Manjima Dhar

Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology

Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells.

High efficiency vortex trapping of circulating tumor cells

Circulating tumor cells (CTCs) are important biomarkers for monitoring tumor dynamics and efficacy of cancer therapy. Several technologies have been demonstrated to isolate CTCs with high efficiency but achieve a low purity from a large background of blood cells. We have previously shown the ability to enrich CTCs with high purity from large volumes of blood through selective capture in microvortices using the Vortex Chip. The device consists of a narrow channel followed by a series of expansion regions called reservoirs. Fast flow in the narrow entry channel gives rise to inertial forces, which direct larger cells into trapping vortices in the reservoirs where they remain circulating in orbits. By studying the entry and stability of particles following entry into reservoirs, we discover that channel cross sectional area plays an important role in controlling the size of trapped particles, not just the orbital trajectories. Using these design modifications, we demonstrate a new device that is able to capture a wider size range of CTCs from clinical samples, uncovering further heterogeneity. This simple biophysical method opens doors for a range of downstream interventions, including genetic analysis, cell culture, and ultimately personalized cancer therapy.

Research highlights: microfluidic point-of-care diagnostics

In this issue we highlight point-of-care (POC) diagnostic technologies to analyze cells, proteins, and small molecules from blood and other body fluids.

Label-free enumeration, collection and downstream cytological and cytogenetic analysis of circulating tumor cells

Circulating tumor cells (CTCs) have a great potential as indicators of metastatic disease that may help physicians improve cancer prognostication, treatment and patient outcomes. Heterogeneous marker expression as well as the complexity of current antibody-based isolation and analysis systems highlights the need for alternative methods. In this work, we use a microfluidic Vortex device that can selectively isolate potential tumor cells from blood independent of cell surface expression. This system was adapted to interface with three protein-marker-free analysis techniques: (i) an in-flow automated image processing system to enumerate cells released, (ii) cytological analysis using Papanicolaou (Pap) staining and (iii) fluorescence in situ hybridization (FISH) targeting the ALK rearrangement. In-flow counting enables a rapid assessment of the cancer-associated large circulating cells in a sample within minutes to determine whether standard downstream assays such as cytological and cytogenetic analyses that are more time consuming and costly are warranted. Using our platform integrated with these workflows, we analyzed 32 non-small cell lung cancer (NSCLC) and 22 breast cancer patient samples, yielding 60 to 100% of the cancer patients with a cell count over the healthy threshold, depending on the detection method used: respectively 77.8% for automated, 60–100% for cytology, and 80% for immunostaining based enumeration.

High-throughput physical phenotyping of cell differentiation

In this report, we present multiparameter deformability cytometry (m-DC), in which we explore a large set of parameters describing the physical phenotypes of pluripotent cells and their derivatives. m-DC utilizes microfluidic inertial focusing and hydrodynamic stretching of single cells in conjunction with high-speed video recording to realize high-throughput characterization of over 20 different cell motion and morphology-derived parameters. Parameters extracted from videos include size, deformability, deformation kinetics, and morphology. We train support vector machines that provide evidence that these additional physical measurements improve classification of induced pluripotent stem cells, mesenchymal stem cells, neural stem cells, and their derivatives compared to size and deformability alone. In addition, we utilize visual interactive stochastic neighbor embedding to visually map the high-dimensional physical phenotypic spaces occupied by these stem cells and their progeny and the pathways traversed during differentiation. This report demonstrates the potential of m-DC for improving understanding of physical differences that arise as cells differentiate and identifying cell subpopulations in a label-free manner. Ultimately, such approaches could broaden our understanding of subtle changes in cell phenotypes and their roles in human biology.

Research highlights: microfluidic single-cell analysis from nucleic acids to proteins to functions

We highlight recent reports using microfluidic systems to perform single-cell analysis. It has been demonstrated on numerous occasions that population averages are often not representative of single-cell behavior. These differences in behavior can be caused by stochastic fluctuations in temporal response, changes in the surrounding instructive environment, or hard-coded genetic changes. Because of the similar length scales, microfluidic approaches have been well-suited to isolating, analyzing, and culturing single-cells to better understand this heterogeneity. Here, we discuss recent works in which microfluidic researchers have extended single-cell characterization approaches, in order to improve analysis from nucleic acids to proteins to final functional behavior. Nucleic acid detection can be amplified beyond what is possible with fluorescence in situ hybridization using droplet-enabled PCR-activated cell sorting. Multiplexed protein detection that overcomes the problems with off-target antibody binding which are associated with traditional immunofluorescence is achieved with single-cell western blotting. Proliferation, migration, and secretion are analyzed in rare circulating tumor cells isolated in microwells. The next steps will be getting these new tools into the hands of a growing number of biologists and developing new tools to report on single-cell epigenetic modifications.

Evaluation of PD-L1 expression on vortex-isolated circulating tumor cells in metastatic lung cancer

Metastatic non-small cell lung cancer (NSCLC) is a highly fatal and immunogenic malignancy. Although the immune system is known to recognize these tumor cells, one mechanism by which NSCLC can evade the immune system is via overexpression of programmed cell death ligand 1 (PD-L1). Recent clinical trials of PD-1 and PD-L1 inhibitors have returned promising clinical responses. Important for personalizing therapy, patients with higher intensity staining for PD-L1 on tumor biopsies responded better. Thus, there has been interest in using PD-L1 tumor expression as a criterion for patient selection. Currently available methods of screening involve invasive tumor biopsy, followed by histological grading of PD-L1 levels. Biopsies have a high risk of complications, and only allow sampling from limited tumor sections, which may not reflect overall tumor heterogeneity. Circulating tumor cell (CTC) PD-L1 levels could aid in screening patients, and could supplement tissue PD-L1 biopsy results by testing PD-L1 expression from disseminated tumor sites. Towards establishing CTCs as a screening tool, we developed a protocol to isolate CTCs at high purity and immunostain for PD-L1. Monitoring of PD-L1 expression on CTCs could be an additional biomarker for precision medicine that may help in determining response to immunotherapies.

Abstract 1582: Isolation of circulating tumor cells and evaluation of PD-L1 expression in metastatic lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA INTRODUCTION Metastatic lung tumors have adopted methods to evade the immune system via overexpression of programmed cell death ligand 1 (PD-L1). PD-L1 binds to PD-1 on T-cells and suppresses its activity. Studies have shown immunotherapy with PD-1 and PD-L1 inhibitors induce clinical response as well as an association with response and tumor PD-L1 level. Thus, tumor PD-L1 expression is a screening criterion for some studies of anti-PD-1 or PD-L1 drugs. Current methods of evaluating PD-L1 expression involve an invasive tumor biopsy. The biopsy itself can be difficult to perform, especially for lung tumors. Biopsies only show one section of the tumor, which may not represent tumor heterogeneity. We show that circulating tumor cells (CTCs) express a wide range of PD-L1 levels, this can aid in screening patients for anti-PD-1/PD-L1 drugs. CTCs slough off from tumors, and can give us a better picture of response that takes into account the heterogeneity at many tumor sites. METHODS We have developed a microfluidic device for rapid, size-based capture of circulating tumor cells (CTCs) from blood. The Vortex HT chip contains parallel channels and rectangular trapping reservoirs. At high flow rate (8mL/min), large cells (>15μm diameter) experience lift forces and become stably trapped in fluid vortices in the reservoirs. Small blood cells do not experience sufficient lift force and can be washed away. We release the trapped cells by lowering the flow rate to dissipate the vortices and collect the cells in a concentrated volume (∼300μL). We use Vortex HT to assess utility of capturing CTCs from patients with non-small cell lung cancer (NSCLC). After collecting the CTCs (30 patient samples), we evaluated the expression of PD-L1 using immunofluorescence with positive controls A549 and H1703, and red blood cells as negative control. We measure cell size, intensity levels of cytokeratin (CK), and CD45 on the collected cells. These metrics allow us to distinguish between CTCs and large leukocytes. Cells larger than 16μm, with high levels of CK, and no CD45 are classified as CTCs. We use a semi-automated algorithm to quantify fluorescence. RESULTS We found significant heterogeneity in PD-L1 expression levels across CTCs from the same patient at one time point. Overall for all samples the PDL1 levels on CTCs ranges from 0 to 4000 total intensity per cell normalized by intensity of PDL1 on H1703. On an individual scale, a sample with 8.5 CTCs/mL had PD-L1 intensities ranging from 300 to 4000. While in another sample with 7 CTCs/ml, the PD-L1 intensities ranged from 0 to 1000. For the CTCs analyzed in these samples, the CD45 levels normalized by CD45 on healthy leukocytes were lower than 0.08 intensity. CONCLUSION Further study can lead to simple, non-invasive methods to predict patient response to immunotherapy. Comparison of CTC PD-L1 levels alongside tumor biopsy results could aid in identifying patients likely to respond to therapy. Citation Format: Manjima Dhar, James Che, Jessica M. Wong, Edward Pao, Victor SH Yu, Melissa Matsumoto, Jonathan Goldman, Edward Garon, Elodie Sollier, Rajan Kulkarni, Dino Di Carlo. Isolation of circulating tumor cells and evaluation of PD-L1 expression in metastatic lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1582. doi:10.1158/1538-7445.AM2015-1582

Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay

Significance The current paradigm in liquid biopsies focuses on circulating tumor cell (CTC) count and genomics to provide clinically actionable information. We push beyond current capabilities to introduce a functional assay that quantifies protease secretion from live CTCs with single-cell resolution. In this assay, an integrated microfluidic device captures CTCs from blood and washes and encapsulates them into nanoliter-scale droplets with fluorogenic substrates within minutes, maintaining physiologic conditions. Our data support the presence of outlier CTCs with high protease activity that may drive metastasis or immune evasion. We reveal an intriguing correlation between CTC protease activity and metastatic progression. Such functional liquid biopsy approaches provide avenues to understand the metastatic process and potential for companion diagnostics. Tumor cells are hypothesized to use proteolytic enzymes to facilitate invasion. Whether circulating tumor cells (CTCs) secrete these enzymes to aid metastasis is unknown. A quantitative and high-throughput approach to assay CTC secretion is needed to address this question. We developed an integrated microfluidic system that concentrates rare cancer cells >100,000-fold from 1 mL of whole blood into ∼50,000 2-nL drops composed of assay reagents within 15 min. The system isolates CTCs by size, exchanges fluid around CTCs to remove contaminants, introduces a matrix metalloprotease (MMP) substrate, and encapsulates CTCs into microdroplets. We found CTCs from prostate cancer patients possessed above baseline levels of MMP activity (1.7- to 200-fold). Activity of CTCs was generally higher than leukocytes from the same patient (average CTC/leukocyte MMP activity ratio, 2.6 ± 1.5). Higher MMP activity of CTCs suggests active proteolytic processes that may facilitate invasion or immune evasion and be relevant phenotypic biomarkers enabling companion diagnostics for anti-MMP therapies.

Abstract B98: Serial evaluation of PD-L1 expression on circulating tumor cells (CTCs)

BACKGROUND: Metastatic non-small cell lung cancer (NSCLC) tumors have adopted methods to evade immune detection and/or clearance. This can occur via overexpression of programmed cell death ligand 1 (PD-L1). Response rate, progression free survival and overall survival with PD-1 inhibitors are greater in tumors with high tumor PD-L1 expression (Garon et al, NEJM 2015, Paz-Ares et al, ASCO 2015). There has been interest in using PD-L1 tumor expression as a treatment selection criterion. Currently available methods of screening involve invasive tumor biopsy followed by histological grading of PD-L1 levels. Biopsies allow sampling from limited sections of the tumor, which may miss heterogeneity. CTC PD-L1 levels could aid in screening patients, and could supplement tissue PD-L1 biopsy results by evaluating a representative sampling from multiple tumor sites which shed cells into the blood. Additionally, following PD-L1 levels on CTCs serially over time may potentially yield information about modulation of tumor PD-L1 expression in the presence of PD-1 or PD-L1 antibodies or other anti-cancer agents. METHODS: We have developed a microfluidic device for rapid, size-based capture of CTCs from blood called Vortex HT chip. The Vortex HT chip utilizes inertial microfluidic flows to isolate CTCs with capture efficiency up to 40% and high purity (>80%). We used the Vortex HT device to capture CTCs from NSCLC patients undergoing PD-1 immunotherapies, both prior to initiating treatment and during treatment. PD-L1 expression was evaluated on CTCs using immunofluorescence staining. CTC number and PD-L1 expression were correlated with treatment response as evaluated by immune related response criteria (irRC) on serial CT scans. We also measured cell size and intensity levels of cytokeratin (CK) and CD45 on the collected cells. We developed a semi-automated algorithm to quantify fluorescence for these different markers on DAPI positive cells collected from each patient sample. We compared these results to PD-L1 expression on the initial tumor biopsy sections, as assayed by immunohistochemical staining and expression quantified with HALO software (Indica Labs). RESULTS: In patients receiving anti-PD-1 antibodies, PD-L1 expression on CTCs could be quantified and compared to a pre-treatment tumor biopsy, as well as to radiographic treatment response. Evaluating patient CTC count and PD-L1 expression at baseline and over time may lead to simple and non-invasive methods to predict response to immunotherapies. As an assay amenable to repeat testing, CTC PD-L1 levels may also be an important pharmacodynamics marker to assess the synergistic potential of combination immunotherapies. Further work is continuing to better understand this predictive biomarker. At the meeting, we will present correlation data between CTC and tumor biopsy PD-L1 expression assays and between the CTC assay and radiographic response to treatment. Citation Format: Jonathan W. Goldman, Manjima Dhar, James Che, Edward B. Garon, Siwen Hu-Lieskovan, Melissa Matsumoto, Brian R. Wolf, James M. Carroll, Matthew J. Crabtree, D. Andrew Tucker, Jennifer Strunck, Elodie Sollier, Rajan Kulkarni, Dino Di Carlo. Serial evaluation of PD-L1 expression on circulating tumor cells (CTCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B98.