. Somatic variant analysis on patient-matched single CTCs and WBCs is ongoing. Conclusions: The molecular investigation of patient matched single CTCs and white blood cells will fundamentally advance understanding on the NSCCL carcinogenic signature of these blood born cells and their potential value as cancer detection and therapy management liquid biopsy-based biomarkers. Citation Format: Mouadh Barbirou, Yariswamy Manjunath, Amanda Miller, Arturo Ramirez, Nolan Ericson, Kevin F. Kevin F. Staveley-O'Carroll, Wes Warren, Guangfu Li, Peter J. Tonellato, Jussuf T. Kaifi. Single circulating tumor cell enumeration and targeted sequencing in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 596.

Usefulness of monitoring circulating tumor cells as a therapeutic biomarker in melanoma with BRAF mutation

BackgroundWhile molecularly targeted therapies and immune checkpoint inhibitors have improved the prognosis of advanced melanoma, biomarkers are required to monitor drug responses. Circulating tumor cells (CTCs) are released from primary and/or metastatic tumors into the peripheral blood. We examined whether CTCs have potential as biomarkers by checking the number of CTCs, as well as the BRAF genotype of individual CTCs, in melanoma patients undergoing BRAF/MEK inhibitor treatment.MethodsCTCs were isolated from peripheral blood using a high-density dielectrophoretic microwell array, followed by labeling with melanoma-specific markers (MART-1 and/or gp100) and a leukocyte marker (CD45). The numbers of CTCs were analyzed in fifteen patients with stage 0–III melanoma. Furthermore, changes in CTC numbers were assessed in five patients with stage IV melanoma at four time points during BRAF/MEK inhibitor treatment, and the BRAF genotype was analyzed in CTCs isolated from one patient.ResultsWe examined CTCs in patients with stage 0–III (five samples per stage: stage 0–I, stage II, and stage III), and detected CTCs even in patients with early disease (stage 0 and I). Interestingly, recurrence occurred in the lymph nodes of one stage I patient 2 years after the detection of a high number of CTCs in the patient’s blood. The total number of CTCs in four of five patients with stage IV melanoma fluctuated in response to BRAF/MEK inhibitor treatment, suggesting that CTC number has potential for use as a drug response marker in advanced disease patients. Interestingly, one of those patients had CTCs harboring seven different BRAF genotypes, and the mutated CTCs disappeared upon BRAF/MEK inhibitor treatment, except for those harboring BRAFA598V.ConclusionsCTCs are present even in the early stage of melanoma, and the number of CTCs seems to reflect patients’ responses to BRAF/MEK inhibitor treatment. Furthermore, genetic heterogeneity of BRAF may contribute to resistance to BRAF/MEK inhibitors. Our findings demonstrate the usefulness of CTC analysis for monitoring responses to targeted therapies in melanoma patients, and for understanding the mechanism of drug resistance.

In vivo isolation of circulating tumor cells in patients with different stages of prostate cancer.

Circulating tumor cells (CTCs) provide accurate information on the clinical stage of cancer progression. The present study examined the clinical validity and feasibility of a new medical device for the in vivo isolation of CTCs from the blood of patients with prostate cancer (PCa). The GILUPI CellCollector® (DC01) was applied in 188 cases. The CTC/prostate-specific antigen (PSA) profile of each patient was checked for therapeutic monitoring of patients with PCa. The CellCollector, which is a unique in vivo approach for the isolation of CTCs, was compared with the CellSearch® system, which is the current standard. Overall survival (OS) and diagnostic performance were evaluated. By in vivo isolation, 78.9% (56/71) of patients with metastatic disease (PCa-m) and 46.3% (24/53) of patients with localized disease (PCa-l) had ≥1 captured CTC. Kaplan-Meier analysis revealed that patients with PCa-m that had ≥5 CTCs had a significantly different OS compared with those with <5 CTCs (27.5 months vs. 37 months; HR 2.6; 95% CI 0.78–8.3). Patients with a higher number of CTCs at all time-points had the shortest median OS of 25 months (HR 1.9; 95% CI 0.4–11.6). The effectiveness of CTC isolation technologies demonstrated that in 65.7% of the applications, patients with cancer were positive for CTCs using the CellCollector. By contrast, the CellSearch system detected CTCs in 44.4% of applications. In vivo isolation of CTCs demonstrated the clinical viability of the CellCollector, related to the current standard for the isolation of CTCs from patients with PCa. The advantage of the in vivo device is that it overcomes the blood volume limitations of other CTC assays. Furthermore, the present study revealed that the CellCollector was well tolerated, and no adverse events (AEs) or serious AEs were reported.

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.

Detection of Circulating and Disseminated Neuroblastoma Cells Using the ImageStream Flow Cytometer for Use as Predictive and Pharmacodynamic Biomarkers.

Circulating tumor cells (CTCs) serve as noninvasive tumor biomarkers in many types of cancer. Our aim was to detect CTCs from patients with neuroblastoma for use as predictive and pharmacodynamic biomarkers. We collected matched blood and bone marrow samples from 40 patients with neuroblastoma to detect GD2 +/CD45- neuroblastoma CTCs from blood and disseminated tumor cells (DTCs) from bone marrow using the Imagestream Imaging flow cytometer (ISx). In six cases, circulating free DNA (cfDNA) extracted from plasma isolated from the CTC sample was analyzed by high-density single-nucleotide polymorphism (SNP) arrays. CTCs were detected in 26 of 42 blood samples (1-264/mL) and DTCs in 25 of 35 bone marrow samples (57-291,544/mL). Higher numbers of CTCs in patients with newly diagnosed, high-risk neuroblastoma correlated with failure to obtain a complete bone marrow (BM) metastatic response after induction chemotherapy (P < 0.01). Ex vivo Nutlin-3 (MDM2 inhibitor) treatment of blood and BM increased p53 and p21 expression in CTCs and DTCs compared with DMSO controls. In five of six cases, cfDNA analyzed by SNP arrays revealed copy number abnormalities associated with neuroblastoma. This is the first study to show that CTCs and DTCs are detectable in neuroblastoma using the ISx, with concurrently extracted cfDNA used for copy number profiling, and may be useful as pharmacodynamic biomarkers in early-phase clinical trials. Further investigation is required to determine whether CTC numbers are predictive biomarkers of BM response to first-line induction chemotherapy.

Circulating tumor cells as a new predictive and prognostic factor in patients with small cell lung cancer.

Background: Small cell lung cancer (SCLC) is the most malignant type of lung cancer characterized by rapid progression, early metastasis and recurrence. In recent years, circulating tumor cells (CTCs) were found to play an important role in tumor invasion, metastasis, recurrence and prognosis.Methods: CTCs were detected in 138 patients with newly diagnosed SCLC from January 2012 to December 2018. Nomogram prediction models were constructed based on prognostic factors screened by multivariate Cox regression analysis and the risk stratification of SCLC patients were performed on basis of nomogram points. A total of 108 patients from January 2012 to December 2016 were assigned to a training group, and 30 patients from January 2017 to December 2018 were included into the validation group for nomogram analysis. This study was approved by ethics committee of Guangzhou First People's Hospital and all subjects provided informed consent.Results: The number of CTCs was associated with age, lymph node metastasis (N), distant metastasis (M), TNM staging, and NSE. The high number of CTC predicted adverse prognosis, and the AUC of time-dependent ROC curve was all high than 0.5. In the training group, after multivariate COX regression screening, the factors in the median survival time (MST) and overall survival (OS) nomogram prediction models were age, TNM, CTC, NSE and treatment mode. The C-index of the nomograms in internal validation for MST and OS was 0.813 and in external validation for MST and OS were 0.885. The AUC of ROC curves for nomogram were high than 0.5. Finally, risk stratification could be effectively performed on the basis of nomogram points.Conclusions: CTC can be served as a predictive and prognostic factor for SCLC, and the nomogram models constructed by CTC and multiple clinical parameters can comprehensively predict the prognosis of SCLC patients and perform risk stratification.

Effects of salinomycin and niclosamide on small cell lung cancer and small cell lung cancer circulating tumor cell lines

SummaryTumor dissemination and recurrence is attributed to highly resistant cancer stem cells (CSCs) which may constitute a fraction of circulating tumor cells (CTCs). Small cell lung cancer (SCLC) constitutes a suitable model to investigate the relation of CTCs and CSCs due to rapid tumor spread and a high number of CTCs. Expansion of five SCLC CTC lines (BHGc7, 10, 16, 26 and UHGc5) in vitro at our institution allowed for the analysis of CSC markers and cytotoxicity of the CSC-selective drugs salinomycin and niclosamide against CTC single cell suspensions or CTC spheroids/ tumorospheres (TOS). Salinomycin exerted dose-dependent cytotoxicity against the SCLC lines but, with exception of BHGc7 TOS, there was no markedly enhanced activity against TOS. Similarly, niclosamide exhibits high activity against BHGc7 TOS and UHGc5 TOS but not against the other CTC spheroids. High expression of the CSC marker CD133 was restricted to three SCLC tumor lines and the BHGc10 CTC line. All SCLC CTCs are CD24-positive but lack expression of CD44 and ABCG2 in contrast to the SCLC tumor lines which show a phenotype more similar to that of CSCs. The stem cell marker SOX2 was found in all CTC lines and SCLC GLC14/16, whereas elevated expression of Oct-3/4 and Nanog was restricted to BHGc26 and UHGc5. In conclusion, the SCLC CTCs established from patients with relapsed disease lack a typical CSC phenotype in respect to chemosensitivity to CSC-selective drugs, surface markers, expression of pluripotent stem cell and transcription factors.

Abstract 2216: Circulating tumor cell isolation based on both physical &amp; 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 &amp; 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.

Effective capture of circulating tumor cells from an S180-bearing mouse model using electrically charged magnetic nanoparticles

BackgroundTechnology enabling the separation of rare circulating tumor cells (CTCs) provides the potential to enhance our knowledge of cancer metastasis and improve the care of cancer patients. Modern detection approaches commonly depend on tumor antigens or the physical size of CTCs. However, few studies report the detection of CTCs by the electrical differences between cancer cells and normal cells.ResultsIn this study, we report a procedure for capturing CTCs from blood samples using electrically charged superparamagnetic nanoparticles (NPs). We found that only positively charged NPs attached to cancer cells, while negatively charged NPs did not. The capture method with positively charged NPs offered a sensitivity of down to 4 CTCs in 1 mL blood samples and achieved a superior capture yield (> 70%) for a high number of CTCs in blood samples (103–106 cells/mL). Following an in vitro evaluation, S180-bearing mice were employed as an in vivo model to assess the specificity and sensitivity of the capture procedure. The number of CTCs in blood from tumor-bearing mice was significantly higher than that in blood from healthy controls (on average, 75.8 ± 16.4 vs. zero CTCs/100 μL of blood, p < 0.0001), suggesting the high sensitivity and specificity of our method.ConclusionsPositively charged NPs combined with an in vivo tumor model demonstrated that CTCs can be distinguished and isolated from other blood cells based on their electrical properties.

Abstract 2112: Cell by cell immuno- and cancer marker profiling for non-small cell lung cancer (NSCLC) tissue sample using non-enzymatic tissue dissociation and high-parameter flow cytometry

Abstract Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 80-85% cases of lung cancer. It has poor prognosis as most NSCLC patients are at advanced stage when diagnosed. The development of immunotherapy blocking the PD-1/PD-L1 ligand pathway, in recent years, has shed light on new treatment for lung cancer. An in-depth understanding of the interaction between immune system and tumor will help to identify novel markers for lung cancer treatment. In this study, we performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples for circulating tumor cells (CTCs). The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization (IncellDx IncellPREPTM). The single cell suspensions were stained simultaneously with multiple (&amp;gt;20) immune check point markers (including PD-1*, PD-L1*, TIM-3*, LAG-3*, and CTLA-4*), cancer markers (such as EGFR* and ALK* fusion protein), and a cell cycle dye. The samples were interrogated on a novel, innovative, high parameter, spectral flow cytometer Cytek AuroraTM. Markers on subsets of immune cell and cancer cell populations were investigated. Our results showed the association of high levels of immune check point marker and cancer marker expressions with the high level of aneuploidy (indicated by DNA index &amp;gt;1.05), and the aggressiveness of the cancer (indicated by the number of CTCs). High numbers of CTCs were associated with aneuploidy, increased Post G0-G1%, and high expression of LAG-3, TIM-3, and PD-1. Multi-parametric flow cytometry allows simultaneous profiling of multiple immune and cancer markers on cancer samples at the single cell level. The knowledge acquired from these studies will enhance our understanding of cancer immune system, cancer cells, and the interaction between immune system and the cancer. It will potentially transform patient diagnosis, disease monitoring, and drug discovery. Our study demonstrated a powerful method to study solid tumors that may provide important information for successful precision cancer immunotherapy. *PD-1: programmed death-1; *PD-L1: programmed death-ligand 1; *TIM-3: mucin domain-3-containing molecule-3; *LAG-3: lymphocyte-activation gene-3; *CTLA-4: cytotoxic T-lymphocyte antigen-4; *EGFR: epidermal growth factor receptor; *ALK: anaplastic lymphoma kinase Citation Format: Xiaoyang Wang, Pin-I Chen, Maria Jaimes, Huimin Gu, Keith Shults, Fariba Fazeli, Janine Fernandez, Vishal Sharma, Kalyan Handique, Bruce K. Patterson. Cell by cell immuno- and cancer marker profiling for non-small cell lung cancer (NSCLC) tissue sample using non-enzymatic tissue dissociation and high-parameter flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2112.

Highlights of This Issue

Uckermann and colleagues use infrared spectroscopy and present a translational approach for fast, label-free optical analysis of the IDH1 genotype of human glioma. Based on the initial research performed on cell lines and tumor cryosections, fresh glioma biopsies were classified regarding their IDH1 genotype with an accuracy of 86%. Because IDH1 mutations are early events in glioma formation with prognostic impact and most likely dichotomizing therapeutic decision making, analysis of the tumor's spectrome might be exploited to complement histopathology and molecular pathology for an in situ diagnostic screen.Clinical utilization of circulating tumor cells (CTC) as a biomarker has been limited by the low sensitivity of CTC assays. Myung and colleagues demonstrate that CTC capture can be improved through two innovative concepts: biomimetic cell rolling and nanotechnology-enabled multivalent binding capture. The investigators show that the new assay can capture high numbers of CTCs in a diverse cohort of patients. Moreover, CTC change is correlated with treatment response and disease status. These findings hold potential for clinical translation.Both EGFR and VEGF are validated targets for treatment of patients with metastatic colorectal cancer (mCRC); however, combinations of monoclonal antibodies targeting both pathways show disappointing results. Mésange and colleagues show that combinations of bevacizumab and erlotinib, a small molecule EGFR inhibitor, have activity in CRC models independent of RAS status and bevacizumab resistance. The results provide a molecular framework to better understand the increased activity of the bevacizumab-erlotinib combination, compared with bevacizumab alone, in the GERCOR DREAM phase III clinical trial. Differential activity of mAb and TKI targeting the same signaling pathway is likely applicable for other tumor types.Attempts at treating glioblastoma (GBM) patients with checkpoint inhibitors have failed to provide a survival benefit. A potential mechanism explaining the failure of PD-1 blockade is based on work showing that GBM-infiltrating T cells increase immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO1) levels. Ladomersky and colleagues tested IDO1 inhibitor and PD-1 mAb treatment, with or without radiotherapy, and found that the combination of all three agents led to long-term survival in a substantial fraction of mice with intracranial GBM. These data provide rationale for the soon-to-open phase I/IIa clinical trial of simultaneous radiotherapy with PD-1 and IDO1 inhibition in GBM patients.

Comparison of isolation platforms for detection of circulating renal cell carcinoma cells.

BackgroundAnalysis of circulating tumor cells (CTCs) has progressed in several tumor entities. However, little is known about CTCs in clear cell renal cell carcinoma (ccRCC) patients. Aim of our studies was to build a stable in vitro fundament for isolation of CTCs in ccRCC.MethodsWe compared the analytical performance of different CTC isolation methods with regard to yield and purity: EpCAM based enrichment, leukocyte depletion and size based enrichment. EpCAM and cytokeratin 8 (KRT8) as biomarker for CTCs expression were evaluated in ccRCC cell lines as well as clinical samples.ResultsWhile the EpCAM based approach failed to successfully isolate tumor cells, CD45 based approaches showed intermediate recovery rates. The cell-size based Parsortix system showed highest recovery rates. EpCAM expression was low or absent in most cell lines as well as in clinical samples, whereas KRT8 was detected as a potential biomarker in ccRCC.ConclusionEpCAM based approaches might miss a high number of CTCs due to low or absent expression of EpCAM in ccRCC, as shown in cell lines as well as in patient samples. We identified the cell-sized based, label independent Parsortix system to be the most effective recovery system for ccRCC CTCs.

Abstract 4816: Circulating tumor cell monitoring, isolation, and culture from a patient with metastatic triple-negative breast cancer for drug screening and creation of a patient-derived xenograft model

Abstract Background. Enumeration, phenotyping and single cell genomics of circulating tumor cells (CTCs) provide three types of information to guide cancer therapy. In some instances, a fourth type is possible: functional analysis in vitro, or in in vivo patient-derived xenografts (PDXs). We used a density-based rare cell separation and analysis system to collect CTCs from the blood of a patient with metastatic triple-negative breast cancer (TNBC) for in vitro culture and high-throughput drug screening and to generate a PDX model. Methods: The patient was enrolled in the ITOMIC-001 study (University of Washington) and after informed consent, CTCs were evaluated prior to initial cisplatin treatment and tracked longitudinally using the AccuCyte – CyteFinder system (RareCyte). Samples containing high numbers of CTCs were placed into 3 different culture media. Cells grown in culture were tested against a panel of anti-cancer drugs and injected into mice to form a PDX model. Results: Nine CTC evaluations were performed over 9.5 months. CTCs were verified by expression of epithelial (cytokeratin and/or EpCAM) and nuclear stains without CD45 expression. After initial treatment with cisplatin, the CTC count per 7.5 mL rose from 4 to 19 cells at 3 months, consistent with the lack of a clinical response, and decreased after LE 011 (CDK4/6 inhibitor) and then glembatumumab vedotin (anti-gpNMB) to 8 and 4 cells at 5 and 7 months respectively. At 9 months the CTC count rose to &amp;gt; 13,000 and 5 days later to &amp;gt; 80,000 shortly before her death. At autopsy there was massive infiltration of the liver and pulmonary vasculature by tumor cells. Cultures in all media showed initial growth, but only one (RPMI + 10% serum) was sustained, forming semi-adherent 3D tumor clusters. 6 million cells were harvested and a drug screen using 160 anti-cancer agents was performed. The CTC line showed sensitivity to several agents. Cells were also injected into the mammary fat pad of immunodeficient mice. In at least one mouse, macroscopic tumors were observed. The CTC cell line has grown continually in culture for over a year. Aliquots of this cell line have been frozen and thawed with no noticeable effect on cell growth. Conclusions: Using a density-based rare cell collection system, we have established a CTC cell line from a TNBC patient with extremely high CTC counts. The line was used to perform a screen for agents active against the tumor cells and to create a PDX model. As in vitro techniques advance, smaller number of CTCs may be effectively cultured and thus allow this approach to be used in real time to find effective drug regiments for individualized cancer therapy. Citation Format: Arturo B. Ramirez, C Anthony Blau, Timothy J. Martins, Elisabeth Mahen, Lacey E. Dobrolecki, Michael T. Lewis, Jackie L. Stilwell, Eric P. Kaldjian. Circulating tumor cell monitoring, isolation, and culture from a patient with metastatic triple-negative breast cancer for drug screening and creation of a patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4816. doi:10.1158/1538-7445.AM2017-4816