Shamileh Fouladdel

High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.

High-Throughput Microfluidic Labyrinth for the Label-free Isolation of Circulating Tumor Cells

We present Labyrinth, a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5xa0mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cellxa0isolation platform to study heterogeneous CTC subpopulations.

Poor Prognosis Indicated by Venous Circulating Tumor Cell Clusters in Early-Stage Lung Cancers

Early detection of metastasis can be aided by circulating tumor cells (CTC), which also show potential to predict early relapse. Because of the limited CTC numbers in peripheral blood in early stages, we investigated CTCs in pulmonary vein blood accessed during surgical resection of tumors. Pulmonary vein (PV) and peripheral vein (Pe) blood specimens from patients with lung cancer were drawn during the perioperative period and assessed for CTC burden using a microfluidic device. From 108 blood samples analyzed from 36 patients, PV had significantly higher number of CTCs compared with preoperative Pe (P < 0.0001) and intraoperative Pe (P < 0.001) blood. CTC clusters with large number of CTCs were observed in 50% of patients, with PV often revealing larger clusters. Long-term surveillance indicated that presence of clusters in preoperative Pe blood predicted a trend toward poor prognosis. Gene expression analysis by RT-qPCR revealed enrichment of p53 signaling and extracellular matrix involvement in PV and Pe samples. Ki67 expression was detected in 62.5% of PV samples and 59.2% of Pe samples, with the majority (72.7%) of patients positive for Ki67 expression in PV having single CTCs as opposed to clusters. Gene ontology analysis revealed enrichment of cell migration and immune-related pathways in CTC clusters, suggesting survival advantage of clusters in circulation. Clusters display characteristics of therapeutic resistance, indicating the aggressive nature of these cells. Thus, CTCs isolated from early stages of lung cancer are predictive of poor prognosis and can be interrogated to determine biomarkers predictive of recurrence. Cancer Res; 77(18); 5194-206. ©2017 AACR.

Heterogeneity of Human Breast Stem and Progenitor Cells as Revealed by Transcriptional Profiling

Summary During development, the mammary gland undergoes extensive remodeling driven by stem cells. Breast cancers are also hierarchically organized and driven by cancer stem cells characterized by CD44+CD24low/− or aldehyde dehydrogenase (ALDH) expression. These markers identify mesenchymal and epithelial populations both capable of tumor initiation. Less is known about these populations in non-cancerous mammary glands. From RNA sequencing, ALDH+ and ALDH−CD44+CD24− human mammary cells have epithelial-like and mesenchymal-like characteristics, respectively, with some co-expressing ALDH+ and CD44+CD24− by flow cytometry. At the single-cell level, these cells have the greatest mammosphere-forming capacity and express high levels of stemness and epithelial-to-mesenchymal transition-associated genes including ID1, SOX2, TWIST1, and ZEB2. We further identify single ALDH+ cells with a hybrid epithelial/mesenchymal phenotype that express genes associated with aggressive triple-negative breast cancers. These results highlight single-cell analyses to characterize tissue heterogeneity, even in marker-enriched populations, and identify genes and pathways that define this heterogeneity.

Selective Photomechanical Detachment and Retrieval of Divided Sister Cells from Enclosed Microfluidics for Downstream Analyses

Considerable evidence suggests that self-renewal and differentiation of cancer stem-like cells, a key cell population in tumorgenesis, can determine the outcome of disease. Though the development of microfluidics has enhanced the study of cellular lineage, it remains challenging to retrieve sister cells separately inside enclosed microfluidics for further analyses. In this work, we developed a photomechanical method to selectively detach and reliably retrieve target cells from enclosed microfluidic chambers. Cells cultured on carbon nanotube-polydimethylsiloxane composite surfaces can be detached using shear force induced through irradiation of a nanosecond-pulsed laser. This retrieval process has been verified to preserve cell viability, membrane proteins, and mRNA expression levels. Using the presented method, we have successfully performed 96-plex single-cell transcriptome analysis on sister cells in order to identify the genes altered during self-renewal and differentiation, demonstrating phenomenal resolution in the study of cellular lineage.

Abstract 3075: Ex-vivo expansion of circulating tumor cells in early lung cancer using a microfluidic model

Circulating tumor cells (CTCs) have gathered much momentum in recent times with emerging micro and nanotechnologies enabling better recoveries of these rare cells. Future research directions in CTCs significantly depend on obtaining meaningful number of CTCs that are available for downstream assays. This can be achieved by expanding CTCs. However, in early stage cancer patients, the numbers of cells that circulate in the peripheral blood are rare. Therefore until now, expanding CTCs remains to be a serious challenge limiting clinical utility. Microfluidic technologies have demonstrated that these rare cells can be recovered with better efficiency, purity and high viability. Herein, we developed a strategy using microfluidic technology to isolate and expand CTCs. We have successfully expanded CTCs isolated from 16 out of 21 early stage lung cancer patients. Expanded CTCs expressed tumor specific markers and formed spheroids in culture. Furthermore, these expanded CTCs carried the identical cancer related mutations (e.g. TP53 mutation) as their primary tumors. We expect that our model opens up the unprecedented avenues to understand biology of these lethal drivers of metastasis offering opportunities for personalized medicine through target drug evaluations. Citation Format: Jennifer Zhuo Zhang, Hiroe Shiratsuchi, Jules Lin, Guoan Chen, Rishindra M. Reddy, Ebrahim Azizi, Shamileh Fouladdel, Andrew C. Chang, Lin Lin, Diane M. Simeone, Max S. Wicha, David G. Beer, Nithya Ramnath, Sunitha Nagrath. Ex-vivo expansion of circulating tumor cells in early lung cancer using a microfluidic model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2014-3075

Characterizing Circulating Tumor Cells Isolated from Metastatic Breast Cancer Patients Using Graphene Oxide Based Microfluidic Assay

The enumeration of circulating tumor cells (CTCs) has shown prognostic importance in patients with breast cancer. However, CTCs are highly heterogeneous with diverse functional properties, which may also be clinically relevant. To provide a comprehensive landscape of the patients disease, further CTC analysis is required. Here, a highly sensitive and reproducible graphene oxide based CTC assay is utilized to isolate and characterize CTCs from 47 metastatic breast cancer patients. The CTCs are captured with high purity, requiring only a few milliliters of blood and enabling efficient enumeration and subsequent analysis at both the protein and the transcription level. The results show that patient clinical outcomes correlate with the associated CTC profile and clearly demonstrate the potential use of the assay in the clinical setting. Collectively, these findings suggest that beyond simple enumeration, CTC characterization may provide further information that improves the diagnosis of the patients disease status for proper treatment decisions. Moreover, this thorough validation study will facilitate the translation of the CTC assay into future clinical applications to broaden the utility of liquid biopsy.

Profiling Heterogeneous Circulating Tumor Cells (CTC) Populations in Pancreatic Cancer Using a Serial Microfluidic CTC Carpet Chip

Although isolation of circulating tumor cells (CTCs) from pancreatic adenocarcinoma patients is feasible, investigating their clinical utility has proven less successful than other cancers due to the limitations of epithelial cellular adhesion molecule (EpCAM)‐only based CTC assays. An integrated technology‐ and biology‐based approach using a microfluidic “Carpet Chip” is presented to study the biological relevance of heterogeneous CTC populations. Both epithelial CTCs (EpCs) and epithelial‐to‐mesenchymal transition (EMT)‐like CTCs (EMTCs) are isolated simultaneously from the whole blood of pancreatic cancer (PaCa) patients (n = 35) by separately targeting two surface markers: EpCAM and CD133. Recovery of cancer cell lines spiked into whole blood is ≥97% with >76% purity. Thirty‐four patients had ≥5 EpCs mL−1 and 35 patients had ≥15 EMTCs mL−1. Overall, significantly higher numbers of EMTCs than EpCs are recovered, reflecting the aggressive nature of PaCa. Furthermore, higher numbers of EMTCs are observed in patients with lymph node involvement compared to patients without. Gene expression profiling of CTCs from 17 patients reveals that CXCR1 is significantly upregulated in EpCs, while known stem cell markers POU5F1/Oct‐4 and MYC are upregulated in EMTCs. In conclusion, successful isolation and genomic profiling of heterogeneous CTC populations are demonstrated, revealing genetic signatures relevant to patient outcomes.

Abstract 1731: Analysis of prostate cancer circulating tumor cells at the mRNA and protein level using a microfluidic graphene oxide chip

While the relative five-year survival rate for men with localized prostate cancer is nearly 100%, it plummets to 28% in the case of distant metastases. This underscores the importance of investigating those cells able to spread and form these metastases: circulating tumor cells (CTCs). Though rare and surrounded by other blood cells, CTCs can be isolated using the sensitive nanomaterial-based microfluidic technology the graphene oxide (GO) Chip. Whole blood samples from 54 prostate cancer patients were obtained with consent and processed on parallel chips for immunofluorescence and RNA analysis. All of the samples were analyzed for CTC enumeration, where captured cells were stained for cytokeratin 7/8 (CK), CD45, and DAPI. CTCs were identified as DAPI+/CK+/CD45- cells. CTCs were detected in all of the samples. Interestingly, 25 samples showed CTC clusters ranging from 2-10 CTCs per cluster. To explore the potential role of EGFR in metastatic progression and CTC survival, a subset of samples was also assayed for EGFR expression with 9/10 samples showing EGFR+ CTCs. RNA extracted from a parallel device was reverse transcribed to cDNA. The cDNA was analyzed via qPCR for expression levels of 96 genes of interest, including housekeeping genes; epithelial and mesenchymal genes; oncogenes and tumor suppressor genes; prostate specific genes; extracellular matrix and inflammatory genes; and others. The results show our ability to examine RNA from CTCs to examine the role of relevant pathways in the cells in transit. With analysis capabilities at both the RNA and protein levels, the GO Chip is an example of a clinically relevant microfluidic technology. Citation Format: Molly Kozminsky, Kathleen C. Day, Guadalupe Lorenzatti Hiles, Shamileh Fouladdel, Ebrahim Azizi, Mark L. Day, Todd Morgan, Sunitha Nagrath. Analysis of prostate cancer circulating tumor cells at the mRNA and protein level using a microfluidic graphene oxide chip [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 1731. doi:10.1158/1538-7445.AM2017-1731

Abstract 939: Single cell mRNA expression profiling reveals heterogeneity of normal and malignant breast stem cell populations

The normal breast contains epithelial populations which are hierarchically organized, a profile recapitulated in breast cancer. At the apex of these hierarchies are stem like cells defined by their capacity to self-renew as well as to generate more differentiated progeny. In breast cancer these cancer stem cells (CSCs) have been shown to be important mediators of tumor metastasis and treatment resistance. Although the phenotypes of normal rodent and human mammary stem cells have been characterized, CSC populations are operationally defined by their capacity for tumor initiation. In addition, putative breast CSC markers such as CD44 + /CD24 low/- and or ALDH + have proven useful in enriching for cells capable of tumor initiation and previous studies have suggested that these markers identify alternative mesenchymal and epithelial stem cell states, respectively. However, it remains unclear whether CSCs exist in discrete states or whether these cells actually represent a continuum reflected in heterogeneity of cell populations defined by expression of these markers. To address this question we utilized single cell mRNA expression profiling of normal and malignant breast stem cells. We first generated a panel of 96 genes representing stem cell and developmental pathways and analyzed expression of these 96 genes utilizing Fluidigm’s C1 and BioMark HD technologies and TaqMan gene expression assays. We also utilized Fluidigm’s Polaris to selectively isolate single cells that were then analyzed by illumina RNA-Seq method. Utilizing these advanced technologies, we demonstrated that both normal and malignant breast stem cells characterized by CD44 + /CD24 low/- and or ALDH + expression are highly heterogeneous. For example, the ALDH isoforms ALDH1A1 and ALDH1A3 were each expressed in some single cells and co-expressed in others. ALDH1A1 expressing single cells appeared to express a unique gene pattern characterized by SLIT/ROBO, PLXNA2, RHOU and HNF1a. These results suggest that at the level of mRNA expression, normal and malignant breast stem cells display a greater heterogeneity than has previously been reported. These results have important implications for breast carcinogenesis as well as for the development of therapeutic strategies designed to target CSCs in breast cancer. Citation Format: Shamileh Fouladdel, Justin Colacino, Ebrahim Azizi, Max S. Wicha. Single cell mRNA expression profiling reveals heterogeneity of normal and malignant breast stem cell populations [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 939. doi:10.1158/1538-7445.AM2017-939