A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells.

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells.

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, a receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 seconds enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

Generation of 3D Spheroids Using a Thiol-Acrylate Hydrogel Scaffold to Study Endocrine Response in ER+ Breast Cancer.

Culturing cancer cells in a three-dimensional (3D) environment better recapitulates in vivo conditions by mimicking cell-to-cell interactions and mass transfer limitations of metabolites, oxygen, and drugs. Recent drug studies have suggested that a high rate of preclinical and clinical failures results from mass transfer limitations associated with drug entry into solid tumors that 2D model systems cannot predict. Droplet microfluidic devices offer a promising alternative to grow 3D spheroids from a small number of cells to reduce intratumor heterogeneity, which is lacking in other approaches. Spheroids were generated by encapsulating cells in novel thiol-acrylate (TA) hydrogel scaffold droplets followed by on-chip isolation of single droplets in a 990- or 450-member trapping array. The TA hydrogel rapidly (∼35 min) polymerized on-chip to provide an initial scaffold to support spheroid development followed by a time-dependent degradation. Two trapping arrays were fabricated with 150 or 300 µm diameter traps to investigate the effect of droplet size and cell seeding density on spheroid formation and growth. Both trapping arrays were capable of ∼99% droplet trapping efficiency with ∼90% and 55% cellular encapsulation in trapping arrays containing 300 and 150 µm traps, respectively. The oil phase was replaced with media ∼1 h after droplet trapping to initiate long-term spheroid culturing. The growth and viability of MCF-7 3D spheroids were confirmed for 7 days under continuous media flow using a customized gravity-driven system to eliminate the need for syringe pumps. It was found that a minimum of 10 or more encapsulated cells are needed to generate a growing spheroid while fewer than 10 parent cells produced stagnant 3D spheroids. As a proof of concept, a drug susceptibility study was performed treating the spheroids with fulvestrant followed by interrogating the spheroids for proliferation in the presence of estrogen. Following fulvestrant exposure, the spheroids showed significantly less proliferation in the presence of estrogen, confirming drug efficacy.

Fluorescent visualization of oil displacement in a microfluidic device for enhanced oil recovery applications.

A microfluidic device was developed to mimic the reservoir pore-scale and track the oil/water phases during air flooding. The chip was generated by combining soft-lithography and NOA81 replication. A unique feature of this approach is the inclusion of fluorescent dyes into the oil/water phases, allowing for real-time visualization of oil recovery without altering the phases' surface properties. As a proof of concept, the air was injected into the water/oil-flooded device for enhanced oil recovery applications.

Characterization of PMI-5011 on the Regulation of Deubiquitinating Enzyme Activity in Multiple Myeloma Cell Extracts.

Deubiquitinating enzyme (DUB)-targeted therapeutics have shown promise in recent years as alternative cancer therapeutics, especially when coupled with proteasome-based inhibitors. While a majority of DUB-based therapeutics function by inhibiting DUB enzymes, studies show that positive regulation of these enzymes can stabilize levels of protein degradation. Unfortunately, there are currently no clinically available therapeutics for this purpose. The goal of this work was to understand the effect of a botanical extract from Artemisia dracunculus L called PMI-5011 on DUB activity in cancer cells. Through a series of kinetic analyses and mathematical modeling, it was found that PMI-5011 positively regulated DUB activity in two model multiple myeloma cells line (OPM2 and MM.1S). This suggests that PMI-5011 interacts with the active domains of DUBs to enhance their activity directly or indirectly, without apparently affecting cellular viability. Similar kinetic profiles of DUB activity were observed with three bioactive compounds in PMI-5011 (DMC-1, DMC-2, davidigenin). Interestingly, a differential cell line-independent trend was observed at higher concentrations which suggested variances in inherent gene expressions of UCHL1, UCHL5, USP7, USP15, USP14, and Rpn11 in OPM2 and MM.1S cell lines. These findings highlight the therapeutic potential of PMI-5011 and its selected bioactive compounds in cancer.

Evaluation of intercellular communication between breast cancer cells and adipose-derived stem cells via passive diffusion in a two-layer microfluidic device.

Breast cancer tumorigenesis and response to therapy is regulated by cancer cell interactions with the tumor microenvironment (TME). Breast cancer signaling to the surrounding TME results in a heterogeneous and diverse tumor microenvironment, which includes the production of cancer-associated fibroblasts, macrophages, adipocytes, and stem cells. The secretory profile of these cancer-associated cell types results in elevated chemokines and growth factors that promote cell survival and proliferation within the tumor. Current co-culture approaches mostly rely on transwell chambers to study intercellular signaling between adipose-derived stem cells (ASCs) and cancer cells; however, these methods are limited to endpoint measurements and lack dynamic control. In this study, a 4-channel, "flow-free" microfluidic device was developed to co-culture triple-negative MDA-MB-231 breast cancer cells and ASCs to study intercellular communication between two distinct cell types found in the TME. The device consists of two layers: a top PDMS layer with four imprinted channels coupled with a bottom agarose slab enclosed in a Plexiglas chamber. For dynamic co-culture, the device geometry contained two centered, flow-free channels, which were supplied with media from two outer flow channels via orthogonal diffusion through the agarose. Continuous fresh media was provided to the cell culture channel via passive diffusion without creating any shearing effect on the cells. The device geometry also allowed for the passive diffusion of cytokines and growth factors between the two cell types cultured in parallel channels to initiate cell-to-cell crosstalk. The device was used to show that MDA-MB-231 cells co-cultured with ASCs exhibited enhanced growth, a more aggressive morphology, and polarization toward the ASCs. The MDA-MB-231 cells were found to exhibit a greater degree of resistance to the drug paclitaxel when co-cultured with ASCs when compared to single culture studies. This microfluidic device is an ideal platform to study intercellular communication for many types of cells during co-culture experiments and allows for new investigations into stromal cell-mediated drug resistance in the tumor microenvironment.

Lignin-graft-PLGA drug-delivery system improves efficacy of MEK1/2 inhibitors in triple-negative breast cancer cell line.

Aim: Few targeted therapies are available for triple-negative breast cancer (TNBC) patients. Here, we propose a novel alkaline-lignin-conjugated-poly(lactic-co-glycolic acid) (L-PLGA) nanoparticle drug delivery system to improve the efficacy of targeted therapies. Materials & methods: L-PLGA nanoparticles (NPs) loaded with the MEK1/2 inhibitor GDC-0623 were characterized, tested in vitro on MDA-MB-231 TNBC cell line and compared with loaded PLGA NPs. Results: Loaded L-PLGA NPs were less than half the size of PLGA NPs, had slower drug release and improved the efficacy of GDC-0623 when tested in vitro. We demonstrated that GDC-0623 reversed epithelial-to-mesenchymal transition in TNBC. Conclusion: Our findings indicate that L-PLGA NPs are superior to PLGA NPs in delivering GDC-0623 to cancer cells for improved efficacy in vitro.

Synthesis and characterization of thiol-acrylate hydrogels using a base-catalyzed Michael addition for 3D cell culture applications.

There is significant interest in developing new approaches for culturing mammalian cells in a three-dimensional (3D) environment due to the fact that it better recapitulates the in vivo environment. The goal of this work was to develop thiol-acrylate, biodegradable hydrogels that possess highly tunable properties to support in vitro 3D culture. Six different hydrogel formulations were synthesized using two readily available monomers, a trithiol (ETTMP 1300 [ethoxylated trimethylolpropane tri(3-mercaptopropionate) 1300]) and a diacrylate (PEGDA 700 [polyethylene glycol diacrylate 700]), polymerized by a base-catalyzed Michael addition reaction. The resultant hydrogels were homogeneous, hydrophilic, and biodegradable. Different mechanical properties such as gelation time, storage modulus (or the elasticity G'), swelling ratio, and rate of degradation were tuned by varying the weight percentage of polymer, the molar ratio of thiol-to-acrylate groups, and the pH of the solution. Cytocompatibility was assessed using two model breast cancer cell lines by both 2D and 3D cell culturing approaches. The hydrogel formulations with a thiol-to-acrylate molar ratio of 1.05 were found to be optimal for both 2D and 3D cultures with MDA-MB-231 cellular aggregates found to be viable after 17 days of 3D continuous culture. Finally, MCF7 cells were observed to form 3D spheroids up to 600 µm in diameter as proof of principle for the thiol-acrylate hydrogel to function as a scaffold for in vitro 3D cell culture. A comparison of the different mechanical properties of the six hydrogel formulations coupled with in vitro cell culture results and findings from previously published hydrogels conclude that the thiol-acrylate hydrogels have significant potential as a scaffold for 3D cell culture.

Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell-permeable, peptide-based reporter.

Deubiquitinating enzymes (DUBs) regulate the removal of the polyubiquitin chain from proteins targeted for degradation. Current approaches to quantify DUB activity are limited to test tube-based assays that incorporate enzymes or cell lysates, but not intact cells. The goal of this work was to develop a novel peptide-based biosensor of DUB activity that is cell permeable, protease-resilient, fluorescent, and specific to DUBs. The biosensor consists of an N-terminal ß-hairpin motif that acts as both a 'protectide' to increase intracellular stability and a cell penetrating peptide (CPP) to facilitate the uptake into intact cells. The ß-hairpin was conjugated to a C-terminal substrate consisting of the last four amino acids in ubiquitin (LRGG) to facilitate DUB mediated cleavage of a C-terminal fluorophore (AFC). The kinetics of the peptide reporter were characterized in cell lysates by dose response and inhibition enzymology studies. Inhibition studies with an established DUB inhibitor (PR-619) confirmed the specificity of both reporters to DUBs. Fluorometry and fluorescent microscopy experiments followed by mathematical modeling established the capability of the biosensor to measure DUB activity in intact cells while maintaining cellular integrity. The novel reporter introduced here is compatible with high-throughput single cell analysis platforms such as FACS and droplet microfluidics facilitating direct quantification of DUB activity in single intact cells with direct application in point-of-care cancer diagnostics and drug discovery.

Luminescent nanomaterials for droplet tracking in a microfluidic trapping array.

The use of high-throughput multiplexed screening platforms has attracted significant interest in the field of on-site disease detection and diagnostics for their capability to simultaneously interrogate single-cell responses across different populations. However, many of the current approaches are limited by the spectral overlap between tracking materials (e.g., organic dyes) and commonly used fluorophores/biochemical stains, thus restraining their applications in multiplexed studies. This work demonstrates that the downconversion emission spectra offered by rare earth (RE)-doped ß-hexagonal NaYF4 nanoparticles (NPs) can be exploited to address this spectral overlap issue. Compared to organic dyes and other tracking materials where the excitation and emission is separated by tens of nanometers, RE elements have a large gap between excitation and emission which results in their spectral independence from the organic dyes. As a proof of concept, two differently doped NaYF4 NPs (europium: Eu3+, and terbium: Tb3+) were employed on a fluorescent microscopy-based droplet microfluidic trapping array to test their feasibility as spectrally independent droplet trackers. The luminescence tracking properties of Eu3+-doped (red emission) and Tb3+-doped (green emission) NPs were successfully characterized by co-encapsulating with genetically modified cancer cell lines expressing green or red fluorescent proteins (GFP and RFP) in addition to a mixed population of live and dead cells stained with ethidium homodimer. Detailed quantification of the luminescent and fluorescent signals was performed to confirm no overlap between each of the NPs and between NPs and cells. Thus, the spectral independence of Eu3+-doped and Tb3+-doped NPs with each other and with common fluorophores highlights the potential application of this novel technique in multiplexed systems, where many such luminescent NPs (other doped and co-doped NPs) can be used to simultaneously track different input conditions on the same platform. Graphical abstract ᅟ.

Biophysical analysis of fluid shear stress induced cellular deformation in a microfluidic device.

Even though the majority of breast cancers respond well to primary therapy, a large percentage of patients relapse with metastatic disease, for which there is no treatment. In metastasis, a tumor sheds a small number of cancerous cells, termed circulating tumor cells (CTCs), into the local vasculature, from where they spread throughout the body to form new tumors. As CTCs move through the circulatory system, they experience physiological forces not present in the initial tumor environment, namely, fluid shear stress (FSS). Evidence suggests that CTCs respond to FSS by adopting a more aggressive phenotype; however, to date single-cell morphological changes have not been quantified to support this observation. Furthermore, the methodology of previous studies involves inducing FSS by flowing cells through the tubing, which lacks a precise and tunable control of FSS. Here, a microfluidic approach is used for isolating and characterizing the biophysical response of single breast cancer cells to conditions experienced in the circulatory system during metastasis. To evaluate the single-cell response of multiple breast cancer types, two model circulating tumor cell lines, MDA-MB-231 and MCF7, were challenged with FSS at precise magnitudes and durations. As expected, both MDA-MB-231 and MCF7 cells exhibited greater deformability due to increasing duration and magnitudes of FSS. However, wide variations in single-cell responses were observed. MCF7 cells were found to rapidly deform but reach a threshold value after 5 min of FSS, while MDA-MB-231 cells were observed to deform at a slower rate but with a larger threshold of deformation. This behavioral diversity suggests the presence of distinct cell subpopulations with different phenotypes.

Development of ß-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination.

Regulation of the ubiquitin-proteasome system (UPS) to treat select types of cancer has become a popular area of drug discovery research. The FDA approval of proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma has led to an increased need for chemical reporters capable of detecting and quantifying protein ubiquitination and the activity of members of the UPS including E3 ubiquitin ligases and the proteasome in the tumor cells of the patients. One limitation of peptide-based reporters is their rapid degradation in the cellular environment by cytosolic peptidases. Conversely, ß-hairpin "protectides" exhibit a pronounced secondary structure that significantly increases their lifetime under cellular conditions. The goal of this work was to develop a family of novel, ornithine-rich protectides that could act as primary degrons serving as substrates for in vitro ubiquitination. The fluorescent peptide-based reporters were demonstrated to be highly resistant to degradation in multiple myeloma cell lysates. The most stable ß-hairpin primary degron, containing a single ornithine residue at the N-terminus, OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH2], demonstrated rapid ubiquitination kinetics and a 20-fold increase in stability when compared with an unstructured primary degron. A screen of E1 and E3 enzyme inhibitors in cell lysates showed that ubiquitination of OWRWR was significantly impaired by inhibitors of the SCF family of E3 ligases. Furthermore, this is the first report demonstrating the use of an ornithine residue on a primary degron as a ubiquitination site. This study serves as a strong foundation for the development of stable, fluorescent, peptide-based reporters capable of quantifying protein ubiquitination and the enzymatic activity of members of the UPS.

Identification of a p53-based portable degron based on the MDM2-p53 binding region.

In recent years the ubiquitin proteasome system (UPS) has garnered increasing interest as a target for chemotherapeutics. Due to the success of the proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma, several new compounds have been developed to target E3 ubiquitin ligases and the proteasome in numerous human cancers. This has increased the need for new analytical methods to precisely measure intracellular enzyme activity in cells. A key component of a desired analytical method is a substrate that is capable of rapid intracellular ubiquitination yet easily incorporated into the next generation of more sophisticated UPS reporters. Portable degradation sequences, or degrons, have the ability to bind to E3 ligases and promote substrate ubiquitination when the sequence is presented in isolation or appended to other entities such as fluorescent peptide-based reporters. Previous work identified an E3 ligase (MDM2)-binding element at p53 amino acids 92-112, which was later demonstrated to be rapidly ubiquitinated in cytosolic lysates effectively functioning as a transportable degron. In this work, a shortened p53 sequence within amino acids 92-112 that displayed rapid ubiquitination kinetics was identified. A nine-member peptide library was synthesized using sequence elements of various sizes and lengths, all based on the initial 22 amino acid long sequence, containing a single ubiquitination site lysine. The ubiquitination kinetics were determined using a combination of gel electrophoresis and analytical high performance liquid chromatography (HPLC) to rank the members of the library and identify the optimal ubiquitination sequence. This analysis identified the five amino acid sequence, KGSYG, corresponding to residues 105-108 with an added N-terminal lysine, as a portable degron since this sequence demonstrated the most rapid ubiquitination kinetics.

Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.

Measuring activity in the ubiquitin-proteasome system: from large scale discoveries to single cells analysis.

The ubiquitin-proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington's disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.