A Portable Controllable Compressive Stress Device to Monitor Human Breast Cancer Cell Protrusions at Single-Cell Resolution.

In vitro devices offer more numerous methods than in vivo models to investigate how cells respond to pressure stress and quantify those responses. Several in vitro devices have been developed to study the cell response to compression force. However, they are unable to observe morphological changes of cells in real-time. There is also a concern about cell damage during the process of harvesting cells from 3D gels. Here we report a device employing transparent, thin gel layers to clamp cells between the interfaces and applied a controllable compression force by stacking multiple layers on the top. In this approach, cells can be monitored for alteration of cellular protrusions, whose diversity has been proven to promote cancer cell dissemination, with single-cell resolution under compression force. Furthermore, p-Rac-1 and rhodamine staining on the device directly to confirm the actin filaments of lamellipodia. The method was able to fulfill real-time live-cell observation at single-cell resolution and can be readily used for versatile cell analysis. MDA-MB-231 and MCF7 breast cancer cells were utilized to demonstrate the utility of the device, and the results showed that the stimuli of compression force induce MDA-MB-231 and MCF7 to form lamellipodia and bleb protrusions, respectively. We envision the device may be used as a tool to explore mechanisms of membrane protrusion transitions and to screen drug candidates for inhibiting cancer cell protrusion plasticity for cancer therapy.

A Microfluidic Single-Cell Cloning (SCC) Device for the Generation of Monoclonal Cells.

Single-cell cloning (SCC) is a critical step in generating monoclonal cell lines, which are widely used as in vitro models and for producing proteins with high reproducibility for research and the production of therapeutic drugs. In monoclonal cell line generation, the development time can be shortened by validating the monoclonality of the cloned cells. However, the validation process currently requires specialized equipment that is not readily available in general biology laboratories. Here, we report a disposable SCC device, in which single cells can be isolated, validated, and expanded to form monoclonal cell colonies using conventional micropipettes and microscopes. The monoclonal cells can be selectively transferred from the SCC chip to conventional culture plates, using a tissue puncher. Using the device, we demonstrated that monoclonal colonies of actin-GFP (green fluorescent protein) plasmid-transfected A549 cells could be formed in the device within nine days and subsequently transferred to wells in plates for further expansion. This approach offers a cost-effective alternative to the use of specialized equipment for monoclonal cell generation.

Active fraction (HS7) from Taiwanofungus camphoratus inhibits AKT-mTOR, ERK and STAT3 pathways and induces CDK inhibitors in CL1-0 human lung cancer cells.

BACKGROUND: The non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. In NSCLC, the oncogenic AKT-mTOR, ERK and STAT3 pathways are commonly dysregulated and have emerged as attractive targets for therapeutic developments. In a relatively limited subset of NSCLC, these pathways driven by mutant EGFR can be treated by the tyrosine kinase inhibitors (TKIs)-mediated targeted therapy. However, for the most NSCLC, more novel targeted agents are imperatively needed. Therefore, we investigated the inhibitory effects of the active fraction HS7 from Taiwanofungus camphoratus, a unique medicinal fungus in Taiwan, on these pathways in CL1-0 EGFR wild-type human NSCLC cells. METHODS: The active fraction HS7 was prepared by n-hexane extraction of T. camphoratus followed by silica gel chromatography. Its effects on the cell viabilities were determined by sulforhodamine B colorimetric assay. Flow cytometry was used to analyze cell-cycle regulation and apoptosis induction. The changes in cellular protein levels were examined by Western blot. RESULTS: The active fraction HS7 vigorously inhibits AKT-mTOR, ERK and STAT3 signaling pathways in CL1-0 cells. At dose of 25 µg/mL, these signaling pathways were almost completely inhibited by HS7, accompanied with induction of cyclin-dependent kinase inhibitors such as p15, p21 and p27. Accordingly, the AKT-mTOR downstream targets p-p70S6K and HIF-1α were also suppressed as well. At this dose, the cell proliferation was profoundly suppressed to 23.4% of control and apoptosis induction was observed. CONCLUSIONS: The active fraction HS7 from n-hexane extract of T. camphoratus exerts multi-targeting activity on the suppression of AKT-mTOR, ERK and STAT3 pathways and induction of p15, p21 and p27 in EGFR wild-type NSCLC cells. This multi-targeting activity of HS7 suggests its potential as an alternative medicine for the treatment of EGFR TKIs resistant NSCLC.

Towards an Endpoint Cell Motility Assay by a Microfluidic Platform.

In vitro cell motility assays are frequently used in the study of cell migration in response to anti-cancer drug treatment. Microfluidic systems represent a unique tool for the in vitro analysis of cell motility. However, they usually rely on using time-lapse microscopy to record the spatial temporal locations of the individual cells being tested. This has created a bottleneck for microfluidic systems to perform high-throughput experiments due to requirement of a costly time-lapse microscopy system. Here, we describe the development of a portable microfluidic device for endpoint analysis of cell motility. The reported device incorporates a cell alignment feature to position the seeded cells on the same initial location, so that the cells' motilities can be analyzed based on their locations at the end of the experiment after the cells have migrated. We show that the device was able to assess cancer cell motility after treatment with a migration inhibitory drug Indole-3-carbinol on MDA-MB-231 breast cancer cells, demonstrating the applicability of our device in screening anti-cancer drug compounds on cancer cells.

A microfluidic dual-well device for high-throughput single-cell capture and culture.

In vitro culture of single cells facilitates biological studies by deconvoluting complications from cell population heterogeneity. However, there is still a lack of simple yet high-throughput methods to perform single cell culture experiments. In this paper, we report the development and application of a microfluidic device with a dual-well (DW) design concept for high-yield single-cell loading (~77%) in large microwells (285 and 485 µm in diameter) which allowed for cell spreading, proliferation and differentiation. The increased single-cell loading yield is achieved by using sets of small microwells termed "capture-wells" and big microwells termed "culture-wells" according to their utilities for single-cell capture and culture, respectively. This novel device architecture allows the size of the "culture" microwells to be flexibly adjusted without affecting the single-cell loading efficiency making it useful for cell culture applications as demonstrated by our experiments of KT98 mouse neural stem cell differentiation, A549 and MDA-MB-435 cancer cell proliferation, and single-cell colony formation assay with A549 cells in this paper.

Elimination of cancer stem-like "side population" cells in hepatoma cell lines by chinese herbal mixture "tien-hsien liquid".

There are increasing pieces of evidence suggesting that the recurrence of cancer may result from a small subpopulation of cancer stem cells, which are resistant to the conventional chemotherapy and radiotherapy. We investigated the effects of Chinese herbal mixture Tien-Hsien Liquid (THL) on the cancer stem-like side population (SP) cells isolated from human hepatoma cells. After sorting and subsequent culture, the SP cells from Huh7 hepatoma cells appear to have higher clonogenicity and mRNA expressions of stemness genes such as SMO, ABCG2, CD133, ß-catenin, and Oct-4 than those of non-SP cells. At dose of 2 mg/mL, THL reduced the proportion of SP cells in HepG2, Hep3B, and Huh7 cells from 1.33% to 0.49%, 1.55% to 0.43%, and 1.69% to 0.27%, respectively. The viability and colony formation of Huh7 SP cells were effectively suppressed by THL dose-dependently, accompanied with the inhibition of stemness genes, e.g., ABCG2, CD133, and SMO. The tumorigenicity of THL-treated Huh7 SP cells in NOD/SCID mice was also diminished. Moreover, combination with THL could synergize the effect of doxorubicin against Huh7 SP cells. Our data indicate that THL may act as a cancer stem cell targeting therapeutics and be regarded as complementary and integrative medicine in the treatment of hepatoma.

Speckle suppression in holographic projection displays using temporal integration of speckle images from diffractive optical elements.

Speckles on images in holographic projection displays (HPDs) were efficiently suppressed by the temporal sum of two diffractive images generated from diffractive optical elements (DOEs). Using a modified iterative Fourier transform algorithm, we obtained pairs of phase-only DOEs that generated the diffractive images with high negative correlation coefficients of -0.827 and -0.490 in the one-dimensional and the two-dimensional simulations, respectively. The suppression ratios of the speckles in the two simulations were 0.301 and 0.457, which were 61% and 35% lower, respectively, than the sum of the two uncorrelated images. We have successfully demonstrated that the sum of two negatively correlated images from DOEs can effectively reduce the image speckles and improve the image quality in HPD systems.

Apoptotic Cell Death and Inhibition of Wnt/ß-Catenin Signaling Pathway in Human Colon Cancer Cells by an Active Fraction (HS7) from Taiwanofungus camphoratus.

Aberrant activation of Wnt/ß-catenin signaling plays an important role in the development of colon cancer. HS7 is an active fraction extracted from Taiwanofungus camphoratus, which had been widely used as complementary medicine for Taiwan cancer patients in the past decades. In this study, we demonstrated the effects of HS7 on the growth inhibition, apoptosis induction, and Wnt/ß-catenin signaling suppression in human colon cancer cells. HS7 significantly inhibited proliferation of HT29, HCT116, and SW480 colon cancer cells in a dose- and time-dependent manner. The apoptosis induction was evidenced by DNA fragmentation and subG1 accumulation, which was associated with increased Bax/Bcl-2 ratio, activation of caspase-3 and cleavage of PARP. By using Tcf-dependent luciferase activity assay, HS7 was found to inhibit the ß-catenin/Tcf transcriptional activities. In addition, HS7 strongly suppressed the binding of Tcf complexes to its DNA-binding site shown in electrophoretic mobility shift assay. This inhibition was further confirmed by the decreased protein levels of Tcf-4 and ß-catenin. The ß-catenin/Tcf downstream target genes, such as survivin, c-myc, cyclin D1, MMP7, and MT1-MMP involved in apoptosis, invasion, and angiogenesis were also diminished as well. These results indicate that Taiwanofungus camphoratus may provide a benefit as integrative medicine for the treatment of colon cancer.

Cytotoxic triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells.

Five lanostane (2, 3, 4, 6 and 8) and three ergostane-type (1, 5 and 7) triterpenes isolated from the fruiting bodies of Antrodia camphorata were evaluated for their in vitro cytotoxic data against various cancer cell types. The three zhankuic acids, 1, 5 and 7 displayed the most potent cytotoxic effect with an IC(50) value of 22.3-75.0microM. The compound 3 was selectively cytotoxic in three colon cancer cell lines (HT-29, HCT-116 and SW-480) and a breast cancer model (MDA-MB-231), whereas 8 only showed its cytotoxicity against MDA-MB-231. None of these isolates was toxic to mammary epithelial (MCF10A) and primary foreskin fibroblast (HS68) cells, two human normal cell lines. The compounds 1, 5 and 7 were also demonstrated to induce apoptosis in HT-29 and SW-480 cells, as confirmed by sub-G1 cell cycle arrest. In HT-29 cells, the expression of apoptosis-associated proteins poly-(ADP-ribose) polymerase cleavage, Bcl-2 and procaspase-3 were suppressed by compounds 1, 5 and 7. A mixture containing 4microM each of compounds 1, 5 and 7 also showed a synergistic cytotoxic effect in HT-29 cells.