Sophoraflavanone G Inhibits RANKL-Induced Osteoclastogenesis via MAPK/NF-κB Signaling Pathway.

Osteoporosis is a common chronic bone metabolism disorder characterized by decreased bone mass and reduced bone density in the bone tissue. Osteoporosis can lead to increased fragility of the skeleton, making it prone to brittle fractures. Osteoclasts are macrophage-like cells derived from hematopoietic stem cells, and their excessive activity in bone resorption leads to lower bone formation than absorption during bone remodeling, which is one of the important factors inducing osteoporosis. Therefore, how to inhibit osteoclast formation and reducing bone loss is an important direction for treating osteoporosis. Sophoraflavanone G, derived from Sophora flavescens Alt and Rhizoma Drynariae, is a flavonoid compound with various biological activities. However, there have been few studies on osteoporosis and osteoclasts so far. Therefore, we hypothesize that genistein G can inhibit osteoclast differentiation, alleviate bone loss phenomenon, and conduct in vitro and in vivo experiments for research and verification purposes.

Spatially controlled diffusion range of tumor-associated angiogenic factors to develop a tumor model using a microfluidic resistive circuit.

Developing a tumor model with vessels has been a challenge in microfluidics. This difficulty is because cancer cells can overgrow in a co-culture system. The up-regulation of anti-angiogenic factors during the initial tumor development can hinder neovascularization. The standard method is to develop a quiescent vessel network before loading a tumor construct in an adjacent chamber, which simulates the interaction between a tumor and its surrounding vessels. Here, we present a new method that allows a vessel network and a tumor to develop simultaneously in two linked chambers. The physiological environment of these two chambers is controlled by a microfluidic resistive circuit using two symmetric long microchannels. Applying the resistive circuit, a diffusion-dominated environment with a small 2-D pressure gradient is created across the two chambers with velocity <10.9 nm s-1 and Péclet number <6.3 × 10-5. This 2-D pressure gradient creates a V-shaped velocity clamp to confine the tumor-associated angiogenic factors at pores between the two chambers, and it has two functions. At the early stage, vasculogenesis is stimulated to grow a vessel network in the vessel chamber with minimal influence from the tumor that is still developed in the adjacent chamber. At the post-tumor-development stage, the induced steep concentration gradient at pores mimics vessel-tumor interactions to stimulate angiogenesis to grow vessels toward the tumor. Applying this method, we demonstrate that vasculogenic vessels can grow first, followed by stimulating angiogenesis. Angiogenic vessels can grow into stroma tissue up to 1.3 mm long, and vessels can also grow into or wrap around a 625 µm tumor spheroid or a tumor tissue developed from a cell suspension. In summary, our study suggests that the interactions between a developing vasculature and a growing tumor must be controlled differently throughout the tissue development process, including at the early stage when vessels are still forming and at the later stage when the tumor needs to interact with the vessels.

Nucleo-cytoplasmic shuttling of 14-3-3 epsilon carrying hnRNP C promotes autophagy.

Translocation of 14-3-3 protein epsilon (14-3-3ε) was found to be involved in Triptolide (Tp)-induced inhibition of colorectal cancer (CRC) cell proliferation. However, the form of cell death induced by 14-3-3ε translocation and mechanisms underlying this effect remain unclear. This study employed label-free LC-MS/MS to identify 14-3-3ε-associated proteins in CRC cells treated with or without Tp. Our results confirmed that heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C) were exported out of the nucleus by 14-3-3ε and degraded by ubiquitination. The nucleo-cytoplasmic shuttling of 14-3-3ε carrying hnRNP C mediated Tp-induced proliferation inhibition, cell cycle arrest and autophagic processes. These findings have broad implications for our understanding of 14-3-3ε function, provide an explanation for the mechanism of nucleo-cytoplasmic shuttling of hnRNP C and provide new insights into the complex regulation of autophagy.

Metastasis and immunosuppression promoted by mtDNA and PD-L1 in extracellular vesicles are reversed by WGP ß-glucan in oral squamous cell carcinoma.

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D-loop and programmed cell death-ligand 1 (PD-L1) in extracellular vesicles (EVs), and attenuate T-cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) ß-glucan in oral squamous cell (OSCC) patients. Arecoline-induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D-loop were analyzed in OSCC cell lines. mtDNA D-loop, PD-L1, IFN-γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP ß-glucan could elevate CD4+ and CD8+ T-cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D-loop and PD-L1, and in turn elicits immune suppression. However, WGP ß-glucan potentially enhances dual effects on T-cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.

A handheld contactless conductivity detector for monitoring the desalting of low-volume virus and cell samples.

Desalting of biosamples is crucial for analytical techniques intolerant to abundant salts. However, there is no simple tool to monitor the desalting of low-volume biosamples so far. Here we developed a handheld capacitively coupled contactless conductivity detector (hC4D) as a miniaturized device to measure the conductivity of 75 µL biosamples. Polyether-ether-ketone (PEEK) tubing was selected as the sample reservoir for sample loading via a pipette. Another pipetting of air pushed the sample solution out of the tubing to recollect the sample. Owing to the low sample consumption and easy sample recollection, hC4D is advantageous for testing expensive biosamples, such as viruses and cells. In addition, the whole process of sample injection, conductivity measurement, recollection, and calibration of conductivity can be completed within 1 min. To verify the feasibility of hC4D, we monitored the desalting progress of gel filtration (GF) of 200 µL blood samples, ultrafiltration (UF) of 300 µL virus samples, and dialysis of 7 mL cell samples. Three rounds of GF and UF completely removed the salts but led to poor sample recovery. In contrast, low concentrations of residual salts remained and better recovery was achieved after two rounds of GF and UF. We further utilized the hC4D to monitor the dialysis and tuned the salt concentration in the cell sample, such that we maintained the viability of cells in a low conductivity environment. These results indicated that hC4D is a promising tool for optimizing the desalting procedure of low-volume biosamples.

Cellular Heterogeneity Facilitates the Functional Differences Between Hair Follicle Dermal Sheath Cells and Dermal Papilla Cells: A New Classification System for Mesenchymal Cells within the Hair Follicle Niche.

Mesenchymal stem cells (MSCs) are known for their self-renewal and multi-lineage differentiation potential, with these cells often being evaluated in the regulation and maintenance of specific cellular niches including those of the hair follicle. Most mesenchymal stem cells in the hair follicles are housed in the dermal papilla (DP) and dermal sheath (DS), with both niches characterized by a broad variety of cellular subsets. However, while most previous studies describing the hair follicle mesenchymal niche treated all DP and DS cells as Hair Follicle Mesenchymal Stem Cells (HF-MSCs), the high number of cellular subsets would suggest that these cells are actually too heterogenous for such a broad definition. Given this we designed this study to evaluate the differentiation processes in these cells and used this data to create a new set of classifications for DP and DS cells, dividing them into "hair follicle mesenchymal stem cells (HF-MSCs)", "hair follicle mesenchymal progenitor cells (HF-MPCs)", and "hair follicle mesenchymal functional cells (HF-MFCs)". In addition, those cells that possess self-renewal and differentiation were re-named hair follicle derived mesenchymal multipotent cells (HF-MMCs). This new classification may help to further our understanding of the heterogeneity of hair follicle dermal cells and provide new insights into their evaluation.

Longitudinal shear wave elasticity measurements of millimeter-sized biomaterials using a single-element transducer platform.

Temporal variations of the extracellular matrix (ECM) stiffness profoundly impact cellular behaviors, possibly more significantly than the influence of static stiffness. Three-dimensional (3D) cell cultures with tunable matrix stiffness have been utilized to characterize the mechanobiological interactions of elasticity-mediated cellular behaviors. Conventional studies usually perform static interrogations of elasticity at micro-scale resolution. While such studies are essential for investigations of cellular mechanotransduction, few tools are available for depicting the temporal dynamics of the stiffness of the cellular environment, especially for optically turbid millimeter-sized biomaterials. We present a single-element transducer shear wave (SW) elasticity imaging system that is applied to a millimeter-sized, ECM-based cell-laden hydrogel. The single-element ultrasound transducer is used both to generate SWs and to detect their arrival times after being reflected from the side boundaries of the sample. The sample's shear wave speed (SWS) is calculated by applying a time-of-flight algorithm to the reflected SWs. We use this noninvasive and technically straightforward approach to demonstrate that exposing 3D cancer cell cultures to X-ray irradiation induces a temporal change in the SWS. The proposed platform is appropriate for investigating in vitro how a group of cells remodels their surrounding matrix and how changes to their mechanical properties could affect the embedded cells in optically turbid millimeter-sized biomaterials.

Postoperative survival of extrahepatic and intrahepatic cholangiocarcinoma after surgery: a population-based cohort.

OBJECTIVES: The study was designed to clarify the difference between extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC) in postoperative cancer-specific death. DESIGN: Patients diagnosed with ECC and ICC after surgery, who are identified from the Surveillance, Epidemiology and End Results programme, are eligible for this retrospective cohort study. SETTING: Survival between groups was compared using the traditional Kaplan-Meier method and the cumulative incidence function (CIF) method. Propensity score-matched (PSM) analysis was conducted to balance the differences in vital variables between groups. The HR and 95% CI for ECC relative to ICC were used to quantify the risk of death. Subgroup analysis was further used to evaluate the stability of the differences between groups. RESULTS: The study included 876 patients with ECC and 1194 patients with ICC. Before PSM, with the Kaplan-Meier method, postoperative overall survival and cancer-specific death for ECC were worse than those for ICC. However, with the CIF method, no difference in postoperative cancer-specific death was found. After PSM, all differences in the considered traits were balanced, and 173 pairs of patients were retained. Survival analysis found that there was no difference in postoperative all-cause death (Kaplan-Meier method, p=0.186) or cancer-specific death (Kaplan-Meier and CIF methods, p=0.500 and p=0.913, respectively), which was consistent with subgroup analysis. CONCLUSIONS: ECC and ICC showed no difference in postoperative cancer-specific death, both in the natural state and in multiple variable-matched conditions. TRIAL REGISTRATION NUMBER: researchregistry4175.

Sonoporation based on repeated vaporization of gold nanodroplets.

BACKGROUND: Gold nanodroplets (AuNDs) have been proposed as agents for photothermal therapy and photoacoustic imaging. Previously, we demonstrated that the sonoporation can be more effectively achieved with synchronized optical and acoustic droplet vaporization. By applying a laser pulse at the rarefactional phase of the ultrasound (US) pulse, the vaporization threshold can be reached at a considerably lower laser average power. However, a large loading quantity of the AuNDs may increase the risk of air embolism. The destruction of phase-shifted AuNDs at the inertial cavitation stage leads to a reduced drug delivery performance. And it also causes instability of echogenicity during therapeutic monitoring. PURPOSE: In this study, we propose to further improve the sonoporation effectiveness with repeated vaporization. In other words, the AuNDs repeatedly undergo vaporization and recondensation so that sonoporation effects are accumulated over time at lower energy requirements. Previously, repeated vaporization has been demonstrated as an imaging contrast agent. In this study, we aim to adopt this repeated vaporization scheme for sonoporation. METHODS: Perfluoropentane NDs with a shell made of human serum albumin were used as the US contrast agents. Laser pulses at 808 nm and US pulses of 1 MHz were delivered for triggering vaporization and inertial cavitation of NDs. We detected the vaporization and cavitation effects under different activation firings, US peak negative pressures (PNPs), and laser fluences using 5- and 10-MHz focused US receivers. Numbers of calcein-AM and propidium iodide signals uptake by BNL hepatocarcinoma cancer cells were used to evaluate the sonoporation and cell death rate of the cells. RESULTS: We demonstrate that sonoporation can be realized based on repeatable vaporization instead of the commonly adopted inertial cavitation effects. In addition, it is found that the laser fluence and the acoustic pressure can be reduced. As an example, we demonstrate that the acoustic and optical energy for achieving a similar level of sonoporation rate can be as low as 0.44 MPa for the US PNP and 4.01 mJ/cm2 for the laser fluence, which are lower than those with our previous approach (0.53 MPa and 4.95 mJ/cm2 , respectively). CONCLUSION: We demonstrated the feasibility of vaporization-based sonoporation at a lower optical and acoustic energy. It is an advantageous method that can enhance drug delivery efficiency, therapeutic safety and potentially deliver an upgraded gene therapy strategy for improved theragnosis.

Laser-scribed graphene nanofiber decorated with oil palm lignin capped silver nanoparticles: a green biosensor.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), requires a high level of attention and is one of the most infectious diseases in the air. Present methods of diagnosing TB remain ineffective owing to their low sensitivity and time consumption. In this study, we produced a green graphene nanofiber laser biosensor (LSG-NF) decorated with oil palm lignin-based synthetic silver nanoparticles (AgNPs). The resulting composite morphology was observed by field-emission scanning electron microscopy and transmission electron microscopy, which revealed the effective adaptation of the AgNPs to the LSG-NF surface. The successful attachment of AgNPs and LSG-NFs was also evident from X-ray diffraction and Raman spectroscopy studies. In order to verify the sensing efficiency, a selective DNA sample captured on AgNPs was investigated for specific binding with M.tb target DNA through selective hybridisation and mismatch analysis. Electrochemical impedance studies further confirmed sensitive detection of up to 1 fM, where a detection limit of 10-15 M was obtained by estimating the signal-to-noise ratio (S/N = 3:1) as 3σ. Successful DNA immobilisation and hybridisation was confirmed by the detection of phosphorus and nitrogen peaks based on X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy. The stability and repeatability of the analysis were high. This approach provides an affordable potential sensing system for the determination of M. tuberculosis biomarker and thus provides a new direction in medical diagnosis.

Functional photoacoustic calcium imaging using chlorophosphonazo III in a 3D tumor cell culture.

This study demonstrates that chlorophosphonazo III (CPZ III) can be used as a contrast agent for photoacoustic calcium imaging. CPZ III can pass across the plasma membrane for labeling intracellular Ca2+ without cytotoxicity. In optical-resolution photoacoustic microscopy (OR-PAM), the photoacoustic (PA) signal intensity was strongly correlated with the presence of CPZ III and Ca2+ at various concentrations. The sensitivity of PA signal reception was enhanced by using an 8 MHz single-element focused ultrasound detector due to their matched frequency characteristics. Differences in the PA signal intensity were successfully found between the core and margin areas of tumorspheres in three-dimensional cell cultures. These findings indicate that CPZ III can serve as a novel PA contrast agent for functional Ca2+ imaging using OR-PAM.

A facile thermometer-like electrophoresis titration biosensor for alternative miRNA assay via moving reaction boundary chip.

Herein, a facile thermometer-like model of electrophoresis titration (ET) biosensor was proposed as an alternative tool for miRNA assay via moving reaction boundary (MRB) chip. For proof-of-concept demonstration, miRNA-122 and catalyzed hairpin assembly (CHA) were chosen as the model analyte and amplification, respectively. In the developed ET system, miRNA triggered the CHA with two hairpin probes (H1, H2) to yield H1-H2 duplexes with negative charges. Under an electric field, the duplexes moved into ET channel, and neutralized the acidic TAE buffer creating an MRB indicated by SYBR Green I (SGI). The model revealed that the MRB distance was as a function of logarithmic miRNA-122 content, indicating a facile sensing model. The relevant experiments were conducted and systemically validated the model of miRNA ET. Under the optimized conditions, the linear range of ET sensor was from 20 fM to 1 nM and the limit of detection (LOD) was 10 fM, showing a more than 100-fold sensitive increase in contrast to the one with a single CHA amplification. The mechanism of sensitive increase was well unveiled by the designed experiments. In addition, the ET biosensor had good selectivity, stability (less than 5% for intra-day and inter-day) and recovery (96%-110%), and was successfully applied for the assay of miRNA-122 and miRNA let-7a in real bio-fluids of serum and cancer cell lysate. Evidently, the proposed biosensor might be used as an alternative assay tool after nucleic acid amplification due to its high simplicity, sensitivity, specificity, linearity, stability and recovery.

Enhanced Radiosensitization for Cancer Treatment with Gold Nanoparticles through Sonoporation.

We demonstrate the megavoltage (MV) radiosensitization of a human liver cancer line by combining gold-nanoparticle-encapsulated microbubbles (AuMBs) with ultrasound. Microbubbles-mediated sonoporation was administered for 5 min, at 2 h prior to applying radiotherapy. The intracellular concentration of gold nanoparticles (AuNPs) increased with the inertial cavitation of AuMBs in a dose-dependent manner. A higher inertial cavitation dose was also associated with more DNA damage, higher levels of apoptosis markers, and inferior cell surviving fractions after MV X-ray irradiation. The dose-modifying ratio in a clonogenic assay was 1.56 ± 0.45 for a 10% surviving fraction. In a xenograft mouse model, combining vascular endothelial growth factor receptor 2 (VEGFR2)-targeted AuMBs with sonoporation significantly delayed tumor regrowth. A strategy involving the spatially and temporally controlled release of AuNPs followed by clinically utilized MV irradiation shows promising results that make it worthy of further translational investigations.

Radiofrequency Ablation (RFA) Combined with Transcatheter Arterial Chemoembolization (TACE) for Patients with Medium-to-Large Hepatocellular Carcinoma: A Retrospective Analysis of Long-Term Outcome.

BACKGROUND The aim of this study was to investigate the prognostic value of radiofrequency ablation (RFA) plus transcatheter arterial chemoembolization (TACE) in hepatocellular carcinoma (HCC) patients with tumor size ranging from 3.0 to 10.0 cm. MATERIAL AND METHODS We retrospectively analyzed data on 201 patients with medium-to-large HCC. According to treatment procedure, the patients were divided into the TACE group (n=124) and the TACE+RFA group (n=77). We recorded data on patient safety, subcapsular hepatic hematoma, large amount of ascites, liver abscess, gallbladder injury, and local skin infection. The overall survival (OS) and progression-free survival (PFS) in the 2 groups were analyzed and compared between groups. RESULTS The median PFS was 4.00 months (3.00-5.00 months) in the TACE group and 9.13 months (6.64-11.62 months) in the TACE+RFA group (P<0.001). Median OS was 12.00 months (8.88-15.13 months) in the TACE group and 27.57 months (20.06-35.08 months) in the TACE+RFA group (P<0.001). In the TACE+RFA group, multivariate Cox regression analysis showed that tumor size ≤5 cm) (HR: 1.952, 95% CI: 1.213-3.143, P=0.006), hepatitis B (HR: 2.323, 95% CI: 1.096-4.923, P=0.028), TACE times (1 or >1) (HR: 1.867, 95% CI: 1.156-3.013, P=0.011), alpha-fetoprotein (AFP) level >200 ng/ml (HR: 2.426, 95% CI: 1.533-3.839, P<0.001), and AST level >40 U/L (HR: 1.946, 95% CI: 1.196-3.166, P=0.007) were independent prognostic factors for overall survival. CONCLUSIONS Combination therapy of TACE with RFA is a safe and effective treatment for patients with medium-to-large HCC, with the long-term beneficial effect of retarding tumor progression and improving PFS and OS.

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography.

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 µL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.

Photoacoustic imaging of cells in a three-dimensional microenvironment.

Imaging live cells in a three-dimensional (3D) culture system yields more accurate information and spatial visualization of the interplay of cells and the surrounding matrix components compared to using a two-dimensional (2D) cell culture system. However, the thickness of 3D cultures results in a high degree of scattering that makes it difficult for the light to penetrate deeply to allow clear optical imaging. Photoacoustic (PA) imaging is a powerful imaging modality that relies on a PA effect generated when light is absorbed by exogenous contrast agents or endogenous molecules in a medium. It combines a high optical contrast with a high acoustic spatiotemporal resolution, allowing the noninvasive visualization of 3D cellular scaffolds at considerable depths with a high resolution and no image distortion. Moreover, advances in targeted contrast agents have also made PA imaging capable of molecular and cellular characterization for use in preclinical personalized diagnostics or PA imaging-guided therapeutics. Here we review the applications and challenges of PA imaging in a 3D cellular microenvironment. Potential future developments of PA imaging in preclinical applications are also discussed.

Synchronized Optical and Acoustic Droplet Vaporization for Effective Sonoporation.

Inertial cavitation-based sonoporation has been utilized to enhance treatment delivery efficacy. In our previous study, we demonstrated that tumor therapeutic efficacy can be enhanced through vaporization-assisted sonoporation with gold nanodroplets (AuNDs). Specifically, the AuNDs were vaporized both acoustically (i.e., acoustic droplet vaporization, ADV) and optically (i.e., optical droplet vaporization, ODV). A continuous wave (CW) laser was used for ODV in combination with an ultrasound pulse for ADV. Although effective for vaporization, the use of a CW laser is not energy efficient and may create unwanted heating and concomitant tissue damage. In this study, we propose the use of a pulsed wave (PW) laser to replace the CW laser. In addition, the PW laser was applied at the rarefaction phase of the ultrasound pulse so that the synergistic effects of ADV and ODV can be expected. Therefore, a significantly lower laser average power can be expected to achieve the vaporization threshold. Compared to the CW laser power at 2 W/cm2 from the previous approach, the PW laser power was reduced to only 0.2404 W/cm2. Furthermore, we also demonstrate in vitro that the sonoporation rate was increased when the PW laser was applied at the rarefaction phase. Specifically, the vaporization signal, the inertial cavitation signal, and the sonoporation rate all displayed a 1-µs period, which corresponded to the period of the 1-MHz acoustic wave used for ADV, as a function of the relative laser delay. The increased sonoporation rate indicates that this technique has the potential to enhance sonoporation-directed drug delivery and tumor therapy with a lower laser power while keeping the cell death rate at the minimum. Photoacoustic imaging can also be performed at the same time since a PW laser is used for the ODV.

Study of diffusive- and convective-transport mediated microtumor growth in a controlled microchamber.

In this paper, we report on using mass transport to control nutrition supply of colorectal cancer cells for developing a microtumor in a confined microchamber. To mimic the spatial heterogeneity of a tumor, two microfluidic configurations based on resistive circuits are designed. One has a convection-dominated microchamber to simulate the tumor region proximal to leaky blood vessels. The other has a diffusion-dominated microchamber to mimic the tumor core that lacks blood vessels and nutrient supply. Thus, the time for nutrition to fill the microchamber can vary from tens of minutes to several hours. Results show that cells cultured under a diffusive supply of nutrition have a high glycolytic rate and a nearly constant oxygen consumption rate. In contrast, cells cultured under convective supply of nutrition have a gradual increase of oxygen consumption rate with a low glycolytic rate. This suggests that cancer cells have distinct reactions under different mass transport and nutrition supply. Using these two microfluidic platforms to create different rate of nutrition supply, it is found that a continuous microtumor that almost fills the mm-size microchamber can be developed under a low-nutrient supply environment, but not for the convective condition. It also is demonstrated that microchannels can simulate the delivery of anti-cancer drugs to the microtumor under controlled mass-transport. This method provides a means to develop a larger scale microtumor in a lab-on-a-Chip system for post development and stimulations, and microchannels can be applied to control the physical and chemical environment for anti-cancer drug screening.

Shear Wave Elasticity Measurements of Three-Dimensional Cancer Cell Cultures Using Laser Speckle Contrast Imaging.

Shear wave elastography (SWE) has been widely adopted for clinical in vivo imaging of tissue elasticity for disease diagnosis, and this modality can be a valuable tool for in vitro mechanobiology studies but its full potential has yet to be explored. Here we present a laser speckle contrast SWE system for noncontact monitoring the spatiotemporal changes of the extracellular matrix (ECM) stiffness in three-dimensional cancer cell culture system while providing submillimeter spatial resolution and temporal resolution of 10 s. The shear modulus measured was found to be strongly correlated with the ECM fiber density in two types of cell culture system (r = 0.832 with P < 0.001, and r = 0.642 with P = 0.024 for cell culture systems containing 4 mg/ml Matrigel with 1 mg/ml and 2 mg/ml collagen type I hydrogel, respectively). Cell migration along the stiffness gradient in the cell culture system and an association between cell proliferation and the local ECM stiffness was observed. As the elasticity measurement is performed without the need of exogenous probes, the proposed method can be used to study how the microenvironmental stiffness interacts with cancer cell behaviors without possible adverse effects of the exogenous particles, and could potentially be an effective screening tool when developing new treatment strategies.

iPhone-imaged and cell-powered electrophoresis titration chip for the alkaline phosphatase assay in serum by the moving reaction boundary.

As a vital enzyme, alkaline phosphatase (ALP) has great clinical significance in diagnoses of bone or liver cancer, bone metastases, rickets, and extrahepatic biliary obstruction. However, there is still no really portable chip for the ALP assay in blood. Herein, a simple electrophoresis titration (ET) model was developed for ALP detection via a moving reaction boundary (MRB). In the model, ALP catalyzed the dephosphorylation of a 4-methylumbelliferyl phosphate disodium salt (4-MUP) substrate in the cathode well to 4-methylumbelliferone ([4-MU]-) with a negative charge and blue fluorescence under UV excitation. After the catalysis, an electric field was used between the cathode and the anode. Under the electric field, [4-MU]- moved into the channel and neutralized the acidic Tris-HCl buffer, resulting in the quenching of [4-MU]- and creating a MRB. The ET system just had an ET chip, a lithium cell, a UV LED and an iPhone used as a recorder, having no traditional expensive power supply and fluorescence detector. The relevant method was developed, and a series of experiments were conducted via the ET chip. The experiments showed: (i) a MRB could be formed between the [4-MU]- base and the acidic buffer, and the MRB motion had a linear relationship with the ALP activity, validating the ET model; (ii) the ET run was not impacted by many interferences, implying good selectivity; and (iii) the ET chip could be used for portable detection within 10 min, implying an on-site and rapid analysis. In addition, the ET method had a relatively good sensitivity (0.1 U L-1), linearity (V = 0.033A + 3.87, R2 = 0.9980), stability (RSD 2.4-6.8%) and recoveries (101-105%). Finally, the ET method was successfully used for ALP assays in real serum samples. All the results implied that the developed method was simple, rapid and low-cost, and had potential for POCT clinical ALP assays.