Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

Ferroportin 3 is a dual-targeted mitochondrial/chloroplast iron exporter necessary for iron homeostasis in Arabidopsis.

Mitochondria and chloroplasts are organelles with high iron demand that are particularly susceptible to iron-induced oxidative stress. Despite the necessity of strict iron regulation in these organelles, much remains unknown about mitochondrial and chloroplast iron transport in plants. Here, we propose that Arabidopsis ferroportin 3 (FPN3) is an iron exporter that is dual-targeted to mitochondria and chloroplasts. FPN3 is expressed in shoots, regardless of iron conditions, but its transcripts accumulate under iron deficiency in roots. fpn3 mutants cannot grow as well as the wild type under iron-deficient conditions and their shoot iron levels are lower compared with the wild type. Analyses of iron homeostasis gene expression in fpn3 mutants and inductively coupled plasma mass spectrometry (ICP-MS) measurements show that iron levels in the mitochondria and chloroplasts are increased relative to the wild type, consistent with the proposed role of FPN3 as a mitochondrial/plastid iron exporter. In iron-deficient fpn3 mutants, abnormal mitochondrial ultrastructure was observed, whereas chloroplast ultrastructure was not affected, implying that FPN3 plays a critical role in the mitochondria. Overall, our study suggests that FPN3 is essential for optimal iron homeostasis.

PRC2-mediated H3K27me3 modulates shoot iron homeostasis in Arabidopsis thaliana.

Plants use intricate mechanisms to adapt to changing iron conditions because iron is essential and also one of the most limiting nutrients for plant growth. Furthermore, iron is potentially toxic in excess and must be tightly regulated. Previously, we showed that chromatin remodeling via histone 3 lysine 27 trimethylation (H3K27me3) modulates the expression of FIT-dependent genes under iron deficiency in roots. This study builds on our previous findings, showing that H3K27me3 also modulates iron regulation in shoots. In the clf mutant, which lacks the predominant H3K27 tri-methyltransferase, we detected increased iron translocation to shoots under iron deficiency as compared to wild type. Transcriptomic analysis of shoots also revealed differential expression of genes consistent with higher iron levels in clf shoots than wild type shoots under iron-deficient conditions. In addition, we verify that YSL1 and IMA1, two genes involved in signaling iron status from shoots to roots, are direct targets of H3K27me3 and reveal iron-dependent deposition of H3K27me3 on these loci. This study contributes to a better understanding of the molecular mechanisms behind iron regulation in plants, as the effect of PRC2-mediated H3K27me3 on iron homeostasis genes expressed in the shoots has not been previously reported to our knowledge.

Lossless immunocytochemistry using photo-polymerized hydrogel thin-films.

Immunocytochemistry (ICC), or immunofluorescence microscopy, is an essential biological technique for phenotyping cells in both research and diagnostic applications. Standard ICC methods often do not work well when the cell sample contains a small number of cells (<10 000) because of the significant cell loss that occurs during washing, staining, and centrifugation steps. Cell loss is particularly relevant when working with rare cells, such as circulating tumor cells, where such losses could significantly bias experimental outcomes. In order to eliminate cell loss in ICC protocols, we present a method to encapsulate the cell sample in a photo-polymerized hydrogel thin-film. The hydrogel thin-film is permeable to antibodies and other ICC reagents, thereby allowing the use of standard ICC protocols without modification. The cell sample is physically constrained by the hydrogel at the bottom surface of a standard (unmodified) imaging microtiter plate, thereby enabling the acquisition of high-quality micrographs regardless of the properties of the cell sample or staining reagents. Furthermore, while standard ICC requires several centrifugation steps during staining and washing, our hydrogel encapsulation method requires only a single centrifugation step. This property greatly reduces the time required to perform ICC protocols and is more compatible with robotic platforms. In this study, we show that standard ICC and Cytospin protocols are extremely lossy (>70% loss) when the sample contains less than 10 000 cells, while encapsulating the cells using a permeable hydrogel thin-film results in a lossless ICC process.

PRC2-Mediated H3K27me3 Contributes to Transcriptional Regulation of FIT-Dependent Iron Deficiency Response.

Iron is an essential micronutrient for nearly all organisms, but excessive iron can lead to the formation of cytotoxic reactive oxygen species. Therefore, iron acquisition and homeostasis must be tightly regulated. Plants have evolved complex mechanisms to optimize their use of iron, which is one of the most limiting nutrients in the soil. In particular, transcriptional regulation is vital for regulating iron in plants, and much work has revealed the role of transcription factors on this front. Our study adds novel insights to the transcriptional regulation of iron homeostasis in plants by showing that chromatin remodeling via histone 3 lysine 27 trimethylation (H3K27me3) modulates the expression of FIT-dependent genes under iron deficiency. We provide evidence that FIT-dependent iron acquisition genes, IRT1 and FRO2, as well as FIT itself are direct targets of PRC2-mediated H3K27me3. In the clf mutant, which lacks the predominant H3K27 tri-methyltransferase, induction of FIT, FRO2, IRT1, and other FIT-regulated genes in roots is significantly higher under iron deficient conditions than in wild type. Furthermore, we observe that clf mutants are more tolerant to iron deficiency than wild type, indicating that gene expression levels appear to be limiting the plants ability to access iron. We propose that H3K27me3 attenuates the induction of FIT-target genes under iron deficiency and hypothesize that this may serve as a mechanism to restrict the maximum level of induction of iron acquisition genes to prevent iron overload.

Interactive Mobile Doctor (iMD) to Promote Patient-Provider Discussion on Tobacco Use among Asian American Patients in Primary Care: A Pilot Study.

INTRODUCTION: This study examined the feasibility, acceptability, and efficacy of an interactive "Mobile Doctor" intervention (iMD) for Korean and Vietnamese American men, population groups with high smoking prevalence rates. METHODS: The iMD delivers 5As (Ask, Advise, Assess, Assist, and Arrange) via tailored in-language video messages on a mobile tablet to Korean and Vietnamese male daily smokers right before a health care visit. A single-group trial was conducted with Korean- and Vietnamese-speaking patients at a federally qualified health center. Outcomes were assessed by self-reported surveys obtained postvisit and 3-month follow-up, and by examining electronic health record (EHR) progress notes from 3 consecutive primary care visits to evaluate impacts. RESULTS: Among 47 male daily smokers (87% participation rate), 98% were limited English proficient and 53% had no intent to quit smoking within 6 months. On average, iMD took 12.9 minutes to complete. All participants reported discussing smoking with their providers during the visit, and more than 90% thought iMD was at least somewhat helpful in their decision about quitting and in communicating with their providers. EHR-documented 5As were significantly higher at the iMD visit for Assess (38.3%), Assist (59.6%), and Arrange (36.2%) compared with other visits without iMD. At 3 months, 51% made at least 1 24-hour quit attempt since the intervention. The self-reported 7-day point prevalence abstinence was 19%. CONCLUSIONS: iMD is feasible and acceptable to Korean and Vietnamese male smokers, including those who were not intending to quit smoking. It is a promising tool for increasing patient-provider discussion of tobacco use and possibly smoking cessation among Asian American male smokers.

Diversity of visuomotor reflexes in two Drosophila species.

For adaptive behavior, an organism must identify and assign subjective value to salient sensory information, but what stimuli are salient could change depending upon the local features of the environment. Insects such as fruit flies (Drosophila), for example, rely on olfactory cues to locate food and oviposition sites. But not all Drosophila species find the same stimuli to be salient: for example, four geographically isolated populations of Drosophila mojavensis, which feed and oviposit on necrotic cacti, show olfactory-driven behavioral preferences for host cacti specific to the local environment of each population [1,2]. We wondered whether visual features specific to certain environments could drive divergent visuomotor responses. We compared the visuomotor reflexes of D. melanogaster, a cosmopolitan generalist found in moderately dense visual environments, with D. mojavensis, a cactophilic specialist found in comparatively sparse visual landscapes. We found that, like D. melanogaster, D. mojavensis steer towards long vertical stripes, such as landscape features [3], but in contrast to D. melanogaster's aversion to small objects [3], D. mojavensis find small objects attractive or of neutral value.

Isolation and genome sequencing of individual circulating tumor cells using hydrogel encapsulation and laser capture microdissection.

Circulating tumor cells (CTCs) are malignant cells released into the bloodstream with the potential to form metastases in secondary sites. These cells, acquired non-invasively, represent a sample of highly relevant tumor tissue that is an alternative to difficult and low-yield tumor biopsies. In recent years, there has been growing interest in genomic profiling of CTCs to enable longitudinal monitoring of the tumor's adaptive response to therapy. However, due to their extreme rarity, genotyping CTCs has proved challenging. Relevant mutations can be masked by leukocyte contamination in isolates. Heterogeneity between subpopulations of tumor cells poses an additional obstacle. Recent advances in single-cell sequencing can overcome these limitations but isolation of single CTCs is prone to cell loss and is prohibitively difficult and time consuming. To address these limitations, we developed a single cell sample preparation and genome sequencing pipeline that combines biophysical enrichment and single cell isolation using laser capture microdissection (LCM). A key component of this process is the encapsulation of enriched CTC sample in a hydrogel matrix, which enhances the efficiency of single-cell isolation by LCM, and is compatible with downstream sequencing. We validated this process by sequencing of single CTCs and cell free DNA (cfDNA) from a single patient with castration resistant prostate cancer. Identical mutations were observed in prostate cancer driver genes (TP53, PTEN, FOXA1) in both single CTCs and cfDNA. However, two independently isolated CTCs also had identical missense mutations in the genes for ATR serine/threonine kinase, KMT2C histone methyltransferase, and FANCC DNA damage repair gene. These mutations may be missed by bulk sequencing libraries, whereas single cell sequencing could potentially enable the characterization of key CTC subpopulations that arise during metastasis.

A Novel Ultrasensitive In Situ Hybridization Approach to Detect Short Sequences and Splice Variants with Cellular Resolution.

Investigating the expression of RNAs that differ by short or single nucleotide sequences at a single-cell level in tissue has been limited by the sensitivity and specificity of in situ hybridization (ISH) techniques. Detection of short isoform-specific sequences requires RNA isolation for PCR analysis-an approach that loses the regional and cell-type-specific distribution of isoforms. Having the capability to distinguish the differential expression of RNA variants in tissue is critical because alterations in mRNA splicing and editing, as well as coding single nucleotide polymorphisms, have been associated with numerous cancers, neurological and psychiatric disorders. Here we introduce a novel highly sensitive single-probe colorimetric/fluorescent ISH approach that targets short exon/exon RNA splice junctions using single-pair oligonucleotide probes (~ 50 bp). We use this approach to investigate, with single-cell resolution, the expression of four transcripts encoding the neuregulin (NRG) receptor ErbB4 that differ by alternative splicing of exons encoding two juxtamembrane (JMa/JMb) and two cytoplasmic (CYT-1/CYT-2) domains that alter receptor stability and signaling modes, respectively. By comparing ErbB4 hybridization on sections from wild-type and ErbB4 knockout mice (missing exon 2), we initially demonstrate that single-pair probes provide the sensitivity and specificity to visualize and quantify the differential expression of ErbB4 isoforms. Using cell-type-specific GFP reporter mice, we go on to demonstrate that expression of ErbB4 isoforms differs between neurons and oligodendrocytes, and that this differential expression of ErbB4 isoforms is evolutionarily conserved to humans. This single-pair probe ISH approach, known as BaseScope, could serve as an invaluable diagnostic tool to detect alternative spliced isoforms, and potentially single base polymorphisms, associated with disease.

Novel Junction-specific and Quantifiable In Situ Detection of AR-V7 and its Clinical Correlates in Metastatic Castration-resistant Prostate Cancer.

BACKGROUND: Androgen receptor splice variant 7 (AR-V7) has been implicated in resistance to abiraterone and enzalutamide treatment in men with metastatic castration-resistant prostate cancer (mCRPC). Tissue- or cell-based in situ detection of AR-V7, however, has been limited by lack of specificity. OBJECTIVE: To address current limitations in precision measurement of AR-V7 by developing a novel junction-specific AR-V7 RNA in situ hybridization (RISH) assay compatible with automated quantification. DESIGN, SETTING, AND PARTICIPANTS: We designed a RISH method to visualize single splice junctions in cells and tissue. Using the validated assay for junction-specific detection of the full-length AR (AR-FL) and AR-V7, we generated quantitative data, blinded to clinical data, for 63 prostate tumor biopsies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We evaluated clinical correlates of AR-FL/AR-V7 measurements, including association with prostate-specific antigen progression-free survival (PSA-PFS) and clinical and radiographic progression-free survival (PFS), in a subset of patients starting treatment with abiraterone or enzalutamide following biopsy. RESULTS AND LIMITATIONS: Quantitative AR-FL/AR-V7 data were generated from 56 of the 63 (88.9%) biopsy specimens examined, of which 44 were mCRPC biopsies. Positive AR-V7 signals were detected in 34.1% (15/44) mCRPC specimens, all of which also co-expressed AR-FL. The median AR-V7/AR-FL ratio was 11.9% (range 2.7-30.3%). Positive detection of AR-V7 was correlated with indicators of high disease burden at baseline. Among the 25 CRPC biopsies collected before treatment with abiraterone or enzalutamide, positive AR-V7 detection, but not higher AR-FL, was significantly associated with shorter PSA-PFS (hazard ratio 2.789, 95% confidence interval 1.12-6.95; p=0.0081). CONCLUSIONS: We report for the first time a RISH method for highly specific and quantifiable detection of splice junctions, allowing further characterization of AR-V7 and its clinical significance. PATIENT SUMMARY: Higher AR-V7 levels detected and quantified using a novel method were associated with poorer response to abiraterone or enzalutamide in prostate cancer.

Robust RNA-based in situ mutation detection delineates colorectal cancer subclonal evolution.

Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth. Current genetic ITH analysis methods do not preserve spatial context and may not detect rare subclones. Here, we address these shortfalls by developing and validating BaseScope-a novel mutation-specific RNA in situ hybridization assay. We target common point mutations in the BRAF, KRAS and PIK3CA oncogenes in archival colorectal cancer samples to precisely map the spatial and morphological context of mutant subclones. Computational modeling suggests that subclones must arise sufficiently early, or carry a considerable fitness advantage, to form large or spatially disparate subclones. Examples of putative treatment-resistant cells isolated in small topographical areas are observed. The BaseScope assay represents a significant technical advance for in situ mutation detection that provides new insight into tumor evolution, and could have ramifications for selecting patients for treatment.

In vitro modeling of HIV proviral activity in microglia.

Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll-like receptor-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-κB inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation.

Microfluidic Separation of Circulating Tumor Cells Based on Size and Deformability.

Circulating tumor cells (CTCs) have been implicated as the seeds of cancer metastasis and therefore have the potential to provide significant prognostic and diagnostic values. Here, we describe a procedure for separating CTCs from whole blood based on size and deformability using the microfluidic ratchet device. This device leverages the ratcheting motion of single cells created as they are deformed through funnel-shaped constrictions using oscillatory flow in order to divert cells based on differences in size and deformability. Subsequent methods for CTC identification and enumeration using immunofluorescence after separation are also described.

Quantitative Whole Genome Sequencing of Circulating Tumor Cells Enables Personalized Combination Therapy of Metastatic Cancer.

Much effort has been dedicated to developing circulating tumor cells (CTC) as a noninvasive cancer biopsy, but with limited success as yet. In this study, we combine a method for isolation of highly pure CTCs using immunomagnetic enrichment/fluorescence-activated cell sorting with advanced whole genome sequencing (WGS), based on long fragment read technology, to illustrate the utility of an accurate, comprehensive, phased, and quantitative genomic analysis platform for CTCs. Whole genomes of 34 CTCs from a patient with metastatic breast cancer were analyzed as 3,072 barcoded subgenomic compartments of long DNA. WGS resulted in a read coverage of 23× per cell and an ensemble call rate of >95%. These barcoded reads enabled accurate detection of somatic mutations present in as few as 12% of CTCs. We found in CTCs a total of 2,766 somatic single-nucleotide variants and 543 indels and multi-base substitutions, 23 of which altered amino acid sequences. Another 16,961 somatic single nucleotide variant and 8,408 indels and multi-base substitutions, 77 of which were nonsynonymous, were detected with varying degrees of prevalence across the 34 CTCs. On the basis of our whole genome data of mutations found in all CTCs, we identified driver mutations and the tissue of origin of these cells, suggesting personalized combination therapies beyond the scope of most gene panels. Taken together, our results show how advanced WGS of CTCs can lead to high-resolution analyses of cancers that can reliably guide personalized therapy. Cancer Res; 77(16); 4530-41. ©2017 AACR.

Microfluidic enrichment of circulating tumor cells in patients with clinically localized prostate cancer.

BACKGROUND: Circulating tumor cells (CTC) have become an important tool in the monitoring of patients with advanced prostate cancer (PC). The role of CTC in localized disease has been addressed by only few studies. However, results of CTC analyses are strongly dependent on the platform used for CTC enrichment and detection. In the present study, a microfluidic platform allowing for antigen-independent enrichment of CTC was investigated for its ability to detect CTC in patients with clinically localized PC. PATIENTS AND METHODS: Blood (2ml) was collected preoperatively from 50 consecutive patients undergoing radical prostatectomy for clinically localized PC. CTC were enriched using a microfluidic ratchet mechanism allowing separation of CTC from white blood cells based on differences in size and deformability. Enriched cells were stained for immunofluorescence with antibodies targeting pancytokeratin, epithelial cell adhesion molecule, and CD45. In 21 patients, we performed staining for the androgen receptor. CTC counts were correlated with clinical and pathological parameters using the Wilcoxon-Mann-Whitney test for continuous parameters and Chi-square test for categorical parameters. RESULTS: CTC were detected in 25 (50%) patients. The median number of CTC in CTC-positive patients was 9 CTC/2ml (range: 1-417). Pancytokeratin positive CTC showed expression of androgen the receptor. We observed no correlation between CTC counts and prostate-specific antigen concentration, tumor stage, lymph node stage, or Gleason grade. CONCLUSION: In a representative cohort of patients with clinically localized PC, CTC can be detected in a considerable proportion of patients when using a new microfluidic ratchet mechanism. This encourages further studies assessing the prognostic effect of antigen-independent enriched CTC in patients with PC.

Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues.

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201-2208, 2016. © 2016 Wiley Periodicals, Inc.

Continuous Flow Deformability-Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets.

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label-free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label-free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10(4) -fold enrichment of target cells relative to leukocytes. In patients with metastatic castration-resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization.

EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low.

PURPOSE: The potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes. RESULTS: Cancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups (p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB. MATERIALS AND METHODS: Ten cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB. CONCLUSIONS: EpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.