Single Cell Population Research Articles

The physical origins of transit time measurements for rapid, single cell mechanotyping.

The mechanical phenotype or 'mechanotype' of cells is emerging as a potential biomarker for cell types ranging from pluripotent stem cells to cancer cells. Using a microfluidic device, cell mechanotype can be rapidly analyzed by measuring the time required for cells to deform as they flow through constricted channels. While cells typically exhibit deformation timescales, or transit times, on the order of milliseconds to tens of seconds, transit times can span several orders of magnitude and vary from day to day within a population of single cells; this makes it challenging to characterize different cell samples based on transit time data. Here we investigate how variability in transit time measurements depends on both experimental factors and heterogeneity in physical properties across a population of single cells. We find that simultaneous transit events that occur across neighboring constrictions can alter transit time, but only significantly when more than 65% of channels in the parallel array are occluded. Variability in transit time measurements is also affected by the age of the device following plasma treatment, which could be attributed to changes in channel surface properties. We additionally investigate the role of variability in cell physical properties. Transit time depends on cell size; by binning transit time data for cells of similar diameters, we reduce measurement variability by 20%. To gain further insight into the effects of cell-to-cell differences in physical properties, we fabricate a panel of gel particles and oil droplets with tunable mechanical properties. We demonstrate that particles with homogeneous composition exhibit a marked reduction in transit time variability, suggesting that the width of transit time distributions reflects the degree of heterogeneity in subcellular structure and mechanical properties within a cell population. Our results also provide fundamental insight into the physical underpinnings of transit measurements: transit time depends strongly on particle elastic modulus, and weakly on viscosity and surface tension. Based on our findings, we present a comprehensive methodology for designing, analyzing, and reducing variability in transit time measurements; this should facilitate broader implementation of transit experiments for rapid mechanical phenotyping in basic research and clinical settings.

Robust detection of alternative splicing in a population of single cells

Robust detection of alternative splicing in a population of single cells

The EGFR signaling pathway controls gut progenitor differentiation during planarian regeneration and homeostasis.

The planarian Schmidtea mediterranea maintains and regenerates all its adult tissues through the proliferation and differentiation of a single population of pluripotent adult stem cells (ASCs) called neoblasts. Despite recent advances, the mechanisms regulating ASC differentiation into mature cell types are poorly understood. Here, we show that silencing of the planarian EGF receptor egfr-1 by RNA interference (RNAi) impairs gut progenitor differentiation into mature cells, compromising gut regeneration and maintenance. We identify a new putative EGF ligand, nrg-1, the silencing of which phenocopies the defects observed in egfr-1(RNAi) animals. These findings indicate that egfr-1 and nrg-1 promote gut progenitor differentiation, and are thus essential for normal cell turnover and regeneration in the planarian gut. Our study demonstrates that the EGFR signaling pathway is an important regulator of ASC differentiation in planarians.

Role of granulocyte macrophage colony stimulating factor in regeneration of the central nervous system.

Role of granulocyte macrophage colony stimulating factor in regeneration of the central nervous system.

Factors regulating nuclear factor-kappa B activation in esophageal cancer cells: Role of bile acids and acid.

Gastroesophageal reflux disease is considered to be a major risk in the development of esophageal adenocarcinoma. Nuclear factor-kappa B (NF-κB) plays important roles in the regulation of several genes coding for cytokines, cell proliferation, and apoptosis. To understand the role of bile and acid in the causation of esophageal cancer, we have examined the effects of bile acids and acid on NF-κB activation in the esophageal epithelial cells OE33 and SKGT-4 qualitatively and quantitatively. Analysis of NF-κB activation in esophageal epithelial cells in response to bile acids and acid was performed by electrophoretic mobility shift assay, Western blotting and the translocation NF-κB was assessed by high content analysis (HCA). Cyclooxygenase-2 (COX-2) promoter activity was assessed by transient transfection assays. This study demonstrated that bile acids and acid activated NF-κB in a dose- and time-dependent manner. HCA analysis was an invaluable method in quantifying NF-κB translocation at the single cell population level following bile or acid treatment. Furthermore, deoxycholic acid (DCA) and acid-induced COX-2 promoter activity, and a mutation in the NF-κB and activator protein-1 (AP-1) binding sites remarkably reduced the reporter gene activity induced by DCA or acid. Our data demonstrate that bile and acid induce NF-κB activation in esophageal cells qualitatively and quantitatively. The induction of COX-2 promoter activity by DCA and acid was mediated via NF-κB and AP-1 transcription. The activation of NF-κB signaling pathway in esophageal cells may contribute to the development of esophageal cancer, and, therefore, modulating of NF-κB pathway may uncover new therapeutic strategies.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs).

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

High-Resolution Genomic Profiling of Disseminated Tumor Cells in Prostate Cancer

Circulating tumor cells and disseminated tumor cells (DTCs) are of great interest because they provide a minimally invasive window for assessing aspects of cancer biology, including tumor heterogeneity, a means to discover biomarkers of disease behavior, and a way to identify and prioritize therapeutic targets in the emerging era of precision oncology. However, the rarity of circulating tumor cells and DTCs poses a substantial challenge to the consistent success in analyzing their molecular features, including genomic aberrations. Herein, we describe optimized and robust methods to reproducibly detect genomic copy number alterations in samples of 2 to 40 cells after whole-genome amplification with the use of a high-resolution single-nuclear polymorphism-array platform and refined computational algorithms. We have determined the limit of detection for heterogeneity within a sample as 50% and also demonstrated success in analyzing single cells. We validated the genes in genomic regions that are frequently amplified or deleted by real-time quantitative PCR and nCounter copy number quantification. We further applied these methods to DTCs isolated from individuals with advanced prostate cancer to confirm their highly aberrant nature. We compared copy number alterations of DTCs with matched metastatic tumors isolated from the same individual to gain biological insight. These developments provide high-resolution genomic profiling of single and rare cell populations and should be applicable to a wide-range of sample sources.

Abstract B1-39: Multi-target drug combinations from single drug responses measured at the level of single cells using Mixture Nested Effects Models (MNEMs) applied to cancer

Abstract Purpose: To identify an optimal drug combination for addressing intratumor heterogeneity by measuring single drug responses at the level of single cells. Background: Drug combination strategies have been derived from drug-drug and drug-target interaction inferences made based on genomic network analysis, such as synthetic lethality network analysis and protein network analysis. A key limitation with these approaches is that they do not account for intratumor heterogeneity. A next generation high throughput single cell technology such as Cytometry Time-of-Flight (CyTOF) has the potential to circumvent this limitation by providing the key information for optimally targeting intratumor heterogeneity within the context of personalized medicine. Methods: We propose a systems level modeling framework for analyzing heterogeneous drug response data across a population of single cells. Our approach, called Mixture Nested Effects Models (MNEM), identifies multi-target drug combinations from single drug effects measured at the level of single cells. MNEM analyzes drug responses among subpopulations (aka clusters) of similar cells, where the subpopulations are defined by cellular surface markers. MNEM infers the nested drug target hierarchy (directed network) from a heterogeneous population of drug effects by combining subpopulation identification, subpopulation matching to infer the drug response pre- and post-treatment, and nested effects modeling for each subpopulation individually or through a mixture of subpopulations. An MNEM infers that Drug A targets are a superset of Drug B targets, if the heterogeneous downstream target effects resulting from Drug B are a noisy subset of those resulting from Drug A. This relationship is represented as a directed edge from Drug A to Drug B. From the graphical model of nested drug effects, a scoring function is superimposed to identify the optimal drug combinations. Results: We apply MNEM to CyTOF drug screening data derived from treating three well-established cancer cell lines: HeLa (ovarian), NCIH460 (lung) and HCT116 (colon) with Mek, mTOR, pP38MAPK, JnK I and PI3K inhibitors at different optimal dose levels in addition to the base line treatment TRAIL, an apoptotic stimulator. MNEM indicates that pP38MAPK and Mek inhibitors are important candidates for targeting the ovarian and lung cancer cell lines. The MNEM analysis from the colon cancer cell line data indicates that several optimal drug regimens may have equivalent efficacy. Survival data from drug combination invitro experiments using clonogenic assays support the results from MNEM. Overall, these analyses suggest that MNEM could be used to optimize drug combinations based on responses from single drugs measured at the single cell level. Conclusion: Intratumor heterogeneity in tumor response, measured at the level of single cells, upon exposure to single drugs may be combined through Mixture Nested Effects Modeling (MNEM) to infer optimal drug combinations. Citation Format: Benedict Anchang, Harris Fienberg, Sean Bendall, Robert Tibshirani, Sylvia K. Plevritis. Multi-target drug combinations from single drug responses measured at the level of single cells using Mixture Nested Effects Models (MNEMs) applied to cancer. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-39.

An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR, DNA DAMAGE CHECKPOINT, AND APOPTOSIS

DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.

Immune Cell Phenotyping Using Flow Cytometry.

Fluorescent immunophenotyping uses fluorescently-conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single-cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single-cell suspension (using multiparameter imaging microscopy, imaging cytometry, and/or flow cytometry). Flow cytometry is an optical, laser-based technology which analyzes the physical and fluorescent properties of cells in suspension in real-time as they flow through the instrument. This approach has a number of advantages over other techniques that can be used for characterizing cell populations in single-cell suspensions, in that it can nonsubjectively interrogate up to millions of cells and acquire data on the presence of different cell subpopulations and phenotypical changes within these populations in seconds. This unit describes basic procedures for the direct and indirect immunofluorescent staining of surface and intracellular proteins that are expressed by lymphoid cells which have been isolated from tissues or blood. Protocols for the resolution of dead cells and for the fixation of cells are also included. This unit also provides essential information relating to the selection and titration of antibodies, fluorochrome choice, spectral overlap and compensation, the use of controls, and the standardization of data acquisition and analysis. It also highlights new technologies and platforms that can be used to interrogate the presence of cell subpopulations and their phenotype to an even greater depth.

Digital signaling decouples activation probability and population heterogeneity.

Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity.

Hydrogel biophysical properties instruct coculture-mediated osteogenic potential.

Cell-based approaches for bone formation require instructional cues from the surrounding environment. As an alternative to pharmacological strategies or transplanting single cell populations, one approach is to coimplant populations that can establish a new vasculature and differentiate to bone-forming osteoblasts. Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential and produce numerous angiogenic growth factors. Endothelial colony-forming cells (ECFCs) are a subpopulation of endothelial progenitor cells capable of vasculogenesis in vivo and may provide endogenous cues to support MSC function. We investigated the contribution of the carrier biophysical properties to instruct entrapped human MSCs and ECFCs to simultaneously promote their osteogenic and proangiogenic potential. Compared with gels containing MSCs alone, fibrin gels engineered with increased compressive stiffness simultaneously increased the osteogenic and proangiogenic potential of entrapped cocultured cells. ECFCs produced bone morphogenetic protein-2 (BMP-2), a potent osteoinductive molecule, and increases in BMP-2 secretion correlated with gel stiffness. Coculture of MSCs with ECFCs transduced to knockdown BMP-2 production abrogated the osteogenic response to levels observed with MSCs alone. These results demonstrate that physical properties of engineered hydrogels modulate the function of cocultured cells in the absence of inductive cues, thus increasing the translational potential of coimplantation to speed bone formation and repair.

CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.

Combining cell-cycle checkpoint kinase inhibitors with the DNA-damaging chemotherapeutic agent gemcitabine offers clinical appeal, with a mechanistic rationale based chiefly on abrogation of gemcitabine-induced G2-M checkpoint activation. However, evidence supporting this mechanistic rationale from chemosensitization studies has not been consistent. Here we report a systematic definition of how pancreatic cancer cells harboring mutant p53 respond to this combination therapy, by combining mathematical models with large-scale quantitative biologic analyses of single cells and cell populations. Notably, we uncovered a dynamic range of mechanistic effects at different ratios of gemcitabine and CHK1 inhibitors. Remarkably, effective synergy was attained even where cells exhibited an apparently functional G2-M surveillance mechanism, as exemplified by a lack of both overt premature CDK1 activation and S-phase mitotic entry. Consistent with these findings, S-G2 duration was extended in treated cells, leading to a definable set of lineage-dependent catastrophic fates. At synergistic drug concentrations, global replication stress was a distinct indicator of chemosensitization as characterized molecularly by an accumulation of S-phase cells with high levels of hyperphosphorylated RPA-loaded single-stranded DNA. In a fraction of these cells, persistent genomic damage was observed, including chromosomal fragmentation with a loss of centromeric regions that prevented proper kinetochore-microtubule attachment. Together, our results suggested a "foot-in-the-door" mechanism for drug synergy where cells were destroyed not by frank G2-M phase abrogation but rather by initiating a cumulative genotoxicity that deregulated DNA synthesis.

Elevated dopamine concentration in light-adapted zebrafish retinas is correlated with increased dopamine synthesis and metabolism.

Probing zebrafish (Danio rerio) retinal cryostat sections, collected either 8h into the light or dark cycle, with an antibody against tyrosine hydroxylase (TH) identified a single population of immunopositive cells in the inner retina. However, the observed labeling patterns were not identical in both sets of tissues - label intensity was brighter in light-adapted tissue. This difference was quantified by probing western blots of retinal homogenates with the same TH antibody, which showed that TH expression increased by 42% in light-adapted tissue. High-performance liquid chromatography with electrochemical detection revealed that the concentrations of both dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) are also elevated in light-adapted zebrafish retinal tissue. Dopamine levels increased by 14% and DOPAC levels increased by 25% when measured in retinal homogenates harvested during the light cycle. These results indicate that dopamine levels in zebrafish retina are significantly increased in light-adapted tissue. The increase in dopamine content is correlated with an increase in both TH and DOPAC, suggesting that changes in dopamine concentration are due to light-adaptive changes in the synthesis, release and metabolism of dopamine. Dopamine concentration is elevated in lighted-adapted zebrafish retinas. This increase is correlated with an increase in both tyrosine hydroxylase (TH) and DOPAC (3,4-dihydroxyphenylacetic acid), suggesting that changes in dopamine concentration are due to light-adaptive changes in the synthesis, release and metabolism of dopamine. This is applicable to studies examining retinal mutants, the role of dopamine in disease or visual system development.

Imaging real-time HIV-1 virion fusion with FRET-based biosensors.

We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells. These biosensors show high sensitivity both spatially and temporally, and allow the real-time recovery of HIV-1 fusion kinetics in both single cells and cell populations simultaneously.

Single-cell messenger RNA sequencing reveals rare intestinal cell types.

Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID we then applied the algorithm to ex vivo-isolated Lgr5-positive stem cells and their direct progeny. We find that Lgr5-positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5-positive secretory cells. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs.

Computational biology: How to catch rare cell types.

The development of an algorithm called RaceID enables the identification of rare cell types by single-cell RNA sequencing, even when they are part of a complex mixture of similar cells. See Letter p.251 Identifying and tracking rare cell types of physiological importance in a mixed population is a challenge. This study, a collaboration between the labs of Hans Clevers and Alexander van Oudenaarden, tackles the issue by applying single-cell mRNA sequencing to mouse intestinal organoids. The authors develop an algorithm, called RaceID, that allows rare cell type identification in a complex population of single cells. They define a marker for rare enteroendocrine cells of the intestine. Analysis of cells from a primary mouse intestine using this method, reveals that the Lgr5-expressing population consists of a homogenous stem cell population with a few rare secretory cells, including Paneth cells.

Abstract 2313: Characterizing intratumoral cellular heterogeneity in human glioblastoma xenografts by 30+ parameter single-cell mass cytometry

Abstract Introduction: Glioblastoma tumors contain dramatic intratumoral heterogeneity at the level of cellular phenotypes. High-parameter single-cell platforms such as mass cytometry offer an opportunity to simultaneously measure tumor subpopulations with unexpected combinations of differentiation markers, as well as stromal cells, vasculature, and infiltrating immune cells. Methods: A subcutaneous patient-derived xenograft from an adult with glioblastoma was enzymatically digested into a single-cell suspension. Half of the cells were treated with epidermal growth factor (EGF) at 20 ng/mL for 15 minutes. Both samples were then stained with a 32-marker antibody panel and measured by single-cell mass cytometry. The antibody panel included surface-expressed markers of stemness and differentiation, as well as intracellular markers to monitor cell cycle, apoptosis, and growth factor signaling. Unsupervised spectral clustering was used to segregate >200,000 tumor cells into 13 tumor cell subsets. Inducible signaling phenotypes were quantified in these subsets by comparing binding of phospho-specific antibodies in the stimulated sample, relative to the unstimulated control. Results: Many previously reported markers of cancer stem cells were detectable, but they did not agree on a single population of cells. Although the CD133+ fraction was rare (<5% of cells), it contained three distinct subpopulations of phenotypically distinct cells. The most informative markers for discriminating the tumor subsets were markers of stemness (i.e. CD133, CD44, Sox2) or integrin expression (i.e. CD49e, CD49f, CD51). The primitive cells (i.e. Sox2+) exhibited higher basal kinase activity across multiple pathways, including markers of cell division (i.e. Histone H3 pSer28). Short-term stimulation with EGF induced distinct responses in each of the different CD133+ subsets. Conclusions: Single-cell phenotyping by mass cytometry produced rich developmental and functional profiles of glioblastoma cell subsets. Subpopulations of primitive (CD133+) cells can exist in a single glioblastoma tumor and exhibit distinct signaling responses to external stimulation. Altogether, these results validate the use of this platform for investigation of the intratumoral cellular heterogeneity of solid neural tumors with this platform. Work is underway to identify correlations between intratumoral cellular phenotypes and upstream driver mutations in a larger cohort. Citation Format: Erin F. Simonds, Garry P. Nolan, William A. Weiss. Characterizing intratumoral cellular heterogeneity in human glioblastoma xenografts by 30+ parameter single-cell mass cytometry. [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 2313. doi:10.1158/1538-7445.AM2015-2313

Abstract 4880: Characterization of genetic concordance between primary tumor cells, circulating tumor cells, and metastatic tumor cells from patients with prostate cancer

Abstract Circulating tumor cells (CTCs) which disseminate from the primary tumor site have been found to be a major source of metastatic tumor. However, CTCs are heterogeneous and most CTC DNA is fragmented due to apoptosis. To better understand the molecular pathogenesis of metastasis, it is imperative to obtain single, non-apoptotic CTCs and investigate the genetic concordance between primary tumor cells, CTCs and metastatic tumor cells. Due to the abundance of normal cells present in the blood and in tumor tissue, isolation of single tumor cells or pure cell populations of these cells is extremely difficult. The DEPArray platform is designated to collect single CTCs after Cell-Search enrichment, and to sort pure intact tumor cells from formalin-fixed, paraffin-embedded (FFPE) tissues. Taking advantage of this technology, we have isolated single CTCs from prostate cancer patients. Whole genome amplification was performed and genomes with high integrity were used to evaluate the hotspot mutations in PIK3CA. For comparison, we also made single cell suspensions from FFPE tissue blocks of primary tumors and metastatic tumor in lymph nodes from the same patients. Immunofluorescence staining for cytokeratin (CK) and vimentin (Vim) was performed, followed by isolation of pure tumor cells (CK+), stromal cells (Vim+) and double-positive cells (CK+/Vim+) from both primary tumor and lymph node samples. We plan to do genetic analysis on these cells and compare the results to determine the genetic concordance between primary tumor cells, CTCs and metastatic tumor cells. By using the DEPArray, we have successfully isolated single CTCs, and populations of tumor cells, stromal cells and double-positive cells from primary and metastatic tumors. We will present new data on the genetic concordance between these cells, which will provide new insight into our understanding of metastasis in prostate cancer. Citation Format: Lixin Yang, Linling Chen, Yafan Wang, Jeremy Jones, Yun Yen, Sofia Loera, Raju Pillai, Peiguo Chu, Dennis Weisenburger. Characterization of genetic concordance between primary tumor cells, circulating tumor cells, and metastatic tumor cells from patients with prostate 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 4880. doi:10.1158/1538-7445.AM2015-4880

Phenotypic diversity of patient-derived melanoma populations in stem cell medium

Melanomas are highly heterogeneous tumors and there is no treatment effective at achieving long-term remission for metastatic melanoma patients. Thus, an appropriate model system for studying melanoma biology and response to drugs is necessary. It has been shown that composition of the medium is a critical factor in preserving the complexity of the tumor in in vitro settings, and melanospheres maintained in stem cell medium are a good model in this respect. In the present study, we observed that not all nodular melanoma patient-derived cell populations grown in stem cell medium were capable of forming melanospheres, and cell aggregates and anchorage-independent single-cell cultures emerged instead. Self-renewing capacity and unlimited growth potential indicated the presence of cells with stem-like properties in all patient-derived populations but immunophenotype and MITF expression exhibited variability. Enhanced MITF expression and activity was observed in melanospheres in comparison with cell aggregates and single-cell culture, and hypoxic-like conditions that increased the ability of single-cell population to form melanospheres enhanced MITF expression and cell pigmentation as well. Thus, MITF seems to be a critical transcription factor for formation of both patient-derived and hypoxia-induced melanospheres. After 2 years of continuous culturing, melanospheres progressively underwent transition into cell aggregates that was accompanied by changes in expression of several MITF-dependent genes associated with melanogenesis and survival and alterations in the composition of subpopulations but not in the frequency of ABCB5-positive cells. Several biological properties of parent tumor are well preserved in patient-derived melanospheres, but during prolonged culturing the heterogeneity is substantially lost when the melanospheres are substituted by cell aggregates. This should be considered when cell aggregates instead of melanospheres are used in the study of melanoma biology and cell response to drugs.