Small Cell Colonies Research Articles

Evaluation of the removal of cyanobacteria and cyanotoxins with a composite hydrogel based on chemically modified gelatin and PVA-containing graphene oxide nanoparticles

After being applied to pharmaceutics removal from water, the newly synthesized composite hydrogel based on chemically modified gelatin and PVA-containing graphene nanoparticles (CHGP-GOn), was now assessed for another application. The hydrogels were able to interact with Microcystis aeruginosa cells, resulting in the formation of small cell colonies and cell lysis related to exposure time. In the removal tests, the best general removal efficiency of cyanobacterial cells was achieved with the highest adsorbent mass at natural pH, achieving values of removal of 90 % for cells, 75 % chlorophyll-a, 63 % and 43 % for turbidity and visible colour removals, respectively. From the kinetic study, the results showed that cell inactivation has achieved removal equilibrium in 19 h, with a qe of 106 × 106 cells/g of CHGP-GOn. Furthermore, with the results from the other parameters, the tests presented a removal equilibrium of just 14 h, there was a removal of 4670 µg/L of Chlorophyll-a and 6450 µg/L/g of MC-LR microcystin per gram of adsorbent. The experimental data best fitted to the Elovich model, indicating possible removal by chemisorption. Analysis of cellular integrity and morphology showed that within just 12 h, few cyanobacterial cells showed membrane disruption and release of intracellular toxins, with an increase in the toxicity medium related to extended exposure time, and presentation of morphological and superficial cellular damage within 24 h. In addition, other characteristic measurements presented an exceptional mechanical strength and resistance that was slightly reduced after swelling. However, showing good development during adsorption tests under agitation, without any detachment of material and an absence of GO leaching. Therefore, it can be concluded that the synthesized hydrogel should be applied in removing M. aeruginosa cells and their toxins from water treatment, serving as an excellent alternative to traditional adsorbents.

Mechanobiology Platform Realized Using Photomechanical Mxene Nanocomposites: Bilayer Photoactuator Design and In Vitro Mechanical Forces Stimulation.

Mechanotransduction is the process by which cells convert external forces and physical constraints into biochemical signals that control several aspects of cellular behavior. A number of approaches have been proposed to investigate the mechanisms of mechanotransduction; however, it remains a great challenge to develop a platform for dynamic multivariate mechanical stimulation of single cells and small colonies of cells. In this study, we combined polydimethylsiloxane (PDMS) and PDMS/Mxene nanoplatelets (MNPs) to construct a soft bilayer nanocomposite for extracellular mechanical stimulation. Fast backlash actuation of the bilayer as a result of near-infrared irradiation caused mechanical force stimulation of cells in a controllable manner. The excellent controllability of the light intensity and frequency allowed backlash bending acceleration and frequency to be manipulated. As gastric gland carcinoma cell line MKN-45 was the research subject, mechanical force loading conditions could trigger apoptosis of the cells in a stimulation duration time-dependent manner. Cell apoptotic rates were positively related to the duration time. In the case of 6 min mechanical force loading, apoptotic cell percentage rose to 34.46% from 5.5% of the control. This approach helps apply extracellular mechanical forces, even with predesigned loading cycles, and provides a solution to study cell mechanotransduction in complex force conditions. It is also a promising therapeutic technique for combining physical therapy and biomechanics.

Endothelialization of an ePTFE vessel prosthesis modified with an antithrombogenic fibrin/heparin coating enriched with bound growth factors.

Early and late thrombosis remain the most frequent reasons for the failure of synthetic cardiovascular grafts. Long-term hemocompatibility of implanted synthetic grafts can be achieved if a natural living endothelium is formed over its blood-contacting surface. Here we present a modification of a standard expanded polytetrafluorethylene (ePTFE) vessel prosthesis by a controlled preparation of a fibrin mesh enriched with covalently bound heparin and noncovalently bound vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Compared to a bare prosthesis, the coated prosthesis showed excellent antithrombogenic properties after contact with heparinized fresh human blood. Human umbilical vein endothelial cells seeded on the inner surface of the coated prosthesis formed a confluent layer in 5 days, whereas only small colonies of cells were scattered on the bare prosthesis. Viability of the cells was promoted mainly by FGF immobilized on the coating. These findings suggest that the coating may prevent acute thrombus formation and support the self-endothelialization of an implanted ePTFE vascular graft in vivo.

Silk Fibroin Promotes the Regeneration of Pancreatic β-Cells in the C57BL/KsJ-Leprdb/db Mouse.

Diabetes mellitus is a chronic metabolic disease, and its progression leads to serious complications. Although various novel therapeutic approaches for diabetes mellitus have developed in the last three decades, its prevalence has been rising more rapidly worldwide. Silk-related materials have been used as anti-diabetic remedies in Oriental medicine and many studies have shown the effects of silk fibroin (SF) in both in vitro and in vivo models. In our previous works, we reported that hydrolyzed SF improved the survival of HIT-T15 cells under high glucose conditions and ameliorated diabetic dyslipidemia in a mouse model. However, we could not provide a precise molecular mechanism. To further evaluate the functions of hydrolyzed SF on the pancreatic β-cell, we investigated the effects of hydrolyzed SF on the pancreatic β-cell proliferation and regeneration in the mouse model. Hydrolyzed SF induced the expression of the proliferating cell nuclear antigen (PCNA) and reduced the apoptotic cell population in the pancreatic islets. Hydrolyzed SF treatment not only induced the expression of transcription factors involved in the pancreatic β-cell regeneration in RT-PCR results but also increased neurogenin3 and Neuro D protein levels in the pancreas of those in the group treated with hydrolyzed SF. In line with this, hydrolyzed SF treatment generated insulin mRNA expressing small cell colonies in the pancreas. Therefore, our results suggest that the administration of hydrolyzed SF increases the pancreatic β-cell proliferation and regeneration in C57BL/KsJ-Leprdb/db mice.

Controllable actuation of photomechanical bilayer nanocomposites for in vitro cell manipulation

Mechanotransduction enables cells to translate external forces and physical constraints into biochemical signals controlling multiple aspects of cell behavior. Methodologies mimicking various mechanical and biomedical forces exposed to cells, have been proposed to investigate cell mechanotransduction. However, it is still a great challenge developing platform towards dynamically multivariate mechanical stimulation addressing individual cells or small colonies of cells. A photomechanical and biocompatible platform for extracellular mechanical stimuli is constructed by soft bilayer nanocomposites composed of polydimethylsiloxane (PDMS) and PDMS/GNPs (graphene nanoplatelets). The fast backlash bending process can give rise to mechanical stimuli to the cells growing on it in a controllable manner by near infrared (nIR) irradiation. Beneficial from the excellent controllability in nIR light intensity and working frequency, the backlash bending velocity, acceleration, frequency, as well as sophisticated bending process can be well controlled, thus extracellular mechanical forces even predesigned loading cycles can be realized. SGC-7901 cells viability, one of the lines in highest incidence Gastric cancer in almost Asian countries, can be restricted by our proposed platform. It will not only provide a solution for studying cell mechanotransduction with more complicated force conditions, but also demonstrate a promising methodology supplementing the physical therapy for fighting against the malignant tumor.

Perspectives on Adjuvant Therapy in Renal Cell Carcinoma

Perspectives on Adjuvant Therapy in Renal Cell Carcinoma

A Miniature Cell Pattern Formation of Ovarian Cancer Cell Lines on Self-Assembling Peptide Nanofiber-Coated Coverslip and In Vitro Chemosensitivity Assay.

Despite of intensive efforts, the tumor progression of ovarian cancer has not yet been fully understood. Various studies suggest that an effective experimental model system In Vitro to mimic microenvironment in vivo and the fabrication of cell pattern formation In Vitro are of great importance for the ovarian cancer research. This study cultivates SKOV3, A2780 and A2780/DDP ovarian cancer cell lines in RADA16-I peptide hydrogel to obtain 3D-precultured cancer cells and fabricate a type of miniature cell pattern formation In Vitro on RADA16-I peptide nanofibercoated coverslip by in situ AFM analysis and chemosensitivity assay in vitro. SKOV3, A2780 and A2780/DDP cells were regularly cultivated in RADA16-I peptide hydrogel, respectively, and present defined cellular characteristics, such as small cell colonies, cell clusters and cell aggregates, etc. On coverslip coated with RADA16-I peptide nanofiber, three 3D-precultured cell lines were flat and spread on coverslips and the regular topographic images by AFM were obtained. The chemosensitivity assay of three ovarian cancer cell lines were significantly different in RADA16-I peptide-coated coverslips compared with 2D dish plates. By 3D cell culture of ovarian cancer cell lines in RADA16-I peptide hydrogel In Vitro, the cell culture model systems was fabricated. The miniature cell pattern formation In Vitro on peptide nanofiber-coated coverslip was particularly useful experiemental model systems to be served as a valuable methodology for studying ovarian cancer biology.

Simple agarose micro-confinement array and machine-learning-based classification for analyzing the patterned differentiation of mesenchymal stem cells

The geometrical confinement of small cell colonies gives differential cues to cells sitting at the periphery versus the core. To utilize this effect, for example to create spatially graded differentiation patterns of human mesenchymal stem cells (hMSCs) in vitro or to investigate underpinning mechanisms, the confinement needs to be robust for extended time periods. To create highly repeatable micro-fabricated structures for cellular patterning and high-throughput data mining, we employed here a simple casting method to fabricate more than 800 adhesive patches confined by agarose micro-walls. In addition, a machine learning based image processing software was developed (open code) to detect the differentiation patterns of the population of hMSCs automatically. Utilizing the agarose walls, the circular patterns of hMSCs were successfully maintained throughout 15 days of cell culture. After staining lipid droplets and alkaline phosphatase as the markers of adipogenic and osteogenic differentiation, respectively, the mega-pixels of RGB color images of hMSCs were processed by the software on a laptop PC within several minutes. The image analysis successfully showed that hMSCs sitting on the more central versus peripheral sections of the adhesive circles showed adipogenic versus osteogenic differentiation as reported previously, indicating the compatibility of patterned agarose walls to conventional microcontact printing. In addition, we found a considerable fraction of undifferentiated cells which are preferentially located at the peripheral part of the adhesive circles, even in differentiation-inducing culture media. In this study, we thus successfully demonstrated a simple framework for analyzing the patterned differentiation of hMSCs in confined microenvironments, which has a range of applications in biology, including stem cell biology.

Palatal periosteum derived mesenchymal stem cells cultured in serum and xeno-free medium

Background: Mesenchymal stem cells (MSC) from palatal periosteum (PP) can be a reliable alternative for tissue engineering applications since it can be easily harvested, healing time is faster with significantly less patient discomfort and risk of complications than invasive bone marrow or fat harvesting procedures. Use of serum and xeno-free (SXF) medium is essential to expand cells for clinical applications. Objectives: To determine the location of MSC within the human PP, their frequency and ability to differentiate into mesenchymal lineage cells when cultured in vitro in SXF medium. Methods: Human PP (n = 3), obtained from patients undergoing surgical exposure of palatally impacted canines, was double-immunostained with MSC-specific antibodies against CD105, CD90 and CD73. Extracted cells were cultured on SXF Essential 8™ medium and analysed for MSC and haematopoietic stem cell (HSC) markers (CD45, CD34, CD11b, CD19, HLADR) using flow cytometry and their multipotency determined in vitro. Findings: MSC were detected within the PP tissue as single or small colonies of cells in close proximity to blood vessels. Flow cytometry analysis revealed that 79.93 ± 1.25% were positive for MSC markers with only 0.46 ± 0.33% positive for the HSC markers. MSC cultured in osteogenic medium were positive for nodule formation, alkaline phosphatase, Alizarin red S, and Von Kossa staining whereas Alcian blue positive micromass cultures and Oil Red O positive vesicles were evident when cultured in chondrogenic and adipogenic medium respectively. Conclusion: This study showed that PP contains MSC which can be easily harvested and successfully expanded in SXF medium suitable for human tissue engineering applications.

Single-Cell Motility Analysis of Stimulated Diatoms using a Microchamber

Motility of diatom cells that were stimulated by irradiation with controlled visible lights were studied using a microchamber. Isolated Navicula sp. cells were deposited in a microchamber made of poly(dimethylsiloxane) in order to realize a long period observation. In the first experiment, the diatom cells were continuously observed for 60 min by an optical microscope. If the microchamber does not appropriately work, the 60 min observation should be difficult, because the cells were moved outside the observation area. In the first 20 min, the cells were irradiated by a halogen lamp. Then, the light shielded by a cut-off filter (cut-off wavelength: 550 nm) irradiated to the sample for the next 20 min. In the final 20 min, the sample was irradiated without the filter again. Brightness was kept at 500 lux for 60 min by a dimming filter to avoid effects of brightness. Then, movements of 17 individual cells were analyzed by a two-dimensional trajectory analysis software. As a result, the velocities of 12 cells were decreased while the light was shielded by the filter (20-40 min). T-test results also suggested that the velocity was significantly decreased. In the second experiments, we prepared small colonies of diatom cells in a microchamber with 12 days incubation. Then, the colonies were irradiated with a mercury lamp with three different cut filters for 100 sec period. In this experiment, we focused on inactive (stopped) cells. The cells were found to start to move after the irradiation. However, after stopping the irradiation, the velocities of the activated cells were decreased gradually. In addition, the decrease was affected by the types of the filters. Our data revealed a possibility of detailed analysis of diatom motility using the microchamber system.

Abstract 1572: Overexpression of Axl delays metastatic outgrowth of prostate cancer bone marrow disseminated tumor cells

Abstract The presence of detectable metastases in the bone marrow of prostate cancer patients represents lethal, incurable disease. These metastases that may emerge years or decades after prostatectomy are derived from small colonies of dormant tumor cells that disseminated from the primary tumor. Our group has previously shown that Gas6/Axl signaling is associated with disseminated tumor cell (DTC) dormancy in mouse models of prostate cancer metastasis. In this study we explored the functional significance of this association and tested the hypothesis that overexpression of Axl in prostate cancer cells would prolong their dormant phase in the bone marrow. Axl overexpression in the AR-positive Axl-negative human prostate cancer cell line C42B slowed proliferation in vitro. Select cells were completely dormant as detected by the lack of EdU incorporation over 40 hours, and this fraction was increased when co-cultured with bone-forming osteoblasts. To assess the functional effects of Axl on prostate cancer progression in vivo, we utilized a mouse model of prostate cancer metastasis in which subcutaneous tumors are established and surgically removed upon reaching 200mm3. At 10 weeks post-tumor removal, mice bearing C42B-Axl tumors did not have overt osteoblastic or osteolytic lesions in their femurs or tibiae, whereas mice bearing C42B-WT tumors had visible lesions by x-ray. Immunohistochemistry staining of mouse femurs and tibiae corroborated these findings. To confirm that the absence of metastases in mice with C42B-Axl tumors was not due to the inability of the cells to disseminate, we used immunofluorescence to detect human cells in the bone marrow of flushed femurs and tibiae. At 12 weeks post-tumor removal, fewer prostate cancer cells were detected in the bone marrow of mice with C42B-Axl tumors compared to those with C42B-WT tumors. Notably however, both groups had detectable prostate cancer cells in the bone marrow, indicating that the absence of C42B-Axl metastasis was not due to failure of primary tumor cells to disseminate. The presence of disseminated C42B-Axl cells in the bone marrow coupled with their failure to develop into clinical metastases at the time of C42B-WT metastatic outgrowth establishes a functional role for the association of Axl expression with DTC dormancy. Axl may be sufficient to induce and maintain dormancy, and ongoing experiments will address the requirement of Axl in this setting. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Kenneth J. Pienta. Overexpression of Axl delays metastatic outgrowth of prostate cancer bone marrow disseminated tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1572.

Microelectromechanical System-Based Sensing Arrays for Comparative in Vitro Nanotoxicity Assessment at Single Cell and Small Cell-Population Using Electrochemical Impedance Spectroscopy.

The traditional in vitro nanotoxicity assessment approaches are conducted on a monolayer of cell culture. However, to study a cell response without interference from the neighbor cells, a single cell study is necessary; especially in cases of neuronal, cancerous, and stem cells, wherein an individual cell's fate is often not explained by the whole cell population. Nonetheless, a single cell does not mimic the actual in vivo environment and lacks important information regarding cell communication with its microenvironment. Both a single cell and a cell population provide important and complementary information about cells' behaviors. In this research, we explored nanotoxicity assessment on a single cell and a small cell population using electrochemical impedance spectroscopy and a microelectromechanical system (MEMS) device. We demonstrated a controlled capture of PC12 cells in different-sized microwells (to capture a different number of cells) using a combined method of surface functionalization and dielectrophoresis. The present approach provides a rapid nanotoxicity response as compared to other conventional approaches. This is the first study, to our knowledge, which demonstrates a comparative response of a single cell and small cell colonies on the same MEMS platform, when exposed to metaloxide nanoparticles. We demonstrated that the microenvironment of a cell is also accountable for cells' behaviors and their responses to nanomaterials. The results of this experimental study open up a new hypothesis to be tested for identifying the role of cell communication in spreading toxicity in a cell population.

Highly sensitive detection of cancer cells using femtosecond dual-wavelength near-IR two-photon imaging

We describe novel imaging protocols that allow detection of small cancer cell colonies deep inside tissue phantoms with high sensitivity and specificity. We compare fluorescence excited in Styryl-9M molecules by femtosecond pulses at near IR wavelengths, where Styryl-9M shows the largest dependence of the two-photon absorption (2PA) cross section on the local environment. We show that by calculating the normalized ratio of the two-photon excited fluorescence (2PEF) intensity at 1200 nm and 1100 nm excitation wavelengths we can achieve high sensitivity and specificity for determining the location of cancer cells surrounded by normal cells. The 2PEF results showed a positive correlation with the levels of MDR1 proteins expressed by the cells, and, for high MDR1 expressors, as few as ten cancer cells could be detected. Similar high sensitivity is also demonstrated for tumor colonies induced in mouse external ears. This technique could be useful in early cancer detection, and, perhaps, also in monitoring dormant cancer deposits.

Biosensors for immune cell analysis-A perspective.

Massively parallel analysis of single immune cells or small immune cell colonies for disease detection, drug screening, and antibody production represents a "killer app" for the rapidly maturing microfabrication and microfluidic technologies. In our view, microfabricated solid-phase and flow cytometry platforms of the future will be complete with biosensors and electrical/mechanical/optical actuators and will enable multi-parametric analysis of cell function, real-time detection of secreted signals, and facile retrieval of cells deemed interesting.

Evanescent photosynthesis: exciting cyanobacteria in a surface-confined light field

The conversion of solar energy to chemical energy useful for maintaining cellular function in photosynthetic algae and cyanobacteria relies critically on light delivery to the microorganisms. Conventional direct irradiation of a bulk suspension leads to non-uniform light distribution within a strongly absorbing culture, and related inefficiencies. The study of small colonies of cells in controlled microenvironments would benefit from control over wavelength, intensity, and location of light energy on the scale of the microorganism. Here we demonstrate that the evanescent light field, confined near the surface of a waveguide, can be used to direct light into cyanobacteria and successfully drive photosynthesis. The method is enabled by the synergy between the penetration depth of the evanescent field and the size of the photosynthetic bacterium, both on the order of micrometres. Wild type Synechococcus elongatus (ATCC 33912) cells are exposed to evanescent light generated through total internal reflection of red (λ = 633 nm) light on a prism surface. Growth onset is consistently observed at intensity levels of 79 ± 10 W m(-2), as measured 1 μm from the surface, and 60 ± 8 W m(-2) as measured by a 5 μm depthwise average. These threshold values agree well with control experiments and literature values based on direct irradiation with daylight. In contrast, negligible growth is observed with evanescent light penetration depths less than the minor dimension of the rod-like bacterium (achieved at larger light incident angles). Collectively these results indicate that evanescent light waves can be used to tailor and direct light into cyanobacteria, driving photosynthesis.

118 INDUCING PLURIPOTENCY IN SOMATIC CELLS FROM THE SNOW LEOPARD (PANTHERA UNCIA), AN ENDANGERED FELID

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMX) encoding either 4 (Oct-4, Sox-2, Klf-4 and cMyc) or 5 (Oct-4, Sox-2, Klf-4, cMyc and Nanog) human transcription factors. Transduction efficiency of the retrovirus was ascertained using pMX-green fluorescent protein transgene expression and averaged 96% from 3 repeated experiments. The reprogramming efficiency of initial colony formation was 0.000308% (37/120 000 cells plated) for 4-factor induction compared with 0.000517% (62/120 000) for 5-factor induction. Transduction with 4 factors resulted in the formation of small colonies of cells, which could not be maintained for more than 4 passages (P4). However, addition of Nanog to the transfection cocktail produced stable iPS cell colonies, which formed as early as Day 3. Colonies of cells were selected at Day 5 and expanded in vitro on mouse embryonic fibroblast feeder cells. The resulting cell line was positive for alkaline phosphatase, Oct-4, Nanog and stage-specific embryonic antigen-4 at P14. Also, RT-PCR confirmed that endogenous Oct-4 and Nanog were expressed by snow leopard iPS cells from P4; although all 5 human transgenes were transcribed at P4, Oct-4, Sox-2 and Nanog transgenes were silenced as early as P14, suggesting that reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the 3 germ layers. This study describes the first derivation of iPS cells from the endangered snow leopard and is also the first report on induced pluripotency in felid species. Our results demonstrate that addition of Nanog to the reprogramming cocktail was essential for derivation of iPS lines in this felid and that iPS cells provide a unique source of pluripotent cells with utility in conservation for cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

Uveal Melanoma Cell Lines Contain Stem-Like Cells That Self-Renew, Produce Differentiated Progeny, and Survive Chemotherapy

Uveal melanoma (UM) cells in high-metastatic risk tumors have an undifferentiated molecular signature indicative of a more primitive cellular phenotype. Given mounting evidence for the existence of cancer stem cells (CSC), the authors investigated whether UM cell lines retain a population of self-renewing tumorigenic cells. Single-cell cloning and spheroid culture studies were used to study the presence of a CSC-like population in two cell lines derived from the primary tumor (Mel270) and metastatic liver lesion (Omm2.5) of the same patient. Mel270 and Omm2.5 cells exhibited distinct clonal morphologies in adherent culture akin to holoclones, meroclones, and paraclones. Holoclones were large colonies of tightly packed small cells, which could be serially passaged (> 10 generations) to produce colonies of all three types; paraclones were small colonies of flattened cells that could be passaged for only one or two generations to produce further paraclones. Mel270 and Omm2.5 cells surviving cisplatin treatment produced significantly more holoclones than untreated cells (P < 0.05), suggesting enrichment for this CSC-like subpopulation. Mel270 and Omm2.5 cells also formed melanomaspheres (MS) when grown at clonal density in nonadherent culture. MS possessed self-renewal capacity to generate further MS and when replated to adherent culture yielded colonies of all three types. Mel270 and Omm2.5 holoclones and MS also demonstrated antigenic heterogeneity expressing markers associated with both a primitive migratory neural crest phenotype, and a more differentiated phenotype. These data suggest the presence in UM cell lines, of a CSC-like subpopulation with enhanced self-renewal and proliferative capabilities that could more appropriately model therapeutic efficacy.

A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (∼1 × 10(4) cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.

Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network.