Circulating Tumor Cells Lines Research Articles

Abstract 1336: An assay for the detection and isolation of single live circulating tumor cells using Accucyte/Rarecyte platform

Abstract Introduction: Circulating tumor cells (CTCs) are critical metastatic drivers for the interface between the primary tumor and the target secondary organs. CTCs are heterogeneous, therefore, investigating their phenotypic, molecular and biological properties on a single cell level warrant a better understanding to metastasis and patient’s response to therapy. AccuCyte® - Rarecyte® system is a density based CTC isolation platform which allows for automated visual identification and retrieval of fixed rare cells from blood. Aims: To establish an assay for the detection and isolation of live single CTCs valid for subsequent molecular and functional characterization. Methods: Normal human whole blood samples were spiked with cells from breast cancer patient-derived CTC lines and were processed using the AccuCyte system. Buffy coats containing spiked CTCs were stained live with a cocktail of PE-594-conjugated antibodies for immune cell markers (CD14, CD16 and CD45) and either a live cell dye or a cocktail of Alexa Fluor 488-conjugated antibodies for cancer cell surface markers (EpCAM, Her2, EGFR and CDH11). Stained cells were then plated on chamber slides in the presence of serum free RPMI media and investigated using RareCyte fluorescence platform. CTCs were identified based on the lack of immune cell marker expression and the presence of the live cell dye (negative selection) or the expression of cancer cell surface markers and the lack of immune cell markers (positive selection). Single CTCs detected by both approaches were retrieved using our optimized interactive picking module. Cells detected by negative selection were tested for their ability to proliferate ex-vivo, whereas those isolated by positive selection were processed for single cell RNA sequencing analyses. Results: Detection rates of our protocols reached up to 85% (12 out of 14 CTCs). Single CTCs isolated by negative selection showed a slower proliferation rate during the first 6 days of culture (1.1 Fold increase relative to Day 0) compared to control cells (2.5 Fold increase). However their proliferation rates were comparable to control cells at day 13 (2.7 Vs. 3.2) and day 21 (2.2 Vs. 2.5). Currently, other live cell dyes are being tested to improve the proliferation ability of CTCs isolated by negative selection. Moreover, single cells which were retrieved by positive selection are currently being validated for RNA sequencing analyses. Conclusion: We have developed a novel assay which allows for the detection and isolation of single viable CTCs. Our assay has two modules; one uses a positive detection approach combined with a two-step cell picking protocol to provide ultrapure single CTCs valid for gene expression analyses. The other module provides an unbiased identification of CTCs using the negative selection approach and a single step of cell picking that provides viable single CTCs able to proliferate in culture. Citation Format: Mohamed Kamal, Shahin Saremi, Remi V. Klotz, Oihana Iriondo, Min Yu. An assay for the detection and isolation of single live circulating tumor cells using Accucyte/Rarecyte platform [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 1336.

Expression of Proteolytic Enzymes by Small Cell Lung Cancer Circulating Tumor Cell Lines.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer which disseminates vigorously and has a dismal prognosis. Metastasis of SCLC is linked to an extremely high number of circulating tumor cells (CTCs), which form chemoresistant spheroids, termed tumorospheres. Intravasation and extravasation during tumor spread requires the activity of a number of proteases to disintegrate the stroma and vascular tissue. Generation of several permanent SCLC CTC lines allowed us to screen for the expression of 35 proteases using Western blot arrays. Cell culture supernatants of two CTC lines, namely BHGc7 and 10, were analyzed for secreted proteases, including matrix metalloproteinases (MMPs), ADAM/TS, cathepsins, kallikreins, and others, and compared to proteases expressed by SCLC cell lines (GLC14, GLC16, NCI-H526 and SCLC26A). In contrast to NCI-H526 and SCLC26A, MMP-9 was highly expressed in the two CTC lines and in GLC16 derived of a relapse. Furthermore, cathepsins (S, V, X/Z/P, A and D) were highly expressed in the CTC lines, whereas ADAM/TS and kallikreins were not detectable. In conclusion, SCLC CTCs express MMP-9 and a range of cathepsins for proteolysis and, aside from tissue degradation, these enzymes are involved in cell signaling, survival, and the chemoresistance of tumor cells.

Autologous cell lines from circulating colon cancer cells captured from sequential liquid biopsies as model to study therapy-driven tumor changes

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However, CTC investigation is hampered by their low number, making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy, and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype, stem-cell like characteristics, angiogenesis potential, an osteomimetic signature and the capacity to escape from the immune system. Moreover, they showed changes in mRNA and protein expression (e.g., DEFA6, ABCB1 and GAL), whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits, treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time.

Anticancer Activity of Fascaplysin against Lung Cancer Cell and Small Cell Lung Cancer Circulating Tumor Cell Lines.

Lung cancer is a leading cause of tumor-associated mortality. Fascaplysin, a bis-indole of a marine sponge, exhibit broad anticancer activity as specific CDK4 inhibitor among several other mechanisms, and is investigated as a drug to overcome chemoresistance after the failure of targeted agents or immunotherapy. The cytotoxic activity of fascaplysin was studied using lung cancer cell lines, primary Non-Small Cell Lung Cancer (NSCLC) and Small Cell Lung Cancer (SCLC) cells, as well as SCLC circulating tumor cell lines (CTCs). This compound exhibited high activity against SCLC cell lines (mean IC50 0.89 µM), as well as SCLC CTCs as single cells and in the form of tumorospheres (mean IC50 0.57 µM). NSCLC lines showed a mean IC50 of 1.15 µM for fascaplysin. Analysis of signal transduction mediators point to an ATM-triggered signaling cascade provoked by drug-induced DNA damage. Fascaplysin reveals at least an additive cytotoxic effect with cisplatin, which is the mainstay of lung cancer chemotherapy. In conclusion, fascaplysin shows high activity against lung cancer cell lines and spheroids of SCLC CTCs which are linked to the dismal prognosis of this tumor type. Derivatives of fascaplysin may constitute valuable new agents for the treatment of lung cancer.

Biophysical analysis of fluid shear stress induced cellular deformation in a microfluidic device.

Even though the majority of breast cancers respond well to primary therapy, a large percentage of patients relapse with metastatic disease, for which there is no treatment. In metastasis, a tumor sheds a small number of cancerous cells, termed circulating tumor cells (CTCs), into the local vasculature, from where they spread throughout the body to form new tumors. As CTCs move through the circulatory system, they experience physiological forces not present in the initial tumor environment, namely, fluid shear stress (FSS). Evidence suggests that CTCs respond to FSS by adopting a more aggressive phenotype; however, to date single-cell morphological changes have not been quantified to support this observation. Furthermore, the methodology of previous studies involves inducing FSS by flowing cells through the tubing, which lacks a precise and tunable control of FSS. Here, a microfluidic approach is used for isolating and characterizing the biophysical response of single breast cancer cells to conditions experienced in the circulatory system during metastasis. To evaluate the single-cell response of multiple breast cancer types, two model circulating tumor cell lines, MDA-MB-231 and MCF7, were challenged with FSS at precise magnitudes and durations. As expected, both MDA-MB-231 and MCF7 cells exhibited greater deformability due to increasing duration and magnitudes of FSS. However, wide variations in single-cell responses were observed. MCF7 cells were found to rapidly deform but reach a threshold value after 5 min of FSS, while MDA-MB-231 cells were observed to deform at a slower rate but with a larger threshold of deformation. This behavioral diversity suggests the presence of distinct cell subpopulations with different phenotypes.

Abstract 3023: Dissecting mechanisms of breast cancer metastasis through patient-derived circulating tumor cells

Abstract During tumor progression, tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred to as circulating tumor cells (CTCs). These CTCs disseminate to other organs, and a subset of these cells will form metastasis. The growing interest for CTCs is confronted with the difficulty associated with their isolation and scarcity of material. To address this challenge, our established CTC lines from breast cancer patients offered a unique opportunity to investigate the signal pathways involved in the metastatic cascade. We assessed the metastatic potential of breast cancer patient-derived CTC lines by injecting them directly into the left ventricle of the heart in NSG mice. CTC lines are capable of generating metastases in most organs that are listed as common sites of metastases from breast cancer, such as the brain, lungs, bones and ovaries. Two patient-derived CTC lines harbored a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in the bones, lungs and ovaries. These mice remained brain-metastases free for up to 8 months. However, in 2 patient-derived CTC lines we demonstrated the brain as the preferential site of metastasis despite their low metastatic potential. Interestingly, in our mouse model, CTC lines recapitulated human cancer metastatic progression, as shown by the clinical data collected from patients where CTC lines were generated. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. To address this we isolated metastatic variants with a preferential tropism for the brain, lungs and bones. Investigating the gene expression signatures of metastatic variants lead us to identify the semaphorin 4D as a possible regulator of the brain metastasis. High SEMA4D expression at the primary site correlated with decreased brain metastasis-free survival in the breast cancer patients. Amplification of the chromosome 9 where the SEMA4D gene resides was also found in CTC-derived brain metastases. The SEMA4D overexpression promoted the ability of CTCs to transmigrate the blood-brain-barrier in vitro. We simultaneously investigated expression changes in stromal cells in organ-specific metastases, and among the pathways with the highest enrichment scores we found TNF and IFN signaling in the brain metastatic microenvironment. Next we used ATAC-seq to assay chromatin accessibility and identified potential regulatory factors mediating the brain metastasis formation. These data provides the evidence for the promising role of CTCs as an early prognostic factor for metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancer. Citation Format: Remi Klotz, Amal Thomas, Sara Restrepo-Vassalli, Oihana Iriondo, Jane Han, Teng Teng, Matthew MacKay, Alan Wang, Negeen Izadian, Byoung-San Moon, Aditya Bardia, James Hicks, Andrew Smith, Min Yu. Dissecting mechanisms of breast cancer metastasis through patient-derived circulating tumor cells [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 3023.

Abstract SY21-01: Molecular and functional characterization of circulating tumor cells in carcinoma patients

Abstract “Real-time liquid biopsy” was introduced in 2010 for the analysis of circulating tumor cells (CTCs) in the blood of cancer patients (1), and this new diagnostic approach has received enormous attention because of its obvious clinical implications for personalized medicine. CTCs are derived from primary tumors and metastatic lesions and, therefore, harbor important information on the molecular characteristics relevant to tumor progression and cancer therapy. Analyses of CTCs have paved new diagnostic avenues with obvious clinical implications for personalized medicine. Key areas of clinical applications of CTCs include detection of cancer, prediction of prognosis in patients with curable disease, monitoring of systemic therapies, and stratification of patients based on the detection of therapeutic targets or resistance mechanisms (2). Much progresses has been made in the development of various technologies to enrich and detect CTCs; however, the discovery and validation of new CTC biomarkers are still in its infancy (3). Different types of CTC characterization studies have been carried out: (i) descriptive studies on isolated individual CTCs requiring a specific isolation process and a process of whole-genome or transcriptome amplification, which are informative on intrapatient heterogeneity; (ii) descriptive studies providing information at the individual cell level, which require only an enrichment but no single-cell isolation; (iii) functional studies on pools of total CTCs used for cell culture and/or xenografts (4). Since current assays cannot distinguish between apoptotic and viable CTCs, it is possible to apply the EPISPOT assay that detects proteins secreted/released/shed from viable epithelial cancer cells (5). Cells are cultured for a short time on a membrane coated with antibodies that capture the secreted/released/shed proteins that are subsequently detected by secondary antibodies labeled with fluorochromes. This functional assay detects viable CTCs at the single-cell level and has been used on hundreds of patients with different tumor types including epithelial tumors (breast, prostate, ovarian, and colon cancer) and melanomas, showing its clinical relevance (6). The fluoro-EPISPOT assay has been optimized for single CTCs, is now named EPIDROP, and should be a more reliable and sensitive technology than those already existing in this field of expertise, allowing the analysis of proteome and secretome simultaneously of unique functional CTCs or of clusters of CTCs. In addition, testing different drugs in an individual patient might lead to a personalized "oncogram" that might help to improve the clinical management of cancer patients. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in cancer patients. Understanding the molecular mechanisms that regulate the biology of metastasis-competent CTCs is of utmost importance in unraveling the formation of metastases and tumor relapse in patients with cancer (3). However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of colorectal cancer patients. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. In 2015, Cayrefourcq et al. described for the first time the establishment of a permanent cell line from CTCs of one metastatic colon cancer patient (7). The cell line, designated "CTC-MCC-41," is stable for more than three years and cells have been characterized at the genome, transcriptome, proteome, and secretome levels. This thorough analysis showed that CTC-MCC-41 cells resemble characteristics of the original tumor cells in the colon cancer patient and display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem-cell like properties and an osteomimetic signature indicating a bone marrow origin. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. Subsequently, Alix-Panabières et al. determined the molecular bases underlying differences between this colon CTC line and well-described cancer cell lines derived from primary tumors and from metastatic sites (8). The results showed clearly that the CTC-MCC-41 displayed a very specific transcription program. Interestingly, among the 1,624 transcripts exclusively upregulated in CTC-MCC-41, key genes related to energy metabolism, DNA repair, and stemness genes were observed. Furthermore, blood samples collected during the course of the systemic therapy and tumor progression from the same metastatic colon cancer patient who gave rise to the CTC-MCC1-41 line have allowed the acquisition of 8 additional colon CTC lines. To our best knowledge, there is no other report on the in vitro expansion of CTCs from cancer patients during the course of their disease and treatment. This unique biologic material represents a chance to understand the clonal selection and resistance mechanisms during tumor progression and cancer treatment. In conclusion, functional analyses of CTCs provide insights for the discovery of new biomarkers to identify the most aggressive CTC subpopulations with stem cell properties. CTC lines could contribute to the development of new drugs to eradicate metastasis-initiator CTCs causing relapses and cancer-related death in individuals with cancer.

Ultra-fast vitrification of patient-derived circulating tumor cell lines.

Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.

Circulating tumor cell interactions with macrophages: implications for biology and treatment.

Cancer and metastasis are closely associated with inflammation. Macrophages are important effector cells in enhancing tumor proliferation, invasion and providing protection against the immune system. Despite advanced knowledge of tumor-macrophage interactions, the role of macrophages in emergence and invasion of circulating tumor cells (CTCs) is not known. A series of six CTC cell lines have been derived from blood of patients with extensive disease small cell lung cancer (ED-SCLC) in our lab, most likely representing a homogenous cell population of the actual metastasis-initiating cells (MIC) of CTCs. SCLC has an unfavorable prognosis due to rapid dissemination and early chemoresistant relapses. SCLC CTCs recruit macrophages and elicit secretion of various cytokines and the six CTC lines express chitinase-3-like-1 (CHI3L1), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) in abundance. CHI3L1 is cytokine/growth factor expressed in inflammation and cancer and found to be correlated to metastasis and a dismal prognosis. In conclusion, SCLC CTCs have acquired the essential means for aggressiveness and invasion in a tumor microenvironment specifically shaped by macrophages and inflammation.

Abstract 1045: Understanding breast cancer metastasis through circulating tumor cells

Abstract Due to the advance in technologies for rare cell isolation, circulating tumor cells (CTC) have recently received vast interest. During tumor progression tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred as CTCs. These CTCs disseminate to other organs and a subset of these cells will be able to form metastasis. The growing interest for CTC is confronted with the difficulty associated with their isolation and characterization. To address this challenge, our lab has recently optimized the ex vivo culture condition and was able to establish CTC lines from breast cancer patients. These CTC lines constitute a unique cell population in the metastatic process and gave us a rare opportunity to investigate the signal pathways involved in each step of the metastatic cascade. To assess the metastatic potential of breast cancer patient-derived CTC lines we utilized an experimental mouse model for metastasis by injecting CTCs directly into the left ventricle of the heart in female immuodeficient NSG mice. The ability of 4 CTC lines to arrest and colonize in organ was monitored by bioluminescence imaging once every 2 weeks for 5 months. CTC lines were capable of generating brain, lung, bone and ovary metastases. Most of those organs are common sites of metastases in breast cancer patients. Two patient-derived CTC lines have a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in multiple organs such as bone, lung and Ovary. These mice remained brain-metastases free for up to 8 months. However, two other patient-derived CTC lines demonstrated the brain as preferential site of metastasis despite their overall low metastatic potential. Interestingly, of 4 breast cancer patients where CTC lines were generated, one developed a metastatic brain tumor and her CTC line has the highest risk of brain metastases in our mouse model. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. We isolated metastatic variants corresponding to a subpopulation of CTCs with a preferential tropism for the brain, lung and bone. Gene expression analysis (RNA-seq) of these variants identified potential gene signatures of breast cancer metastasis. The pathways with the highest enrichment scores were glioblastoma multiform, ceramide biosynthesis and PCP pathway for the brain metastatic variants and were interferon and mTOR signaling for the lung metastatic variants. Next we used a method for assaying chromatin accessibility (ATAC-seq) and identified potential regulatory regions mediating the organ tropism in breast cancer. Together our data provide the evidence of a promising role of CTC as an early prognostic factor in metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancers. Citation Format: Remi Klotz, Thomas Amal, Alan Wang, Matthew Mackay, Kathleen Heller, Lin Li, Maxwell Serowoky, Grace Lee, Jane Han, Andrew Smith, Min Yu. Understanding breast cancer metastasis through circulating tumor cells [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 1045. doi:10.1158/1538-7445.AM2017-1045

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 > 13,000 and 5 days later to > 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

Insights into mechanisms of tumor dissemination from circulating tumor cell lines of small cell lung cancer

Insights into mechanisms of tumor dissemination from circulating tumor cell lines of small cell lung cancer

Simultaneous detection of MCF-7 and HepG2 cells in blood by ICP-MS with gold nanoparticles and quantum dots as elemental tags

In this work, we demonstrate a novel method based on inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticles (Au NPs) and quantum dots (QDs) labeling for the simultaneous counting of two circulating tumor cell lines (MCF-7 and HepG2 cells) in human blood. MCF-7 and HepG2 cells were captured by magnetic beads coupled with anti-EpCAM and then specifically labeled by CdSe QDs-anti-ASGPR and Au NPs-anti-MUC1, respectively, which were used as signal probes for ICP-MS measurement. Under the optimal experimental conditions, the limits of detection of 50 MCF-7, 89 HepG2 cells and the linear ranges of 200-40000 MCF-7, 300-30000 HepG2 cells were obtained, and the relative standard deviations for seven replicate detections of 800 MCF-7 and HepG2 cells were 4.6% and 5.7%, respectively. This method has the advantages of high sensitivity, low sample consumption, wide linear range and can be extended to the simultaneous detection of multiple CTC lines in human peripheral blood.

Abstract 1547: Circulating tumor cells from a syngeneic mouse model of hepatocellular carcinoma demonstrate epithelial-mesenchymal transition, decreased MHC I expression, and increased CCR7 expression

Abstract Metastasis is the leading cause of death in cancer patients, worldwide. A better understanding of how cancer cells detach from the primary tumor, escape from immunosurveillance while in circulation, and successfully colonize distant parts of the body, is needed. Circulating tumor cells (CTCs) are excellent tools to study this process. Using a syngeneic hepatocellular carcinoma mouse model, we were able to isolate cancer cells from the primary tumor and CTCs from the blood and establish novel primary tumor-derived cell lines and novel CTC lines. Wound healing assays revealed that CTCs have greater migratory capacity than the cancer cells from the primary tumors. However, MTT assays revealed that CTCs had less proliferative capacity than the cancer cells from the primary tumors. Furthermore, molecular characterization of the cancer cells from primary tumors and CTCs using Western blotting showed a decreased expression of E-cadherin and an increased expression of fibronectin by CTCs, suggesting that epithelial-to-mesenchymal transition (EMT) takes place in CTCs in comparison to cancer cells from primary tumors. To address the question of how CTCs may evade immunosurveillance, we investigated the profile of expression of two cell surface proteins involved in immune system function: the major histocompatibility complex class I (MHCI)- required for cytotoxic T cell activation and subsequent cancer cell killing, and chemokine receptor 7 (CCR7)- exploited by cancer cells for immune evasion. Our analysis revealed that there is decreased cell surface expression of MHCI and increased expression of CCR7 in the CTCs in comparison to the cancer cells from primary tumors. These findings indicate that although CTCs are not more proliferative, they have acquired significantly increased migratory capacity than cancer cells in primary tumors. This may be explained by their demonstration of EMT. Furthermore, their loss of MHC I expression and their gain of CCR7 expression may help with evasion of killing by cytotoxic T cells and result in enhanced metastasis. Citation Format: Jeannette A. Huaman, Ubayed Muhith, Dibash K. Das, Michelle Naidoo, Olorunseun O. Ogunwobi. Circulating tumor cells from a syngeneic mouse model of hepatocellular carcinoma demonstrate epithelial-mesenchymal transition, decreased MHC I expression, and increased CCR7 expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1547.

Small cell lung cancer: Circulating tumor cells of extended stage patients express a mesenchymal-epithelial transition phenotype

ABSTRACTSmall cell lung cancer (SCLC) is distinguished by aggressive growth, early dissemination and a poor prognosis at advanced stage. The remarkably high count of circulating tumor cells (CTCs) of SCLC allowed for the establishment of permanent CTC cultures at our institution for the first time. CTCs are assumed to have characteristics of cancer stem cells (CSCs) and an epithelial-mesenchymal transition (EMT) phenotype, but extravasation of tumors at distal sites is marked by epithelial features. Two SCLC CTC cell lines, namely BHGc7 and BHGc10, as well as SCLC cell lines derived from primary tumors and metastases were analyzed for the expression of pluripotent stem cell markers and growth factors. Expression of E-cadherin and β-Catenin were determined by flow cytometry. Stem cell-associated markers SOX17, α-fetoprotein, OCT-3/4, KDR, Otx2, GATA-4, Nanog, HCG, TP63 and Goosecoid were not expressed in the 2 CTC lines. In contrast, high expression was found for HNF-3β/FOXA2, SOX2, PDX-1/IPF1 and E-cadherin. E-cadherin expression was restricted to the 2 CTCs and 2 cell lines derived from pleural effusion (SCLC26A) and bone metastases (NCI-H526), respectively. Thus, these SCLC CTCs established from extended disease SCLC patients lack expression of stem cell markers which suppress the epithelial phenotype. Instead they express high levels of E-cadherin consistent with a mesenchymal-epithelial transition (MET or EMrT) and form large tumorospheres possibly in response to the selection pressure of first-line chemotherapy. HNF-3β/FOXA2 and PDX-1/IPF1 expression seem to be related to growth factor dependence on insulin/IGF-1 receptors and IGF-binding proteins.

Second-line therapy for small cell lung cancer: exploring the potential role of circulating tumor cells.

Small cell lung cancer (SCLC) is an aggressive and invasive variant of lung tumors. SCLC, especially in advanced stages, is distinguished by extremely high numbers of circulating tumor cells (CTCs) in comparison to other malignancies. CTCs are operative in tumor spread and are currently enumerated to assess prognosis and response to cytotoxic therapy. Chemosensitivity of SCLC CTCs compared to primary tumors and metastases is not known. Establishment of two SCLC CTC cell lines, namely BHGc7 and BHGc10, allowed the in vitro characterization of their chemosensitivity to the second-line chemotherapeutics topotecan and epirubicin in comparison to a range of SCLC cell lines. The SCLC CTC cell lines exhibited an approximately 7- and 12-fold increased chemosensitivity to epirubicin compared to topotecan, respectively, in in vitro cytotoxicity assays. In comparison to a panel of six SCLC cell lines, the two CTC lines showed a significantly higher chemosensitivity to epirubicin (range, 3- to 16-fold) and topotecan (range, 2.2- to 14.4-fold), respectively. CTC cell lines derived from SCLC patients with recurrent disease exhibit high chemosensitivity to epirubicin vs. topotecan and show considerable more cytotoxicity in response to both compounds in comparison to a panel of SCLC cell lines. Thus, a decrease in the number of CTCs in response to second-line chemotherapy in SCLC patients may overestimate the effect on resident SCLC lesions and metastases.

Small cell lung cancer: Recruitment of macrophages by circulating tumor cells

ABSTRACTTumor-associated macrophages (TAMs) play an important role in tumor progression, suppression of antitumor immunity and dissemination. Blood monocytes infiltrate the tumor region and are primed by local microenvironmental conditions to promote tumor growth and invasion. Although many of the interacting cytokines and factors are known for the tumor-macrophage interactions, the putative contribution of circulating tumor cells (CTCs) is not known so far. These specialized cells are characterized by increased mobility, ability to degrade the extracellular matrix (ECM) and to enter the blood stream and generate secondary lesions which is a leading cause of death for the majority of tumor patients. The first establishment of two permanent CTC lines, namely BHGc7 and 10, from blood samples of advanced stage small cell lung cancer (SCLC) patients allowed us to investigate the CTC-immune cell interaction. Cocultures of peripheral blood mononuclear cells (PBMNCs) with CTCs or addition of CTC-conditioned medium (CTC-CM) in vitro resulted in monocyte-macrophage differentiation and appearance of CD14+, CD163weak and CD68+ macrophages expressing markers of TAMs. Furthermore, we screened the supernatants of CTC-primed macrophages for presence of approximately 100 cytokines and compared the expression with those induced by the local metastatic SCLC26A cell line. Macrophages recruited by SCLC26A-CM showed expression of osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1), IL-8, chitinase3-like 1 (CHI3L1), platelet factor (Pf4), IL-1ra and matrix metalloproteinase-9 (MMP-9) among other minor cytokines/chemokines. In contrast, BHGc7-CM induced marked overexpression of complement factor D (CFD)/adipsin and vitamin D-BP (VDBP), as well as increased secretion of OPN, lipocalin-2 (LCN2), CHI3L1, uPAR, MIP-1 and GDF-15/MIC-1. BHGc10, derived independently from relapsed SCLC, revealed an almost identical pattern with added expression of ENA-78/CXCL5. CMs of the non-tumor HEK293 cell line revealed no induction of macrophages, whereas incubation of PBMNCs with recombinant CHI3L1 gave positive results. Thus, the specific contributions of CTCs in SCLC affect CFD/adipsin, possibly involved in immunity/cachexia, VDBP which gives rise to group-specific component protein-derived macrophage-activating factor (GcMAF), GDF-15/MIC-1 which enhances the malignant phenotype of tumor cells and ENA-78/CXCL5 which attracts angiogenic neutrophils. In conclusion, CTCs are competent to specifically manipulate TAMs to increase invasiveness, angiogenesis, immunosuppression and possibly lipid catabolism.

Cell lines from circulating tumor cells.

Circulating tumor cells (CTCs) in the blood of cancer patients have received increasing attention as new diagnostic tool enabling “liquid biopsies” [1]. The perspective to avoid invasive tissue biopsies in the future and obtain similar or even more information by a “simple” blood test has enormous implications in cancer diagnostics [2]. In contrast to the wealth of descriptive studies demonstrating the clinical relevance of CTCs as biomarker, the extremely low concentration of CTCs in the peripheral blood of most cancer patients challenges further functional studies. A prerequisite for functional analyses was therefore the recent advances in our ability to culture CTCs in vitro or expand the CTC pool in vivo using xenografts. The recent manuscript by Cayrefourcq et al. focused on how to establish a colon CTC line and its key characteristics have been reported [3]. In this study, they applied a negative selection for CTC enrichment on blood samples from 71 patients with metastatic colon cancer and cultured all of the CD45(−) remaining cells in non-adherent culture conditions. The authors have provided the experimental proof that CTCs isolated from the blood of a colon cancer patient are able to give rise to a permanent cell line [3]. To our best knowledge, no other group has published the establishment of a permanent cell CTC line or even transient CTC cultures from patients with colon cancer. One reason is that the frequency of CTCs is lower in peripheral blood of colon cancer patients as compared to breast or prostate cancer patients, making it even more difficult to find and grow CTCs in colon cancer. Besides its capacity to expand ex vivo for more than 2 years, the CTC-MCC-41 line showed (i) epithelial properties with stem-cell like characteristics, (ii) an intermediate epithelial/mesenchymal phenotype, (iii) a potential to induce quickly in vitro angiogenesis, (iv) an osteomimetic signature and (v) tumorigenic properties in SCID mice. Importantly, this CTC line shares the main features of the original primary tumor and lymph nodes metastasis of the colon cancer patient. Recent advances in cell culture technologies have opened a new avenue to develop primary cell cultures or permanent cell lines from CTCs. The first publications on CTC cultures dealt with blood from metastatic breast cancer patients. First, Dario Marchetti's group has established primary cultures from CTCs of breast cancer patients with brain metastases [4]. In EpCAM(−) CTCs, a potential signature of brain metastasis comprising “brain metastasis selected markers (BMSMs)” HER2(+)/EGFR(+)/HPSE(+)/Notch1(+) was identified [4]. CTC lines expressing the BMSM signature were highly invasive and able to generate brain and lung metastases in nude mice. Second, Daniel Haber's group has reported on oligoclonal cultures (sustained in vitro for > 6 months) of CTCs isolated from six patients with metastatic breast cancer [5]. Third, in prostate cancer, Gao et al. used a 3D organoid system and succeeded to develop a long-term culture from the peripheral blood of one patient with castration-resistant metastatic disease and high CTC counts and PSA levels [6]. Fourth, Sunitha Nagrath's group focused on lung cancer and developed a novel in situ capture and culture methodology for ex vivo expansion of CTCs using a 3D co-culture model [7]. Figure 1 Establishment of a colon CTC line CTC lines might be used to test new therapeutic approaches. In this context, novel immunotherapeutic strategies have been developed and antibodies against proteins that block the immune response of T-cells such as PD-L1 or CTLA4 have been approved for treatment of cancer patients. Recently, we could show, for the first time, that PD-L1 is frequently expressed on a variable subset of CTCs obtained from metastatic breast cancer patients [8]. Further functional analysis of this interesting subset might reveal CTC-specific immunosuppressive properties. Taken together, the establishment of functional CTC cell line models is now feasible. These models might be useful to test new cancer drugs specifically targeting CTCs. However, to achieve this important goal it is crucial to increase the efficiency of establishing CTC cell lines and xenografts and extend the current studies to patients at earlier stages. The low concentration of CTCs in earlier stage patients might be a bottleneck to identify “metastasis-initiator cells” that may be resistant as well to the immune system by expressing PD-L1. Thus, increasing the yield of current CTC assays along with the development of more efficient culture strategies seems to be of utmost importance.

Receptor tyrosine kinase expression of circulating tumor cells in small cell lung cancer.

Small cell lung cancer (SCLC) has a poor prognosis and is found disseminated at first presentation in the majority of cases. The cell biological mechanisms underlying metastasis and drug resistance are not clear. SCLC is characterized by high numbers of circulating tumor cells (CTCs) and we were able to expand several CTC lines ex vivo and to relate chitinase-3-like-1/YKL-40 (CHI3L1) as marker. Availability of expanded SCLC CTC cells allowed for a screening of receptor tyrosine kinases (RTKs) expressed. The metastatic CHI3L1-negative SCLC cell line SCLC26A, established from a pleural effusion was used for comparison. The CTC cell line BHGc10 was found to exhibit increased expression of RYK, AXL, Tie-1, Dtk, ROR1/2, several ephrins (Eph) and FGF/EGF receptors compared to SCLC26A. All of these RTKs have been associated with cell motility, invasion and poor prognosis in diverse cancer entities without knowledge of their association with CTCs. The identification of RYK, AXL and ROR1/2 as pseudokinases, lacking activity, seems to be related to the observed failure of RTK inhibitors in SCLC. These kinases are involved in the noncanonical WNT pathway and their expression in SCLC CTCs represents a cancer stem cell-like phenotype.

Metastatic breast cancer cells in lymph nodes increase nodal collagen density.

The most life-threatening aspect of breast cancer is the occurrence of metastatic disease. The tumor draining lymph nodes typically are the first sites of metastasis in breast cancer. Collagen I fibers and the extracellular matrix have been implicated in breast cancer to form avenues for metastasis. In this study, we have investigated extracellular matrix molecules such as collagen I fibers in the lymph nodes of mice bearing orthotopic human breast cancer xenografts. The lymph nodes in mice with metastatic MDA-MB-231 and SUM159 tumor xenografts and tumor xenografts grown from circulating tumor cell lines displayed an increased collagen I density compared to mice with no tumor and mice with non-metastatic T-47D and MCF-7 tumor xenografts. These results suggest that cancer cells that have metastasized to the lymph nodes can modify the extracellular matrix components of these lymph nodes. Clinically, collagen density in the lymph nodes may be a good marker for identifying lymph nodes that have been invaded by breast cancer cells.